annual scientific exchange - harvard university · ama pra statement the ast designates this...

December 2-6, 2009Marriott World Center

Orlando, Floridawww.ast-ase.org

Program & Abstracts

American Society of Transplantation

Scientific Exchange

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Annual Scientific Exchange December 3-6, 2009

Marriott World Center Orlando, Florida

Table of Contents

Schedule Overview.............................................................................................................. ii ASE Program Planning Committee................................................................................... iii Invited Faculty List ............................................................................................................. iv Objectives, Accreditation, Certificates .............................................................................. v General Information ........................................................................................................... vi Exhibits .............................................................................................................................. vii Awards ................................................................................................................................ ix Sponsors/Supporters.......................................................................................................... x Pre-meeting Symposium Program.................................................................................. xiii ASE Program .................................................................................................................... xiv Poster Guide ................................................................................................................. xxviii Abstracts.............................................................................................................................. 1 Author Index ...................................................................................................................... 39 Keyword Index ................................................................................................................... 43

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Program Planning Committee

Executive Committee

Joren C. Madsen, MD, DPhil, Co-chair Harvard Medical School

Massachusetts General Hospital

Mohamed H. Sayegh, MD, Co-chair Harvard Medical School

Brigham and Women’s Hospital

Nader N. Najafian, MD Transplant Research Center, Harvard Medical School

David M. Rothstein, MD University of Pittsburgh

Committee

Mark L. Barr, MD University of Southern California

Gheetha Chalasani, MBBS

University of Pittsburgh

William E. Harmon, MD Harvard Medical School

Children’s Hospital Boston

Peter S. Heeger, MD Mount Sinai School of Medicine

Suzanne T. Ildstad, MD University of Louisville

Alan D. Kirk, MD, PhD

Emory University

Alexander HK Kroemer, MD University Hospital Regensburg

Jerzy Kupiec-Weglinski, MD, PhD David Geffen School of Medicine at UCLA

James F. Markmann, MD, PhD

Massachusetts General Hospital

Timothy M. Millington, MD Massachusetts General Hospital

Susan Moffatt-Bruce, MD, PhD

The Ohio State University

Flavio Vincenti, MD University of California, San Francisco

Hans-Dieter Volk, MD

University Medicine - Charite, Berlin

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Invited Faculty The following distinguished faculty serve as breakfast symposium presenters, session moderators, program

organizers, and/or keynote speakers.

E. Edward Baetge, PhD Novocell, Inc Mark L. Barr, MD University of Southern California Jeffrey Bluestone, PhD UCSF Diabetes Center Andrew M. Cameron, MD, PhD The Johns Hopkins University Gheetha Chalasani, MBBS University of Pittsburgh Peter Chin-Hong, MD University of California, San Francisco A. Benedict Cosimi, MD Harvard Medical School Massachusetts General Hospital Deborah M. Crowe, PhD DCI Laboratory Lara Danziger-Isakov, MD, MPH Cleveland Clinic Children's Hospital Anil Dhawan, MD King's College Hospital Rene Duquesnoy, PhD University of Pittsburgh Medical Center Sandy Feng, MD, PhD University of California San Francisco Jacques Galipeau, MD Emory University Robert S. Gaston, MD University of Alabama at Birmingham John S. Gill, MD, MS University of British Columbia, Vancouver Daniel R. Goldstein, MD Yale University Julia Greenstein, PhD Juvenile Diabetes Research Foundation

William E. Harmon, MD Harvard Medical School Children’s Hospital Boston Peter S. Heeger, MD Mount Sinai School of Medicine Kevan Herold, MD Yale University School of Medicine Donald E. Hricik, MD Case Western Reserve University Suzanne T. Ildstad, MD University of Louisville Hartmut Jaeschke, PhD University of Kansas Medical Center Maryl R Johnson, MD University of Wisconsin-Madison Alan D. Kirk, MD, PhD Emory University Stuart Knechtle, MD Emory University School of Medicine Daniel Kreisel, MD, PhD Washington University in St. Louis Alexander HK Kroemer, MD University Hospital Regensburg Jerzy Kupiec-Weglinski, MD, PhD David Geffen School of Medicine at UCLA Joren C. Madsen, MD, DPhil Harvard Medical School Massachusetts General Hospital Roslyn B. Mannon, MD University of Alabama at Birmingham James F. Markmann, MD, PhD Massachusetts General Hospital Herwig-Ulf Meier-Kriesche, MD University of Florida Stephen D. Miller, PhD Northwestern University Medical School

Timothy M. Millington, MD Massachusetts General Hospital Susan Moffatt-Bruce, MD, PhD The Ohio State University Barbara Murphy, MD Mount Sinai School of Medicine Nader N. Najafian, MD Transplant Research Center Harvard Medical School Camille Nelson Kotton, MD Harvard Medical School Massachusetts General Hospital Scott M. Palmer, MD, MHS Duke University Medical Center Chirag Parikh, MD, PhD Yale University David M. Rothstein, MD University of Pittsburgh Daniel R. Salomon, MD The Scripps Research Institute Abraham Shaked, MD University of Pennsylvania Elizabeth Trachtenberg, MS, PhD Children’s Hospital and Research Center Oakland Jacques P. Tremblay, PhD University of Laval Andrew D. Wells, PhD University of Pennsylvania School of Medicine Hans-Dieter Volk, MD University Medicine – Charite, Berlin

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Objectives, Accreditation, Certificates About the Program Each year ASE will focus on a single topic. In 2009 the topic is the spectrum of immunosuppression and tolerance. The science presented at ASE will cover research from the entire spectrum of immunosuppression and tolerance in relationship to basic science, laboratories, preclinical models, and clinical trials. Who Should Attend ASE is designed for all transplant professionals at all levels of experience, with an emphasis on young investigators. Accreditation Physicians The American Society of Transplantation (AST) is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing medical education for physicians. AMA PRA Statement The AST designates this educational activity for a maximum of 25.75 AMA PRA Category 1 Credit(s)™. Physicians should only claim those credits that he/she actually spent in the educational activity. Goals and Objectives At the conclusion of this activity, participants will be able to: • Determine the best immunosuppressive strategy for producing an optimal outcome. • Describe the pros and cons of the proliferation of minimization immunosuppression trials. • Identify what updates are needed to clinical tolerance protocols. • Determine what the extensive research on biomarkers tells us about predicting outcomes based on

immunosuppressive strategies. • Discuss the latest in basic research and preclinical animal models. Statement of Disclosure All faculty and planners participating in this continuing medical education program sponsored by AST are expected to disclose to the program audience any/all relevant financial relationships with commercial entities related to the content of their presentation(s). Faculty with relevant financial relationships are listed in the disclosure document in your conference bag. All potential conflicts of interest have been resolved satisfactorily. All others indicated they had no such relevant financial relationships. Abstract disclosures, if any, are listed at the end of each abstract. Physician Credit After the meeting, physicians will receive a link via email that will direct you to a CME Web site. You will complete a brief evaluation, claim your credit, and print your certificate. Other Transplant Professionals Physician credit is the only type of credit being offered for this meeting. You will receive an email after the meeting that will direct you to an evaluation form and you will be able to print out a general certificate of attendance.

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General Information Name Badge Your name badge must be worn at all times to gain access to the ASE Learning Pavilion, educational sessions, breakfast symposia, and other activities. Registered guests must also wear their name badges at all times. The registration desk is located outside of the ASE Learning Pavilion. Pre-meeting Symposium The pre-meeting symposium is separate from the main ASE meeting and carries an additional registration fee. There is no continuing education credit offered for the pre-meeting symposium; goals and objectives are listed below. If you only registered for the pre-meeting symposium, you will not be allowed access to the ASE Learning Pavilion (exhibit hall) or ASE sessions. Please see the registration desk to register for the ASE meeting. After attending this program, participants will be able to understand and discuss:

• The basic immunology of solid organ transplantation including T-cell and B-cell recognition of alloantigens, signaling pathways involved in lymphocyte activation and the pathologic manifestations of rejection injury in solid organ transplants

• The mechanisms by which cellular and/or humoral immunity and inflammation acutely damage transplanted kidneys, livers, hearts, lungs and other solid organs

• The long-term complications of transplantation relative to chronic immunosuppressive therapy and chronic rejection

• The principles of modern immunosuppression and new strategies in immunosuppression. • Emerging areas in clinical and basic science transplantation research that may provide novel targets for

immunosuppression and tolerance Posters and Oral Poster Exchange Sessions Posters will be displayed in three separate poster sessions. The poster exchange session is on Thursday, December 3, and the oral poster exchange sessions are on Friday, December 4 and Saturday, December 5. For the oral poster exchange sessions, the posters that are on display in the ASE Learning Pavilion during the day will be discussed in four concurrent sessions that evening. Poster Exchange Session (poster session I) – Thursday, December 3 Poster hanging hours: 1:00 pm – 4:00 pm Poster display hours: 4:45 pm – 6:15 pm Attendance hours: 5:00 pm – 6:00 pm Tear down: 6:15 pm – 7:00 pm Oral Poster Exchange Sessions – Friday, December 4 (poster session II) and Saturday, December 5 (poster session III) Poster hanging hours: 8:00 am – 10:00 am Poster display hours: 10:00 am – 6:15 pm Oral Poster Exchange presentation time: 6:15 pm – 7:15 pm Tear down: 4:30 pm – 7:30 pm Find posters in the ASE Learning Pavilion by referring to the “P” numbers listed in the program next to each poster each day. The poster abstracts can be found by their abstract number near the back of this book. Speaker Ready Room The speaker ready room is located in Grand Ballroom 7B. Hours are listed below. Please check in as soon as you arrive at the meeting, but no later than 3 hours prior to the start of your session. All slides must be uploaded in the Speaker Ready Room. Personal laptops cannot be used in the session rooms. Slides will not be loaded in the session rooms. Wednesday, December 2, 11:00 am – 6:00 pm Thursday, December 3, 9:00 am – 6:00 pm Friday, December 4, 6:30 am – 6:00 pm Saturday, December 5, 6:30 am – 6:00 pm Sunday, December 6, 6:30 am – 10:30 am

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Awards

Congratulations to the following recipients of the ASE Young Innovator Award: Francesca D'Addio

Sussan Dejbakhsh-Jones Qing Ding

Boonsong Kiangkitiwan Karen M. Kim Tetsu Oura

Simon Urschel Xiaozhi Zhao

Additionally, AST leadership is proud to support the following young professionals

with a travel stipend to ASE: Carlos E. Benitez

Harish C. Chandramoorthy Luis P. Fernandez

Mandy L. Ford Julia Fang Gao Jay A. Graham Yanfei Huang

Haseeb Ilias Basha Haofeng Ji

Li Li Delia Lozano Porras

K. A. Mohammad Deepti Saini Hua Shen

Miao Wang Toshihiko Watanabe

Jun Yang Wensheng Zhang

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ASE Supporters

The AST Board of Directors and the ASE Program Planning Committee thank the following companies who have provided support for this educational activity:

Company Support of this activity

Astellas Pharma US, Inc. $100,000

CSL Behring $20,000

The AST Board of Directors and the ASE Planning Committee thank the following companies for supporting the evening activities held in conjunction

with this meeting:

Company Support

Bristol-Myers Squibb $100,000

Genzyme $100,000

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AST Supporters

The AST Board of Directors and the Development Committee thank the following companies who have confirmed their commitment to support

the AST’s many educational activities:

DIAMOND Astellas Pharma US, Inc.

PLATINUM

Roche Laboratories Inc.

GOLD Wyeth

SILVER

Bristol-Myers Squibb Company Novartis Pharmaceuticals Corporation

BRONZE

Genzyme Corporation

PATRON Isotechnika Inc.

SUPPORTER

ViraCor Laboratories

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AST’s Annual Scientific Exchange December 2-6, 2009

Marriott World Center Orlando, Florida, USA

Pre-meeting Symposium: Update in Solid Organ Transplantation

Grand Ballroom 7A

Wednesday, December 2, 2009 11:00 am – 6:00 pm Registration Palms Ballroom Foyer 12:50 pm – 1:00 pm Opening Remarks Donald E. Hricik, MD 1:00 pm – 3:00 pm Update Session I Chair: Roslyn B. Mannon, MD 1:00 pm Basic Transplant Immunology: T Cells, B Cells, and Other Components of Alloimmunity Peter S. Heeger, MD 1:30 pm Novel Targets for Immunosuppression and Tolerance Allan D. Kirk, MD, PhD 2:00 pm Outcomes in Organ Transplantation: All Organs John S. Gill, MD, MS 2:30 pm Trends in Immunosuppression: All Organs Donald E. Hricik, MD 3:00 pm Break 3:15 pm – 5:45 pm Update Session II Chair: Donald E. Hricik, MD 3:15 pm – 5:15 pm Long-term Complications of Transplantation

3:15 pm Kidney Roslyn B. Mannon, MD 3:45 pm Liver Sandy Feng, MD, PhD 4:15 pm Heart Mark L. Barr, MD 4:45 pm Lung Scott M. Palmer, MD

5:15 pm New Immunosuppressants in the Pipeline Herwig-Ulf Meier-Kriesche, MD 5:45 pm Adjourn

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AST’s Annual Scientific Exchange December 2-6, 2009

Marriott World Center Orlando, Florida, USA

ASE Scientific Program

All sessions held in Palms Ballroom: Royal unless otherwise noted.

Learning Pavilion and posters are located in Palms Ballroom: Sago and Sabal.

Thursday, December 3, 2009 8:00 am – 7:15 pm Registration Palms Ballroom Foyer 1:45 pm – 2:00 pm Welcome Remarks Joren C. Madsen, MD, DPhil and Mohamed H. Sayegh, MD 2:00 pm – 2:45 pm Keynote Presentation Tolerance: Lessons from the Immune Tolerance Network Jeffrey A. Bluestone, PhD 2:45 pm – 4:45 pm Oral Plenary Exchange I Translational Science I and Clinical Science I: Biomarkers/Monitoring Moderators: Joren C. Madsen, MD, DPhil and Mohamed H. Sayegh, MD 2:45 pm Inducing Tolerance in Clinical Kidney Transplantation (Abstract #1) J. Scandling 3:00 pm Identification of a Peripheral Blood Transcriptional Biomarker Panel for Diagnosis and Prediction of Renal Allograft Tolerance (Abstract #6) Li Li 3:15 pm Gene Arrays May Predict Stable Clinical Kidney Tolerance and Support Safe Immunosuppression Withdrawal (Abstract #3) Minne M. Sarwal 3:30 pm Elevated HLA-G and ILT4 Expression on Circulating Dendritic Cell Subsets in Pediatric Liver Transplant Recipients Successfully Weaned Off Immunosuppression (Abstract #2) Antonino Castellaneta 3:45 pm Pre-Transplant Analysis of Whole Blood Toag-1 Gene Expression and CD4+CD45RO-CD62L- TEMRA Frequencies Identifies High Risk Patients (Abstract #4) Birgit Sawitzki 4:00 pm Pre-Transplant Immune Regulation Due to Fetal and Maternal Antigen Exposure (Abstract #5) William J. Burlingham 4:15 pm Clinical Outcome of Liver Transplant Recipients after Successful Immunosuppression Withdrawal (Abstract #7) Panagiotis Tryphonopoulos

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4:30 pm Use of Transcriptional Biomarkers To Identify Liver Transplant Recipients Who Can Successfully Discontinue Immunosuppressive Therapy (Abstract #8) Carlos E. Benitez 4:30 pm – 6:30 pm Learning Pavilion Open 4:45 pm – 6:15 pm Poster Exchange and Welcome Reception See page xxviii for a list of posters on display 6:30 pm ASE “UNconference” Grand Ballroom 8A and 8B

Friday, December 4, 2009 7:00 am – 7:15 pm Registration Palms Ballroom Foyer 7:00 am – 8:20 am Concurrent Joint Breakfast Symposia

AST-ILTS Symposium

Inflammation and Tolerance in Liver Transplantation Grand Ballroom 7A Program Organizers: Andrew Cameron, MD, PhD Jerzy Kupiec-Weglinski, MD James Markmann, MD, PhD Session Co-chairs: Dr. Cameron and Dr. Markmann Innate Immunity in Liver Inflammation and Hepatocellular Damage Hartmut Jaeschke PhD From Bench to Bedside: Can We Prevent the Ischemia/Reperfusion Insult in Liver Transplant Recipients? Jerzy Kupiec-Weglinski, MD The Liver Tolerance Conundrum Avi Shaked, MD

AST-ASN Symposium

Biomarkers that Predict Outcome in Renal Transplantation Grand Ballroom 8A Program Organizers: William E. Harmon, MD Nader N. Nafian, MD David Rothstein, MD Session Co-chairs: Dr. Harmon and Dr. Rothstein Markers Involved in Immediate vs. Delayed Graft Function Chirag Parikh, MD, PhD Markers of Tolerance: Data from the European Consortium Hans-Dieter Volk, MD Markers of Chronic Allograft Nephropathy Daniel R. Salomon

AST-ISHLT Symposium

Role of Innate Immunity in Thoracic Transplantation Grand Ballroom 8B Program Organizers: Mark L. Barr, MD Susan Moffatt-Bruce, MD, PhD Scott Palmer, MD Session Co-chairs: Dr. Moffatt-Bruce and Dr. Palmer Innate Immune Responses in Allograft Rejection Daniel R. Goldstein, MD Implications of Innate Immunity in Achieving Tolerance Joren C. Madsen, MD, DPhil Role of Neutrophilic Inflammation in Orthotopic Murine Lung Transplant Rejection Daniel Kreisel, MD, PhD

8:30 am – 10:15 am Oral Plenary Exchange II Innate Immunity Moderators: Daniel Goldstein, MD and Alexander HK Kroemer, MD 8:30 am DHMEQ, a New NF-κB Inhibitor, Suppresses Innate and Subsequent Allo-Immune Responses, and Permits a Long-Term Allogeneic Islet Engraftment Together with Tacrolimus (Abstract #9) Masaaki Watanabe

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8:45 am T Cell Ig Mucin (TIM) Pathways Regulate Liver Ischemia and Reperfusion Injury (Abstract #10) Yoichiro Uchida 9:00 am Withdrawn (Abstract #11) 9:15 am The Role of PD-1:B7-H1 T Cell Co-Stimulation Signaling in Liver Innate Pro- Inflammatory Immune Response (Abstract #12) Haofeng Ji 9:30 am DAP12 Expression in Liver Dendritic Cells Controls Their Maturation and the Induction of Tr-1 Cells (Abstract #13) Tina L. Sumpter 9:45 am Relationship between NK Cells, Regulatory T Cells, and Effector T Cells in the Development of Coronary Allograft Vasculopathy (CAV) in Mouse Heart Transplants (Abstract #14) Tsutomu Hirohashi 10:00 am The Role of Complement Activation on Tissue Perfusion and Oxygenation in Airway Allograft Rejection (Abstract #15) K A. Mohammad 10:00 am – 6:15 pm Learning Pavilion Open 10:15 am – 11:00 am Networking Break in Learning Pavilion 11:00 am – 12:30 pm Oral Plenary Exchange III Preclinical and Translational Sciences II Moderators: John S. Gill, MD and James F. Markmann, MD, PhD 11:00 am IFN-γ Negatively Regulates While IL-6 and IL-1β Promote the Induction of Hepatitis C Virus (HCV) Specific Th17 Cells in Liver Transplant Recipients with HCV Recurrence (Abstract #16) Haseeb Ilias Basha 11:15 am Donor-Specific B-Cell Tolerance after ABO-Incompatible Heart Transplantation Is Facilitated by Limited B-Cell Memory in Early Childhood (Abstract #17) Simon Urschel 11:30 am Development of Immune Responses to Self Antigens (K-α1 Tubulin & CollagenV) Following Human Lung Transplant Plays a Pivotal Role in the Pathogenesis of Chronic Rejection (Abstract #18) Deepti Saini 11:45 am Donor-Reactive Memory T Cells in Cynomolgus Monkeys Are Critical to the Rejection of and Tolerance to Allografts (Abstract #19) Gilles Benichou Noon Non-Human Primate Lung Allografts Show Enhanced Immunogenicity as Compared to Analogous Kidney Allografts (Abstract #20) Akihiro Aoyama (presented onsite by Timothy Millington) 12:15 pm Withdrawn (Abstract #21)

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12:30 pm – 1:50 pm Poster Luncheon in Learning Pavilion 2:00 pm – 3:45 pm Oral Plenary Exchange IV Clinical Science II Moderators: Stuart Knechtle, MD and Susan Moffatt-Bruce, MD, PhD 2:00 pm Withdrawn (Abstract #22) 2:15 pm Antibody-Mediated Rejection (AMR) after Pancreas and Pancreas-Kidney Transplantation (Abstract #23) Erika B. Rangel 2:30 pm Autoantibody Conversion Is a Risk Factor for Recurrence of Type 1 Diabetes (T1DR) after Simultaneous Pancreas-Kidney (SPK) Transplantation (Abstract #24) George W. Burke 2:45 pm Primary Outcomes from a Randomized, Phase III Study of Belatacept Versus Cyclosporine in Kidney Transplant Recipients (BENEFIT Study) (Abstract #25) Thomas Pearson 3:15 pm A Single Center Study of the Use of Campath-1H Induction in Adult Liver Transplantation: Long-Term Follow Up of 203 Patients (Abstract #26) Panagiotis Tryphonopoulos 3:30 pm The Impact of Sirolimus on Hepatitis C Recurrence after Liver Transplantation (Abstract #27) Sonal Asthana 3:45 pm Primary Outcomes from a Randomized, Phase III Study of Belatacept Versus Cyclosporine in ECD Kidney Transplants (BENEFIT-EXT Study) (Abstract #28) Sander Florman 3:45 pm – 4:30 pm Networking Break in Learning Pavilion 4:30 pm – 6:00 pm Oral Plenary Exchange V B Cells and T Cells Moderators: Peter S. Heeger, MD and Allan D. Kirk, MD, PhD 4:30 pm Prolonged Allograft Survival and Impaired Alloreactive CD8 T Lymphocyte Memory Response in Sensitized Recipients in the Absence of Humoral Immunity (Abstract #29) Haofeng Ji 4:45 pm B Cells Promote Development of Alloreactive Memory T Cells (Abstract #30) Harish C. Chandramoorthy 5:00 pm In Vivo BLyS Neutralization Induces Robust Transplantation Tolerance by Down-Regulating APC Costimulation and Polarizing T-Cell Cytokine Profiles (Abstract #31) Ronald Parsons 5:15 pm The Novel Role of B7.1, as an Alternate PDL1 Receptor, in Regulating Alloimmune Responses In Vivo (Abstract #32) Jun Yang (presented onsite by Anil Chandraker)

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5:30 pm An IL-21 Receptor-Targeted Antagonizing IL-21/Fc Protein Promotes Allograft Tolerance (Abstract #33) Wensheng Zhang 5:45 pm Anti-LFA-1 Synergizes with CD28/CD154 Blockade To Inhibit Anti-Donor Cytotoxicity and Promote Graft Survival in Recipients Possessing Donor- Reactive Memory T Cells (Abstract #34) Mandy L. Ford 6:15 pm – 7:15 pm Concurrent Oral Poster Exchange Sessions Oral Poster Exchange 1 – Experimental Tolerance Canary 1 Moderators: Mark L. Barr, MD and Hans-Dieter Volk, MD 6:15 pm P1 Skin Graft Rejection: Direct or Semi-Direct Allorecognition? (Abstract #76) Georges Tocco 6:25 pm P2 Withdrawn (Abstract #77) 6:35 pm P3 Infection with the Intracellular Bacterium, Listeria Monocytogenes, Transiently Overrides Established Allograft Tolerance (Abstract #78) Tongmin Wang 6:45 pm P4 Cardiac Allograft Survival Is Prolonged by the Induction of Mixed Hematopoietic Chimerism Two Months after Heart Transplant (Abstract #79) Timothy M. Millington 6:55 pm P5 Regeneration of Naïve-Lymphocytes from Heart-Thymus Grafts Occurs at a Low Level and Is Associated with Prolonged Survival but Not Tolerance (Abstract #80) Timothy M. Millington 7:05 pm P6 Sensitization Prior to Composite Tissue Allotransplantation in Rat Model Does Not Result in Hyperacute Rejection (Abstract #81) Shengli Wu Oral Poster Exchange 2 – T Cells and T Regs I Canary 2 Moderators: Deborah M. Crowe, PhD and Roslyn B. Mannon, MD 6:15 pm P7 Expansion of CD4+CD25+FoxP3+ Regulatory T Lymphocytes with Low-Dose Anti-Thymocyte Globulin Leads to Prolonged Survival of Cardiac Allografts but Not Tolerance (Abstract #82) Timothy M. Millington 6:25 pm P8 Easiness To Generate Is Compensated with Limited Therapeutic Applicability (Abstract #83) Arthur Andakyan (presented onsite by Sigrid Burruss) 6:35 pm P9 Generation of Human Tregulatory Cells with the Capacity To Suppress Islet Allograft Rejection in a Xenogeneic Mouse Model (Abstract #84) Alicia N. McMurchy 6:45 pm P10 Effect of FTY720 Treatment on CD4+ T Cell-Endothelial Cell Interactions in Ischemia/Reperfusion Injury of the Pancreas (Abstract #85) Dirk Uhlmann

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6:55 pm P11 Withdrawn (Abstract #86) 7:05 pm P12 Ex-Vivo Expanded CD4+CD25+ Treg Cells Suppress T and B Immune Response (Abstract #87) Avneesh K. Singh Oral Poster Exchange 3 – T Cells and T Regs II Canary 3 Moderators: Alexander HK Kroemer, MD and Jerzy Kupiec-Weglinski, MD, PhD 6:15 pm P13 Both Rejection and Tolerance of Allogeneic Transplants Can Occur in the Absence of Secondary Lymphoid Organs (Abstract #88) Gilles Benichou 6:25 pm P14 Suppressing Antigen-Bearing Dendritic Cells by Double Negative Regulatory T Cells (Abstract #89) Julia Fang Gao 6:35 pm P15 Mechanisms of Immunological Tolerance Induced by Double Negative Regulatory T Cells in a Semiallogeneic Model of Graft-Versus-Host Disease (Abstract #90) Stephen C. Juvet 6:45 pm P16 Synergistic Effect of CsA and Tregs in Preventing Memory T Cell Induced Tolerance Abrogation (Abstract #91) Anja Siepert 6:55 pm P17 Indoleamine 2,3-Dioxygenase (IDO) and Treg Support Are Critical for CTLA4Ig Mediated Tolerance Induction to Solid Organ Allografts (Abstract #110) Robert Sucher (formerly P11 in poster session III) 7:05 pm P18 A Novel Strategy and Unique Model for Composite Tissue Allotransplantation Tolerance (Abstract #93) Rishi Jindal (presented onsite by Xin Xiao Zheng) Oral Poster Exchange 4 – Experimental Tolerance, Innate Immunity, Tolerance Protocols Canary 4 Moderators: Kevan Herold, MD and Stephen D. Miller, PhD 6:15 pm P19 Studies on Some Immune Properties of the Pancreatic Progenitor Cells Derived from Human Fetal Pancreas (Abstract #94) Man Ting Ma 6:25 pm P20 HMGB1 Regulates Tubular Epithelial Cell (TEC) Expression of MCP-1 in Ischemic Kidney Injury (Abstract #95) Arthur Lau 6:35 pm P21 Serum Anti-MHC Immunoglobulins Ineffective in Prevention of Acute Graft Rejection (Abstract #96) Arthur Andakyan (presented onsite by Sigrid Burruss) 6:45 pm P22 Withdrawn (Abstract #97) 6:55 pm P23 Lentiviral Transduction of Face and Hand Allografts: Implications for Immunomodulation of Composite Tissue (Abstract #98) Angelo A. Leto Barone

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7:05 pm P24 Allogeneic Porcine CD4+CD25

+ T Cells Regulate the Development of

Transplant Arteriosclerosis in Porcinized Mice (Abstract #99) Gregor Warnecke 7:30 pm Dinner Satellite Symposium Novel Immunomodulatory Strategies to Improve Long-Term Transplant Outcomes Grand Ballroom 8A and 8B This CME-Certified dinner symposium is presented for attendees of the AST Annual Scientific Exchange. This event is sponsored by Education Outcomes Science and supported by Bristol- Myers Squibb. This is not an official function of the Annual Scientific Exchange.

Saturday, December 5, 2009 7:00 am – 7:15 pm Registration Palms Ballroom Foyer 7:00 am – 8:20 am Concurrent Joint Breakfast Symposia

AST-TTS Symposium

Everything You Need to Know about H1N1 Grand Ballroom 7A Program Organizer: Camille Nelson Kotton, MD Session Co-Chairs: William E. Harmon, MD Camille Nelson Kotton, MD Best Vaccination, Prophylaxis, and Treatment Practices for the Solid Organ Transplant Population Camille Nelson Kotton, MD Optimal Diagnostics and Infection Control: Novel H1N1 and Pediatric Solid Organ Transplant Lara Danziger-Isakov, MD Preventing Donor Derived Infections in the Time of Influenza Peter Chin-Hong, MD

AST-JDRF Symposium

Merging the Stem Cell Experience with Organ Transplantation: Lessons to Be Learned from Islet Cells Grand Ballroom 8A Program Organizers: Julia Greenstein, PhD Suzanne Ildstad, MD James Markmann, MD, PhD Session Co-Chairs: Dr. Greenstein and Dr. Ildstad Novel Therapies for Treatment of T1D Kevan Herold, MD ECDI-fixed Allogeneic Splenocytes Induce Long-term Tolerance Without Lymphoid Ablation in Islet Transplantation Stephen D. Miller, PhD Making Beta Cells from Stem Cells E. Edward Baetge, PhD

AST-ASHI Symposium

Novel Approaches in Transplant Medicine via Applied Genetics Grand Ballroom 8B Program Organizers: Deborah M. Crowe, PhD Alexander HK Kroemer, MD Hans-Dieter Volk, MD, PhD Session Co-chairs: Dr. Crowe and Dr. Kroemer HLA and KIR Polymorphisms in Natural Killer Cell Repertoire Selection and Modulation of Effector Function Elizabeth Trachtenberg, PhD Epigenetic Regulation Andrew D. Wells , PhD Structurally Based HLA Matching in Identifying Acceptable and Permissible HLA Mismatches Rene J. Duquesnoy, PhD

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8:30 am – 10:15 am Oral Plenary Exchange VI Regulatory T cells Moderators: A. Benedict Cosimi, MD and Daniel Kreisel, MD, PhD 8:30 am Withdrawn (Abstract #35) 8:45 am Pretransplant Infusion of Donor B Cells Enhances Donor-Specific Allograft Survival through Activating Double Negative Regulatory T Cells (Abstract #36) Julia Fang Gao 9:00 am CD14 and CD36 Restrain Inducible T reg Generation and Prevent Costimulatory Blockade Extension of Allograft Survival (Abstract #37) Hua Shen 9:15 am Inhibition of the Kinase, GSK-3beta, Potentiates the Suppressive Function of Regulatory T Cells by Decreasing Cellular Death (Abstract #38) Jay A. Graham 9:30 am Treg Treatment Leads to Heart and Skin Graft Tolerance in a Non- Cytotoxic Murine Mixed Chimerism Model (Abstract #39) Nina Pilat 9:45 am Spontaneous Renal Allograft Acceptance in Mice Requires Foxp3 Cells (Abstract #40) Masahiro Miyajima 10:00 am Mice with Constitutively Active STAT5b Generate Powerful and Stable Treg (Abstract #41) Qiang Zhou 10:00 am – 6:15 pm Learning Pavilion Open 10:15 am – 11:00 am Networking Break in Learning Pavilion 11:00 am – 12:30 pm Oral Plenary Exchange VII Translational Science III Moderators: Barbara Murphy, MD and Hans-Dieter Volk, MD 11:00 am Immunoprofiling after Profound T Cell Depletion and Minimization of Immunosuppression in Pediatric Kidney Transplantation: A Study of CCTPT/NIAID (Abstract #42) William Harmon 11:15 am Broad Autoantibody Responses in Chronic Humoral Rejection of Human Kidney Allografts (Abstract #43) Emmanuel Zorn 11:30 am A Peripheral Blood 12 Gene-Set for Diagnosis of Pediatric Liver Allograft Tolerance (Abstract #44) Li Li 11:45 am Withdrawn (Abstract #45) 12:00 pm Withdrawn (Abstract #46) 12:15 pm Antibodies to Non-HLA Antigens Are Associated with Chronic Allograft Injury (Abstract #47) R. Dinavahi

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12:30 pm – 1:50 pm Poster Luncheon in Learning Pavilion 2:00 pm – 3:45 pm Oral Plenary Exchange VIII Basic Science: Stem Cells, Facilitiation Cells and Mixed Chimerism Moderators: Geetha Chalasani, MBBS and Suzanne T. Ildstad, MD 2:00 pm Embryonic Stem Cell-Based Therapy for Transplantation Tolerance (Abstract #48) Luis P. Fernandez 2:15 pm Kidney-Derived Mesenchymal Stem Cells Induce Tolerogenic Dendritic Cells That Inhibit Alloresponses and Prevent Allograft Rejection (Abstract #49) Yanfei Huang (presented onsite by Karl Womer) 2:30 pm Induction of Chimerism in Diabetic NOD Mice Results in Immune Tolerance of Donor Islets, Reduction of the Required Amount of Donor Islets for Reversing Diabetes, and Excellent Long-Term Graft Function (Abstract #50) Miao Wang 2:45 pm Hematopoietic and Pluripotent Stem Cells Acquired during Pregnancy Give Rise to Wide-Spread Maternal Microchimerism Required for Tolerance to Non-I nherited Maternal Antigens (Abstract #51) Partha Dutta 3:00 pm Evidence That Innate Immunity Is Tolerized in Mixed Chimeras (Abstract #52) Hong Xu 3:15 pm Chimeric Treg Potently Enhance Donor Chimerism and Tolerance: The Acquisition of FoxP3 Expression Is Directly Correlated with Treg Function In Vivo (Abstract #53) Yiming Huang 3:30 pm Induction of Local Hyporesponsiveness for Cell Transplants (Abstract #54) Shiguang Qian 3:45 pm – 4:30 pm Networking Break in Learning Pavilion Room: Palms Ballroom - Sago and Sabal 4:30 pm – 6:00 pm Oral Plenary Exchange IX Clinical Science III Moderators: Andrew Cameron, MD, PhD and Herwig-Ulf Meier-Kriesche, MD 4:30 pm Predicting Recurrence after Liver Transplantation in Patients with Hepatocellular Carcinoma Exceeding the Up-to-Seven Criteria (Abstract #55) Francesco D'Amico 4:45 pm Renal Benefit of Belatacept Versus Cyclosporine in Kidney Transplant Patients Is Not Impacted by Acute Rejection (BENEFIT Study) (Abstract #56) Barbara Bresnahan 5:00 pm Alemtuzumab Induction May Exacerbate Antibody-Mediated Rejection in Pediatric Renal Transplant Recipients (Abstract #57) George C. Wu 5:15 pm The Effect of Alemtuzumab Induction on CMV Seroconversion and Infection after Valganciclovir Prophylaxis in Solid Organ Transplant Recipients (Abstract #58) Yashaswini Rangan

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5:30 pm Long-Term Experience in Patients Receiving Infliximab (Abstract #59) Undine A. Gerlach 5:45 pm Improvement of Glycometabolic Control after Islet Transplantation Is Associated with a Better Function of Endothelial Progenitor Cells in Type 1 Diabetic Patients (Abstract #60) Alessandra Petrelli 6:15 pm – 7:15 pm Concurrent Oral Poster Exchange Sessions Oral Poster Exchange 5 – Tolerance/Immunological Monitoring and Biomarkers Canary 1 Moderators: Joren C. Madsen, MD, PhD and Chirag Parikh, MD, PhD 6:15 pm P1 Establishment of Local Tolerance for Cell Transplants, toward Clinical Application (Abstract #100) Lina Lu 6:25 pm P2 High Levels of IDO-Expressing CD16+ Peripheral Cells, and Tregs in Graft Biopsies from Kidney Transplant Recipients under Belatacept Treatment (Abstract #101) Janette Furuzawa-Carballeda 6:35 pm P3 Endothelial Cells Co-Cultured with Embryoid Bodies Enhance Insulin-Producing beta-Cell Differentiation through BMP-2 Signaling (Abstract #102) Dodanim Talavera-Adame 6:45 pm P4 Effect of Rituximab on the Regulation of Sphingomyelinase-Like Phosphodiesterase 3b-Precursor To Prevent FSGS Recurrence after Renal Transplantation (Abstract #103) Junichiro Sagheshima 6:55 pm P5 Indoleamine 2,3-Dioxygenase (IDO) Immune Status Monitoring Post- Transplantation (Abstract #104) Vikas Dharnidharka 7:05 pm P6 Liver Transplant Recipients Successfully Weaned Off Immunosuppression Lack Donor-Specific Anti-HLA Antibodies (Abstract #105) Antonino Castellaneta Oral Poster Exchange 6 – T Cells and T Regs III Canary 2 Moderators: Suzanne T. Ildstad, MD and Allan D. Kirk, MD, PhD

6:15 pm P7 Pro-Inflammatory Cytokines Modulate the Ability of Regulatory T Cells To Control Alloimmunity (Abstract #106) Giorgio Raimondi (presented onsite by Tina Sumpter) 6:25 pm P8 Transplantation Tolerance to Non-Inherited MHC Class I Maternal Antigens: New Insights Using TCR Transgenic Models (Abstract #107) Gilles Benichou 6:35 pm P9 Pregnant Level Estrogen and Progestone Affect the Regulatory T Cells in Mouse (Abstract #108) Xing-Guang Lin 6:45 pm P10 Withdrawn (Abstract #109)

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6:55 pm P11 CD200 Immunomodulation Increases Tregs and Prolongs CTA Survival (Abstract #92) Rishi Jindal (formerly P17 in poster session II) 7:05 pm P12 Anti-HLA Abs along with Defensins Contribute to BOS Pathogenesis (Abstract #111) Deepti Saini Oral Poster Exchange 7 – Minimization Strategies and Chronic Rejection Canary 3 Moderators: Mark L. Barr, MD and Barbara Murphy, MD 6:15 pm P13 Minimization of Immunosuppression in Primary Adult Liver Transplantation with Steroid-Free Regimen Using Low Dose of Tacrolimus with Mycophenolate Mofetil after Daclizumab Induction (Abstract #112) Olivier Boillot 6:25 pm P14 Feasibilty of Long-Term Sirolimus Monotherapy after Liver Transplantation (Abstract #113) Dirk Uhlmann 6:35 pm P15 Excellent Outcomes with Alemtuzumab Induction and Steroid-Free Maintenance Compared to Interleukin-2 Receptor Antagonist or Thymoglobulin with Prednisone Maintenance (Abstract #114) Jimmy A. Light (presented onsite by Elliot Zahtz) 6:45 pm P16 Alemtuzumab Induction, Steroid-Free Maintenance Immunosuppression in African-Americans vs Non-African Americans (Abstract #115) Jimmy A. Light (presented onsite by Elliot Zahtz) 6:55 pm P17 The Significance of Immune Cell Function Testing in Renal Transplantation (Abstract #116) Vaughn E. Whittaker 7:05 pm P18 Bronchoalveolar Lavage CD4+ Fox P3+ T Cells Are Decreased in Lung Transplant Recipients with Bronchiolitis Obliterans Syndrome (Abstract #117) Sangeeta M. Bhorade Oral Poster Exchange 8 - Clinical Science, Chronic and Antibody-mediated Rejection Canary 4 Moderators: Robert S. Gaston, MD and Maryl R. Johnson, MD 6:15 pm P19 Liver Function Tests Do Not Correlate with Liver Biopsy Results in Pediatric Patients Who Have Undergone Liver Transplantation (Abstract #118) Jason D. Fraser (presented onsite by Walter Andrews) 6:25 pm P20 Antibody Mediated Rejection Secondary to Anti-DP Antibodies in a Kidney Transplant Recipient (Abstract #119) Maria P. Martinez Cantarin 6:35 pm P21 Machine Perfusion in Kidney Transplantation: Lower Rate of Acute Rejection in the Presence of Longer Pump Time, Regardless of Immunosuppression (Abstract #120) Gaetano Ciancio (presented onsite by Giselle Guerra) 6:45 pm P22 Socioeconomic Status and Acute Rejection in Kidney Transplant Recipients (Abstract #121) S. Lodhi

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6:55 pm P23 The Effect of Change or Modulation of Maintain Immunosuppressive Regimen on Long-Term Transplant Result (Abstract #122) Myoung Soo Kim 7:05 pm P24 Unusual Lymphoma and Concurrent Presentation of Graft Arteriopathy in a Hand Transplant Recipient Three Years Post Transplant (Abstract #123) Christina L. Kaufman 7:30 pm Dinner Satellite Symposium Rejection in Kidney Transplantation: Evolving Concepts and Implications for Clinical Management Grand Ballroom 8A and 8B This CME-Certified dinner symposium is presented for attendees of the AST Annual Scientific Exchange. This event is sponsored by CTI and supported by Genzyme. This is not an official function of the Annual Scientific Exchange.

Sunday, December 6, 2009 7:00 am – 1:00 pm Registration Palms Ballroom Foyer 7:00 am – 8:20 am Concurrent Joint Breakfast Symposia

AST-ASTS Symposium

Tolerance: A View From the Front Line Room: Grand Ballroom 7A Program Organizers: Alan D. Kirk, MD, PhD Timothy M. Millington, MD Sandy Feng, MD, PhD Session Co-chairs: Dr. Kirk and Dr. Millington What Makes a Tolerant Recipient: Conditions Influencing the Development of Tolerance Sandy Feng, MD, PhD What Makes a Pro-tolerant Therapy Stuart Knechtle, MD Clinical Tolerance: What the Clinical Experience Tells Us About What Needs to be Done Experimentally A. Benedict Cosimi, MD

AST-CTS Symposium

Cell Transplantation and Immune Modulation Room: Grand Ballroom 8A Program Organizers: Geetha Chalasani, MD Peter S. Heeger, MD Ole Isacson, PhD Session Co-chairs: Dr. Chalasani and Dr. Heeger Mesenchymal Stem Cells in Suppression of Inflammation, Tissue Repair and Immune Modulation Jacques Galipeau, MD Hepatocyte Transplantation Experience in Patients Anil Dhawan, MD Mixed Chimerism and Immunological Tolerance in Myoblast Transplantation Jacques P. Tremblay, PhD

8:30 am – 10:15 am Oral Plenary Exchange X Basic Science: T Cells and Aging and the Alloimmune Response Moderators: Timothy M. Millington, MD and Daniel R. Salomon, MD 8:30 am Donor Age Is Associated with Elevated Numbers of Passenger Leukocytes Augmenting the Recipients Immune Response (Abstract #61) Christian Denecke

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8:45 am Recipient Age Is Associated with an Impaired CD4+

Effector and Intact Regulatory T-Cell Response (Abstract #62) Christian Denecke 9:00 am Delayed Acute Allograft Rejection in Aged Transplant Recipients Is Associated with Age-Dependent Increase of Memory T Cells with Impaired Function (Abstract #63) Xupeng Ge 9:15 am MHC Class II Gene Therapy: A Novel Approach to Donor-Specific Tolerance without Sustained Immunosuppression (Abstract #64) Christian LeGuern 9:30 am Opposing Roles for T Cell-NF-κB and TLR Signals in the Fate of Islet Allografts (Abstract #65) Delia Lozano Porras 9:45 am Regulatory Function of Galectin-9 in Allograft Rejection and Acquired Tolerance (Abstract #66) Toshihiko Watanabe 10:00 am Withdrawn (Abstract #67) 10:15 am – 10:45 am Break 10:45 am – 12:45 pm Oral Plenary Exchange XI Young Innovators Moderators: Nader N. Najafian, MD and David M. Rothstein, MD 10:45 am ABO Incompatible Heart Transplantation in Early Childhood: Tolerance towards the Donor Blood Group Is Associated with Reduced De Novo HLA- Sensitization (Abstract #68) Simon Urschel 11:00 am TIM-1 Defines a Novel Population of Inducible Regulatory B Cells That Underlies Prolongation of Allograft Survival by Anti-TIM1 (Abstract #69) Qing Ding (presented onsite by David Rothstein) 11:15 am Varying Effects of Donor Brain Death on Lung, Kidney, and Heart Allograft Tolerance in Miniature Swine (Abstract #70) Karen M. Kim 11:30 am The Link between PDL1 Costimulatory Pathway and T17 in Fetomaternal Tolerance (Abstract #71) Francesca D'Addio 11:45 am The Impact of Donor-Specific Antibody (DSA) and Conjoint DSA/C4D on Graft Survival during the First Year Post-Renal Transplantation and One Year Later (Abstract #72) Boonsong Kiangkitiwan 12:00 pm The Role of Proinflammatory Cytokine IL-6 in Alloimune Responses (Abstract #73) Xiaozhi Zhao 12:15 pm Changes in T Cell Subsets in Patients with Tolerance and Chimerism after Renal and Hematopoietic Cell Transplantation (Abstract #74) Sussan Dejbakhsh-Jones

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12:30 pm Marked Hepatic Allograft Survival Prolongation by ASKP1240 (4D11) in Non- Human Primates (Abstract #75) Tetsu Oura 12:45 pm – 1:00 pm Young Innovator Award Presentations and Closing Remarks Joren C. Madsen, MD, DPhil, Mohamed H. Sayegh, MD, and Maryl R. Johnson, MD

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Poster Exchange (Poster Session I)

Thursday, December 3 4:45 pm – 6:15 pm

Board # Abstract # Title Presenting

Author

P1 124 Comparison of Standard and High Dose Rabbit Antithymocyte Globulin Induction on Renal Allograft Outcomes in High Risk Recipients with Slow Graft Function

Winston Ally

P2 125 Pharmacodynamic Monitoring of IMPDH Activity To Optimize MMF Therapy after Renal Transplantation

Ferdi Sombogaard

P3 126 The Value of Chronic Renal Allograft Injury Score for the Conversion from Cycspoline to Tacrolimus or Sirolimus in Chronic Allograft Nephropathy

Jiqiu Wen

P4 127 Efficacy of Biopsy as a Method of Detecting Early Kidney Transplant Rejections

Mohammed Shenaq

P5 128 What Is Optimal Induction Therapy for Expanded Criteria Donor Kidneys?

Paul Tso

P6 129 Withdrawn

P7 130 Withdrawn

P8 131 Withdrawn

P9 132 Withdrawn

P10 133 Single Dose Alemtuzumab Induction Followed by Steroid Free Immunosuppression in Filipino Cohort

Beverly Aguila

P11 134 Successful Immunosuppression Minimization in Pediatric Liver Transplantation

Udeme Ekong

P12 135 Transplant Arteriopathy and Immunosuppression Minimization in Hand Transplant Recipients

Warren Breidenbach

P13 136

African-American Patients on a Steroid-Free Immunosuppression Regimen Using Alemtuzumab Have Excellent Outcomes Compared to Steroid-Containing Immunosuppressive Regimens Using Interleukin-2 Receptor Antagonist or Thymoglobulin Based Inductions

Elliot Zahtz

P14 137 The Indication of Conversion from Cyclosporine to Sirolimus in Chronic Allograft Nephropathy

Jiqiu Wen

P15 138 Bortezomib for Treatment of Antibody Mediated Rejection (AMR) in Intestinal Transplantation

Kyle Soltys

P16 139 Withdrawn

P17 140 Withdrawn

P18 141 Complication Rates after Cardiac Catheterization in Patients with Cirrhosis

Tarun Narang

P19 142 Post-Transplant Lymphoproliferative Disorders in Children: Recent Outcomes and Response to Dual Rituximab/Chemotherapy Combination

Vikas Dharnidharka

P20 143 Withdrawn

P21 144 Withdrawn

P22 145 Withdrawn

P23 146 Effect of Intrahepatic Stainable Iron on Cardiac Dysfunction Post Orthotopic Liver Transplantation

Tarun Narang

P24 147 Withdrawn

P25 148 Withdrawn

P26 149 Chronic Rejection Results in Different Changes of Urine Metabolite and Protein Patterns Than Immunosuppressant Toxicity in the Rat

Uwe Christians (presented onsite by Amanda Crunk)

P27 150 Sirolimus, but Not Everolimus, Enhances Tacrolimus Nephrotoxicity in the Rat

Uwe Christians (presented onsite by Amanda Crunk)

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P28 151 IL-21 and IL-21 Receptor (IL-21R): Potential Targets for Immunotherapy in Highly Sensitized Kidney Transplant Patients

Reza Tavana

P29 152 Chronic Humoral Rejection of Human Kidney Allografts Is Associated with Matrix Metalloproteinase-2 Accumulation in Glomeruli and Its Release in the Urine

Waichi Wong

P30 153 Withdrawn

P31 154 In Vitro Assessment of Alloreactivity in Pediatric Living Related Liver Transplantation

Udeme Ekong

P32 155 ATP in CD4+ T Cells as an Independent Predictor for Infection and Rejection: Preliminary Results from a Pediatric Liver Transplant Study

Gunnar Brandhorst

P33 156 Signatures of Cytomegalovirus Protective Immunity in the Setting of Immunosuppression

Aneesh Mehta

P34 157 Withdrawn

P35 158 Withdrawn

P36 159 TOL101; a Novel αβ TCR Targeting Monoclonal Antibody Daniel Getts

P37 160 Effect of AEB-071 on Prevention of Acute Heart Allograft Rejection in the Rat

Myoung Soo Kim

P38 161 Peripheral Tolerance in Porcine Lung Transplantation Induced by Splenocyte Infusion and Low Dose Irradiation - Modifications of the Protocol for Improved Clinical Feasibility

Karla Dreckmann

P39 162 Novel Transplantation Approach for Correction of Type 1 Diabetes without Insulin or Immunosuppression

Subhadra Gunawardana

P40 163 Treatment with ILT3-Fc Improves Allogeneic Pancreatic Islet Graft Survival in hu-NOD/SCID Mice

George Vlad

P41 164 Withdrawn

P42 165 Immune Active Effect of Chemokine RANTES on Human Peripheral Mononuclear Cells

Xiao Gu

P43 166 Withdrawn

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Oral Poster Exchange Sessions 1-4 (Poster Session II) Friday, December 4

Posters on display: 10:00 am – 6:15 pm, Oral sessions: 6:15 pm – 7:15 pm

Board # Abstract # Title Presenting Author

P1 76 Skin Graft Rejection: Direct or Semi-Direct Allorecognition? Georges Tocco

P2 77 Withdrawn

P3 78 Infection with the Intracellular Bacterium, Listeria Monocytogenes, Transiently Overrides Established Allograft Tolerance

Tongmin Wang

P4 79 Cardiac Allograft Survival Is Prolonged by the Induction of Mixed Hematopoietic Chimerism Two Months after Heart Transplant

Timothy Millington

P5 80 Regeneration of Naïve T-Lymphocytes from Heart-Thymus Grafts Occurs at a Low Level and Is Associated with Prolonged Survival but Not Tolerance

Timothy Millington

P6 81 Sensitization Prior to Composite Tissue Allotransplantation in Rat Model Does Not Result in Hyperacute Rejection

Shengli Wu

P7 82 Expansion of CD4+CD25+FoxP3+ Regulatory T Lymphocytes with Low-Dose Anti-Thymocyte Globulin Leads to Prolonged Survival of Cardiac Allografts but Not Tolerance

Timothy Millington

P8 83 Easiness To Generate Is Compensated with Limited Therapeutic Applicability

Arthur Andakyan

P9 84 Generation of Human Tregulatory Cells with the Capacity To Suppress Islet Allograft Rejection in a Xenogeneic Mouse Model

Alicia McMurchy

P10 85 Effect of FTY720 Treatment on CD4+ T Cell-Endothelial Cell Interactions in Ischemia/Reperfusion Injury of the Pancreas

Dirk Uhlmann

P11 86 Withdrawn

P12 87 Ex-Vivo Expanded CD4+CD25+ Treg Cells Suppress T and B Immune Response

Avneesh Singh

P13 88 Both Rejection and Tolerance of Allogeneic Transplants Can Occur in the Absence of Secondary Lymphoid Organs

Gilles Benichou

P14 89 Suppressing Antigen-Bearing Dendritic Cells by Double Negative Regulatory T Cells

Julia Fang Gao

P15 90 Mechanisms of Immunological Tolerance Induced by Double Negative Regulatory T Cells in a Semiallogeneic Model of Graft-Versus-Host Disease

Stephen Juvet

P16 91 Synergistic Effect of CsA and Tregs in Preventing Memory T Cell Induced Tolerance Abrogation

Anja Siepert

P17 92 Indoleamine 2,3-Dioxygenase (IDO) and Treg Support Are Critical for CTLA4Ig Mediated Tolerance Induction to Solid Organ Allografts

Robert Sucher (formerly P11 in poster session III)

P18 93 A Novel Strategy and Unique Model for Composite Tissue Allotransplantation Tolerance

Rishi Jindal

P19 94 Studies on Some Immune Properties of the Pancreatic Progenitor Cells Derived from Human Fetal Pancreas

Man Ting Ma

P20 95 HMGB1 Regulates Tubular Epithelial Cell (TEC) Expression of MCP-1 in Ischemic Kidney Injury

Arthur Lau

P21 96 Serum Anti-MHC Immunoglobulins Ineffective in Prevention of Acute Graft Rejection

Arthur Andakyan

P22 97 Withdrawn

P23 98 Lentiviral Transduction of Face and Hand Allografts: Implications for Immunomodulation of Composite Tissue

Angelo Leto Barone

P24 99 Allogeneic Porcine CD4

+CD25

+ T Cells Regulate the

Development of Transplant Arteriosclerosis in Porcinized Mice Gregor Warnecke

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Oral Poster Exchange Sessions 5-8 (Poster Session III)

Saturday, December 5 Posters on display: 10:00 am – 6:15 pm, Oral sessions: 6:15 pm – 7:15 pm

Board # Abstract # Title Presenting Author

P1 100 Establishment of Local Tolerance for Cell Transplants, toward Clinical Application

Lina Lu

P2 101 High Levels of IDO-Expressing CD16+ Peripheral Cells, and Tregs in Graft Biopsies from Kidney Transplant Recipients under Belatacept Treatment

Janette Furuzawa-Carballeda

P3 102 Endothelial Cells Co-Cultured with Embryoid Bodies Enhance Insulin-Producing beta-Cell Differentiation through BMP-2 Signaling

Dodanim Talavera-Adame

P4 103 Effect of Rituximab on the Regulation of Sphingomyelinase-Like Phosphodiesterase 3b-Precursor To Prevent FSGS Recurrence after Renal Transplantation

Junichiro Sagheshima

P5 104 Indoleamine 2,3-Dioxygenase (IDO) Immune Status Monitoring Post-Transplantation

Vikas Dharnidharka

P6 105 Liver Transplant Recipients Successfully Weaned Off Immunosuppression Lack Donor-Specific Anti-HLA Antibodies

Antonino Castellaneta

P7 106 Pro-Inflammatory Cytokines Modulate the Ability of Regulatory T Cells To Control Alloimmunity

Giorgio Raimondi

P8 107 Transplantation Tolerance to Non-Inherited MHC Class I Maternal Antigens: New Insights Using TCR Transgenic Models

Gilles Benichou

P9 108 Pregnant Level Estrogen and Progestone Affect the Regulatory T Cells in Mouse

Xing-Guang Lin

P10 109 Withdrawn

P11 110 CD200 Immunomodulation Increases Tregs and Prolongs CTA Survival

Rishi Jindal (formerly P17 in poster session II)

P12 111 Anti-HLA Abs along with Defensins Contribute to BOS Pathogenesis Deepti Saini

P13 112 Minimization of Immunosuppression in Primary Adult Liver Transplantation with Steroid-Free Regimen Using Low Dose of Tacrolimus with Mycophenolate Mofetil after Daclizumab Induction

Olivier Boillot

P14 113 Feasibilty of Long-Term Sirolimus Monotherapy after Liver Transplantation

Dirk Uhlmann

P15 114 Excellent Outcomes with Alemtuzumab Induction and Steroid-Free Maintenance Compared to Interleukin-2 Receptor Antagonist or Thymoglobulin with Prednisone Maintenance

Jimmy Light

P16 115 Alemtuzumab Induction, Steroid-Free Maintenance Immunosuppression in African-Americans vs Non-African Americans

Jimmy Light

P17 116 The Significance of Immune Cell Function Testing in Renal Transplantation

Vaughn Whittaker

P18 117 Bronchoalveolar Lavage CD4+ Fox P3+ T Cells Are Decreased in Lung Transplant Recipients with Bronchiolitis Obliterans Syndrome

Sangeeta Bhorade

P19 118 Liver Function Tests Do Not Correlate with Liver Biopsy Results in Pediatric Patients Who Have Undergone Liver Transplantation

Jason Fraser

P20 119 Antibody Mediated Rejection Secondary to Anti-DP Antibodies in a Kidney Transplant Recipient

Maria Martinez Cantarin

P21 120 Machine Perfusion in Kidney Transplantation: Lower Rate of Acute Rejection in the Presence of Longer Pump Time, Regardless of Immunosuppression

Gaetano Ciancio

P22 121 Socioeconomic Status and Acute Rejection in Kidney Transplant Recipients

S Lodhi

P23 122 The Effect of Change or Modulation of Maintain Immunosuppressive Regimen on Long-Term Transplant Result

Myoung Soo Kim

P24 123 Unusual Lymphoma and Concurrent Presentation of Graft Arteriopathy in a Hand Transplant Recipient Three Years Post Transplant

Christina Kaufman

DECEMBER 3-6, 2009

ABSTRACTS

TRANSLATIONAL SCIENCE I AND CLINICAL SCIENCE I: BIOMARKERS/MONITORING

1

Abstracts

All presenters are required to disclose relevant confl icts of interest.All such disclosures are published within the Abstract Book following each abstract.

Any presenters who have nothing to disclose have been omitted from the disclosure listing.

mDC1, TOL patients expressed higher HLA-G and ILT4 than in the PW, but not the MI group (MFI HLA-G: 39.0±5.8 vs 34.0±3.1- p<0.03; MFI ILT4: 35.0±5.4 vs 28.0±3.8- p<0.007).Conclusion: Higher expression, in TOL liver transplant patients, of HLA-G and ILT4 on circulating DC subsets suggests a possible tolerogenic role of these immunoregulatory molecules, and also suggests the possibility of using these molecules as potential biomarkers in immunosuppression management or reduction in liver transplant recipients.

Abstract# 3Gene Arrays May Predict Stable Clinical Kidney Tolerance and Support Safe Immunosuppression Withdrawal. Minne M. Sarwal,1 Li Li,1 Szu-chuan Hsieh,1 Robert Lowsky,1 Stephan Busque,1 John Scandling,1 Sussan Jones,1 Samuel Strober.1 1Pediatrics, Medicine, Immunology and Surgery, Stanford University School of Medicine, Stanford, CA, USA.BACKGROUND. 10 HLA-matched patients were enrolled in a combined kidney and hematopoietic cell txp (HCT) tolerance induction protocol (NEJM, 358(4), 2008). Kidney txp recipients got 5 ATG and 10 TLI doses and donor HCT. IS was withdrawn after 6 months if chimerism persists, with no GVHD or rejection.AIM. To determine whether serial monitoring of PBMCs of HLA-matched combined kidney transplant and HCT recipients by gene arrays can predict successful withdrawal of immunosuppression and continued good graft function.METHODS. PBMC samples were analyzed before, monthly for 6 mo and every 3 mo for the fi rst 2 yrs post-txp by Agilent arrays. Matching scores for the tolerant pattern are based on prediction scores from 0% (failure) to 100% (full tolerance match) by analysis of cross-sectional expression data from non-study patients with operational tolerance (off IS for >2 years). Each serial sample is given a prediction score based on the results of the array analysis.RESULTS. To date, 7 of 10 patients have met drug withdrawal criteria; 5 of these are off drugs for 8 to 35 months, and 2 are being tapered. Serial gene array monitoring showed a change in gene expression in PBMCs from a non-tolerant pattern to a tolerant pattern in 5 patients in whom immunosuppressive drugs were successfully withdrawn . Gene array studies showed a tolerance gene pattern before the drug withdrawal. In contrast, 3 patients tested for gene expression changes who failed to meet withdrawal criteria had a persistent non-tolerant pattern after transplantation.CONCLUSION. Tolerant PBMC gene patterns may predict the development of stable tolerance after a combined kidney and HCT tolerance induction protocol. These tolerance patterns can develop as early as 1 month post-transplantation and can predict safe immunosuppression withdrawal and excellent graft function. Failure of the tolerance gene pattern can correlate with failure of tolerance induction and select patients who need to be maintained on chronic immunsuppression.

Abstract# 4Pre-Transplant Analysis of Whole Blood Toag-1 Gene Expression and CD4+CD45RO-CD62L- TEMRA Frequencies Identifi es High Risk Patients. Birgit Sawitzki,1 Undine Gerlach,2 Hiroe Yamaguchi,1 Katrin Vogt,1 Stefanie Haase,1 Dieter Volk,1,3 Petra Reinke,3,4 Andreas Pascher.2 1Institute of Medical Immunology, Charite University Medicine, Berlin, Germany; 2Department of Transplant Surgery, Charite University Medicine, Berlin, Germany; 3Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, Germany; 4Department of Nephrology, Charite University Medicine, Berlin, Germany.Introduction: Achieving long-term, drug-free graft acceptance is still an unsolved problem in clinical transplantation. Barriers to transplantation tolerance are a specifi c immune memory already acquired in adult human patients and heterologous immunity. We could recently show that Tolerance associated gene 1 (Toag-1) is highly expressed in PBMCs and grafts of tolerance developing recipients (Sawitzki et. al. AJT 2007) and can predict CMV mediated tolerance abrogation. Here we investigated whether analysis of whole blood Toag-1 gene expression and memory subpopulations allows identifi cation of high risk patients experiencing acute rejections episodes during the fi rst 6 months following liver transplantation.Methods and Results: We have examined whole blood Toag-1 gene expression and peripheral CD4+/CD8+ T cell subpopulations in samples of 24 liver transplant patients prior to and at frequent intervals after transplantation. Results were compared to samples of age matched healthy controls. 10 out of the 24 patients developed signs of acute rejection within the fi rst 6 months after transplantation. Whole blood Toag-1 gene expression was signifi cantly decreased in rejecting patients as compared to non-rejecting patients and healthy controls (0.054±0.026 vs 0.115±0.038, p=0.0035). We also detected a signifi cantly elevated frequency of CD4+CD45RO-

CD62L- terminal differentiated effector memory T cells (TEMRA) prior to and early after transplantation in patients developing acute rejections (14.5±5.6 vs 3.9±1.8, p=0.02) as compared to non-rejecting patients. These differences were exclusive

ORAL PLENARY EXCHANGE I - TRANSLATIONAL SCIENCE I AND CLINICAL SCIENCE I:

BIOMARKERS/MONITORING

Abstract# 1Inducing Tolerance in Clinical Kidney Transplantation. J. Scandling, S. Busque, S. Dejbakhsh-Jones, C. Benike, L. Li, T. Holmes, J. Shizuru, R. Lowsky, E. Engleman, M. Sarwal, S. Strober. Stanford University, Stanford, CA, USA.Immune tolerance and persistent mixed chimerism can be achieved reproducibly after combined organ and hematopoietic cell transplantation in mice conditioned with total lymphoid irradiation and antithymocyte globulin. We studied the safety and reproducibility of this approach in clinical transplantation.Methods: Ten patients, fi ve men and fi ve women, age range 23 to 61 years, underwent HLA-matched kidney transplantation followed by conditioning with 10 doses of total lymphoid irradiation and 5 doses of antithymocyte globulin. This was followed by infusion of donor CD34+ hematopoietic progenitor cells and T cells. Criteria for withdrawal of anti-rejection drug therapy at > 6 months after kidney transplantation were stable chimerism for at least 6 months, and absence of rejection and graft versus host disease.Results: All patients had excellent graft function, with serum creatinine range 0.8 to 1.3 mg/dL at last observation 9 to 48 months following transplantation. Length of stay for transplant hospitalization was 4 to 7 days. The range of the nadir white blood cell count was 0.6 to 3.4 x103/mm3, occuring from day 3 to day 84 after kidney transplantation. Three patients required hospital readmission within the fi rst year, one each for neutropenic fever, ureteral stricture and acute rejection. Two patients developed varicella zoster and one primary CMV disease. Two patients developed rejection episodes, and one patient developed immediately recurrent focal segmental glomerulosclerosis. All three remain on conventional maintenance immunosuppression.Seven patients met withdrawal criteria. Five with chimerism persisting for at least 6 months have been completely withdrawn from anti-rejection drugs for 8 to 36 months, and two are being tapered. Surveillance biopsy prior to drug withdrawal showed 0 to 10% (grade I, lowest score) interstitial fi brosis/tubular atrophy. Blood cells from patients off drugs showed development of specifi c unresponsiveness to donor alloantigens, “tolerance” profi les on gene micoarrays, early high ratios of regulatory versus conventional naive T cells, and early high levels of chimerism among NK cells.Conclusion: Total lymphoid irradiation and antithymocyte globulin safely promoted the development of persistent chimerism and tolerance in clinical HLA-matched kidney with hematopoietic cell transplantation. Assays were identifi ed that can assist in the safe withdrawal of immunosuppressive drugs.

Abstract# 2Elevated HLA-G and ILT4 Expression on Circulating Dendritic Cell Subsets in Pediatric Liver Transplant Recipients Successfully Weaned Off Immunosuppression. Antonino Castellaneta,1,2 George V. Mazariegos,1,2 Michael DeVera,2 Rakesh Sindhi,1,2 Navdeep Nayyar,1 Adriana Zeevi,2 Angus W. Thomson.2 1Children’s Hospital of Pittsburgh, University of Pittsburgh; 2Starzl Transplantation Institute, University of Pittsburgh.Background and Aim: Human leukocyte antigen (HLA)-G belongs to the family of non-classical HLA class I molecules and is expressed as 7 different isoforms, four membrane-bound (HLA-G1 to -G4) and three solubles (HLA-G5 to -G7). It has been reported that the specifi c interaction of HLA-G with its receptor, Ig-like transcript (ILT)4, on dendritic cells (DC) down-regulates their T cell co-stimulator ability. High serum sHLA-G level, after transplantation, has been associated with lower incidence of severe heart graft rejection, and normal liver and kidney graft function. No data are available concerning the expression on DC of HLA-G and ILT4 in liver transplant patients. The aim of this study was to analyze the expression of HLA-G and ILT4 on circulating DC subsets in three clinically stable pediatric liver recipient groups: operationally tolerant (TOL; n=7); prospective drug weaning (PW; n=12) and maintenance immunosuppression (MI; n=10).Patients and methods: Twenty-nine pediatric liver transplant recipients were eligible for the study. PBMC were stained with a lin mAb cocktail. They were also stained with the DC subset-specifi c mAbs, blood DC Ag (BDCA)-1 (CD1; clone AD5-8E7) for monocytoid (m)DC and BDCA-2 (CD303; clone AC144) for plasmacytoid (p)DC, as well as for HLA-G (87G) and ILT4 (CD85d). DC precursor subsets were identifi ed as: mDC1 (lin-BDCA-1+BDCA-2-) and pDC (lin-BDCA-1-BDCA-2+).Results: Multi-color fl owanalysis showed that both circulating pDC and mDC expressed surface HLA-G and ILT4. In TOL patients, pDC expressed higher HLA-G and ILT4 than in the MI, but not the PW group (MFI HLA-G: 36.0±3.8 vs 28.0±6.0- p<0.02; MFI ILT4: 70.0±12.0 vs 49.0±16.0- p<0.02). With regard to

TRANSLATIONAL SCIENCE I AND CLINICAL SCIENCE I: BIOMARKERS/MONITORING

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for the CD4 compartment and were not observed for CD8+ T cells. Furthermore, our analysis revealed no differences in CD25 highCD127low Tregs between rejecting and non-rejecting patients at any time point studied.Conclusions: Whole blood Toag-1 gene expression and CD4+ TEMRA frequencies allow pre-transplant identifi cation of high risk patients. This should be implemented into personalized adapted therapies possibly resulting in reduced incidences of acute rejections episodes and improved long term graft function.DISCLOSURE: Sawitzki, Birgit: Grant/Research Support, gene marker; Volk, Dieter: Grant/Research Support, gene marker.

Abstract# 5Pre-Transplant Immune Regulation Due to Fetal and Maternal Antigen Exposure. William J. Burlingham,1 Ewa Jankowska-Gan,1 Lynn D. Haynes,1 Astrid G. van Halteren,2 Miranda P. Dierselhuis,2 Els A. Goulmy.2 1Surgery-Division of Transplantation, University of Wisconsin, Madison, WI, USA; 2Immunohematology, Leiden University Medical Centre, Leiden, Netherlands.Background: Maternal and fetal stem cells are exchanged between mother and offspring. Microchimerism [Mc] is therefore the norm for a healthy mammalian immune system. The consequences of Mc for solid organ transplant tolerance and rejection, however, have been controversial. Recent data suggest that the type of Mc [multi- vs. uni-lineage] is determined by the balance between regulatory and effector T cells. We hypothesized that pre-transplant allo-specifi c regulation [Treg>Teff] exists, and is correlated with multi-lineage Mc of maternal or fetal origin.Methods: Six HLA-A2+ healthy blood donors, and 39 live donor-recipient pairs 1d prior to RTx were tested for alloantigen-specifi c regulation as measured by ≥ 50% suppression of recall antigen [EBV or TT]-induced footpad swelling in the trans-vivo DTH assay. Tregs were triggered either by a) crude sonicate of cells from an HLA-identical sibling [multiple mismatched minor H Ags], or HLA –mismatched [MHC + minor H Ags] relative; or b)HLA-A2-restricted minor H allopeptides HA-1[VLHDDLLEA] or HY[FIDSYICQV]. Minor H Ag-specifi c CD8 CTL and Tregs were detected in PBMC of healthy donors using HLA-A2/HA-1 and HLA-A2/HY tetramers; Mc, using real-time PCR for HY and HA-1 DNA.Results: Both CTL- and Treg- mediated alloimmunity against familial minor H antigens were present in healthy females and males. Tregs were detected within isolated tetramerdim staining fractions and functioned in a CTLA-4–dependent fashion. A Treg bias to maternal HA-1 was observed in 4/5 males; 4/10 mothers of sons had a Treg bias to HY, while 6/10 had a HY/CTL bias. Isolated PBMC subsets revealed Mc in both regulators [n=3] and non-regulators [n=3]; however Mc in HLA class II+ APC was only found in regulators. Remarkably,14/14 (100%) HLA-ID siblings and 14/25 (56%) HLA haploidentical family members regulated to their prospective donor’s minor H, or minor + MHC Ags. Pre-Tx regulation between recipient and donor was bi-directional in 7/7 HLA-ID pairs, but unidirectional in 9/11 (both exceptions: child to mother) HLA haploidentical pairs. All 9 bi-directional regulators retained a Treg bias post-transplant and had excellent [median sCr=1.1 mg/dl] renal function at 3yr.Conclusions: Pre-existing bi-directional regulation to familial minor H antigens is common in HLA-ID sibling pairs, and may be associated with multilineage Mc.

Abstract# 6Identifi cation of a Peripheral Blood Transcriptional Biomarker Panel for Diagnosis and Prediction of Renal Allograft Tolerance. Li Li,1 Sue Heish,1 Lihua Ying,1 Tara Sigdel,1 Minnie Sarwal.1,2 1Pediatric, Stanford University, Stanford, CA, USA; 2Immunology, Stanford University, Stanford, CA, USA.Aim: To identify and validate a peripheral blood transcriptional biomarker gene-panel for diagnosis and prediction of operational tolerance in a heterogeneous cohort of adult kidney transplant recipients.Methods: 32 peripheral blood samples were analyzed by oligonucleotide Agilent arrays from 3 different phenotypes from different transplant centers: 16 operational tolerant (TOL) kidney transplant patients without medications for more than 1 year, 10 with chronic graft injury and on triple maintenance immunosuppression (CAN), and 6 healthy donors (HD). Samples were used as a training set to defi ne a minimum gene-set for diagnosis of operational tolerance. The microarray samples were tested by Q-PCR to build a model for prediction of tolerance by logistic regression and tested on an independent sample set containing 8 TOL patients. Bioinformatics analysis including GeneSpring, AILUN, SAM, PAM, logistic regression and IPA were used.Results: Twenty-one unique genes were identifi ed (FDR<5%) by prediction analysis of microarrays (PAM) and statistical analysis of microarray (SAM) as a minimum gene for TOL. These genes are enriched in immune cell traffi cking and 13/21 genes are regulated by TNF. Multinomial logistic regression modeling by Q-PCR for all samples identifi ed 3 genes as a minimal gene set for TOL in the array data-set with the ability to predict TOL in a blinded independent data-set with 100% sensitivity, 80% specifi city, 89% PPV and 100% NPV. 2 out of 3 genes genes are highly expressed in B cells and dendritic cells.

Conclusion: A highly regulated minimal gene-set in peripheral blood has been validated across multiple patients groups and transplant centers. This gene-set can be used as a non-invasive monitoring tool for screening patients with stable operational tolerance after kidney transplantation. Serial measurement of expression for this gene-set in peripheral blood opens the door for deliberate immunosuppression minimization after transplantation, and should be further tested for its utility in also monitoring patients on tolerance induction protocols in kidney transplantation.

Abstract# 7Clinical Outcome of Liver Transplant Recipients after Successful Immunosuppression Withdrawal. Panagiotis Tryphonopoulos,1 Phillip Ruiz,1 Debbie Weppler,1 David M. Levi,1 Seigo Nishida,1 Jang Moon,1 Akin Tekin,1 Gennaro Selvaggi,1 Eddie Island,1 Seung H. Shin,1 Tamara Defranc,1 Alex Volsky,1 Barbara Sotolongo,1 Manuel Carreno,1 Maria B. Torres,1 Andreas Tzakis.1 1Surgery-Transplant, University of Miami, Miami, Fl, USA.Background: Successful attempts to discontinue immunosuppresion post-transplantation have been reported both in kidney and liver transplant recipients.Methods: In an immunosuppression withdrawal study we previously performed in our center, we managed to wean off immunosuppression 22 adult liver transplant recipients (tolerant group) while in the remaining 72 patients complete withdrawal of immunosuppression was not possible due to rejection (rejector group). We analyzed the long term follow up of these patients after the end of the withdrawal study.Results: Patient-graft survival: until the end of the follow up period there were four mortalities in the tolerant group and fi fteen mortalities in the rejector group. There were not any re-transplants in the 2 groups. Rejection episodes: After the end of the withdrawal study, in the rejector group, 18 patients presented 18 clinically suspected and 4 biopsy proven mild (n=1), moderate (n=2) or severe (n=1) rejection episodes. Twenty two patients have achieved a tolerant state for 7+0.39 years after immunosuppression withdrawal. A tolerant patient presented a moderate rejection 5.3 years after immunosuppression withdrawal and was restarted on maintenance immunosuprression. Renal function: There was a trend for serum creatinine to be lower in tolerant transplant patients. In the rejector group, since the end of the withdrawal study fi ve patients received a kidney transplant and three more are on dialysis. Twelve patients had sirolimus (n=4) or MMF (n=6) added to their immunosuppressive regimen and six more were converted to sirolimus/MMF immunosuppression for nephrotoxicity. There were no cases of renal replacement in the tolerant group until the end of the follow up period. Neoplasms: In the rejector group, eleven patients presented neoplasms since the end of the withdrawal study vs four in the tolerant group.Conclusion: About 20% of liver transplant patients can be successfully weaned off immunosuppression with a better quality of life and less immunosuppression-related complications. This tolerant state however is not permanent and rejection can occur even a long time after immunosuppression withdrawal.

Abstract# 8Use of Transcriptional Biomarkers To Identify Liver Transplant Recipients Who Can Successfully Discontinue Immunosuppressive Therapy. Carlos E. Benitez,1 Juan J. Lozano,1 Marc Martínez-Llordella,1 Isabel Puig-Pey,1 Marta Lopez,1 Anna Rodríguez,1 Giuseppe Tisone,2 Jacques Pirenne,3 Jan Lerut,4 Antoni Rimola,1 Alberto Sanchez-Fueyo.1 1Liver Transplant Unit, Hospital Clinic de Barcelona/IDIBAPS, Barcelona, Spain; 2Liver Transplant Center, University Tor Vergata, Rome, Italy; 3Abdominal Transplant Surgery, University Hospitals Leuven, Leuven, Belgium; 4Abdominal and Transplantation Surgery, University Hospitals St Luc, Leuven, Belgium.Approximately 20% of stable liver transplant recipients can completely discontinue immunosuppressive therapy (IS) without undergoing rejection.These patients are considered as operationally tolerant and differ from recipients requiring indefi nite IS in their peripheral blood gene expression patterns. We present here the preliminary results of a joint publicly (EU-funded RISET consortium) and privately (TcLand Expression) sponsored clinical trial to assess the capacity of transcriptional profi ling to identify among stable liver recipients those who can successfully discontinue IS. METHODS: 102 stable liver recipients >3 years after transplantation were enrolled after undergoing collection of baseline peripheral blood samples and liver biopsy. IS was gradually discontinued over a 6-9 month period and all patients were followed-up for at least 12 additional months. Recipients who did not develop graft rejection over this 18-24-month period were considered tolerant. In order to predict the outcome of the weaning protocol we conducted Affymetrix whole-genome expression studies on baseline blood samples using a previously described group of 25 genes associated with liver allograft tolerance (JCI 2008;118-2845). RESULTS: 71 recipients have completed the study, out of which 53 have rejected and 18 have been classifi ed as tolerant with a mean post-weaning follow-up of 28 months; 25 patients are still in progress and 6 have dropped-out of the study.The use of the group of 25 genes correctly predicted the outcome of 85% of the 71 recipients (sensitivity 53%, specifi city 95%). The expression of these genes was measured in a second blood sample collected 18 months after enrollment and found to be

INNATE IMMUNITY

3

Abstracts

highly stable over time. CONCLUSIONS: The use of a previously described gene expression classifi er accurately identifi es, before IS weaning is attempted, a subset of liver recipients with a very high likelihood of being tolerant. These results open the door to the development of a clinically applicable diagnostic test of tolerance in liver transplantation.DISCLOSURE: Sanchez-Fueyo, Alberto: Grant/Research Support, TcLand Expression.

ORAL PLENARY EXCHANGE II - INNATE IMMUNITY

Abstract# 9DHMEQ, a New NF-κB Inhibitor, Suppresses Innate and Subsequent Allo-Immune Responses, and Permits a Long-Term Allogeneic Islet Engraftment Together with Tacrolimus. Masaaki Watanabe,1 Kenichiro Yamashita,1 Hirofumi Kamachi,1 Daisuke Kuraya,1 Yasuyuki Koshizuka,1 Susumu Shibasaki,1 Michitaka Ozaki,1 Kazuo Umezawa,2 Michiaki Matsushita,1 Satoru Todo.1 1First Department of Surgery, Hokkaido University, Sapporo, Hokkaido, Japan; 2Department of Applied Chemistry, Keio University, Yokohama, Kanagawa, Japan.Background: Immediate and late allograft loss after islet transplantation (ITx) remains uncontrollable. Innate immune responses, mediated by molecules such as high mobility group box1 (HMGB1) that is released from damaged cells and tissues, elicit immediate islet graft loss and strengthen allo-immune responses leading to graft rejection. Activation of the transcription factor, NF-κB plays a key role during this process.Aim: We examined the effect of a new NF-κB inhibitor, DHMEQ, on innate and subsequent adaptive responses in mouse allo-ITx.Method: BALB/c (H-2d) mice pancreatic islets (600) were transplanted to STZ induced diabetic C57BL/6 (H-2b) mice via the portal vein. Islet recipient was given either vehicle (Control), DHMEQ (20 mg/kg/day, i.p.) for 14 days (Group 1), tacrolimus (FK: 1.5 mg/kg/day, i.p.) for 14 days (Group 2) or DHMEQ (3 days)+FK (14 days thereafter) (Group 3). The effect of DHMEQ on HMGB1 stimulated mouse dendritic cells (DCs) was examined in vitro.Results: Islet graft survival rate on day 100 was 0%, 11% and 38% in the Control, Groups 1 and 2, respectively. In contrast, all animals remained normoglycemic (GSR: 100%) in Group 3 (Fig 1A). DHMEQ treatment suppressed serum HMGB1 level at 6 hours after ITx (Fig. 1B), and the allo-immune response as assessed by IFN-γ ELISPOT assay on day 6 was inhibited in the Group 3 animals as compared to other groups. HMGB1 driven DC activation and pro-infl ammatory cytokine production were inhibited by DHMEQ (Fig. 1C). These DCs further suppressed allogeneic T cell proliferation (Fig. 1D).Conclusion: Induction DHMEQ treatment inhibited HMGB1-mediated innate and subsequent allo-immune responses, and allowed a long-term allogeneic islet engraftment in conjunction with tacrolimus. DHMEQ plus tacrolimus is an attractive strategy to achieve a successful long-term engraftment in ITx.

Abstract# 10T Cell Ig Mucin (TIM) Pathways Regulate Liver Ischemia and Reperfusion Injury. Yoichiro Uchida,1 Maria Cecilia S. Freitas,1 Bibo Ke,1 Nader Najafi an,2 Ronald W. Busuttil,1 Jerzy W. Kupiec-Weglinski.1 1Surgery, Dumont-UCLA Transplant Center, Los Angeles, CA, USA; 2Transplantation Research Center, Brigham and Women’s Hospital, Boston, MA, USA.Background: TIM gene family molecules, expressed by Th1 and Th2 cells, regulate host immunity/tolerance. Although CD4+ T cells mediate innate immunity-dominated ischemia and reperfusion injury (IRI), the underlying mechanisms remain to be defi ned. This study investigates the role of TIM signaling in liver IRI. Methods: Partial warm ischemia was produced in the liver lobes of B6 mice for 90min, followed

by 6-24h of reperfusion. Mice were treated with antagonistic anti-TIM-1 Ab (RMT 1-10; 0.5mg i.p.) or agonistic anti-TIM-3 Ab (RMT3-23; 0.5mg i.p.) at day 1 prior to the ischemia insult. At 6h or 24h of reperfusion, sALT levels were assessed. The expression of pro-infl ammatory cytokines (TNF-α, IL-6, IL-1β), chemokines (CXCL-1, -2) and immune-related genes (IFN-γ) was screened by qRT-PCR. Liver samples were studied for histology/immunohistology, and apoptosis (TUNEL). Spleen T cells and RAW 264.7 macrophages co-cultures were performed. Results: Anti-TIM-1 Ab treatment reduced sALT levels (IU/L), as compared with controls (26,638±1,874 vs 10,474±3,146; p<0.01 at 6h, and 6,212±1,323 vs 2,873±1,595; p<0.01 at 24h). In contrast, anti-TIM-3 Ab treatment signifi cantly exacerbated the hepatocelluar damage (sALT: 27,333±1,607 vs 3,6383±4,995; p<0.01 at 6h, and 5,796±945 vs 8,297±1,514; p<0.01 at 24h). The induction of TNF-α, IL-6, IL-1β, CXCL-1,-2 and IFN-γ was reduced in anti-TIM-1 Ab; on the contrary, it was increased in anti-TIM-3 Ab treatment group. Histological examination has revealed that unlike in controls, anti-TIM-1 Ab treatment ameliorated hepatocellular damage, decreased neutrophil infi ltration, inhibited the accumulation of T cells/ macrophages/TIM-4 positive cells, and reduced apoptosis. Conversely, anti-TIM-3 Ab exacerbated the hepatocellular damage and facilitated local accumulation of T cells/macrophages. In in-vitro studies, anti-TIM-1 Ab supplement signifi cantly suppressed IFN-γ production in Con A-stimulated spleen T cells, and diminished TNF-α/IL-6 expression in macrophage/spleen T cell co-cultures. Conclusion: This study provides evidence for the novel role of TIM-1, 4 and 3 signaling in the mechanism of liver IRI. Indeed, TIM regulates not only T cell activation but may also affect macrophage function in the local innate-dominated infl ammation response. TIM pathway may represent the key mediator in IR-triggered organ damage.

Abstract# 11Withdrawn

Abstract# 12The Role of PD-1:B7-H1 T Cell Co-Stimulation Signaling in Liver Innate Pro-Infl ammatory Immune Response. Haofeng Ji,1 Xiuda Shen,1 Feng Gao,1 Bibo Ke,1 Yoichiro Uchida,1 Yuan Zhai,1 Ronald W. Busuttil,1 Jerzy W. Kupiec-Weglinski.1 1Department of Surgery, Dumont-UCLA Transplant Center, Los Angeles, CA, USA.Background: We have shown that CD4+ T cells play a key role in the pathogenesis of liver ischemia and reperfusion injury (IRI). Stimulation of programmed cell death 1 (PD-1) triggers negative signaling critical for immunoregulatory functions in the activation of local immune responses induced by hepatic IRI. We hypothesized that in vivo PD-1 engagement after administration of recombinant PD-L1Ig, a dimeric Ig fusion protein, or anti-PD-L1 mAb (9G2) may affect liver IRI. Methods and Results: B6 mice were subjected 90 min partial warm hepatic ischemia, followed by 6 h reperfusion. The PD-L1Ig or anti-PD-L1 mAb (0.5 mg IV at day -1 and 0) was administered prior to the onset of ischemia. Unlike in control group, PD-L1Ig treated mice were fully protected from hepatic IRI, as evidenced by sALT levels (227 vs. 4958 U/L; p=0.0005). Their liver architecture was preserved, showing minimal sinusoidal congestion without edema/vacuolization or necrosis (H/E staining). I/R induced hepatic pro-infl ammatory cytokine (TNF-α, IL-1β, IL-6, IFN-γ), and chemokine (CCL2, CXCL5, CXCL10) programs were signifi cantly reduced in PD-L1Ig group. Local neutrophil accumulation and activity, assessed by myeloperoxidase (MPO) levels, were also attenuated after PD-L1Ig treatment (0.23 vs. 1.13 U/g; p<0.05). The hepatocellular apoptosis, as shown by TUNEL staining, was selectively diminished in PD-L1Ig group. In marked contrast, pretreatment with anti-PD-L1 mAb increased sALT levels, and has led to signifi cant liver sinusoidal congestion/cytoplasmic vacuolization, and severe hepatocellular necrosis. In in vitro cultures, addition of PD-L1Ig to T cells stimulated with anti-CD3/anti-CD28 abolished IFN-γ secretion (29 pg/ml vs. 445.9 pg/ml activation group, p<0.001). We then employed stimulated T cells and macrophages (RAW264.7) to mimic in vivo T cell-Kupffer cell cascade. Interestingly, in such a co-culture system, PD-L1Ig supplement decreased TNF-α/IL-6 secretion [125.9 pg/ml vs. 1212.5 pg/ml in control group (TNF-α), p<0.001]; [3.8 pg/ml vs. 455 pg/ml in control group (IL-6), p<0.001]. Conclusion: Our novel fi ndings: 1) provide evidence that PD-1 activated lymphocytes, defective in their ability to mount effi cient pro-infl ammatory responses, or transmit pro-infl ammatory signal to macrophages, protect livers against I/R-induced damage; 2) offer the rationale for refi ned therapeutic strategy to combat IRI.

Abstract# 13DAP12 Expression in Liver Dendritic Cells Controls Their Maturation and the Induction of Tr-1 Cells. Tina L. Sumpter,1 Heth R. Turnquist,1 Angus W. Thomson.1,2 1Surgery, University of Pittsburgh, Pittsburgh, PA, USA; 2Immunology, University of Pittsburgh, Pittsburgh, PA, USA.Inherent mechanisms that control DC maturation are poorly understood. Liver-resident DC, implicated in tolerance induction, are phenotypically immature, IL-10hi IL-12lo , and exhibit impaired LPS responses (‘endotoxin tolerance’), providing a natural model for investigating DC maturation and subsequent T cell tolerance induction. Mouse liver myeloid (m) DC express the membrane adaptor DNAX-activating protein of 12kDa (DAP12), that dampens co-stimulatory

PRECLINICAL AND TRANSLATIONAL SCIENCES II

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molecule and infl ammatory cytokine expression by bone marrow-derived mDC. In this study, we evaluated DAP12 function in purifi ed C57BL/6 liver mDC (CD11c+CD11b+NK1.1-B220-) using DAP12 siRNA. DAP12-silenced DC increased CD80, CD86, B7-H1 and B7-H2 expression, but retained low levels of MHC class II, ICOSL, B7-H3 and B7-H4 compared to DC transfected with negative control siRNA. Loss of DAP12 elevated IL-12p70, TNFα and IL-6 secretion but diminished IL-10 secretion. Exogenous IL-10 lowered CD80 and CD86 expression on DC lacking DAP12, supporting an autocrine function for DC-secreted IL-10 in maintaining DC immaturity. Loss of DAP12 corresponded with elevated STAT3 phosphorylation but diminished IRAK-M expression. We hypothesized that the decreased IRAK-M expression observed in DAP12silenced DC would enhance LPS driven maturation, and override ‘endotoxin tolerance’. While LPS stimulation failed to enhance CD80 and CD86 expression in DAP12-silenced liver DC beyond that seen with DAP12 silencing alone, , IL-12p70 secretion and expression of the Th1-polarizing molecule, Jagged 1 increased. Consistent with their increased maturation, liver DC lacking DAP12enhanced proliferation of allogeneic CD4+ and CD8+ T cells (BALB/c) . The Enhanced proliferation did not correlate with changes in the incidence of Foxp3+CD4+ T cells, but correlated with a decreased IL-10 in MLR supernatants, suggesting a defi cit in generation of Type-1 regulatory T cells (Tr1). Stimulation of T cells with LPS-treated DAP12-silenced liver DC enhanced IFNγ but diminished IL-4 secretion in MLR. These data reveal dependence of liver DC maturation and inherent endotoxin tolerance on DAP12 expression. They also identify a role for DAP12 in regulation of IL-10 production by DC and consequent induction of Tr1 cells. Elucidation of molecules upstream of DAP12 may yield novel therapeutic targets to promote DC-based tolerance for transplant recipients.

Abstract# 14Relationship between NK Cells, Regulatory T Cells, and Effector T Cells in the Development of Coronary Allograft Vasculopathy (CAV) in Mouse Heart Transplants. Tsutomu Hirohashi,1 Catharine M. Chase,1 Alessandro Alessandrini,1 Robert B. Colvin,2 Paul S. Russell,1 Joren C. Madsen.1,3 1Department of Surgery, Massachusetts General Hospital, Boston, MA, USA; 2Department of Pathology, Massachusetts General Hospital, Boston, MA, USA; 3MGH Transplant Center, Massachusetts General Hospital, Boston, MA, USA.Purpose:We previously reported that inhibition of both NK cells and CD4 T cells was necessary to prevent CAV in parent to F1 heart allografts at day 56. However, the relationship between NK cells and CD4 T cells remained unclear. Therefore, we have undertaken further experiments to study this relationship.Methods:Hearts from C57BL/6 (H-2b) donors were transplanted heterotopically to BALB/c x C57BL/6 recipients (H-2bxd). Recipients were given either anti-CD25 antibody(PC61) or PC61and anti-NK1.1 antibody(PK136). Recipients were pretreated at a dose of 250 ug of PC61 on days -7 and -1, and in the PC61 and PK136 treatment group, PK136 was also given to recipients at a dose of 200ug on day -6. Following heart transplantation, PC61 was given on +5 and thereafter once/wk, and PK136 was given on day +1 and thereafter once/wk. Animals were sacrifi ced on day 21, and evaluated for CAV by morphometry (neointimal index, NI). In these experiments, animals treated with either PK136 alone or anti-CD4 antibody (GK1.5) treatment are also included.Results:In controls (no treatment) CAV developed by 21 days in 71% (5/7) but was not very severe, with an average NI of 22.8% (range 5.4-47.2%). In the anti-CD25 antibody treated group the frequency of CAV was similar, 100% (12/12), but was signifi cantly more severe, with an NI of 82% (range 37-100, p<0.005 vs. control). Anti-NK1.1 completely eliminated CAV at 21 days (0% with CAV, 0/3), and did so even when combined with anti-CD25 (0% with CAV, 0/7; average NI 4.7%, range 2-9.6%), the latter signifi cantly less than anti-CD25 alone (p<0.0005). Anti-CD4 mAb treatment group induced CAV, with 100% developing CAV (5/5) with an average NI of 35% (range 18-56%) (not signifi cantly different from the control untreated group, but signifi cantly less than anti-CD25 alone (p<0.01).Conclusions: These data indicate that inhibition of NK cells is suffi cient to prevent the early phase of CAV, that CD4 Teff cells amplify CAV in the absence of Treg, and that even in the absence of CD4 T cells, CAV can be induced by other cell types along with NK cells.

Abstract# 15The Role of Complement Activation on Tissue Perfusion and Oxygenation in Airway Allograft Rejection. Mohammad A. Khan, S. Tomlinson, J. Xinguo, M. R. Nicolls. Medicine, Stanford University, Palo Alto, CA, USA; Medical University of South Carolina, Charleston, SC, USA.Introduction: Microvascular loss may be a root cause for chronic rejection in all solid organ transplants. Previous research implicates complement as a potential mediator of microvascular injury during rejection However, complement has never been examined as an actual cause of graft hypoxia and ischemia. The orthotopic tracheal

transplant (OTT) model is ideal for studying the role of complement activation and regulation in the rejection-associated airway ischemia implicated in chronic lung transplant rejection. The current study addresses the impact of complement defi ciency in C3-/- B6 mice and complement inhibition (CR2-Crry; C3 convertase blocker) in OTT recipients with specifi c respect to tissue perfusion and oxygenation. The expression of the complement regulatory protein, CD55, on graft vascular endothelium was also evaluated during rejection.Methods: B6 (H-2b), and C3-/- (H-2b) mice were transplanted with Balb/C (H-2d) mice trachea and evaluated at multiple time points during rejection. Some B6 recipients were also treated with CR2-Crry (0.25 mg i.p. Q3d). Tissue pO2 and vascular fl ow were assessed by bioprobe and FITC-lectin perfusion. CD55 expression was evaluated by confocal microscopy.Results: Perfusion to rejecting allografts stops on d10 and is restored 5-7d later by the ingrowth of recipient-derived vessels. Loss of perfusion is correlated with a signifi cantly lower pO2 (p<0.01). C3 deposition occurs on graft vascular endothelium begins prior to microvascular destruction on d6. Compared to controls, C3-/- and CR2-Crry- treated allograft recipients had signifi cantly higher tissue pO2 (p<0.0001) at all times following transplantation. C3-/- recipients exhibited only a transient period of ischemia during rejection and a rapid return of perfusing vessels relative to C3-replete recipients. Endothelial CD55 expression was signifi cantly decreased on d10 and coincided with the loss of microvascular patency in wild type recipients.Conclusions: C3 defi ciency and inhibition led to signifi cantly attenuated ischemia and hypoxia that our group has previously implicated as a cause of airway fi brosis1. Furthermore, decreased expression of a protein, which normally limits C3 activation (CD55), occurred simultaneously with the loss of tissue perfusion. Thus, both complement activation and dysregulation during rejection are implicated in the microvascular loss associated with chronic rejection.1 JCI 2007; 117:3774-85

ORAL PLENARY EXCHANGE III - PRECLINICAL AND TRANSLATIONAL SCIENCES II

Abstract# 16IFN-γ Negatively Regulates While IL-6 and IL-1β Promote the Induction of Hepatitis C Virus (HCV) Specifi c Th17 Cells in Liver Transplant Recipients with HCV Recurrence. Haseeb Ilias Basha,1 Nancy Steward,1 William Chapman,1 Jeffrey Crippin,1 T. Mohanakumar.1 1Surgery, Internal Medicine, Path&Immunology, Washington Univ in St. Louis.T helper type 17 (Th17) cells have been implicated in the pathogenesis of multiple infl ammatory conditions signifi cantly associated with fi brosis. In this study, we characterized the role of HCV specifi c Th17 cells and the immune mechanisms contributing to their induction in patients with recurrent HCV induced allograft fi brosis following liver transplantation (LTx). Fifty seven LTx recipients with HCV recurrence and 15 normal subjects as well as 9 HCV negative LTx recipients as controls were analyzed for the frequency of CD4+T cells secreting IFNγ, IL-17 and IL-10 in response to HCV derived proteins(NS3,NS4,NS5 and CORE) by ELISPOT. Serum cytokines and chemokines were measured by LUMINEX. In contrast to LTx recipients with no or mild allograft fi brosis, patients with recurrent HCV induced allograft infl ammation and advanced fi brosis demonstrated a lack of HCV specifi c Th1 immunity indicated by a marked decrease in IFN-γ secreting T cells, a profound increase in IL-4 and IL-5 levels, and a signifi cant increase in the frequency of HCV specifi c Th17 cells as well as increased levels of pro-infl ammatory mediators IL-17, IL1-β, IL-6 and MCP-1. Concurrently, patients with advanced allograft fi brosis demonstrated a signifi cant increase in the frequency of Foxp3+ regulatory T cells which specifi cally inhibited IFN-γ but not IL-17 secretion from HCV specifi c CD4+ effector T cells. Furthermore, IFN-γ negatively regulated the IL-6 and IL-1β dependent induction of HCV specifi c Th17 cells. These fi ndings suggest that recurrent HCV infection in LTx recipients induces an infl ammatory cytokine milieu characterized by increased IL-6, IL-1β and decreased IFN-γ levels that favors the development of HCV specifi c Th17 cells. The induction of HCV specifi c Th17 cells that are resistant to regulatory T cells can mediate an aggressive infl ammatory pathology and increased progression to allograft cirrhosis in LTx recipients with HCV recurrence.

Abstract# 17Donor-Specifi c B-Cell Tolerance after ABO-Incompatible Heart Transplantation Is Facilitated by Limited B-Cell Memory in Early Childhood. Simon Urschel,1 Lauren A. Ryan,1 Ingrid M. Larsen,1 Mylvaganam Jeyakanthan,1 Lori J. West.1 1Department of Pediatrics, Cardiac Transplant Research, University of Alberta, Edmonton, AB, Canada.Background:ABO-incompatible heart transplantation in early childhood leads to persistent tolerance of the donor blood group antigens in the majority of patients. In the same age range, immune responses to polysaccharide antigens from infectious agents are impaired. CD21 (complement receptor 2) is a crucial component in the activation of

PRECLINICAL AND TRANSLATIONAL SCIENCES II

5

Abstracts

B-cells towards polysaccharide antigens. We sought to determine relevant differences in the presence of memory B-cells and CD21 expression in early childhood.Methods:Lymphocytes from peripheral blood of children undergoing ABO-compatible or incompatible heart transplantation were analysed for expression of surface markers with 7-colour fl ow-cytometry staining. B-cells were subtyped for their expression of CD27, IgM, IgD and CD21; expression levels of CD81, CD5 and CD1d were quantifi ed. In vitro proliferation assays were performed on carboxyfl uorescein-succinimidyl-ester (CFSE) labelled spleen cells of children and adults, testing responses to stimulation with mitogens as well as erythrocytes and synthetic ABO antigens.Results:55 peripheral blood samples were analyzed from children (median age 14.6 months [0.03-51.3]), 53% after ABO-incompatible transplantation. A signifi cant correlation was found between the prevalence of both IgMhigh (CC: 0.45, p=0.001) and switched IgMlow (CC: 0.31 p=0.027) memory B-cells and age throughout the fi rst 5 years of life. CD21high-expressing cells were less frequent in the fi rst 6 months but showed constant presence thereafter, while CD81 expression was not dependent on age. In adult spleen samples CD21 high-expressing marginal zone B-cells comprised the main pool of CD27+ memory B-cells, whereas in infants this reservoir was nearly absent. In CFSE-assays memory B-cells proliferated signifi cantly more than CD27- B-cells, especially when stimulated as isolated B-cell cultures without T-cell support. When stimulated with non-self blood group antigens, specifi c proliferation was exclusively found in CD27+IgM+ B-cells.Conclusions:CD27+ memory B-cells are crucial for T-independent immune response to polysaccharides such as blood group antigens. Their absence or restriction in early childhood provides the necessary environment to develop tolerance towards the donor blood group. This may be further facilitated by lower expression of the complement receptor 2 (CD21).

Abstract# 18Development of Immune Responses to Self Antigens (K-α1 Tubulin & CollagenV) Following Human Lung Transplant Plays a Pivotal Role in the Pathogenesis of Chronic Rejection. Deepti Saini,1 Sabarinathan Ramachandran,1 Michael Liu,1 Venkataswarup Tiriveedhi,1 Nancy Steward,1 Aviva Aloush,1 Ramsey Hachem,3 Elbert Trulock,1 Alexander Patterson,1 T. Mohanakumar.1,2 1Surgery, Washington University School of Medicine, Saint Louis, MO, USA; 2Pathology and Immunology, Wash. Univ. Sch. of Med., Saint Louis, MO, USA; 3Internal Medicine, Wash. Univ. Sch. of Med., Saint Louis, MO, USA.Immune responses to epithelial cell antigens have been considered to play an important role in the pathogenesis of chronic rejection, Brochiolitis Obliterans Syndrome (BOS), following human lung transplant (LTx). There is agreement that immune responses against mismatched HLA as evidenced by acute rejection episodes, and repeated viral infections predispose to the development of BOS. The goal is to determine whether allo-immune responses lead to the development of immune responses to two self antigens (K-1 tubulin and CollagenV (ColV)) and BOS following human LTx.Development of antibodies (Abs) to donor mismatched HLA during the post-Tx period was analyzed using monthly serum samples by solid phase assays. In addition, development of Abs to two self antigens, K-α1 tubulin and ColV, was determined using an ELISA developed in our laboratory. Donor specifi c HLA Abs (DSA) were detected in 42% of LTx recipients (103 patients) during post-LTx, of which 30.09% also developed Abs to self antigens K-α1 tubulin and ColV. Serial analysis demonstrated that development of DSA preceded development of Abs to self antigens. More signifi cant is that while DSA were usually transient, Abs to self antigens remained persistent once developed. Patients with clinically diagnosed BOS were more likely to be HLAAb+ and epithelial Ab+ than BOS- patients (80% vs. 30% BOS- patients). However, 12.6% of epithelial Ab+ patients had no detectable DSA suggesting that cellular immune responses to mismatched MHC or anti-viral responses can also lead to development of Abs to self antigens and BOS. In addition cellular responses to K-α1 tubulin and ColV were determined using ELISPOT. Peripheral blood lymphocytes from BOS+ but not BOS- patients showed proliferation against both K-α1 tubulin and ColV with higher IFNγ and IL-17 and decreased IL-10 production.In summary, our results strongly suggest that an allo-immune response leading to exposure of cryptic determinants of self antigens and a Th17 immune response to these self antigens play a pivotal role in the immunopathogenesis of chronic rejection post-human LTx.

Abstract# 19Donor-Reactive Memory T Cells in Cynomolgus Monkeys Are Critical to the Rejection of and Tolerance to Allografts. Ognjenka Nadazdin,1 Svjetlan Boskovic,1 Georges Tocco,1 Joren Madsen,1 Roger Wiseman,2 David O’Connor,2 Tatsuo Kawai,1 Benedict Cosimi,1 Gilles Benichou.1 1Surgery, MGH/Harvard Medical School, Boston, MA, USA; 2Primate Research Center, University of Wisconsin, Madison, Madison, WI, USA.Alloreactive memory T cells present in primates are thought to prevent transplant tolerance induction. Therefore it is crucial to characterize these memory T cells, elucidate the mechanisms by which they hinder tolerance and design strategies to block or delete these cells in recipients before transplantation. To address this, we performed an in-depth analysis of the structural and functional properties of allospecifi c memory T cells in Cynomolgus monkeys tested before and after placement of kidney and cardiac allotransplants. After transplantation, we observed a massive expansion of allospecifi c memory T cells, which could be detected for up to 6 months after rejection of the allografts. In a series of monkeys that underwent mixed chimerism induction (irradiation, depletion of lymphoid cells and bone marrow transplantation), only memory T cells survived and expanded homeostatically upon reconstitution of blood and secondary lymphoid tissues. Next, over one hundred donor/recipient combinations were tested pre-transplantation for memory T cell donor-specifi c reactivity using ELISPOT. Memory T cell alloreactivity mediated via direct but not pathway of allorecognition was detected in all combinations. In addition, we found that all MHC class II-matched pairs displayed a low memory alloreactivity. In turn, while MHC mismatching was often associated with a high memory alloreactivity, some recipients exhibited a low anamnestic responsiveness to fully mismatched donors. Finally we observed that recipients with low frequencies of donor-specifi c memory T cells pre-transplantation were more susceptible to tolerance via mixed chimerism and displayed long-term kidney allograft survival. Interestingly, memory T cells underwent similar homeostatic expansions in both tolerant and rejecting monkeys while donor-specifi c memory T cells were activated only in rejecting monkeys. In summary, our results support the view that the presence of high frequencies of donor-specifi c memory T cells pre-transplantation results in tolerance resistance. Our results also show that transplant tolerance achieved via mixed chimerism is associated with selective inhibition of the activation/expansion of donor-specifi c memory T cells.

Abstract# 20Non-Human Primate Lung Allografts Show Enhanced Immunogenicity as Compared to Analogous Kidney Allografts. Akihiro Aoyama,1 Choo Y. Ng,1 Timothy M. Millington,1 Svjetlan Boskovic,1 John C. Wain,1 Joren C. Madsen,1 Tatsuo Kawai,1 James S. Allan.1 1Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.BACKGROUND: We have reported induction of renal allograft tolerance in non-human primates using a mixed-chimerism technique. Here, we applied this same technique to lung allotransplantation in cynomolgus monkeys.METHODS: Nine pairs of cynomolgus monkeys were used for left single-lung transplantation. Donors and recipients were ABO-compatible and mismatched at the MHC class I and class II loci. The pre-transplant conditioning regimen consisted of total body irradiation (1.5 Gy×2), thymic irradiation (7Gy), and anti-thymocyte globulin. The recipients underwent lung and bone marrow transplantation on day 0, followed by anti-CD154 mAb (6 doses over 12 days) and a one-month course of cyclosporine. Because acute cellular rejection was found in three of the initial four lung recipients, the regimen was modifi ed by adding anti-CD8 mAb (days 0 and 2) in the subsequent two recipients. Post-transplant administration of α1-antitripsin was also added in the last 3 recipients. The results were compared with 8 recipients that received kidney allografts using the same conditioning regimen. Chimerism in peripheral blood was determined with fl ow cytometry.RESULTS: Multi-lineage chimerism was detected in eight lung recipients and the other could not be technically assessed. Granulocyte chimerism developed up to 88.1 ± 7.6% in the lung recipients and 69.8 ± 23.5% in the kidney recipients (p = .055), and lymphocyte chimerism up to 7.6 ± 4.4% in lung recipients and 4.1 ± 2.2% in kidney recipients (p = 0.067). Two lung allografts were lost due to causes unrelated to rejection. The other seven lung recipients rejected their allografts by day 85 (85, 62, 55, 33, 26, 22, and 21 days), while the same regimen induced long-term allograft survival without the need for maintenance immunosuppression in the kidney recipients (>2500, >2000, 837, 755, 401, 373, 206, and 58 days) (p < 0.01).CONCLUSIONS: Despite the successful induction of mixed chimerism in recipients of fully mismatched lung allografts, we have not observed long-term graft survival, as has been seen in an analogous kidney model. We suspect that this organ-specifi c difference in the ability to induce graft tolerance relates to the fact that the lung, being a barrier organ, exists in a more infl amed state, which exerts a deleterious infl uence on the antigenicity, immunogenicity, and tolerogenicity of this organ.


CLINICAL SCIENCE II

6

ORAL PLENARY EXCHANGE IV - CLINICAL SCIENCE II


Abstract# 23Antibody-Mediated Rejection (AMR) after Pancreas and Pancreas-Kidney Transplantation. Erika B. Rangel,1 Denise M .A. C. Malheiros,2 Maria Cristina R. Castro,1 Irina Antunes,1 Margareth A. Torres,3 Fabio Crescentini,4 Tercio Genzini,4 Marcelo Perosa.4 1Nephrology, Albert Einstein Hospital, Sao Paulo, SP, Brazil; 2Pathology, Albert Einstein Hospital, Sao Paulo, SP, Brazil; 3Human Laboratory of Histocompatibility, Albert Einstein Hospital, Sao Paulo, SP, Brazil; 4Surgery, Albert Einstein Hospital, Sao Paulo, SP, Brazil.Background: Antibody mediated-rejection (AMR) requires specifi c diagnostic tools and treatment and is associated with lower graft survival.Methods: We prospectively screened C4d in both pancreas and kidney for cause biopsies and reported its pattern of deposition, the grafts outcome, and the correlation with laboratorial data. Optic microscopy, immunofl uorescence for C4d staining in frozen tissue and donor-specifi c antibody search by Luminex® were performed in 33 kidney biopsies (21 patients) and 35 pancreas biopsies (27 patients). Serum amylase and lipase, amylasuria, fasting blood glucose and 2-hour capillary glucose were analyzed for both acute T-cell mediated rejection (TCMR) and AMR.Results: We found that 27.3% of kidney biopsies and 43% of pancreas biopsies showed C4d staining (66.7% and 53.3% diffuse in peritubular and interacinar capillaries, respectively). Isolated exocrine dysfunction was the main indication for pancreas biopsy (54.3%) and was followed by both exocrine and endocrine dysfunctions (37.1%) and isolated endocrine dysfunction (8.6%). Laboratorial parameters were comparable between T-cell mediated rejection and AMR: amylase 151.5 vs. 149 U/L (P = 0.075), lipase 1120 vs. 1288.5 U/L (P = 0.83), amylasuria variation 46.5 vs. 61 % (P = 0.97), FBG 69 vs. 97 mg/dL (P = 0.20) and 2-hour CG maximum 149.5 vs. 197.5 mg/dL (P = 0.49), respectively. Amylasuria values after treatment correlated with pancreas allograft loss (P = 0.015). On average, acute AMR of either pancreas or kidney allograft was treated with a mean of 6.8 sessions of plasmapheresis (range 3 to 11 sessions) and 2.2 doses of intravenous Immunoglobulin 1g/kg (range 1 to 4 doses).Conclusion: These data suggest that C4d staining should be routinely investigated when pancreas allograft dysfunction is present due to its high detection in cases of rejection.

Abstract# 24Autoantibody Conversion Is a Risk Factor for Recurrence of Type 1 Diabetes (T1DR) after Simultaneous Pancreas-Kidney (SPK) Transplantation. Stavros Diamantopoulos,2 Gloria Allende,2 Gaetano Ciancio,1 Junichiro Sageshima,1 Linda Chen,1 Alberto Pugliese,2 George W. Burke.1 1Surgery, Division of Transplantation, University of Miami Miller School of Medicine, Miami, FL, USA; 2Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, USA.We studied the prevalence of GAD65 and IA-2 autoantibodies (AAb) before transplant and on follow-up (range 0.22-15.98 years, mean 5.4 + SD 3.59 years) in 200 patients with type 1 diabetes (T1D) who received SPK transplants. Fifty-one percent (103/200) were AAb- on follow-up, 27.5% (55/200) had at least one AAb before transplant persisting on follow-up and 22% (42/200) converted to AAb positivity. Over time, 36 patients (18%) returned to hyperglycemia (HG) while 164 (82%) remained normoglycemic (NGT). AAb conversion was more frequent in HG (18/36, 50%) compared to NGT patients (24/164, 14.6%, p=0.00001). AAb persistence was similar in the two groups (7/36, 19.4% HG vs 48/164, 29.3% NGT, p=0.3). There were more AAb- NGT patients (92/164, 56.1%) than HG (11/36, 30.6%, p=0.006). AAb conversion and persistence were associated with an odds ratio (OR) of 6.2 and 5.41 for developing HG, respectively, compared to AAb-. 13 patients in the HG group had T1DR confi rmed by biopsy, and 11/13 were AAb+. Twelve other patients had milder diabetes with an undetermined cause, of whom 9 were AAb+ with 5 converters. The remaining 11 HG patients developed hyperglycemia in relation to chronic pancreas rejection: 4/11 were AAb+ vs 11/13 T1DR patients, p= 0.03, OR=9.62), of which 3 were converters. Thus, AAb conversion can help predicting T1DR but not chronic rejection (p=0.44, OR=1.6) in SPK patients. By comparison of Kaplan-Meier survival curves, more patients experienced HG or T1DR on follow-up among AAb converters than among AAb- patients (p=0.003 comparing the entire HG group, p=0.0001 excluding the patients with rejection, and p=0.02 only comparing the T1DR to the NGT patients). Thus, AAb conversion is a risk factor for the development of T1DR in SPK recipients

Abstract# 25Primary Outcomes from a Randomized, Phase III Study of Belatacept Versus Cyclosporine in Kidney Transplant Recipients (BENEFIT Study). Thomas Pearson,1 Flavio Vincenti,2 Josep Grinyo,3 Bernard Charpentier,4 Jose Medina Pestana,5 Lionel Rostaing,6 Yves Vanrenterghem,7 Gregory Di Russo,8 Pushkal Garg.8 1Emory Univ. School of Medicine; 2UCSF; 3Univ. Hosp. of Bellvitge; 4Bicetre Hosp.; 5Hospital do Rim e Hipertensao Unifesp; 6Univ. Hosp. Toulouse; 7Univ. Hosp. Leuven; 8Bristol-Myers Squibb; 9Bristol-Myers Squibb.Introduction: Belatacept, a co-stimulation blocker, is developed as an immunosuppressant for kidney transplant (tx) recipients to avoid renal and extra-renal toxicities of calcineurin inhibitors that impact long-term patient/graft survival. BENEFIT assessed belatacept-based regimens vs a cyclosporine-based regimen (CsA) in kidney tx recipients.Methods: BENEFIT is a 3-year, randomized, Ph III study in adults receiving a kidney tx from a living or deceased donor (LD, DD) with an anticipated CIT< 24 hrs. Patients were randomized 1:1:1 to a more intensive (MI) or less intensive (LI) regimen of belatacept or CsA; all patients received basiliximab induction, MMF, and corticosteroids. Co-primary endpoints were composite patient/graft survival, composite renal impairment (measured GFR (mGFR) < 60 mL/min/1.73 m2 at month 12 or a decrease in mGFR ≥ 10 mL/min/1.73 m2 from month 3 to month 12), and incidence of acute rejection (AR).Results: 666 patients were randomized and transplanted. 58% received LD tx; 42% from DD. Patient/graft survival with belatacept regimens was non-inferior to CsA (95.4% MI; 96.5% LI; 92.8% CsA) at month 12. Renal function was superior in belatacept vs CsA patients with fewer patients reaching the composite renal impairment endpoint (55% MI; 54% LI; 78% CsA; p<0.0001 MI or LI vs CsA) and the mGFR at Month 12 (65 mL/min MI, 63 mL/min LI, and 50 mL/min CsA; p<0.0001 MI or LI vs CsA). The incidence of AR was 22%, 17%, and 7% in the MI, LI, and CsA groups; the LI regimen was non-inferior to CsA. AR in belatacept patients had limited impact on graft survival and the relative renal benefi t of belatacept. NODAT trended lower and CV risk factors were lower with belatacept. Infection and overall malignancy rates were similar across arms; PTLD was observed in 1(0.5%), 2(0.9%), and 1(0.5%) patients in the MI, LI, and CsA groups in the fi rst year.Conclusions: At 1 year, belatacept regimens demonstrated superior renal function and similar patient/graft survival vs CsA, despite an increase in AR in the early post-tx period. Belatacept represents a promising, non-nephrotoxic therapy option in kidney tx patients.DISCLOSURE: Vincenti, Flavio: Grant/Research Support, Novartis; Grinyo, Josep: Other, Bristol-Myers Squibb, Advisory Board Member; Vanrenterghem, Yves: Other, Astellas, Advisory Board Member; Di Russo, Gregory: Employee, Bristol-Myers Squibb; Garg, Pushkal: Employee, Bristol-Myers Squibb.

B CELLS AND T CELLS

7

Abstracts

Abstract# 26A Single Center Study of the Use of Campath-1H Induction in Adult Liver Transplantation: Long-Term Follow Up of 203 Patients. Panagiotis Tryphonopoulos,1 Phillip Ruiz,1 Debbie Weppler,1 Seigo Nishida,1 David M. Levi,1 Jang Moon,1 Gennaro Selvaggi,1 Akin Tekin,1 Eddie Island,1 Barbara Sotolongo,1 Alex Volsky,1 Leopoldo Arosemena,1 Paul Martin,1 Ernesto Pretto,1 Manuel Carreno,1 Seung H. Shin,1 Tamara Defranc,1 Maria B. Torres,1 Andreas Tzakis.1 1Surgery-Transplant, University of Miami, Miami, Fl, USA.Background: We investigated the effect of Campath -1H induction in adult liver transplantation in our center.Methods: We performed a retrospective analysis of all adult patients who underwent liver transplantation using C1H induction and low dose Tacrolimus maintenance immunosuppression at our center. C1H was not administered to Hepatitis-C patients . Patients transplanted until April 2004 received their fi rst dose of C1H pre-operatively, those transplanted afterwards received the fi rst C1H infusion immediately after the transplant.Results: Two hundred and three patients received a liver allograft with C1H induction immunosuppression, 74 received the fi rst dose pre-operatively, 129 immediately post-operatively. Patient and graft survival were 91.62% and 89.16% at one year and 86.87% and 80.43% at seven years respectively. Freedom from biopsy proven rejections was 74.93% at 7 years post-transplant. There was no signifi cant difference in patient, graft survival or incidence of rejection between patients that received the fi rst dose of Campath-1H before or after the transplant or if patients received one or two doses post transplant. Average Tacrolimus trough levels were between 5-7 ng/ml for the fi rst 2 years post transplantation and less than 5 ng/ml thereafter. One hundred and twenty six patients did not receive maintenance steroids. The remaining seventy seven patients received steroids for their autoimmune disease (n=13), treatment of rejection (n=59), or for other reasons (n=5).The overall rate of infections was19.57% at 5 years. Five patients received a kidney graft and an additional patient is currently on dialysis.Immunosuppression withdrawal: Two patients are off immunosuppression for a period of 5 months and 3 months respectively, 38 months and 90 months post-transplantation.Conclusion: Excellent short and long term results are achieved with Campath-1H induction and half dose Tacrolimus ISP in non-HCV liver transplant recipients. Postoperative administration of C1H seems to be just as effective as preop.

Abstract# 27The Impact of Sirolimus on Hepatitis C Recurrence after Liver Transplantation. Sonal Asthana,1 Christian Toso,1 Glenda Meeberg,1 David L. Bigam,1 Andrew J. Shapiro,1 Andrew Mason,1 Norman M. Kneteman.1 1Division of Multiorgan Transplantation, University of Alberta Hospital, Edmonton, AB, Canada.Background: While some immunosuppression strategies may accelerate hepatitis C virus (HCV) recurrence after liver transplantation (LT), the impact of sirolimus is not known.Methods: One hundred and forty-one consecutive patients, who had undergone a fi rst LT for HCV cirrhosis, were analyzed retrospectively, including 88 with de novo sirolimus therapy. The risk of biopsy-proven HCV recurrence and patient survival were assessed, using known and suspected risk factors for HCV recurrence as co-variates (transplant era, donor and recipient ages, MELD score, cold ischemia time, immunosuppressant drugs, and steroid-treated rejection).Results: Overall, 73.1% of the cohort developed biopsy-proven HCV recurrence. The incidence was not signifi cantly different for patients on sirolimus (75 % vs. 69.8%, p=0.5), and there was no difference found for time to recurrence, mean activity or fi brosis scores. Donor age and acute rejection episodes (HR 1.02 (95 % CI 1.01-1.03), p=0.03; and HR 2.8 (95% CI 1.8-4.3), p<0.01) were the only factors affecting HCV recurrence. Sirolimus treatment did not alter patient survival.Among patients treated with SRL-based IS, higher drug-AUC levels were associated with a trend to lower disease activity and fi brosis at diagnosis, but higher SRL levels were associated with shorter recurrence-free survival (p=0.038).Conclusion: This analysis suggests that de novo sirolimus immunosuppression does not signifi cantly affect timing and severity of post-transplant HCV recurrence.

Abstract# 28Primary Outcomes from a Randomized, Phase III Study of Belatacept Versus Cyclosporine in ECD Kidney Transplants (BENEFIT-EXT Study). Antoine Durrbach,1 Sander Florman,2 Christian Larsen,3 Jose Medina Pestana,4 Yves Vanrenterghem,5 Flavio Vincente,6 Alan Block,7 Pushkal Garg,7 Josep Grinyo.8 1Bicetre Hospital; 2Mount Sinai School of Medicine, New York, NY; 3Emory University School of Medicine; 4Hospital do Rim e Hipertensao Unifesp; 5University Hospital Leuven; 6UCSF; 7Bristol-Myers Squibb; 8University Hospital of Bellvitge.Introduction: Belatacept, a selective co-stimulation blocker, is being evaluated as an immunosuppressant in renal allograft recipients to avoid the renal and extra-renal toxicities of calcineurin inhibitors. As recipients of extended criteria donor (ECD) kidneys are at elevated risk of graft dysfunction and loss, they may particularly benefi t from a non-nephrotoxic option such as belatacept.Methods: BENEFIT-EXT is a 3-year, randomized, Ph III study in adults receiving an ECD kidney transplant. Patients were randomized 1:1:1 to receive a more intensive (MI) or less intensive (LI) regimen of belatacept or CsA; all patients received basiliximab induction, MMF, and corticosteroids. At 12 months, the two co-primary endpoints were: composite patient/graft survival and composite renal impairment (measured GFR < 60 mL/min/1.73 m2 at month 12 or a decrease in measured GFR ≥ 10 mL/min/1.73 m2 from month 3 to month 12). Secondary endpoints included the incidence of acute rejection (AR).Results: 543 patients were randomized and transplanted. Patient/graft survival with belatacept was non-inferior to CsA (86% MI, 88% LI, 85% CsA) at month 12. Renal function was superior in belatacept MI vs CsA patients with fewer patients reaching the composite renal end point (71% MI, 76% LI, and 85% CsA; p=0.002 MI vs CsA; p=0.06 LI vs CsA) and by the measured GFR at Month 12 (52 mL/min MI, 50 mL/min LI, and 45 mL/min CsA; p=0.008 MI vs CsA; p=0.10 LI vs CsA). The prevalence of AR was 17%, 18%, and 14% in the MI, LI, and CsA groups. NODAT trended lower and CV risk factors were lower with belatacept. The overall rates of infection and malignancy were comparable between groups. PTLD was observed in 1 (0.5%) and 2 (0.9%) patients in the MI and LI groups and in none in the CsA group in the fi rst 12 months.Conclusions: Belatacept regimens demonstrated better renal function, with comparable patient/graft survival and AR compared to a CsA-based regimen in ECD kidney transplant recipients. Belatacept represents a promising immunosuppressant therapy in ECD kidney transplant recipients.DISCLOSURE: Larsen, Christian: Other, Bristol-Myers Squibb, Clinical Trials; Vanrenterghem, Yves: Other, Astellas, Advisory Board Member; Vincente, Flavio: Other, Astellas Pharma US Inc, Research Contract; Block, Alan: Employee, Bristol-Myers Squibb; Garg, Pushkal: Employee, Bristol-Myers Squibb; Grinyo, Josep: Other, Bristol-Myers Squibb, Advisory Board Member.

ORAL PLENARY EXCHANGE V - B CELLS AND T CELLS

Abstract# 29Prolonged Allograft Survival and Impaired Alloreactive CD8 T Lymphocyte Memory Response in Sensitized Recipients in the Absence of Humoral Immunity. Haofeng Ji,1 Xiuda Shen,1 Feng Gao,1 Yuan Zhai,1 Ronald W. Busuttil,1 Jerzy W. Kupiec-Weglinski.1 1Department of Surgery, Dumont-UCLA Transplantation Center, Los Angeles, CA, USA.Background: The sensitized patients can develop an accelerated form of allograft (Tx) rejection that is mediated by humoral and/or T cell-mediated immune responses. Unlike in naive counterparts, alloreactive CD8 activation and Tx rejection are usually resistant to most of the immunosuppressive regimenss. Methods and Results: In our model of cardiac graft rejection in sensitized recipients, C57BL/6 WT or C57BL/6-Igh-6tm1Cgn/J (BKO) mice were fi rst challenged with MHC-incompatible Balb/c skin. We found that alloreactive CD8+CD62LhighCD44high(Tmem) differentiation and CD8+CD62LlowCD44high (Teff) activation were signifi cantly reduced in BKO recipients (vs. WT) during 40-60 day follow-up. Interestingly, CD8 T lymophocyte development and homeostasis, tracked by IL2R, IL7R, and IL15R expression, on Tmem and Teff was impaired in BKO hosts. The sensitized recipients were challenged at day 40 post skin Tx, with Balb/c hearts in conjunction with CD154 blockade (MR1, 0.5mg IV day 0), with or w/o CD4 (GK1.5) or CD8 (2.43) depleting mAb (day -2, -1, 0; 0.5 mg IV). Cardiac Tx were rejected within 3.5 days in sensitized WT mice. In BKO, the survival increased to 6 days (p<0.001). Unlike MR1-resistant in WT (4 days), CD154 blockade prolonged cardiac Tx survival in BKO sensitized hosts (8 days, p<0.001). Long-term cardiac Tx survival was observed in BKO sensitized recipients after CD154 blockade plus CD8, but not CD4 T cell depletion. In BKO hosts, CD4 depletion w/o or with MR1 resulted in cardiac Tx rejection in 8 and 10 days, resp. (vs. 6 and 8 days in BKO w/o or with MR1 alone; p<0.05). After CD8 depletion, 50% of cardiac Tx survived >100 days (vs. 6 days w/o CD8 depletion in BKO; or vs. 10 days with CD8 depletion in WT; p<0.001). Strikingly, 100% of cardiac Tx survived >100 days after concomitant CD8 depletion and MR1 treatment (vs. 8 days after MR1 alone in BKO; p<0.001). Conclusion: This is the fi rst kinetic

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study of CD8 Teff activation, Tmem development, and IL2R, IL7R, and IL15R expression-supported homeostasis in sensitized cardiac Tx recipients, with or w/o humoral immunity. Our fi ndings provide evidence that: 1) allo-Ab secreted B cells do affect CD8 Teff activation and Tmem development. 2) B cell defi ciency prolongs cardiac Tx survival in sensitized host (vs. WT). 3) CD8, but not CD4 T cell depletion results in long-term cardiac Tx acceptance in BKO sensitized hosts.

Abstract# 30B Cells Promote Development of Alloreactive Memory T Cells. Yue-Harn Ng,1 Harish C. Chandramoorthy,1 Qi Li,1 Qiang Zeng,1 Autumn Marlowe,1 Rosemary Hoffman,1 Geetha Chalasani.1 1Medicine & Immunology and Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA.B cells and memory T cells contribute to acute and chronic allograft rejection. We tested whether B cells promote differentiation of alloreactive T cells to memory T cells and contribute to allograft rejection. Methods and Results: B-cell defi cient µMT and wild-type (wt) mice reject skin allografts with similar graft survival (MST 18 days, n=6/gp). Despite comparable alloreactive effector T cells (at rejection), memory CD4 and CD8 T cells (> 8 weeks after rejection) were ten and twelve fold fewer, respectively, in µMT than in wt mice. To test if antigen presentation by B cells promotes differentiation of alloreactive T cells, syngeneic bone marrow chimeras of irradiated µMT recipients and µMT, wt, and/or Ab1-/-β2m-/- (MHCko, lack MHC I & II expression on all cells) donors were utilized. B cells but not other APCs in µMT+MHCko chimeras lack expression of MHC I & II and are thus impaired in antigen presentation. Heart, but not skin allograft rejection was delayed in µMT+MHCko than in µMT+ wt chimeras (MST = 23 days & 15 days, respectively, p=0.03, n = 5/grp). Alloreactive memory T cells after skin allograft rejection were fewer in µMT+ MHCko (7-fold fewer CD4, p = 0.02, & 5-fold fewer CD8, p = 0.003, n = 5/grp) than in µMT+ wt chimeras, showed impaired allogeneic cell lysis, and did not accelerate skin allograft rejection. To further test if defi cient CD4 T cell help contributed to impaired CD8 T cell memory in µMT+ MHCko chimeras, alloreactive T cell differentiation was studied in µMT+ β2m-/- chimeras that lack expression of MHC I on B cells and can present antigen to CD4 T cells but not CD8 T cells. Alloreactive effector and memory T cells were comparable between µMT+ β2m-/- and µMT+ wt chimeras suggesting that development of CD8 T cell memory does not require antigen presentation by B cells but requires CD4 T cell differentiation that is dependent upon antigen presentation by B cells. Conclusions: Heart allograft rejection and development of alloreactive memory T cells was impaired in the absence of antigen presentation by B cells. Antigen presentation by B cells to CD4 T cells was required for optimal CD4 T cell differentiation to provide help for generation of alloreactive CD8 memory T cells. These results emphasize the role of B cells in alloimmune responses and highlight the need for B-cell targeted therapies for lasting allograft survival.

Abstract# 31In Vivo BLyS Neutralization Induces Robust Transplantation Tolerance by Down-Regulating APC Costimulation and Polarizing T-Cell Cytokine Profi les. Ronald Parsons,1 Ming Yu,1 Kumar Vivek,1 Ghazal Zekavat,1 Susan Rostami,1 Robert Redfi eld,1 Brigitte Koeberlein,1 Thi-Sau Migone,2 Michael Cancro,1 Ali Naji,1 Hooman Noorchashm.1 1University of Pennsylvania School of Medicine; 2Human Genome Sciences.We previously demonstrated that in vivo BLyS neutralization depletes mature peripheral B cells and, in conjunction with a 2-week course of rapamycin (rapa), promotes transplantation tolerance to murine islet allografts (B6->BALB/c MST>251 days; BALB/c->B6 MST >168 days). Treatment with neutralizing anti-BLyS (aBLyS) mAb depletes mature follicular B cells. This depletion is accompanied by a relative expansion of tolerance susceptible transitional B cells for up to 9 weeks post-transplantation.Here, we report that the combined aBLyS/Rapa regimen caused a marked attenuation of CD4+ T cell proliferation after in vitro stimulation with anti-CD3 and anti-CD28. At 3, 5, 7, 11, and 31 weeks after initiation of treatment there was a signifi cant reduction in CD4+ T cell proliferation (p<0.05) in this assay. Interestingly, the combined aBLyS/Rapa therapy also promoted a signifi cant reduction in soluble interleukin(IL)-2 and a signifi cant increase in IL-10 production in vitro. Furthermore CD4+ T-cell proliferation in mice treated with the aBLyS/Rapa regimen was only partially restored when co-cultured with untreated syngeneic splenocytes. Moreover, we found that CD4+ T cells from splenocytes of untreated mice proliferated with only half the intensity when co-cultured with splenocytes from mice treated with the aBLyS/Rapa regimen. We have also observed a marked reduction in the proliferation of purifi ed T cells from untreated mice that were co-cultured with purifi ed antigen presenting cells from mice treated with the aBLyS/Rapa regimen (<0.05). Interestingly, the aBLyS/Rapa regimen polarized T-cell activation profi les toward IL-5, IL-10, and transforming growth facter-beta production, consistent with the T-regulatory type 1 cell phenotype.In summary, in vivo BLyS neutralization, combined with a transient course of Rapamycin, was associated with a signifi cantly reduced CD4 T cell division profi le and a polarization of the activated CD4+ T cell cytokine profi le towards a T regulatory

phenotype. This modulation of CD4+ T cell activation profi le was imposed by the APC compartment of aBLyS/Rapa treated mice and was not T-cell intrinsic. These data indicate that in vivo BLyS neutralization may be a novel and effective means of promoting transplantation tolerance.DISCLOSURE: Migone, Thi-Sau: Employee, Human Genome Sciences, Inc.; Cancro, Michael: Grant/Research Support, Human Genome Sciences, Inc.

Abstract# 32The Novel Role of B7.1, as an Alternate PDL1 Receptor, in Regulating Alloimmune Responses In Vivo. Jun Yang, Leonardo V. Riella, Susanne Robles, Mohamed H. Sayegh, Anil Chandraker. Transplantation Research Center, Brigham and Women’s Hospital & Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA.We have recently shown that the interaction between B7.1 and PDL1 is functional in inhibiting alloimmune responses in vivo (ATC 2009). In this study we further expanded our investigation to the roles and mechanisms of this interaction in a bm12 into B6 MHC class II mismatched cardiac transplant model in mice.We transplanted bm12 hearts into WT, B7.1 or B7.2-defi cient (KO) B6 mice and treated the recipients with anti-PDL1 mAb. While PDL1 blockade accelerated allograft rejection in WT (MST=34.5 d, p=0.0032) and B7.2KO (MST=48.5, p=0.04) recipients, it failed to do so in B7.1KO recipients, all of whose grafts survived long-term, similar to those of untreated WT recipients. Furthermore, bm12 hearts transplanted into PD1KO recipients survive long-term (>56 d). However, when blocking anti-PDL1 mAb was administered to PD1KO mice, we observed an accelerated rejection (MST=35.5 d). The acceleration of rejection in PD-1 KO recipients by PDL1 double blockade was associated with increased frequency of IFN-gamma and IL-4 producing cells (p<0.05). However, this blockade did not signifi cantly affect the percentage of effector T cells and regulatory T cells.These data clearly indicate that PDL1 interaction with B7.1 plays a critical role in regulating alloimmune responses. This pathway may affect the function of T cells and act in the effector phase rather than affect the activation, differentiation and proliferation of T cells. Our results have potentially important clinical implications since human trials with chronic B7 T cell costimulatory pathway blockade are ongoing in kidney transplant recipients; and such blockade may have a negative impact on long-term allograft outcome and tolerance induction.

Abstract# 33An IL-21 Receptor-Targeted Antagonizing IL-21/Fc Protein Promotes Allograft Tolerance. Wensheng Zhang,1 Dong Zhang,1 Joshua Lee,1 Miaoda Sehn,1 Yan Tian,1 W. P. Andrew Lee,1 Xinxiao Zheng.1 1Division of Plastic and Reconstructive Surgery, Thomas E. Starzl Transplantation Institute, University of Pittsburgh, Pittsburgh, PA, USA.Regulatory T (Treg) cells play a critical role in the maintenance of the immune system’s homeostasis. IL-21 emerges as a key modulator of TGF-beta signaling, leading to the reciprocal differentiation of Treg and Th17 cells. In this study, we investigated the role of IL-21 in the allograft response using a MHC mismatched murine transplant model. Two novel IL-21 receptor specifi c antagonists were created as high affi nity, long-lasting mutant IL-21/Fc immunoligands, where a single (smIL-21/Fc) or double (dmIL-21/Fc) amino acid were substituted in the 4th helix of IL-21. Wild-type IL-21 fusion protein was also created (wtIL-21/Fc). To investigate the effect of IL-21 antagonists fusion proteins on cell proliferation, we co-cultured anti-CD3 stimulated C57BL6 splenocytes with wtIL-21/Fc, smIL-21/Fc and dmIL-21/Fc. wtIL-21/Fc, but not dmIL-21/Fc, greatly augmented anti-CD3 driven T cell proliferation. To examine the effect of mIL-21/Fc fusion protein on the balance of Treg and Th17 differentiation, we analyzed by FACS the conversion of Foxp3 and IL-17 from Naïve CD4+Foxp3- T cells, which co-cultured with LPS-matured BM DC, TGF-beta and IL-21, in the presence or absence of dmIL-21/Fc. dmIL-21/Fc blocked the effect of IL-21 on favoring TGF-beta-driven Th17 cells induction and enhanced the Foxp3+ Treg cells conversion. We next treated diabetic B6AF1 mice at the time of DBA/2 islet cell transplantation with 5 ug /mouse of wtIL-21/Fc, smIL-21/Fc, or dmIL-21/Fc for 8 days. smIL-21/Fc treatment led to prolonged graft survival (MST > 55 days) and dmIL-21/Fc treatment led to indefi nite graft survival > 120 days in 2 of 3 mice. Since we have been able to induce permanent graft survival in an MHC mismatched islet allograft model using IL-21 antagonizing monotherapy, IL-21 and/or IL-21R+ cells are likely important components of the allograft response. mIL-21/Fc may offer a new therapeutic approach for the selective targeting of alloimmune T cells and promoting allograft tolerance.

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Abstracts

Abstract# 34Anti-LFA-1 Synergizes with CD28/CD154 Blockade To Inhibit Anti-Donor Cytotoxicity and Promote Graft Survival in Recipients Possessing Donor-Reactive Memory T Cells. Mandy L. Ford,1 Craig M. Steeds,1 Maylene E. Wagener,1 Jennifer M. Robertson,1 Samantha S. Hanna,1 Allan D. Kirk,1 Christian P. Larsen.1 1Surgery, Emory University, Atlanta, GA, USA.Blockade of the CD28 and CD40 pathways represents a potent method of inhibiting primary alloreactive T cell responses following transplantation. However, numerous studies have demonstrated that the presence of pre-existing memory T cells profoundly reduces the effi cacy of CD28/CD40 pathway blockade-based therapies. LFA-1 is an integrin that is upregulated on memory T cells relative to naïve T cells. We hypothesized that combined blockade of CD28, CD40, and LFA-1 pathways in recipients possessing donor-reactive memory T cells might result in prolonged graft survival as compared to CD28/CD40 pathway blockade alone. In order to address this question we made use of a transgenic model system wherein recipients possessing donor-reactive T cells specifi c for a surrogate minor antigen were transplanted with donor tissue. Specifi cally, TCR transgenic T cells (OT-I) specifi c for SIINFEKL/Kb were adoptively transferred into naïve B6 recipients, which were then infected with ovalbumin- (SIINFEKL) expressing Listeria monocytogenes (LM-OVA) or wild-type Listeria as a control. At memory, mice received a skin graft expressing ovalbumin (mOVA), and therefore also the SIINFEKL epitope. Engraftment of mOVA skin on LM-OVA memory recipients resulted in rejection with accelerated kinetics relative to LM-infected controls (MST=13d vs 20d). Following treatment with CTLA-4 Ig and anti-CD154, 9/10 LM-OVA infected recipients experienced costimulation blockade-resistant rejection (MST 19 d), while LM-infected controls went onto long-term graft survival of > 80 days (p<0.001). In contrast, 8/10 recipients treated with CTLA-4 Ig, anti-CD154, and anti-LFA-1 experienced long term graft survival of >80 days (p<0.001). In order to address the mechanism of anti-LFA-1-induced prolongation in graft survival, in vivo cytotoxicity was measured by adoptively transferring donor-antigen-bearing (CFSEint) or syngeneic (CFSEhi) CD45.1+ target cells into graft recipients. We found that CD28/CD154 and LFA-1 pathway blockade synergized in attenuating donor-specifi c cytotoxicity on a systemic level. These results suggest that anti-LFA-1 may impact the ability of donor-reactive memory T cells to differentiate into competent effector CTL following secondary stimulation, and thus to mediate rejection following transplantation.

ORAL PLENARY EXCHANGE VI - REGULATORY T CELLS


Abstract# 36Pretransplant Infusion of Donor B Cells Enhances Donor-Specifi c Allograft Survival through Activating Double Negative Regulatory T Cells. Julia Fang Gao,1 Megan S. Ford McIntyre,2 Ramesh B. Vanama,2 Stephen C. Juvet,2 Xujian Li,2 Edward Y. Kim,2 Li Zhang.2 1Department of Immunology, University of Toronto, Toronto, ON, Canada; 2Multi-Organ Transplantation Program, Toronto General Research Institute, University Health Network, Toronto, ON, Canada.Pretransplantation donor specifi c transfusion or donor lymphocyte infusion (DLI) has been shown to enhance donor-specifi c allograft survival in rodents, primates and humans. However, the cell subset that is critical for the DLI effect and the mechanisms involved remain elusive. In this study, we monitored various donor cell subsets after DLI in a single MHC class I locus-mismatched skin transplantation mouse model. We found that donor B cells, but not dendritic cells, are the major surviving donor antigen presenting cells in recipients following DLI. Interestingly, infusing purifi ed donor B cells but not B cell-depleted splenocytes resulted in signifi cantly enhanced donor-specifi c skin allograft survival. Furthermore, mice receiving purifi ed B cells showed a signifi cant decrease in both percentage and total number of donor-specifi c CD8+ T cells concomitant with an increase in antigen-specifi c αβ-TCR+CD4-CD8-NK1.1- double negative T regulatory cells (DN Tregs). In addition, we found that donor B cells were able to donate alloantigen to DN Tregs and prime DN Treg proliferation in an antigen-specifi c fashion. These data demonstrate that donor B cells enhance allograft survival by activating recipient DN Tregs, which may suppress and/or eliminate anti-donor CD8+ T cells. These fi ndings provide novel insights into the mechanisms underlying DLI-induced transplant tolerance and suggest that DN Tregs have great potential as an antigen-specifi c immune therapy to enhance allograft survival.

Abstract# 37CD14 and CD36 Restrain Inducible T reg Generation and Prevent Costimulatory Blockade Extension of Allograft Survival. Hua Shen, Daniel Robert Goldstein. Internal Medicine, Yale University, New Haven, CT, USA.Emerging evidences suggest that the innate immune system plays a critical role in initiation of adaptive immune response during organ transplantation. We examined the impact of two innate receptors, CD14 and CD36, on alloimmunity. We found that without immune modulation, wild-type C57BL/6 (WT) and CD14-/-CD36-/- mice rejected allogeneic BALB/c skin grafts at a similar tempo. We next repeated the experiment but this time we administered peri-operative anti-CD45RB and anti-CD154, a protocol that induces immune regulation to allografts. In this case, we noted that double defi cient mice, but not single mutant mice, exhibited a large and signifi cantly delayed time to allograft rejection (median survival time 105 days) vs. wild type recipients (52 days, p < 0.0012). This phenotype was associated with defective T cell responses as CD14-/-CD36-/- transplant recipients that were treated with costimulatory blockade exhibited defective anti-donor T cell IFN-g responses as compared to WT counterparts. Furthermore, CD14/CD36 signaling restrained T reg generation as double defi cient recipients exhibited higher proportions of CD4+Foxp3+ post transplant and anti-CD45RB + anti-CD154 treatment (22.3% ± 1.09%) as compared to similarly treated WT recipients (18% ± 1.95%), despite similar proportions of T regs prior to transplantation (11-12%). Functionally, we found that that adoptive transfer of CD4+ T-cells from CD14-/-CD36-/- mice treated with anti-CD45RB and anti-CD154 to RAG OTII TCR transgenic recipients, which had previously accepted BALB/c skin grafts, induced a signifi cantly slower tempo of allograft rejection (MST= 24 days, p = 0.033) as compared to RAG OTII recipients transferred with WT T cells (MST = 20 days). In contrast to the above T cell data, DC alloimmune priming function was not dependent on CD14/CD36 as irradiated whole splenic cells or bone marrow DCs from WT and CD14-/-CD36-/- mice had induced similar allogeneic T cell proliferation. These results suggest that the combined expression of CD14 and CD36 restrains T reg generation after treatment of anti-CD45RB and anti-CD154 and impairs allograft survival.

Abstract# 38Inhibition of the Kinase, GSK-3beta, Potentiates the Suppressive Function of Regulatory T Cells by Decreasing Cellular Death. Jay A. Graham,1 Michael A. Fray,1 Stephanie de Haseth,1 Kang-Mi Lee,1 Moh-Moh Lian,1 Catharine M. Chase,1 Joren C. Madsen,1 James F. Markmann,1 Gilles Benichou,1 Robert B. Colvin,2 A. B. Cosimi,1 Shaoping Deng,1 Alessandro A. Alessandrini.1 1Surgery, Massachusetts General Hospital, Boston, MA, USA; 2Pathology, Massachusetts General Hospital, Boston, MA, USA.INTRODUCTION: Considerable interest has developed in designing allotransplant tolerance regimens involving the manipulation of regulatory T cells (Tregs). Yet the mechanism by which Tregs suppress the immune response is not well defi ned, nor are the signaling pathways involved in suppression fully understood. Recent data has shown that inhibition of GSK-3beta by SB216763, a specifi c GSK-3beta inhibitor, leads to a signifi cant increase and stabilization of Treg function, in part by prolonging cellular survival and FoxP3 expression in Tregs possibly through anti-apoptotic proteins like Bcl-XL. Moreover, we showed increased islet allograft survival in recipients treated with SB216763, hinting at the powerful possibilities of potential Treg cell therapy in transplantation. RESULTS: We show that SB216763 potentiates Treg function in vitro, as judged by increased suppression of Teff CD4+CD25- proliferation when Teff CD4+CD25-cells were incubated at a 1:1 ratio with Tregs in 5 µM SB216763 (86% inhibition vs 65% without the drug). Moreover, the Tregs in coculture with SB216763 had a slower diminishment in intracellular FoxP3 signal indicating a probability of enhanced suppression in vitro. Furthermore, regulatory T cells, grown in the presence of low levels of IL-2, exhibited a 40% decrease in cell number after three days in culture. Cells treated with GSK-3beta inhibitor exhibited a 19% drop in cell number during the same time period. Moreover, the temporal kinetics of Bcl-XL loss is signifi cantly slower in the treated group. Collectively these fi ndings suggest that the GSK-3beta inhibitor, SB216763, may work through Bcl-XL to protect Tregs from apoptosis in vitro. Lastly, diabetogenic B6 recipients of C3H islets were injected daily with 100 µM SB216763 post-operatively. Interestingly, these islets had a 2-fold slower rejection time than untreated B6 recipients as demonstrated by prolonged euglycemia. CONCLUSION: We have shown that inhibition of GSK-3beta results in increased suppression activity by Treg cells. Analysis of SB216763-treated Treg cells shows that there is a stabilization of beta-catenin and that this in part may account for increased functionality of Tregs.

TRANSLATIONAL SCIENCE III

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Abstract# 39Treg Treatment Leads to Heart and Skin Graft Tolerance in a Non-Cytotoxic Murine Mixed Chimerism Model. Nina Pilat,1 Ulrike Baranyi,1 Christoph Klaus,1 Elmar Jaeckel,2 Rupert Oberhuber,3 Gerald Brandacher,3 Ferdinand Muehlbacher,1 Thomas Wekerle.1 1Medical University Vienna, Vienna, Austria; 2Hannover Medical School, Germany; 3Innsbruck Medical University, Austria.Background: The mixed chimerism approach is a promising strategy for the induction of transplantation tolerance but non-cytotoxic bone marrow (BM) transplantation protocols allowing routine clinical application are still an unmet need. We explored whether the therapeutic use of regulatory T-cells (Tregs) allows the development of such protocols.Methods: C57BL/6 recipients received 20x106 fully mismatched Balb/c BM cells (d0) under the cover of costimulation blockade (anti-CD154 mAb d0, CTLA4Ig d+2) and short-course rapamycin (d-1/0/+2) with or without approx. 4x106 polyclonal WT B6 Tregs (d0). CD4 cells retrovirally transduced with FoxP3 (FoxP3-Tregs), TGFbeta-induced (iTregs) and activated natural Tregs (nTregs) were evaluated. No irradiation and no cytotoxic drugs were administered. Multilineage chimerism was followed by fl ow-cytometry. Skin and primarily vascularized hearts were grafted 1-2 months post-BMT.Results: Mice receiving additional Treg treatment with any of the tested Treg populations developed long-term multilineage chimerism, whereas recipients of the same regimen without Tregs did not (follow-up 5-7 months; 6/7 long-term chimeras with FoxP3-Tregs; 3/4 chimeras with nTregs, 5/6 chimeras with iTregs; 0/13 chimeras without Tregs; P = 0.0002 for FoxP3-Tregs; P = 0.0059 for nTregs; P = 0.0005 for iTregs). Treg-treated recipients showed long-term survival of donor skin (>100 days 7/7 for FoxP3 Tregs, p<0.0002; 5/6 for iTregs p<0.0043; third party skin was promptly rejected) and donor heart (>150 days) allografts. Humoral and in vitro tolerance were demonstrated by the absence of anti-donor Abs and specifi c unresponsiveness in MLR-assays. Treg treatment without rapamycin failed to induce chimerism and tolerance (8/8 chimeras with rapamycin; 0/8 without; P = 0.0002). FoxP3-Tregs were effective also at substantially lower, clinically more relevant doses (3/3 chimeras with 1.5x106, 2/2 chimeras with 0.5x106).Conclusions: Polyclonal recipient Tregs lead to engraftment of conventional doses of fully allogeneic BM with unique potency, obviating the need for cytoreductive recipient treatment. Lasting mixed chimerism and robust heart and skin graft tolerance are achieved. These fi ndings have important implications for the development of non-toxic chimerism-based tolerance protocols for clinical use.

Abstract# 40Spontaneous Renal Allograft Acceptance in Mice Requires Foxp3 Cells. Masahiro Miyajima,1 Catharine M. Chase,1 Jay A. Graham,1 Evan A. Farkash,2 Alessandro Alessandrini,1 Joren C. Madsen,1 Paul S. Russell,1 Robert B. Colvin.2 1Transplantaion Unit, Massachusetts General Hospital, Boston, MA, USA; 2Pathology Service, Massachusetts General Hospital, Boston, MA, USA.Spontaneous acceptance of fully MHC incompatible kidney allografts was described in mice over 30 years ago, but the process remains enigmatic. We reasoned that understanding the mechanisms involved might inform strategies to promote allograft acceptance in humans. We tested the hypothesis that Treg might be responsible in B6.Foxp3DTR mice that have the diphtheria toxin (DT) receptor gene inserted into the 3’untranslated portion of the Foxp3 gene (kind gift of A. Rudensky). We performed 11 DBA/2 to B6.Foxp3DTR life-sustaining kidney transplants according to our standard method. Administration of DT for two consecutive days i.p. at 50 µg/kg to 7 B6.Foxp3DTR+/y recipients of DBA kidneys from 3 weeks to 3 months post-transplant, resulted prompt depletion of circulating Foxp3+ cells in the circulation and lymphoid organs and triggered rejection of the allograft as assessed by a sudden increase in BUN 6-7 days post-treatment. None of the controls showed a similar rise in BUN, including B6.Foxp3DTR+/y recipients of DBA/2 kidneys given vehicle or no treatment, B6 WT recipients of DBA/2 kidney given DT, and B6.Foxp3DTR+/y recipients of B6.Foxp3DTR+/y isografts given DT. ELISPOT assays (IFNg producing spleen T cells in 24 hrs) revealed that elimination of Foxp3+ cells increased the alloreactive clonal frequency of the recipients to donor up to 40-fold compared to self-reactivity. In contrast, recipients with accepted grafts had DBA reactive clones similar to that reactive to self. DT treatment of B6.Foxp3DTR+/y isograft recipients or naïve B6.Foxp3DTR+/y mice also produced a marked increase in DBA/2 clonal reactivity compared with self reactivity, suggesting latent alloreactivity in naïve mice is held in check by Foxp3 cells. Control accepted DBA/2 allografts showed periarterial nodules rich in Foxp3+CD3+ cells and proliferating cells (Ki67+), whereas the DT-treated B6.Foxp3DTR+/y recipients showed classic features of acute cellular rejection, including a diffuse infi ltrate rich in T cells and Ki67+ cells, tubulitis and endarteritis; few Foxp3 cells were present in the infi ltrate and the nodules were no longer evident. We conclude that Foxp3+ cells play a necessary role in establishing and maintaining spontaneous acceptance of fully MHC incompatible renal allografts and act by inhibiting alloreactivity.

Abstract# 41Mice with Constitutively Active STAT5b Generate Powerful and Stable Treg. Qiang Zhou,1 Wenda Gao,1 Michael A. Farrar,2 Terry B. Strom.1 1The Transplant Institute, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; 2Center for Immunology and Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA.The inability to maintain robust Foxp3 expression among induced-Treg (iTreg) in infl ammatory environments is a bottle-neck that may impede Treg based adoptive therapy. IL-2 induced Foxp3 expression depends on STAT5 phosphorylation. Activated STAT5 binds to the Foxp3 promoter and 1st intron and triggers Foxp3 expression in the absence of exogenous TGF-b signaling. Mutations (298 His to Arg & 715 Ser to Phe) generate a constitutively active form of STAT5b (STAT5b-CA). In STAT5b-CA transgenic mice, there is a 4 fold increase in frequency of Foxp3+ Treg as compared to WT. To investigate if constitutively active STAT5b expression in T cells promotes Treg differentiation, and enhances stability of Treg phenotype and immunoregulatory function, we analyzed the phenotype and regulatory functions of Treg from STAT5b-CA transgenic mice. Using ex vivo Treg polarizing conditions, in vitro conversion by TGF-b yielded two fold more induced Foxp3+ Tregs in STAT5b-CA transgenic mice than WT mice. Under Th17 polarizing conditions, STAT5b-CA transgenic mice generate more iTreg than WT mice. We anticipate fewer Th17 cells will be found in this system. STAT5b-CA transgenic mice more robustly express Foxp3 and TGF-b, but fewer Th17 promoting, e.g., RoRgt, IL-17A, IL-17F transcripts in (1) resting natural Treg, (2) anti-CD3/anti-CD28 activated nTreg, (3) TGF-b induced-Treg (iTreg), and (4) naïve T cells cultured in Th17 polarizing or Th0 conditions. When STAT5b-CA transgenic iTregs were transferred to DBA/2 skin grafted B6.Rag1-/- mice (n=4), all grafts survived throughout the 70 day observation period, while transferring Wt iTregs resulted in rejection before 40 days in all cases. As in the in vitro experiments, the transferred CD4+ Foxp3+ and CD4+ Foxp3- T cells recovered from transgenic T cells manifested more robust expression of Treg promoting genes and lower expression of Th17 polarizing genes than recovered WT iTregs. These fi ndings suggest constitutively active STAT5b facilitates Foxp3+Treg differentiation and enhances stability of their molecular and immune regulatory phenotype. As STAT5b-CA transgenic T cells generate stable and effi cient iTregs, we are interested in exploring means to enhance STAT5b expression in human CD4+ T cell as an approach to promote adoptive cell therapy.

ORAL PLENARY EXCHANGE VII - TRANSLATIONAL SCIENCE III

Abstract# 42Immunoprofi ling after Profound T Cell Depletion and Minimization of Immunosuppression in Pediatric Kidney Transplantation: A Study of CCTPT/NIAID. William Harmon,1 Fanny Benitez,1 Nader Najafi an,2 Mohamed Sayegh.1,2 1Transplantation Research Center, Children’s Hospital Boston, Boston, MA, USA; 2Transplantation Research Center, Brigham and Women’s Hospital, Boston, MA, USA.Despite excellent short-term outcomes for recipients of pediatric kidney transplantation, long-term success is compromised by complications of chronic immunosuppression and chronic allograft nephropathy. We thus undertook a study of minimal maintenance immunosuppression using a steroid-free, calcineurin inhibitor withdrawal protocol in 4 pediatric renal transplant centers. Campath-1H® was used for induction and MMF and tacrolimus immunosuppression were used for 2-3 months, followed by withdrawal of tacrolimus and replacement with sirolimus. One-year acute rejection rate was 17% and only 2 grafts were lost, one due to recurrent FSGS and the other to medication non-adherence. There were no deaths and more importantly no cases of PTLD or other serious infections. Twenty-eight subjects had serial assessments of anti-HLA Class I and II antibody by Luminex up to 2 years post transplantation. Five subjects (18%) developed new anti-HLA antibody, 1 against Class I, 3 against Class II and 1 against both Class I and II. Twenty-three subjects had evaluation of lymphocyte recovery for at least one year post transplantation. Peripheral blood monocytes were monitored by fl ow cytometry and analyzed by FACSCalibur cytometry at 8 times point up to 24 months post transplant. Phenotypes examined in CD4+ and CD8+ T cell subsets included naive, Central memory and Effector memory; as well as T Regulatory Cells (Tregs). CD8+ T-cell percentages rebounded in 6 months, but CD4+ T-cells took 18 months to reach pre-transplant levels. Naive CD4+ cells rebounded more quickly than CD4+ memory and Central memory was more reduced than Effector memory. Patterns in CD8+ cells were similar. Percentages of Tregs increased immediately post transplant and remained high until at least 8 months. This pattern of recovery in children was different from the typical pattern in adults after profound T cell depletion in at least 2 ways: pediatric subjects had depletion of both memory and naive T cells with quicker recovery of naive cells, whereas adults typically have sparing of memory T cells in comparison to

TRANSLATIONAL SCIENCE III

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naive cells. Also, the memory T cells spared in children were mostly Effector rather than Central. Taken together with Tregs sparing, these observations suggest that this protocol could permit less maintenance immunosuppression in children.DISCLOSURE: Harmon, William: Other, Genzyme, Consultant; Najafi an, Nader: Other, Bristol Myers Squibb, Consultant; Sayegh, Mohamed: Other, Genzyme, Consultant.

Abstract# 43Broad Autoantibody Responses in Chronic Humoral Rejection of Human Kidney Allografts. Fabrice Porcheray,1 Julie DeVito,1 Susan L. Saidman,2 Beow Yeap,3 Timothy C. Girouard,2 Rosemary Paine,1 Lixuan Xue,1 Ian Dargon,1 Robert B. Colvin,2 Waichi Wong,4 Emmanuel Zorn.1 1Transplantation Biology Research Center, Massachusetts General Hospital, Boston, MA, USA; 2Department of Pathology, Massachusetts General Hospital, Boston, MA, USA; 3MGH Cancer Center, Massachusetts General Hospital, Boston, MA, USA; 4Renal Unit, Department of Medicine, Massachusetts General Hospital, Boston, MA, USA.Chronic humoral rejection (CHR) is a major complication of kidney transplantation recipients. The cause of CHR is still unknown. Although the presence of anti-donor HLA antibodies is well documented in this pathology, autoantibodies have also been reported. Yet, the lack of comprehensive studies has limited our understanding of this autoimmune component and its contribution to CHR. Using ELISA and immunocytochemistry assays, we assessed the development of autoantibodies to a broad range of autoantigens in 25 kidney transplant recipients with CHR and 25 with stable graft function. We observed that the majority of CHR patients but not controls had developed antibody responses to one or several autoantigens.

To further characterize these autoantibody responses, we assessed the patient serum reactivity to 8000 recombinant human proteins using a protein microarray platform. These assays were carried out with samples collected from 5 CHR and 5 control patients and revealed a burst of autoimmunity at the time of CHR.

Remarkably, microarray analysis showed only minimal overlap between profi les, indicating that each patient had developed autoantibodies to a unique set of antigenic targets. The breadth of autoantibody responses, together with the absence of consensual targets, suggests that these may result from systemic B cell deregulation. Further studies are warranted to examine the contribution of these autoantibodies to the pathophysiology of CHR.

Abstract# 44A Peripheral Blood 12 Gene-Set for Diagnosis of Pediatric Liver Allograft Tolerance. Li Li,1 Anita Talisetti,1 Sue Hsieh,1 Ken Cox,1 Carlos Esquivel,2 Waldo Concepcion,2 Minnie Sarwal.1,2 1Pediatrics, Stanford University, Stanford, CA, USA; 2Surgery, Stanford University, Stanford, CA, USA.Aim: To identify transcriptional biomarkers for and mechanisms of operational liver tolerance in pediatric recipients.Methods: 47 unique whole blood samples from 4 demographically matched patient phenotypes were run on Agilent Whole Human Genome 44K microarrays: 9 operational tolerant pediatric liver transplant patients were on no medications from 3.4-12.6 years (P-TOL); 2 patients had a history of PTLD > 5 years prior. Additional liver transplant recipient groups were: 13 with biopsy proven acute rejection (AR), 8 on low-dose prograf monotherapy/ minimal immunosuppression (MIS), and 13 stable patients on dual immunosuppression. Additionally we also processed samples from 6 healthy donors (HD). Standardized bioinformatic analyses were applied and signifi cant TOL genes were mapped by AILUN to published data on Affymetrix arrays from an operational tolerance study in adult liver transplant recipients (A-TOL; Llordella et al).Results: Twelve unique genes were identifi ed (FDR<5%) by prediction analysis of microarrays (PAM) as a minimum gene set to cross-validate and predict P-TOL with 100% sensitivity and 84% specifi city. These genes are enriched in liver regeneration and 9/12 genes are regulated by TGF-β1. The tolerant specifi c genes are highly expressed in T cells, CD34+ endothelial and NK cells. 2/8 MIS (∼25%), 12/13 STA (92%) and 0/6 HD patients samples were predicted as TOL based on prediction probability scores >50%. These genes also correctly predicted 76% of the 17 A-TOL samples and 86% of the 21 non-TOL samples in the adult study. The most signifi cant 100 genes from the A-TOL published study could not back predict any of the P-TOL samples in the tolerance class.Conclusion: Specifi c peripheral transcriptional programs can be identifi ed in operational tolerance in pediatric recipients of liver allografts, distinct from those previously identifi ed in adult operationally tolerant liver recipients, and may provide a means to non-invasively monitor patients in a serial manner for immunosuppression minimization. These genes are highly expressed in specifi c peripheral blood lymphocyte subsets, and their coordinated regulation by specifi c cytokines may support the maintenance of operational tolerance in children, following liver transplantation.



Abstract# 47Antibodies to Non-HLA Antigens Are Associated with Chronic Allograft Injury. R. Dinavahi,1 S. Pinney,1 E. Akalin,1 S. Ames,1 J. S. Bromberg,1 G. DeBoccardo,1 S. Lerner,1 B. Murphy,1 B. Schroppel,1 V. Sehgal,1 T. Mohanankumar,2 D. Salomon,3 S. Horvath,4 P. S. Heeger.1 1Recanati Miller Transplantation Institute, NY, NY; 2Washington University School of Medicine, St Louis, MO; 3Scripps Institute, La Jolla, CA; 4UCLA, Los Angeles, CA.Emerging evidence suggests that in addition to alloimmunity, autoreactive T cells and autoantibodies may contribute to chronic graft injury. To test this, we obtained sera from 17 kidney, 25 heart and 16 lung transplant recipients with and without chronic injury (CI), 5 hemodialysis (HD) patients and 8 healthy controls(HC). Sera were tested for reactivity to >8000 non-HLA antigens using a protein microarray. Number and specifi city of the reactivities were compared among groups. Sera obtained from stable kidney transplant (SKF) recipients (Cr < 1.5mg/dL, no proteinuria) reacted to 277 different antigens than sera obtained from HC, and 97 distinct antigens compared to HD patients. Sera from stable heart transplant recipients reacted to 234 more antigens than HC and sera from stable lung transplant recipients reacted to 139 distinct antigens compared to sera from HC (p<0.05). Furthermore, <32% of the differential reactivities in the heart transplant cohort were shared by the kidney transplant cohort or lung transplant cohort (p<0.05) and only 3% of reactivities were found in all three groups. Comparing antibody profi les in kidney recipients with CI to those with SKF, we found a distinct pattern of reactivities in those with CI. Several reactivities were directed at kidney specifi c ion transporters. Reactivities found in kidney recipients were distinct from those found in heart or lung recipients with CI. Dot blot assays using 9 of the antigens validated the detected differential reactivities by an alternative method. The results confi rmed stronger reactivity to the antigens that were preferentially detected by array in patients with CI and confi rmed no reactivity to control antigen. Mapping the targeted antigens to canonical response pathways refl ected reactivity to antigens within pathways implicated in allograft injury and fi brosis. Our results indicate that antibodies reactive to non-HLA antigens develop commonly posttransplant, that the specifi cities are in part organ specifi c and that unique antibody profi les are associated with CI. If prospectively validated, the

BASIC SCIENCE: STEM CELLS, FACILITIATION CELLS AND MIXED CHIMERISM

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fi ndings support the hypothesis that autoantibodies contribute to the pathogenesis of chronic rejection and suggest that such antibodies could be used as biomarkers of incipient graft injury.

ORAL PLENARY EXCHANGE VIII - BASIC SCIENCE: STEM CELLS,

FACILITIATION CELLS AND MIXED CHIMERISM

Abstract# 48Embryonic Stem Cell-Based Therapy for Transplantation Tolerance. Luis P. Fernandez,1 Paulo N. Martins,1 Sharon K. Germana,1 Anne-Laurence Blanc,1 Christian LeGuern.1 1Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School, Boston, MA, USA.Bone marrow (BM)-derived dendritic cells (DC) are instrumental to the induction of thymocyte tolerance to self and possibly allogeneic antigens. However, the potent immunogenicity of both allogeneic BM cells and ex vivo-derived DC has so far curtailed DC-based therapies for tolerance to allogeneic transplants. Unlike DC or BM cells, embryonic stem cells (ESC) are poorly immunogenic and can differentiate in vitro into DC from early DC-committed progenitors (ES-DCp). Thus, the goal was to examine whether allogeneic ES-DCp would be as easily accepted as ESC, sustain DC differentiation in vivo and down-modulate host T cell responses to donor antigens. ES-DCp were defi ned in ESC cultures by rtPCR detection of myeloid and DC lineage markers; in vivo DC differentiation from ES-DCp was monitored by immunohistochemistry and rtPCR. Host responses to donor alloantigens were assessed by mixed lymphocyte reaction. Results showed that short treatments of pluripotent ESC with GM-CSF and IL-3 switched on early genetic programs of DC development including PU.1, CX3CR1, and CD11b genes in 5% of cells while maintaining undetectable levels of MHC class I and II proteins. Accordingly, unpurifi ed ES-DCp from C57BL/6 (B6; H-2b) mice were readily accepted when injected under the kidney capsule of BALB/c (H-2d) hosts minimally treated with a single dose of anti-CD4 mAb. Accepted ES-DCp proceeded to DC differentiation in situ and yielded CD11c+ MHC class II+ cells, which also seeded the thymus, potentially modulating alloresponses. Indeed, T cell activation by donor B6 antigens was selectively dampened in recipients of ES-DCp as compared to that of ESC recipients. Through treatments with poorly immunogenic DC precursors that generate potentially tolerogenic DC, our data sets the groundwork for an original and promising approach toward the induction of allogeneic T cell tolerance. ESC-based DC therapy for transplantation tolerance is all the more justifi ed in light of recent advances on the generation of pluripotent cells from human fi broblasts.

Abstract# 49Kidney-Derived Mesenchymal Stem Cells Induce Tolerogenic Dendritic Cells That Inhibit Alloresponses and Prevent Allograft Rejection. Yanfei Huang,1 Cassie B. Zhang,1 Miriam Ruiz,1 Asif Zakaria,1 Paolo Fiorina,2 M. Javeed Ansari,2 Reza Abdi,2 Hamid Rabb,1 Karl L. Womer.1 1Department of Medicine, Johns Hopkins University; 2Brigham and Women’s Hospital and Children’s Hospital Boston and Harvard Medical School.Bone marrow mesenchymal stem cells (MSC) ameliorate acute kidney injury through mechanisms aside from transdifferentiation and have powerful immune modulatory effects. We hypothesized that similar cells exist within the adult kidney and are important to immune responses during injury and repair. Using a clinically applicable technique, we isolated mouse kidney-derived stromal cells (KSC) with growth properties and immunophenotype consistent with MSC, including ability to differentiate into mesodermal cell lineages, including osteocytes and adipocytes. Furthermore, in co-culture, KSC increased the generation of bone marrow-derived DC with dramatically reduced MHC II expression (MFI: 113±6.5 vs 679±125; p<0.01), yet increased CD80 expression (MFI: 195±18 vs 85±4.7; p<0.01), and after LPS stimulation, increased IL-10 production (135.7+12.4 pg/ml vs 46.1+6.9 pg/ml; p<0.01), and inability to stimulate CD4+ T cell proliferation in allogeneic culture (2.1±0.1% vs 53±1.3%; p<0.01), as determined by CFSE staining. Effects on DC were demonstrated in mixed and transwell cultures and after addition of KSC conditioned medium alone. Thus, immunomodulatory effects of KSC on DC differentiation appear predominately due to soluble factors, yielding an IL-10 producing DC with very low expression of MHC class II but increased CD80 expression, a phenotype consistent with semi-mature DC important for maintenance of peripheral tolerance. We next studied the ability of these KSC-DC to induce tolerance in an allogeneic model of transplantation. Bone marrow-derived DC from previous KSC co-culture were transplanted with islets under the kidney capsule of STZ-induced diabetic mice. The median graft survival for groups administered (i) saline, (ii) untreated donor-derived DC, (iii) KSC-treated donor-derived DC, and (iiii) KSC-treated recipient-derived DC were 14, 23, 48, and 19 days respectively, with KSC treated- donor- DC resulting in signifi cant delay of graft rejection (P<0.01). These novel fi ndings extend our in vitro results and provide a potential mechanism by which resident progenitor cells in the kidney could alter local alloresponses in

vivo after transplantation. Our fi ndings also raise the possibility of KSC expansion ex vivo for use as cellular therapy in kidney transplantation.

Abstract# 50Induction of Chimerism in Diabetic NOD Mice Results in Immune Tolerance of Donor Islets, Reduction of the Required Amount of Donor Islets for Reversing Diabetes, and Excellent Long-Term Graft Function. Miao Wang, Jeremy J. Racine, Ivan Todorov, Defu Zeng. Departments of Diabetes Research and Hematopoietic Cell Transplantation, Beckman Research Institute of City of Hope, Duarte, CA, USA.Islet transplantation through portal injection of donor islets into host liver could be curative for type 1 diabetes, but graft rejection, shortage of donor islets, and functional defect of long-term grafts remain as major obstacles. In current studies, we tested whether induction of chimerism under a radiation-free and graft versus host disease (GVHD) preventative anti-CD3-based conditioning regimen could solve these problems. Accordingly, late-stage diabetic NOD mice were conditioned with anti-CD3/CD8 mAb and transplanted with bone marrow cells (BMT) from FVB/N donors to induce chimerism. Graded numbers of donor islets from luciferase transgenic mice were implanted into liver and pancreas following BMT. The control mice were treated with only Edmonton protocol of anti-IL-2, Sirolimus, and Tacrolimus. Islet graft survival was monitored with in vivo bioluminescent imaging and blood glucose levels of the recipients. Islet graft function was measured with RT-PCR of β cell functional genes; the proliferation of graft β cells was measured with Brdu-labeling and sequential CldU and IdU labeling. We found that while 900 islets reversed diabetes in control mice for short-term, 100 islets were suffi cient to reverse diabetes in chimeric recipients for long-term, indicating that induction of chimerism not only provides immune tolerance but also markedly the reduced requires donor islet dose for reversal of diabetes. We also found that, when 50 islets were implanted in pancreas or liver of the chimeric recipients, grafts in pancreas but not in liver reversed diabetes in the majority of recipients; graft islet β cells replicated much better in pancreas than in liver; gene expression levels of insulin, GLUT-2, GCK, and PDX-1 of the long-term graft islet β cells from pancreas was close to before transplantation and much higher than grafts from liver. These results indicate that graft islet β cells replicate and function better in pancreas than in liver. Taken together, induction of chimerism under the radiation-free and GVHD preventative anti-CD3-based conditioning regimen not only provide immune tolerance to donor islet grafts but also reduced the required amount of donor islets for reversal of diabetes. In addition, this regimen makes pancreas of autoimmune diabetic recipients a favorable site for donor islets.

Abstract# 51Hematopoietic and Pluripotent Stem Cells Acquired during Pregnancy Give Rise to Wide-Spread Maternal Microchimerism Required for Tolerance to Non-Inherited Maternal Antigens. Partha Dutta,1 Steve M. Schumacher,1 Melanie Dart,1 William J. Burlingham.1 1Department of Surgery, University of Wisconsin-Madison, Madison, WI, USA.Background: Kidney transplants from sibling donors mismatched for noninherited maternal antigens (NIMA) fare signifi cantly better than those mismatched for noninherited paternal antigens (NIPA). Nonetheless, the basis for this effect, in particular the relationship of microchimerism (Mc) and tolerance to transplants remains highly controversial. To address this issue, we used a mouse model. BDF1 female and B6 male backcross offspring (H-2b/b) exposed to NIMA-H-2d can accept a fully allogeneic, DBA/2 heart allograft, whereas non-exposed NIPAd-controls (B6 female and BDF1 male backcross) uniformly reject.Hypothesis: Hematopoietic and pluripotent maternal stem cells acquired by the offspring result in a multilineage Mc needed to tolerize both class I and class II-specifi c T cells as well as T cells specifi c for tissue-restricted minor H antigens.Methods and Results: With a very sensitive (to 10-5) quantitative PCR technique, we found that the presence of NIMA-specifi c TGF-β-producing Tregs was strongly correlated with the level of maternal microchimerism (MMc). Though MMc was rarely detected in unfractionated bone marrow (BM) and spleen, MHCII+ cells such as CD11b+ and CD11c+ cells and CD4+ cells contained maternal cells indicating a multi-lineage MMc. We detected c-kit+ multipotent maternal stem cells in the freshly isolated bone marrow of 80% NIMAd-exposed mice, but not in the BM of the NIPAd control mice, which suggests transplacental migration of the maternal stem cells during the pregnancy. However, only 30-40% of the offspring had suffi cient multi-lineage Mc to induce a high level of NIMA-specifi c Tregs. Maternal mesenchymal stem cells in the BM of the NIMAd-exposed offspring were also detectable, indicating that the offspring not only have hematopoietic but also non-hematopoietic stem cells. We sorted different cells fractions from the heart: lineage+ cells, lineage- c-kit- cells, and lineage- c-kit+ cells to fi nd which cell populations carry maternal cells. Interestingly, we found all of the populations carry some degree of MMc.Conclusions: We conclude that a variety of maternal stem cells are acquired by 80% of the offspring; however, only 50% of them retain long-lasting MMc. Long-lived class II+ APC of maternal origin and c-kit- lin- cells may both be required for NIMA-induced tolerance to heart allografts.

CLINICAL SCIENCE III

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Abstracts

Abstract# 52Evidence That Innate Immunity Is Tolerized in Mixed Chimeras. Hong Xu,1 Ziqiang Zhu,1 Lala R. Hussain,1 Yiming Huang,1 Jun Yan,2 Suzanne T. Ildstad.1 1Institute for Cellular Therapeutics, University of Louisville, Louisville, KY, USA; 2James Brown Cancer Center, University of Louisville, Louisville, KY, USA.Hematopoietic mixed chimerism refers to the coexistence of hematolymphopoietic cells of donor and recipient origin. Mixed chimeras with as little as 1% donor chimerism are tolerant to transplanted tissue. In the present study, we examined chimeras for evidence of innate immune tolerance. Allogeneic bone marrow transplants were performed between C57BL/6 (H2b) and BALB/c (H2d) mice. Recipient C57BL/6 mice were nonmyeloablatively conditioned with anti-αβ-TCR, anti-CD154, and sirolimus and transplanted with 15 x 106 BALB/c marrow cells after varying doses of cGy of total body irradiation (TBI). Pretreatment of recipients with anti-CD154 and sirolimus with or without T cell lymphodepletion reduced the TBI requirement to 100 cGy for establishing stable multilineage mixed chimerism. The mixed chimeras accepted donor islet allografts up to 6 months. We evaluated adaptive immune tolerance by MLR for T cell tolerance and fl ow cytometric cross-match (FCXM) assay for B cell (humoral) tolerance. Lymphocytes from mixed chimeras did not respond to host and donor alloantigens, yet were reactive to MHC-disparate third party alloantigens (B10.BR, H2k), suggesting functional donor-specifi c T-cell tolerance. In FCXM assays, no antibodies against donor and host were detected in mixed chimeras at 1, 3 and 6 moths after BMT, suggesting humoral tolerance. Innate immune tolerance was examined using an in vivo cytotoxicity assay. T cell defi cient mice (TCRβ/δ-/-) and wildtype B6 mice served as controls. The rate of elimination of donor cells 1 day after cell infusion in TCRβ/δ-/- mice was similar to wildtype B6 controls, indicating that acute cellular cytotoxicity was likely not T cell mediated but rather mediated by innate immune function. As T cell activation requires at least 3 days, the effectors mediating donor cell rejection would be the innate cells: macrophages, neutrophils, or NK cells. Mixed chimeras showed no cytotoxicity to donor cells but a similar rapid killing rate to third-party B10.BR cells, suggesting tolerance in the innate immune cells was achieved in mixed chimeras. In conclusion, mixed chimeras prepared with nonmyeloablative conditioning exhibit tolerance for innate immunity as well as adaptive T and B cell immune responses.

Abstract# 53Chimeric Treg Potently Enhance Donor Chimerism and Tolerance: The Acquisition of FoxP3 Expression Is Directly Correlated with Treg Function In Vivo. Yiming Huang,1 Larry D. Bozulic,1 Thomas Miller,1 Lala-Rukh Hussain,1 Yujie Wen,1 Hong Xu,1 Suzanne T. Ildstad.1 1University of Louisville, Institute for Cellular Therapeutics, Louisville, KY, USA.We previously reported that CD8α

+ plasmacytoid precursor dendritic cell facilitating cells (p-preDC FC) induce the generation of chimeric regulatory T cells (Treg) in vivo. To evaluate the function of FC-induced chimeric Treg, we tested the ability of chimeric Treg to enhance engraftment and donor chimerism following transplantation. Splenocytes were harvested from mixed chimeras 2, 3, 4 and 5 weeks after HSC plus FC transplantation from B6 donors into conditioned NOD recipients. CD8-/CD4+/CD25bright chimeric Treg were sorted from the spleens of chimeras and 50,000 chimeric Treg plus 10,000 B6 HSC were transplanted into NOD recipients conditioned with 950 cGy of TBI. All secondary recipients of 2 week chimeric Treg (n = 7) or 3 week chimeric Treg (n = 8) plus HSC expired before 30 days after transplantation, suggesting that at these time points the Treg are not functional. Three of fi ve recipients of 4 week chimeric Treg engrafted, with an average of 18% donor chimerism (range: 1.7% - 79%). Only one of fi ve (20%) NOD mice receiving 4 week chimeric Treg exhibited durable (> 100 days) engraftment. In striking contrast, 100% of recipients (n = 4) given 5 week chimeric Treg + HSC engrafted and survived over 100 days. All of the recipients showed donor cell chimerism in excess of 90% (range: 84% - 95%). These mixed chimeras exhibited durable engraftment and multilineage donor chimerism for T cells (CD8, CD4, β-TCR), NK cells (NK1.1DX5), B cells (CD19), DC (CD11c), macrophage (Mac-1), and granulocytes (Gr-1). The level of FoxP3 expression was recently reported to correlate with suppressive function of Treg. We therefore compared the expression of FoxP3 in 2 week vs. 5 week chimeric Treg from mouse spleen. There was a signifi cant increase in the level of FoxP3 expression in 5 week chimeric Treg of spleen compared to 2 week chimeric Treg (86.9 ± 1.8 vs 38.9 ± 7.6, respectively; P = 0.001). Taken together, these data suggest that 5 week chimeric Treg are more effi cient in suppressing immune responses and potently enhance engraftment of allogeneic B6 HSC in NOD recipient compared with naïve B6 Treg. FoxP3 gene expression is associated with the functional development of suppressive capacity of CD4+/CD25+ Treg in vivo.

Abstract# 54Induction of Local Hyporesponsiveness for Cell Transplants. Shiguang Qian,1,2 Horng-Ren Yang,2 Cheng-Hsu Chen,1 John J. Fung,2 Lina Lu.1,2 1Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA; 2General Surgery, Transplantation Center, Cleveland Clinic, Cleveland, OH, USA.Solid organ transplantation has been applied for decades, but outcomes of cell transplants remain disappointing. Similarly in animal models, mouse liver transplants are spontaneously accepted, but hepatocyte transplants acutely rejected, suggesting a crucial role of non-parenchymal cells (NPC) in protecting parenchymal cells. By testing various liver NPC, we have identifi ed the potent T cell inhibitory activity of hepatic stellate cells (HpSC), liver stromal cells responsible for fi brosis. BALB/c islets (300) mixed with 3 x 105 HpSC (B6) transplanted under renal capsule of STZ-induced diabetic B6 recipients achieved long survival islet grafts in 6/11 (MST >60d), compared to islets only (MST=13d, p<0.01). Removal of the grafted kidney resulted in rapid glycemia. HpSC exerted multi-inhibitory effects, including marked apoptotic death in graft infi ltrating antigen-specifi c effector T cells and expansion of CD4+Foxp3+ Treg cells (up to ∼40%). Both effects required intact IFN-γ signaling, evidenced by lose of islet protective effect when IFN-gR1-/- HpSC were used. B7-H1, a product molecule of IFN-g signaling, was responsible for induction of T cells apoptosis, but had no effect on expansion of Treg cells. The local vs. systemic effect was determined by in vivo CTL activity using donor targets-BALB/c splenocytes labeled with low CFSE and B6 splenocytes labeled with high CFSE as syngeneic targets. The specifi c lysis of targets was identifi ed by the numbers of CFSElow and CFSEhigh cells. CTL against BALB/c targets in normal B6 mice was 62.5%, refl ecting an allogeneic CTL activity in normal recipients. Transplant with islet allografts alone enhanced the specifi c CTL to 82%, similar to co-transplant with HSpC (80.6%), suggesting lack of systemic immunosuppressive effect. This was supported by failure of HpSC to protect islet allografts transplanted in the other side kidney. These results suggest that elimination of effector T cell in islet allografts is critical in establishing local hyporesponsiveness. The potent immunosuppressive activity was only demonstrated in specifi c stromal cells, because co-transplantation with same number of bone marrow-derived stromal cells, mesenchymal stem cells (MSC) only slightly prolonged survival of islet allografts (MST 28d). In conclusions, induction of local hyporesponsiveness by co-transplantation with specifi c stromal cells can protect cell transplants.

ORAL PLENARY EXCHANGE IX - CLINICAL SCIENCE III

Abstract# 55Predicting Recurrence after Liver Transplantation in Patients with Hepatocellular Carcinoma Exceeding the Up-to-Seven Criteria. Francesco D’Amico,1 Myron Schwartz,2 Alessandro Vitale,3 Sasan Roayaie,2 Swan Thung,2 Maria Guido,4 Umberto Cillo.1 1Unità di Chiurgia Epatobiliare e Trapianto Epatico, Azienda-Università di Padova, Padova, Italy; 2Recanati-Miller Transplantation Institute, Mount Sinai School of Medicine, Padova, Italy; 3Unità di Chirurgia Oncologica, Istituto Oncologico Veneto, IRCCS, Padova, Italy; 4Department of Medical and Diagnostic Sciences, Pathology Section, Università di Padova, Padova, Italy.Background/aim. The up-to-seven (Up-to-7) criteria (with seven being the result of the sum of size and number of tumours for any given HCC) have been recently proposed to identify potential candidates for liver transplantation (LT) among patients exceeding the Milan criteria. The aim of this study is to compare the ability in predicting recurrence of available pathologic staging systems (Milan vs. UCSF, vs. Up-to-7).Methods. A study population of 479 HCC transplanted patients was identifi ed from prospectively-collected databases at Mount Sinai Medical Center (New York, NY) and the University of Padua (Padua, Italy). The best pathologic staging system was identifi ed by using the Log-rank, the proportion of separation (PSEP), and Cox analyses. Pathologic tumor characteristics (tumors number, size, sum of diameters, macroscopic and microscopic vascular invasion, grading) were then tested by uni- and multivariate Cox analyses in the prognostic subgroups within and beyond the calculated criteria.Results. Up-to-7 criteria performed as the best pathologic staging system, being the calculated 1, 3, 5-year recurrence probabilities 4%, 8%, 14% within (n = 355) and 22%, 45%, 51% beyond (n = 124) the criteria (p<.0001), and the calculate PSEP = 0.27 (95% CI= 0.23-0.31).At multivariate analysis, only biological variables (vascular invasion and tumor grade) significantly predicted recurrence beyond Up-to-7 criteria. A 3-stage pathologic staging system with a potential to be applied in the preoperative setting was thus created: within Up-to-7 (recurrence rate = 8%); beyond Up-to-7 without macrovascular invasion and poorly differentiated grade (recurrence rate = 24%); beyond Up-to-7 with macrovascular invasion and/or poorly differentiated grade (recurrence rate = 45%).

CLINICAL SCIENCE III

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Conclusions. HCC patients within pathologic Up-to-7 criteria were associated to a low risk of recurrence after LT. Beyond these criteria, however, a signifi cant proportion of patients with a good HCC biological profi le had an acceptable risk of recurrence.

Abstract# 56Renal Benefit of Belatacept Versus Cyclosporine in Kidney Transplant Patients Is Not Impacted by Acute Rejection (BENEFIT Study). Barbara Bresnahan,1 Flavio Vincenti,2 Josep Grinyo,3 Bernard Charpentier,4 Gregory Di Russo,5 Pushkal Garg,5 Christian Larsen.6 1Univ of Wisconsin; 2USCF; 3Univ. Hospital of Bellvitge; 4Bicentre Hospital; 5Bristol-Myers Squibb; 6Emory Univ. School of Medicine.Introduction: Belatacept-based immunosuppression is associated with superior renal function and comparable patient/graft survival in two phase III trials in kidney transplant recipients vs cyclosporine (CsA). In one trial (BENEFIT; non-ECD recipients), there was a higher incidence of acute rejection (AR) with belatacept-based regimens vs CsA. This descriptive analysis characterizes AR and its impact on 1-year outcomes.Methods: BENEFIT is a 3-year, randomized, phase III study in adults receiving a kidney TX from a live or deceased donor (anticipated cold ischemia time < 24 hours). Patients were randomized to a more (MI) or less intensive (LI) regimen of belatacept or CsA; all received basiliximab induction, MMF, and corticosteroids.Results: In the ITT analysis at 1 year, 22% (MI), 17% (LI), and 7% (CsA) exhibited AR. Banff grade ≥IIb AR occurred in 10% (MI), 5% (LI), and 1% (CsA) of patients. Most AR occurred within the fi rst 3 months. Few patients had >1 episode of AR (2.7%, 1.7%, and 0.9% for MI, LI, and CsA) and sub-clinical rejection was observed infrequently (4%-5% across groups) at year 1 biopsy. Donor-specifi c antibody production was lower in belatacept treated patients. The most common AR treatment was corticosteroids; 6% (MI), 4% (LI), and 0% (CsA) of patients had corticosteroid resistant AR. Initial T-cell depletion was given in 6% (MI), 4% (LI), and 1% (CsA). Approximately 50% of patients with AR in the belatacept groups remained on belatacept. Few patients (n = 1 MI, n = 2 LI, n = 2 CsA) were adjudicated to have graft loss due to AR. Of the patients with AR, 94%, 92%, and 94% in the MI, LI, and CsA groups survived with a functioning graft, compared to 96%, 97%, and 93% without AR. Among AR patients at month 12, measured GFR (mGFR) was 62 ml/min/1.73m2 (MI), 61 ml/min/1.73m2 (LI), and 48 ml/min/1.73m2 (CsA); for patients without AR, mGFR was 66 ml/min/1.73m2 (MI), 65 ml/min/1.73m2 (LI), and 51 ml/min/1.73m2 (CsA).Conclusions: The relative benefi t of belatacept regimens on renal function was maintained vs CsA in patients who had AR, despite higher rates and grades of AR. The impact of rejection on graft function and survival at 1 year was limited and longer-term effects will continue to be assessed over the 3-yr trials.DISCLOSURE: Vincenti, Flavio: Grant/Research Support, Novartis; Grinyo, Josep: Other, Bristol-Myers Squibb, Advisory Board Member; Di Russo, Gregory: Employee, Bristol-Myers Squibb; Garg, Pushkal: Employee, Bristol-Myers Squibb; Larsen, Christian: Other, Bristol-Myers Squibb, Clinical Trials.

Abstract# 57Alemtuzumab Induction May Exacerbate Antibody-Mediated Rejection in Pediatric Renal Transplant Recipients. George C. Wu,1 Kenneth V. Lieberman,1 Leigh M. Ettinger,1 Joan M. Abrams,1 Michael E. Shapiro.1 1Organ Transplantation and Pediatric Nephrology, Hackensack University Medical Center, Hackensack, NJ, USA.Background Alemtuzumab is a humanized anti-CD52 panlymphocytic monoclonal antibody that is used as an induction agent in approximately 10% of all renal transplant recipients. There are a few studies that have demonstrated its safety and effi cacy. However, more reports have shown that alemtuzumab use has resulted in worse death-censored graft survival and an increase in acute rejection rates. A recent study also demonstrated that alemtuzumab augments B-cell activation. In this retrospective chart review, we report our experience with alemtuzumab induction in our pediatric renal transplant recipients.Methods Between August 1, 2008 and August 31, 2009, 8 pediatric renal transplant patients at our institution received alemtuzumab induction. Seven patients were low-risk recipients and one patient was highly-sensitized, who received Rituximab pre-transplant for desensitization. They all received Tacrolimus, Mycophenolate Mofetil and Prednisone as their immunosuppressive regimen post-transplant. We reviewed their charts for any complications and recorded their absolute lymphocyte counts (ALC) pre-transplant, immediately post-transplant and at 1 month.Results Out of the 8 patients, 2 low-risk recipients (25%) developed biopsy-proven antibody-mediated rejection (AMR) within 1 month of transplant. One of these two patients had complete graft failure, which required a transplant nephrectomy. The other was successfully treated with IVIG, plasmapheresis and Rituximab. The remaining 6 patients continued to have normal graft function at mid-term follow-up. No patients developed any opportunistic infections or any cellular rejections. All patients had their ALC drop to less than 2% after alemtuzumab was given. No correlation was observed between the level of the ALC and patients who had AMR.

Conclusion Alemtuzumab is supposed to deplete both mature T and B cells. However, in our small single center experience, despite a very low ALC immediate post-transplant, 25% of all alemtuzumab-induced pediatric renal transplant recipients developed AMR. This observation supports the view that alemtuzumab potentially augments B-cell activation and should be used with caution as an induction therapy for the pediatric renal transplant population. In addition, a low ALC may not be an accurate prediction that antibody-mediated rejection cannot occur. More studies need to be done to confi rm such observations.

Abstract# 58The Effect of Alemtuzumab Induction on CMV Seroconversion and Infection after Valganciclovir Prophylaxis in Solid Organ Transplant Recipients. Yashaswini Rangan,1 Sarah Parmelee,2 Michael C. Chobanian.2 1Nephrology, Dartmouth Hitchcock Medical Center, Lebanon, NH, USA; 2Solid Organ Transplantation, Dartmouth Hitchcock Medical Center, Lebanon, NH, USA.In 2005, Alemtuzumab was introduced as the induction therapy for the majority of solid organ transplant recipients at our institution. A retrospective analysis of both CMV infection and seroconversion rates in recipients who were at high risk for CMV transmission from donor to recipient (D+ R-) was performed.Review of all recipients 2005-present, identifi ed 198 patients that had undergone alemtuzumab induction. Of these, 54.5% were seronegative for CMV (R-). Of these potential high risk individuals, 43 (39.8%) received organs from CMV seropositive donors (D+). Maintenance immunosuppression was tacrolimus alone or tacrolimus + mycophenolate mofetil in the majority of the recipients (35/43).Recipients who were D+ R- received oral valganciclovir for a period up to 12 months unless it was discontinued for severe neutropenia (ANC < 750 /mm3). No recipient developed CMV infection while taking valganciclovir.Of the individuals who were D+ R-, 9/43 (20.9%) developed CMV infection or syndrome at a mean of 13.5 +/- 3.5 months post transplantation. 8/9 seroconverted to CMV IgG positivity after treatment for the infection at a mean of 15.2 +/- 2.8 months post transplantation. Serologic status for 1 individual was unknown. 4/43 (9.3%) seroconverted without overt signs of infection or viremia at a mean of 12.2 +/- 7.3 months post transplantation. 1 patient was lost to follow up. 29/43 (67.4%) patients showed no evidence of seroconversion. 19 of these 29 patients had completed prophylaxis with oral valganciclovir and had not seroconverted at a mean of 23.68 +/- 9.7 months following transplantation. The remaining recipients were still taking valganciclovir because they had not completed 12 months of prophylaxis.Northern New England recipients are at risk for CMV infection post transplantation due to the high percentage of CMV positive donors and CMV negative recipients. Alemtuzumab induction appears not to be associated with an increased risk of development of CMV infection while on or after stopping prophylaxis. However, there is an increased risk of not successfully seroconverting to CMV IgG positivity for as long as 1 year after stopping prophylaxis.We speculate that intensive induction with alemtuzumab may alter the immune response to CMV, thereby creating a longer term risk of CMV infection despite 1 year of prophylaxis with oral valganciclovir.

Abstract# 59Long-Term Experience in Patients Receiving Infl iximab. Undine A. Gerlach,1 Johann Pratschke,1 Peter Neuhaus,1 Andreas Pascher.1 1Department for General, Visceral, and Transplantation Surgery, Charité, Universitaetsmedizin Berlin, Campus Virchow Klinikum, Berlin, Germany.We report on 9 out of 24 intestinal or multivisceral transplant recipients, who received infl iximab for steroid- and OKT3-resistant rejection, accompanied by ulcerative infl ammation of the distal ileal graft after intestinal transplantation (ITX).Infl iximab application started at 20.2 ± 15.5 months [0,5;40] after transplantation at a dose of 5 mg/kg body weight. The average number of treatment periods per patient was 5 ± 4.4 [1;11], the number of applications within one treatment period: 1.9 ± 1.4 [1;7], the overall number of applications: 9.4 ± 5.6 [2;17]. Time intervals between applications within one treatment period was 8.2 ± 6.7 days [1;20], between treatment periods: 5.5 ± 9.3 months [1;48].7 patients received infl iximab for late-onset rejection at 23.0 ± 12.8 [6;36] months after transplantation, responding instantly with improvement of ulceration and graft histology. Lately one patient successfully received Infl iximab for early-onset OKT 3 resistant rejection which led to a reduction of infl ammatory signs and a complete resolution of rejection. In one patient though, OKT 3-refractory early-onset rejection 15 days after ITX resulted in graft loss and death despite infl iximab. Adverse events upon infl iximab were EBV-infections (n=3), treatment: tapering of immunosuppression, Pneumonia (n=1), treatment: antibiotics and a local cutaneous mycosis (n=1), treatment: local excision. In long-term surveillance, there were no CMV-infections or malignancies like PTLD associated with infl iximab.In conclusion, TNF α-inhibitors extend therapeutic options for late-onset steroid or OKT 3-refractory rejection and chronic infl ammatory graft alterations in intestinal allograft recipients.

BASIC SCIENCE: T CELLS AND AGING AND THE ALLOIMMUNE RESPONSE

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Abstracts

Abstract# 60Improvement of Glycometabolic Control after Islet Transplantation Is Associated with a Better Function of Endothelial Progenitor Cells in Type 1 Diabetic Patients. Alessandra Petrelli,1,2 Andrea Vergani,1,2 Massimo Venturini,2 Michele Carvello,2 Roberto Bassi,2 Daniela Belloni,2 Alessandro Del Maschio,2 Antonio Secchi,2 Paolo Fiorina.1,2 1Transplantation Research Center, Harvard Medical School, Boston, MA, USA; 2Internal Medicine Department, San Raffaele Scientifi c Institute, Milan, Italy.Background: Endothelial progenitor cells (EPC) are bone-marrow derived cells that are functionally committed to vascular repair. It is known that EPCs are dysfunctional in diabetes. Since patients affected by type 1 diabetes (T1D) do have lower number of EPCs, we aimed at studying if restoration of euglycemia after islet transplant is associated with an improvement of EPCs number and function. Methods: Circulating EPCs (CD133+/KDR+) and their rate of apoptosis were studied by FACS in a cross-sectional study in patients with T1D, islet transplanted patients (ITA) and healthy controls (C). CFU-ECs (colony forming unit endothelial cells) were generated in vitro and their endothelial origin was confi rmed by immunofl uorescence (CD34+ and Tie2+ cells). CFUs obtained from the three groups were quantifi ed and the percentage of apoptotic cells was evaluated after 7 days of culture. Moreover, a modifi ed migration assay was set-up to assess the migratory ability of EPCs in presence/absence of SDF1. Finally, vascular endothelial function was assessed with the use of endothelial dependent (EDD) and nitrates dependent (NDD) dilatation by ultrasound technique. Results: Peripheral EPCs number did not differ among groups (CD133+/KDR+: 0.3±0.1 vs. 0.3±0.2 vs. 0.6±0.5 %, ns, T1D, C and ITA respectively), as well as EPCs apoptotic percentage (4.1±2.0 vs. 0.8±0.4 vs. 1.2±0.4 %, ns). Differently, the number of CFUs obtained from T1D, but not from ITA, was reduced compared to C (p=0.01). This reduction was inversely related to HbA1c level (R=-0.4, p=0.01). An increase in CFU apoptosis was evident in T1D compared to ITA and C (p<0.05), while no statistical difference was found in Bcl2/Bax ratio. We found a similar SDF1-mediated EPCs migration ability among the three groups. Finally, T1D, but not ITA, had a lower EDD compared with C (p=0.0001). We even found a negative correlation between EDD and HbA1c (R=-0.40, p=0.03). Conclusions: EPC function is altered in T1D and islet transplantation, by improving glycometabolic control, normalizes EPC function despite the intake of immunosuppressants. Furthermore, EDD, which represents a reliable parameter of vascular function, is impaired in T1D and while being normal in ITA.

ORAL PLENARY EXCHANGE X - BASIC SCIENCE: T CELLS AND AGING AND THE ALLOIMMUNE RESPONSE

Abstract# 61Donor Age Is Associated with Elevated Numbers of Passenger Leukocytes Augmenting the Recipients Immune Response. Christian Denecke,1,2 Xupeng Ge,2 Irene Kim,2 Daman Bedi,2 Anke Jurisch,2 Stefan G. Tullius.2 1Dept of Surgery, Charite Virchow Clinic, Berlin, Germany; 2Transplant Surgery Research Lab, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA.Organs from elderly donors are more frequently utilized for transplantation. We postulated that advanced donor age will impact the recipient’s immune response. We investigated underlying causes of an increased immunogenicity with advanced donor age and analyzed the immune response in a mouse heart transplant model. Age dependent changes in naive hearts and heart allografts from 3, 12 and 18mths old bm12 donors were examined. Subsequently, the recipients immune response was analyzed.Elderly, non-manipulated hearts contained overall signifi cantly elevated frequencies of CD4+and CD8+ T-cells and DCs (CD11c+) (p<0.05).While survival rates were comparable between all groups, numbers of activated DCs (CD11c+I-Ab+and CD11c+CD40+) had signifi cantly increased in recipient spleens after transplantation of 18mths old heart grafts at day 14 (day 14:p<0.05). In parallel, frequencies of effector/memory phenotype T-cells (CD4+CD44highCD62Llow and CD8+CD44highCD62Llow), IL-2+ and IFNg+ CD4+ T-cells but also Tregs (CD4+CD25+FoxP3+ ) were signifi cantly elevated with increasing age (p< 0.01). Proliferation as measured by Mixed Lymphocyte Reaction (MLR; p=0.0002) and T-cell alloreactivity (IFNg-production in ELISPOT) gradually inclined with advancing donor age (p<0.05) indicating an enhanced immunogenicity of older organs.At 8 weeks, recipients of old grafts demonstrated signifi cantly higher frequencies of Tregs and CD4+IL-10+ T-cells but fewer effector/memory phenotype T-cells (p<0.05). Still, proliferation (MLR) and alloreactive IFNg-production signifi cantly inclined with increasing donor age (p<0.01). In parallel, enhanced CD4+ and CD8+ T-cell infi ltrates and an intense Ki67 positivity of GICs in 18mths old heart grafts were detected by immunhistochemical staining. Intragraft gene expression of FasL, Ki67 and in particular ICAM-1 were signifi cantly elevated, thereby sustaining an intense rejection response against elder grafts (p<0.05). These results emphasize an intensifi ed immune response towards organs from old donors.

In summary, a higher number of passenger leukocytes with advanced donor age contributed to an increased immunogenicity of old grafts. The augmented recipients immune response to these grafts may be sustained by intense ICAM-1 expression and stronger intragraft cell proliferation.

Abstract# 62Recipient Age Is Associated with an Impaired CD4+ Effector and Intact Regulatory T-Cell Response. Christian Denecke,1,2 Xupeng Ge,2 Irene Kim,2 Daman Bedi,2 Anke Jurisch,2 Stefan G. Tullius.2 1Dept of Surgery, Charite Virchow Clinic, Berlin, Germany; 2Division of Transplant Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA.Elderly recipients represent the most rapid growing segment of patients on the waiting list. We examined age dependent T-cell functions in a transgenic mouse transplant model.Effector T-cell phenotype, -function, cytokine production and regulatory T-cell function were analyzed in 3 and 18mths old B6 mice. In an in vivo transplant model, BL/6 nude mice were reconstituted with 2x10 6 young or old transgenic alloantigen-specifi c CD4+T-cells and engrafted with bm12 skin grafts. T-cell phenotype and cytokine secretion were analyzed in all lymphatic compartments.Splenocytes of naïve old B6 mice contained signifi cantly higher frequencies of CD4+T-cells with an effector/memory phenotype (CD4+CD44highCD62Llow; p<0.005). However, in vitro proliferation (MLR: p<0.005) and IFNγ-production (ELISPOT (p<0.001) were signifi cantly reduced in aged mice.Older wild-type (WT) skin transplant recipients showed a modifi ed immune response. While graft survival in elderly native recipients was prolonged (11 vs. 13 d, p<0.05), increasing recipient age was associated with an impaired proliferation and IFNγ-production. Signifi cantly diminished numbers of early activated (CD69+) T-cells, IFNγ+ - and IL-2+ T-cells were observed. In parallel, regulatory functions remained age-independent as alloantigen-specifi c CD4+CD25+FoxP3+ T-cells isolated from sensitized old mice demonstrated a dose-dependent well preserved suppressor function.We then examined the impact of age in a transgenic skin transplant model analyzing the alloantigen –specifi c CD4+ T-cell response: Rejection kinetics were also prolonged with increasing recipient age in this model (young vs. old: 10.1 vs. 14.3d, p<0.05) Besides, the T-cell response was signifi cantly different: Fewer numbers of activated CD4+CD25+ and effector/memory phenotype T-cells (CD4+CD44highCD62Llow) (dln) were found after transfer of old T-cells (p<0.05). As a consequence, intragraft CD4+ T-cell infi ltration was reduced.compared to recipients of young T-cells.In summary, elderly mice showed an overall impaired effector T-cell response. In vivo allospecifi c CD4+ T-cell activation was delayed in elderly transplant recipients while regulatory -T-cell function remained preserved. These experimental fi ndings may have important clinical implications for an age-adapted immunosuppression.

Abstract# 63Delayed Acute Allograft Rejection in Aged Transplant Recipients Is Associated with Age-Dependent Increase of Memory T Cells with Impaired Function. Xupeng Ge,1 Anke Jurisch,1 Damanpreet S. Bedi,1 Christian Denecke,1 Gulcin Demirci,2 Xian Chang Li,2 Stefan G. Tullius.1 1Division of Transplant Surgery, Brigham and Women’s Hospital, Boston, MA, USA; 2The Transplant Institute, Beth Israel Deaconess Medical Center, Boston, MA, USA.Clinically, older recipients represent more than 50% of the overall amount of patients waiting for a renal transplant. We postulated that recipient age will affect the overall immune responses to organ transplants and impact graft survival.DBA hearts (3 months) were transplanted into C57Bl/6 recipients (age: 3, 18 months). Age-matched native C57Bl/6 served as controls. Animals were followed until grafts discontinued functioning. Age-dependent immune responses were evaluated in parallel.Heart allografts in young mice were rejected after 6.8±0.7 days with characteristic morphological signs of acute rejection. In sharp contrast, allograft survival was signifi cantly prolonged in elderly recipients (10.0±1.3 days, p<0.0001). Splenocytes from older mouse demonstrated signifi cantly increased rates of apoptosis (Anexin V/7-AAD staining: CD4+: 36.2% vs 17.6%; CD8+: 21.6% vs 12.6%; p<0.05). At the same time, CD44high/CD62Llow effector/memory T cells had signifi cantly increased (FACS: CD4+: 25.5% vs 16.1%; CD8+: 11.8% vs 5.0%, P<0.05). In contrast, proliferation of CD4+/CD8+ T cells were reduced in MLR (71.2% and 49.5%, respectively, p<0.05). Old and young T-regs demonstrated comparable suppressive effects in MLR at various ratios. Next, we evaluated if regulatory T cells also contributed to the observed delayed rejection. While young CD4+T-cell proliferated signifi cantly in MLR following the depletion of Tregs, increase in proliferation of older CD4+ T-cells remained minimal (97 vs. 15%, p<0.05).We conclude that delayed allograft rejection is associated with a compromised memory T-cell response, reduced proliferation and increased apoptosis of CD4+/CD8+ T-cells while regulatory T-cell function remained intact. Immunosuppression will need to consider age-adapted modifi cations of the immune response in transplant patients.

YOUNG INNOVATORS

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Abstract# 64MHC Class II Gene Therapy: A Novel Approach to Donor-Specifi c Tolerance without Sustained Immunosuppression. Sharon Germana,1 Luis Fernandez,1 Paulo Martins,1 Kaela Goldstein,1 Maria Denaro,2 Kathleen Hehir,2 Julian Down,2 Gilles Benichou,1 Christian LeGuern.1 1TBRC/TC, Surgery, MGH/HMS, Boston, MA, USA; 2Genetix Pharmaceuticals Inc., Cambridge, MA, USA.The mechanism of peptide presentation by MHC class II (MHCII) for T cell activation is well-known, but that of MHCII regulation of activated T cells is poorly understood. Studies have established the benefi cial effect of donor/recipient MHCII matching on graft survival, providing us with a model to study MHCII regulation of T cell alloreactivity. This project was designed to 1. Examine whether MHCII molecules could down-regulate T cell responses to allografts in a preclinical model; 2. Decipher the mechanism of this control in murine models and 3. Design in non-human primates, conditions to translate this approach to the bedside. Experiments involved transfer of donor-type MHCII genes in bone marrow cells (BMC) of recipients of subsequent transplants that were only matched to the transferred MHCII. Transgene expression was tested by rtPCR and fl owcytometry, and T and B cell responses were assessed by MLR, CML, Elispot assays and Ab detection. Graft survival was monitored according to clinical and histopathologic parameters. Experiments performed in a miniature swine kidney transplantation model showed that transfer of a single donor MHCII-DR or DQ did not affect graft-specifi c T cell responses pre-transplantation but resulted in donor-specifi c tolerance of allogeneic kidney grafts (N= 6 and 4, respectively). Long-term accepted transplants had no sign of chronic rejection in the absence of immuno-suppression. Similar survivals were found for murine heart transplants (> 175 days in 80% of recipients; N=12, no chronic allograft vasculopathy). Mechanistic studies demonstrated that CD4+CD25+FoxP3+ regulatory T cells carried tolerance and that graft MHCII was crucial to donor-specifi c tolerance as MHCII-defi cient and 3rd party MHCII hearts were rejected by treated recipients (MST=12 ± 3 days, N= 8 and 6, respectively). Clinically-approved lentivirus vectors have been made to express Cynomolgus Macaque MHC-DRB*1002 with high effi ciency in allogeneic BMC. Pilot transductions performed on CD34+ progenitors demonstrated >50% transduction rate, 14 days after infection, paving the way for ongoing in vivo experiments. Collectively, these data indicate that MHCII gene therapy might be a promising clinical alternative to non-specifi c immunosuppression for induction of donor-specifi c tolerance.

Abstract# 65Opposing Roles for T Cell-NF-κB and TLR Signals in the Fate of Islet Allografts. Delia Lozano Porras,1 Ying Wang,1 Maria-Luisa Alegre.1 1Department of Medicine, University of Chicago, Chicago, IL, USA.Islet transplantation has the potential to cure Type 1 Diabetes (T1D), a chronic lifelong disease. A major obstacle limiting its effi cacy is allograft rejection. At present, many immunosuppressive regimens inhibit NF-κB globally to maintain graft acceptance. We hypothesized that inhibiting NF-κB specifi cally in T cells would result in long-term islet allograft acceptance. To study the role of NF-κB in alloreactive T cells, IκBα∆N-Tg (C57Bl/6, H2b) mice that express an NF-κB super-repressor specifi cally in the T cell lineage were rendered diabetic with a single high dose of streptozotocin (SHD-STZ). These mice were then transplanted with BALB/c (H2d) islet allografts. Blood glucose levels were monitored to determine islet allograft survival. Inhibiting NF-κB specifi cally in T cells resulted in long-term islet allograft survival. To understand the mechanism of islet allograft acceptance, CFSE-labeled DO11.10 x IκBα∆N-Tg splenocytes were adoptively transferred into syngeneic BALB/c recipient mice transplanted with BALB/c islets expressing membrane-bound ovalbumin. NF-κB-impaired T cells proliferated less than wildtype T cells in response to islet antigen and fewer antigen-specifi c T cells were recovered from DO11.10 x IκBα∆N-Tg mice. However, histological examination of IκBα∆N-Tg mice at day 40 post islet transplantation revealed recruitment of IκBα∆N-Tg CD4+ and CD8+ T cells surrounding the islets, in the absence of islet destruction. These data suggest that the NF-κB-impaired T cells lack a signal(s) that renders them capable of islet destruction. To investigate the signals that would promote transition between peri-insulitis and destructive islet invasion, IκBα∆N-Tg (C57Bl/6) mice that had accepted BALB/c islet allografts were subsequently transplanted with donor skin. Skin grafts were acutely rejected by IκBα∆N-Tg mice and precipitated the rejection of the primary islet allografts. Furthermore, in IκBα∆N-Tg mice with established islet allografts, administration of the TLR9 agonist, CpG, was suffi cient to trigger islet allograft rejection in the majority of the animals. Together, our data suggest that impaired NF-κB activity in T cells results in long-term islet allograft acceptance but not donor-specifi c tolerance, and that TLR9 signals in mice bearing stable transplants can trigger allograft rejection in a manner independent from NF-κB activation in T cells.

Abstract# 66Regulatory Function of Galectin-9 in Allograft Rejection and Acquired Tolerance. Toshihiko Watanabe,1 Olaf Boenisch,1 Hideo Yagita,2 Hisaya Akiba,2 Nader Najafi an.1 1Nephrology, Children’s Hospital Boston, Boston, MA, USA; 2Immunology, Juntendo University, Tokyo, Japan.Galectin-9 (Gal-9), a molecule widely expressed in various tissues, regulates Th1 cell function through its receptor TIM-3. Function and mechanism of targeting Gal-9 was investigated in in vivo models of murine vascularized cardiac transplantation.RG9-1, a novel blocking antibody against Gal-9, was administered i.p. to recipients in various donor/recipient strain combinations (500µg on day 0; 250µg on day 2, 4, 6, 8, 10 post TX). Graft survival was assessed by palpation, frequencies of IFN-γ-, IL-4-, IL-5-, IL-6-, and IL-17-producing splenocytes were measured by ELISPOT.RG9-1 did not affect graft survival in wild-type (MST 7 vs 7 days; n=10/5) or CD4-defi cient (MST >100 vs >100 days; n=5/10) B6 recipients of fully MHC-disparate allografts from BALB/c donors, but accelerated rejection in CD8-defi ceint recipients (MST 9.5 vs 12 days; n=8/7; p=0.02). Moreover, RG9-1 accelerated rejection in recipients lacking CD28:B7 costimulation (CD28-defi cient: MST 8 vs 15.5 days; n=11/8; p=0.002; B7-1/2-defi cient: MST 51.5 vs >100 days; n=6/8; p=0.01). RG9-1-treated mice showed increased frequencies of IFN-γ- (461±48 vs 270±27 spots/500.000 responder cells; n=10/10;p=0.01), IL-4- (241±43 vs 71±10; p=0.0006), and IL-6- (136±19 vs 79±9; p=0.01) producing splenocytes and a trend towards higher frequencies of IL-17-producing splenocytes (5.1±0.8 vs 3.1±0.5; p=0.08). Furthermore, Gal-9 blockade abrogates acquired tolerance induced by CTLA4-Ig in B6 recipients of BALB/c grafts (MST 41 vs 92 days; n=9/8; p=0.07).Single MHC class I/II-mismatched allografts survive long-term, but develop chronic allograft vasculopathy. RG9-1 did not affect graft survival in a CD8+ T cell-mediated MHC-I-mismatch model (bm1 into B6; MST >56 vs >56 days), but precipitated rejection in a CD4+ T cell-mediated MCH-II-mismatch model (bm12 into B6; MST 48 vs >56 days; n=6/6; p=0.03). Accelerated rejection was accompanied by increased frequencies of IFN-γ-, IL-17-, IL-6-, and IL-4-producing splenocytes compared to control animals.These data are the fi rst to establish the regulatory functions of Gal-9 in alloimmune responses in solid organ transplantation models. The inhibitory actions require the presence of CD4+ T cells and may be secondary to modulation of T helper cell differentiation. The regulatory function of the intact TIM-3:Galectin-9 pathway seems to be independent of B7:CD28 costimulation and to be essential for the induction of tolerance by CTLA4-Ig.


ORAL PLENARY EXCHANGE XI - YOUNG INNOVATORS

Abstract# 68ABO Incompatible Heart Transplantation in Early Childhood: Tolerance towards the Donor Blood Group Is Associated with Reduced De Novo HLA-Sensitization. Simon Urschel,1 Patricia M. Campbell,2 Steven R. Meyer,3 Ingrid M. Larsen,1 Kim Derkatz,1 Kathryn Tinckam,4 Julia Nuebel,5 Julia Birnbaum,5 Theresa Kauke,5 Yashu J. Coe,1 Jeffrey M. Platt,6 Lori J. West.1 1Pediatrics, University of Alberta, Edmonton, AB, Canada; 2Nephrology, University of Alberta; 3Cardiac Surgery, University of Alberta; 4University of Toronto, Canada; 5Pediatric Cardiology, Ludwig Maximilians University, Munich, Germany; 6University of Ann Arbor, USA.Introduction:In heart transplanted (HTx) patients the presence of HLA antibodies is associated with acute rejection, earlier transplant-related coronary artery disease and decreased graft survival. Children after ABO incompatible HTx in infancy mostly show absence of specifi c antibodies towards the donor blood group. This antigen specifi c tolerance may be associated with an altered immune response towards HLA.Methods:We determined the incidence of de novo HLA antibodies in 122 pediatric patients after thoracic transplantation (28 ABO incompatible) and 36 matched controls using FlowPRA® or ELISA in. Donor specifi city (DSA) was determined with single antigen bead fl ow analysis.Results:Median age at Tx was 1.7 yrs (range 1 day to 17.8 yr). Testing was assessed at a median of 3.48 years after Tx (range 0.6 to 17.1 yr). Antibodies against HLA-class I antigens were found in 21 patients (17.2%), against class II in 18 (14.8%) in 10 (8.2%) of these against both. DSA were identifi ed in six patients, all against class II, one additional class I. Patients with DSA were signifi cantly older at time of transplantation (Median: 11.3 yrs, p=0.043). In patients with pre-transplant cardiac surgeries class II antibodies were signifi cantly more frequent. None of the ABO incompatible patients had DSA, and class II- antibodies were signifi cantly less

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frequent than in patients transplanted ABO-compatible. This was true also when comparing the two groups within the patients transplanted within the fi rst 2 years of life.Conclusions:We found de novo HLA-antibodies in pediatric patients and healthy children less frequently than reported in adults. DSA were only present in patients transplanted at older age. Absence of DSA may be due to young age at transplantation while the lower presence of class II antibodies following ABO-incompatible transplantation refl ects a modulated B-cell response to HLA accompanying the donor blood group tolerance. Our fi ndings show for the fi rst time that in vivo T-independent immune response can modulate response to T-dependent antigens.

Abstract# 69TIM-1 Defi nes a Novel Population of Inducible Regulatory B Cells That Underlies Prolongation of Allograft Survival by Anti-TIM1. Qing Ding,1 Nader Najafi an,2 Songyan Deng,1 Mohamed H. Sayegh,2 David Rothstein,3 Melissa Yeung. 1Yale Medical School; 2Harvard Medical School; 3University of Pittsburgh.TIM-1 is a novel costimulatory molecule, in the T-cell Ig and mucin domain-containing family, that plays an important role in regulation of immune responses. The low affi nity anti-TIM-1 mAb, RMT1-10 (RMT) + Rapa induces long-term allograft survival (GS) through mechanisms that depend on Th2 deviation. However, TIM-1 is expressed on only 1-2% of Th1, Th2, or Treg cells, in vivo. We now report the novel fi nding that 5-10% of B cells in naïve mice constitutively express TIM-1. Moreover, TIM-1 expression is markedly increased in RMT-treated allograft recipients reaching 20-40% of B cells, while T cell expression remains minimal. In BALB/c recipients of B6 islets, RMT alone increases GS (MST 12d to 28d, with 30% >120d GS). Surprisingly, after B cell depletion (anti-CD20), or in B-defi cient JHD (BALB/c) recipients, anti-TIM-1 dramatically accelerated islet rejection (MST: from 12d to 6d). This suggests that B cells, and possibly TIM-1+ B cells, are a critical target for anti-Tim1-mediated GS. In this regard, we found that TIM-1+ B cells exhibit >10-fold higher IL-10 and IL-4 expression than TIM-1- B cells (6-8% vs. 0.3-0.8%, respectively). In accord, the Th2 shift induced by RMT required B cells, and reconstitution of B-defi cient mice with wt B cells restored both prolongation of GS and Th2 deviation by RMT. We found that IL-4R-/- mice have few TIM-1+ B cells, and these remain reduced after transplantation + RMT compared to wt mice. Transfer of IL-4R- B cells (limited TIM-1) into JHD mice no longer rescues prolongation of GS by RMT. Critically, transfer of TIM-1+ but not TIM-1- B cells from allograft recipients into otherwise untreated JHD recipients, markedly prolongs GS (MST 14d TIM1- vs. >60d TIM1+ B cells), indicating that TIM-1+ B cells are regulatory (Breg). While TIM-1 identifi es most IL-10+ cells within the recently defi ned “B10” Breg cells (CD5+CD1dHi), only 20% of TIM-1+ B cells are in this subset, and unlike B10 cells, TIM1+ Breg express and require IL-4 as well as IL-10 for their activity. Thus, TIM-1 defi nes a new subset of Breg, inducible through TIM1 ligation, that are required for altered T cell responses and allograft survival observed after anti-TIM-1 therapy.

Abstract# 70Varying Effects of Donor Brain Death on Lung, Kidney, and Heart Allograft Tolerance in Miniature Swine. Karen M. Kim,1 Gregory R. Veillette,1 Timothy M. Millington,1 Akihiro Aoyama,1 Andrew J. Meltzer,1 David H. Sachs,1 Joren C. Madsen,1 James S. Allan,1 Bruce R. Rosengard.1 1Transplantation Biology Research Center, Massachusetts General Hospital, Boston, MA, USA.Donor brain death results in an intense infl ammatory state that culminates in the activation of the immune system. Here we summarize our fi ndings regarding its effect on tolerance induction in previously established models of lung, kidney, and heart allograft tolerance in MHC-inbred miniature swine.Methods: Orthotopic lung transplants were performed across a full MHC-mismatch with 12 days of high-dose FK-506 (mean level 35-55 ng/mL). Kidney and combined heart-kidney (immediate orthotopic kidney followed by heterotopic heart) transplants were performed across a class I MHC-mismatch with 12 days of high-dose CsA (mean level 400-800 ng/mL). Brain death was induced by intracranial infl ation of a 30 mL Foley catheter and confi rmed by a Cushing’s refl ex and apnea test. After 4 hrs of donor brain death, the lung, kidney, or heart was harvested and transplanted. Control animals underwent routine transplantation, or historical controls were used for comparison. Recipients were followed with routine labs, biopsies, and organ-specifi c studies. In vitro studies (CML, MLR, qPCR) were performed.Results: Animals receiving lungs from brain-dead donors (N=4) all rejected their grafts (ISHLT grade 4/4) by POD 45 in comparison to 5 of 6 control animals that demonstrated long-term acceptance (>365 days) of lungs from healthy donors. In contrast, animals receiving kidneys from brain-dead donors (N=4) all accepted their grafts >100 days (Cr 0.8-1.6 mg/dL at sacrifi ce). Three of 4 animals that received heart-kidney allografts from brain-dead donors similarly accepted their grafts >100 days, with 1 animal developing ISHLT grade 3a/4 rejection of the heart and ACR type 2 of the kidney before expiring of renal failure on POD 47. CML assays for tolerant animals showed donor-specifi c hyporesponsiveness. qPCR revealed differential intragraft gene expression after brain death, with upregulation of IL-1 and IL-6 in lung tissue and marked upregulation of IL-6 in heart tissue.

Conclusions: Donor brain death appears to inhibit tolerance induction in a swine model of lung transplantation but not kidney transplantation, and likely does not inhibit tolerance induction of heart allografts in a swine model of combined heart-kidney transplantation. The variable effects may be due to organ-specifi c changes that occur as a result of the infl ammatory milieu of brain death.

Abstract# 71The Link between PDL1 Costimulatory Pathway and T17 in Fetomaternal Tolerance. Francesca D’Addio,1 La-Tonya Adams,1 Bechara Mfarrej,1 Indira Guleria.1 1Transplantation Research Center, Renal Division, Brigham & Women’s Hospital and Children’s Hospital, Transplantation Research Center - Brigham & Women’s Hospital and Children’s Hospital, Boston, MA, USA.Background:Programmed death-1 (PD1) and its ligand (PDL1), a novel negative costimulatory pathway, plays an important role in peripheral tolerance. PDL1, expressed at the uteroplacental interface, is necessary to maintain fetomaternal tolerance and PDL1 deficiency/blockade results in compromised pregnancy. Regulatory T cells (Tregs) have been demonstrated to take part in mediating fetomaternal tolerance and express PDL1. More recently, the novel IL-17 producing T cells (T17) have been recognized as a barrier in inducing tolerance in transplantation. We investigated the mechanisms of PDL1 mediated regulation of fetomaternal tolerance using an alloantigen specifi c CD4 TCR transgenic mouse model system (ABM-tg mouse).Methods: Thy1.1B6 female mice mated with bm12 males were treated with anti-PDL1 and followed for fetal resorption. 3x106 ABMCD4+Thy1.2+ cells were adoptively transferred into Thy1.1B6 plugged females previously mated with bm12 males to track behaviour of alloantigen-specifi c immune response.Results: PDL1 blockade signifi cantly increased fetal resorption and resulted in reduced litter size. Flow cytometry analysis at 11.5 days showed a signifi cant increase in Thy1.2+ effector T cells (Teff) and a decrease in Thy1.2+ Tregs with a Treg / Teff ratio in favor of Teff with anti-PDL1 treatment. A decreased PDL1 expression on peripheral Thy1.2+ Tregs and at the fetomaternal interface with anti-PDL1 treatment was observed. Interestingly, anti-PDL1 treated mice showed higher production of IL-17 both in the periphery (spleen) and in the placenta. Neutralization of IL-17 in vivo abrogated anti-PDL1 effect in reducing fetal survival rate.Conclusion: Our study demonstrates that the PD1-PDL1 costimulatory pathway orchestrates fetomaternal interface by controlling the pathogenic cytokine IL-17. Neutralization of IL-17 by anti-IL-17 mAb abrogated the effect of anti-PDL1 in reducing fetal survival rate. This is the fi rst report utilizing an alloantigen specifi c model that establishes a link between PDL1, T17 cells and fetomaternal tolerance.

Abstract# 72The Impact of Donor-Specifi c Antibody (DSA) and Conjoint DSA/C4D on Graft Survival during the First Year Post-Renal Transplantation and One Year Later. Boonsong Kiangkitiwan,1 Debra Kukuruga,2 Matthew Cooper,3 Heather Hurley,4 Cinthia Drachenberg,5 David Klassen,1 Abdolreza Haririan.1 1Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA; 2Immunogenetics Laboratory, University of Maryland School of Medicine, Baltimore, MD, USA; 3Department of Surgery, University of Maryland School of Medicine, Baltimore, MD, USA; 4Pharmacy, University of Maryland School of Medicine, Baltimore, MD, USA; 5Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, USA.We reviewed 297 donor renal allograft recipients who were screened for DSA and underwent biopsy for cause between January 2005 and December 2007. Data were retrieved through manual chart review and inquiry into electronic databases.Patient and transplant-related characteristics of the entire cohort are summarized in Table 1.Age (yr) 51.4±13.0 Re-transplantation (%) 15.5Male (%) 66.7 HLA MM 4.2±1.6African-American (%) 54.9 Peak PRA <10%/10-40/>40% 70.1/9.6/20.4Deceased donor (%) 76.4 BSX/ATG/Camp* (%) 44.8/30.7/19.3*Basiliximab/Thymoglobulin/Campath-H1We found the association of the four different combinations of DSA with graft survival as the following; during the fi rst post-transplant year the presence of any DSA was predictive of higher risk of graft loss. Adjustment for gender, race, re-transplantation, and acute cellular rejection did not affect these associations. However, after one year, only positivity for class II DSA, either alone or with class I, was predictive of worse graft survival. Adjustment for the potential confounders only modifi ed the strength of this association. When using the conjoint DSA and C4d to predict graft survival. After adjustment for the potential confounders, we found that during the fi rst year the presence of DSA with or without C4d positivity was associated with higher risk of graft failure. Interestingly, one year later only patients with positive C4d/DSA had worse graft survival.In summary, only positivity for class II DSA alone was associated with poor graft survival after the fi rst year post-transplant. When combining DSA with C4d, the presence of DSA irrespective of C4d status was an independent predictor of worse

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graft survival during the fi rst year. In contrast, after one year of transplant, only patients with both C4d and DSA positivity experienced worse outcome.

Abstract# 73The Role of Proinfl ammatory Cytokine IL-6 in Alloimune Responses. Xiaozhi Zhao,1 Olaf Boenisch,1 Bechara Mfarrej,1 M. Javeed Ansari,2 John Iacomini,1 Laurence A. Turka,3 Mohamed H. Sayegh,1 Xueli Yuan.1 1Renal Division, Brigham and Women’s Hospital, Boston, MA, USA; 2Division of Nephrology and Organ Transplantation, Northwestern University Feinberg School of Medicine, Chicago, IL, USA; 3Renal Electrolyte & Hypertension Division, University of Pennsylvania, Philadelphia, PA, USA.Given the importance of IL-6 in controlling Th17 and Treg development, we examined the function of IL-6 in regulating allograft rejection and tolerance. Following transplantation of BALB/c cardiac allografts into WT, IL-17-/- or IL-6-/- mice on a C57BL/6 background, all recipients rejected their graft around 7 days after surgery, and splenocytes from WT mice produced signifi cant amounts of INF-γ and IL-6 (1865±616.7 and 556.5±100.3 pg/ml). Interestingly, the amount of INF-γ produced was greater in IL-17-/- and IL-6-/- mice (12211.7±4683.8 and 9065±5471.2 pg/ml), when compared to WT mice (P<0.01) suggesting that defi ciency in IL-17 and IL-6 may favor a Th1 response. IL-6 production was similar in WT and IL-17-/- recipients. An increase in CD25+Foxp3+ CD4 T-regs and reduction in CD44hiCD62Llow CD8 effector cells was observed in the peripheral blood of IL-6-/- recipients. Administration of single CTLA4Ig signifi cantly prolonged allograft survival in WT and IL-17-/- mice although they eventually rejected their grafts (median survival time: 22 and 24 days respectively). Strikingly, a single dose of CTL4-Ig was suffi cient to allow for long-term survival (>100days) of allografts in all IL-6-/- recipients. CTLA4Ig signifi cantly inhibited INF-γ production by cells from WT, IL-17-/- and IL-6-/- recipients , but not IL-6 production in WT and IL-17-/- recipients. In IL-17-/- mice, and to a greater extent in IL-6-/- mice, CTLA4Ig treatment resulted in a reduction in graft infi ltration. No signifi cant difference could be seen in the number of Tregs and CD8 effectors in the peripheral blood among different treated groups. In comparison to WT IL-6+/+ Tregs, IL-6-/- Tregs demonstrated stronger ability in inhibiting INF-γ production by effector cells (examined with Luminex), although IL-6 defi ciency did not change the frequency of INF-γ producing cells (ELISPOT data). These data suggest that IL-6, rather IL-17, is critically important in allograft rejection and tolerance induction. Targeting IL-6 and its signaling pathway could promote Treg function and therefore be a promising strategy to prevent allograft rejection prevention and overcome resistance to tolerance induction.

Abstract# 74Changes in T Cell Subsets in Patients with Tolerance and Chimerism after Renal and Hematopoietic Cell Transplantation. Sussan Dejbakhsh-Jones,1 Jhon Scanding,1 Stephan Busque,1 Claudia Benike,1 Aditi Mukhopadhyay,1 Edgar Engleman,1 Samuel Strober.1 1Medicine, Stanford University, Stanford, CA, USA; 2Medicine, Stanford University, Stanford, CA, USA; 3Medicine, Stanford University, Stanford, CA, USA; 4Medicine, Stanford University, Stanford, CA, USA; 5Medicine, Stanford University, Stanford, CA, USA; 6Medicine, Stanford University, Stanford, CA, USA; 7Medicine, Stanford University, Stanford, CA, USA.AIMS: We have attempted to achieve mixed chimerism and tolerance in humans after transplantation of combined HLA-matched kidney and hematopoietic cells using conditioning with total lymphoid irradiation (TLI) and antithymocyte globulin (ATG). This regimen can induce tolerance and prevent GVHD in laboratory animals using combined organ and bone marrow transplantation.METHODS: We enrolled ten patients in which the TLI/ATG conditioning regimen was given for 10 days after HLA-matched kidney transplantation, and cryopreserved GCSF mobilized blood mononuclear donor cells were infused.RESULTS: Seven patients developed persistent chimerism for at least 6 months. Five with chimerism for more than12 months have been withdrawn from immunosuppressive drugs and had excellent graft function for at least 8 to 36 months thereafter.Immediately after conditioning with TLI/ATG (at the time of the donor cell infusion) lymphodepletion was maximum (less than 100 lymphocytes /mm^3), and the absolute number of all T cell subsets declined markedly . However the number of naïve CD4+ and CD8+ T cells declined to a much greater extent than the CD4+CD25+FOXP3+ Treg cells or Vß11+Va24+(NK) T cells.Whereas the ratio of Treg/naïve CD4+ T cells pre transplant was about 1:10, the ratio after conditioning was up to 10:1 in patients who developed persistent chimerism and tolerance with specifi c unresponsiveness to donor cells in the MLR. Patients who failed to develop tolerance had Treg/naïve CD4+ T cell ratios of less than 1:1 at the same time point. The ratio of NKT cells to naïve T cell pre transplant was about 1:300 and rose to 1:10 after conditioning. There was a return of the absolute numbers of all T cell subsets to pre transplant levels during the fi rst 6 to 12 months, and CD8+T cells returned more rapidly than CD4+ T cells.CONCLUSION: The changes in the balance of T cell subsets favoring regulatory CD4+CD25+FOXP3+ T cells was associated with the successful induction of tolerance in the current study in association with persistent chimerism.

Abstract# 75Marked Hepatic Allograft Survival Prolongation by ASKP1240 (4D11) in Non-Human Primates. Tetsu Oura,1 Kenichiro Yamashita,1 Tomomi Suzuki,1 Daisuke Fukumori,1 Masaaki Watanabe,1 Gentaro Hirokata,1 Kenji Wakayama,1 Toru Miura,2 Kazumichi Okimura,2 Koushi Maeta,2 Masahiko Taniguchi,1 Tsuyoshi Shimamura,1 Hironori Haga,3 Kanako Kubota,3 Akira Shimizu,4 Fumihiko Sakai,5 Hiroyuki Furukawa,1 Satoru Todo.1 1Surgery, Hokkaido University, Japan; 2Kyowa Hakko Kirin Co.Ltd, Japan; 3Surgical Pathology, Hokkaido University, Japan; 4Pathology, Nippon Medical School, Japan; 5Astellas Pharma Inc., Japan.Background: Previously, we reported that CD40-CD154 co-stimulation blockade by ASKP1240 (4D11), a fully human anti-CD40 mAb, prolonged renal allograft survival in monkeys.Aim: The effect of 4D11 was examined on hepatic allografts in non-human primates.Methods: Orthotopic liver transplantation (LTx) was performed among cynomolgus monkey pairs (blood-type: identical, MLR-SI: >5). For the induction treatment group, 4D11 (10 mg/kg) was given i.v. on days 0, 4, 7, 11 and 14. Weekly 4D11 (5 mg/kg) injection was continued thereafter for up-to 6 months in the maintenance treatment group. Graft survival, graft histology and allo-immune responses (IFN-g ELISPOT assay and anti-donor Ab formation) were assessed.Results: Both induction and maintenance 4D11 treatments markedly prolonged hepatic allograft survival. In the induction treatment group, cellular and humoral responses were inhibited during treatment period. However, IFN-g producing cells and anti-donor-IgG Ab increased after cessation of 4D11, and grafts failed due to chronic rejection. In contrast, by maintaining 4D11 treatment, both direct and indirect cellular responses were completely abolished even after drug cessation. Some animals experienced cholangitis, while there was no evidence of rejection, and one graft is still surviving. No drug-related side effect was noted.Conclusion: CD40 blockade using 4D11 is an attractive modality for immunosuppression in LTx.

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Abstracts

Ten monkeys received heterotopic heart transplants followed by one to four months of immunosuppression with Tacrolimus, mycophenolate mofetil and methylprednisolone. They then underwent a nonmyeloablative conditioning regimen consisting of whole-body and thymic irradiation, equine anti-thymocyte globulin (ATG), anti-CD8 antibody and costimulatory blockade, followed by bone marrow transplantation (BMT).In group A (n=4), induction of chimerism was delayed for 4-6 weeks. BMT was followed by 90 days of Tacrolimus monotherapy. One monkey developed chimerism and its graft survived for 122 days after BMT. No chimerism was detected in the remaining animals, two of which developed PTLD and one bacterial sepsis. In group B (n=3), induction of chimerism was delayed for 17-22 weeks. BMT was followed by 60 days of Tacrolimus. Two monkeys developed chimerism. One of these died of likely viral infection 29-days post BMT while the other rejected its graft 63 days after BMT. In group C (n=3), recipients were treated with rabbit ATG and splenectomy at the time of heart transplant, followed by eight weeks of immunosuppression. BMT was followed by 28 days of cyclosporine. One monkey died of PTLD 28 days after BMT. One monkey’s graft rejected 96 days after BMT and the other has no evidence of rejection 60 days after BMT.Although rejection-free graft survival was maintained for more than one month after the cessation of immunosuppression in group C, cellular and humoral rejection ultimately occurred and was associated with a marked increase in CD8+ effector memory T-lymphocytes beginning 5-6 weeks after BMT. We hypothesize that this population may be responsible for the failure of this strategy to tolerize cardiac allograft recipients.

Abstract# 80 Poster Board #-Session: P5-IIRegeneration of Naïve T-Lymphocytes from Heart-Thymus Grafts Occurs at a Low Level and Is Associated with Prolonged Survival but Not Tolerance. Timothy M. Millington,1 Aseda Tena,1 Svjetlan Boskovic,1 Tatsuo Kawai,1 John Wain,1 James S. Allan,1 David H. Sachs,1 Joren C. Madsen.1 1Surgery, Massachusetts General Hospital, Boston, MA, USA.Purpose: The thymus induces tolerance to self-antigens and may also be important in induction of tolerance to alloantigens after transplantation of a heart-thymus graft with lymphodepletion. We assessed the extent to which the thymic graft participates in immune reconstitution.Procedures: Heterotopic en bloc heart-thymus transplants were performed on fully mismatched cynomolgus macaques (n=10). Most animals (n=8) also underwent thymectomy. Antithymocyte globulin was given at high (10-20 mg/kg, n=3) or low (6mg/kg, n=3) doses. The remaining four animals received only tacrolimus. Heart transplants alone were also performed with (n=2) and without (n=4) thymectomy. Thymopoiesis was detected by quantifying signal joint T cell receptor excision circles using PCR. Memory (CD95 +) and naïve (CD95-/CD28+) T-cells were quantifi ed by FACS.Results: In heart-thymus recipients, mean graft survival after tacrolimus levels became subtherapeutic was 64.3 days in animals conditioned with ATG and 16.3 days in unconditioned animals, compared to 36.8 and 13 days respectively in the heart-alone group. Peripheral lymphodepletion after administration of ATG was accompanied by a reduction in sjTREC to undetectable levels and expansion of CD95+ lymphocytes. Low sjTREC counts and numbers of naïve T-cells were detectable by day 60 but as rejection developed, sjTREC disappeared. When the native thymus was left in place, sjTREC counts and naïve populations recovered to pre-treatment levels whereas in thymectomized animals receiving only a heart graft, sjTREC levels remained undetectable.Conclusion: Early reconstitution of the immune system after lymphodepletion occurs predominantly by proliferation of memory lymphocytes. Transient reconstitution of naïve lymphocytes by a transplanted thymus occurred but was not suffi cient to induce tolerance to a transplanted heart.

Abstract# 81 Poster Board #-Session: P6-IISensitization Prior to Composite Tissue Allotransplantation in Rat Model Does Not Result in Hyperacute Rejection. Shengli Wu,1 Hong Xu,1 Olayemi M. Ikusika,1 Ashley Ocker,1 Suzanne T. Ildstad.1 1Institute for Cellular Therapeutics, University of Louisville, Louisville, KY, USA.Objective: Presensitization to donor alloantigens results in hyperacute rejection of renal allografts. The role of antibody-mediated rejection in composite tissue allotransplantation (CTA) is largely unstudied. Currently, the donor-recipient matching process for CTA closely follows the standard practices for solid organ transplantation. Thus, a positive crossmatch is a contraindication to CTA. The purpose of this study was to determine if a positive crossmatch is indeed a contraindication to CTA.Methods: Major histocompatibility-mismatched rat strains, ACI and WF, were used as donor and recipient, respectively. WF rats were presensitized to ACI antigens by skin transplantation. The recipient serum was collected at baseline and weekly for 5 weeks and fl ow crossmatch assays were performed to demonstrate evidence of donor-specifi c antibody production. Sensitized WF rats or unsensitized control rats received

ORAL POSTER EXCHANGE 1 - EXPERIMENTAL TOLERANCE

Abstract# 76 Poster Board #-Session: P1-IISkin Graft Rejection: Direct or Semi-Direct Allorecognition? Georges Tocco,1 Cavit D. Kant,1 Pilhan Kim,2 Sarah E. Connolly,1 Susan Shea,1 Charles Lin,2 Seok-Hyun Yun,2 Gilles Benichou.1 1Surgery/Transplant Center, Massachusetts General Hospital/Harvard Univ., Boston, MA, USA; 2Wellman Center for Photomedicine, Massachusetts General Hospital/Harvard Univ., Boston, MA, USA.Through the use of in vivo microscopy, we investigated the cellular traffi cking from the transplant to the host (passenger leukocytes) after primarily vascularized (skin and heart) or non-vascularized transplantation (skin). Transgenic mice displaying the fl uorescent GFP protein on all of their cells or on MHC-II+ cells were used as donors to visualize donor cellular traffi cking in the host. As early as day 1 after placement of vascularized transplants (skin or heart), donor MHC-II+ cells were found in all observed recipient’s lymph nodes (graft draining and non-draining) and spleen. In contrast, for non-primarily vascularized skin grafts, very few donor cells (4-8 altogether) could be detected only in the recipient draining lymph nodes and only at later time points (5 days and beyond post-transplant). At a time point when no donor cells could be detected in recipient lymphoid organs (day 3-4), Elispot analyses revealed that T cells were already activated through the direct pathway. Apparently, at these early time points (3-4 days), directly activated T-cells could not have been activated by passenger leukocytes. In addition, it is unlikely that the very low numbers of donor APCs found in the host’s draining lymph nodes at later time points (day 5 and beyond) following the placement of non-vascularized skin grafts would be suffi cient to trigger the large polyclonal direct alloresponse (1-5 % of T cell repertoire). These fi ndings challenge the current dogma that after non-primarily vascularized skin transplantation, antigen-presenting cells (APCs) of donor origin (passenger leukocytes) migrating to the recipient lymph nodes represent the driving force behind the direct alloresponse. We surmise that recipient APCs, having captured donor MHC molecules (“cross-dressing”), may activate allospecifi c T-cells recognizing intact donor alloMHC molecules through the “semi-direct” pathway in the recipient lymphoid organs.

Abstract# 77 Poster Board #-Session: P2-IIWithdrawn

Abstract# 78 Poster Board #-Session: P3-IIInfection with the Intracellular Bacterium, Listeria Monocytogenes, Transiently Overrides Established Allograft Tolerance. Tongmin Wang,1 Emily B. Ahmed,1 Luqiu Chen,2 Jing Xu,1 Jing Tao,1 Chyung-Ru Wang,3 Maria-Luisa Alegre,2 Anita S. Chong.1 1Department of Surgery, The University of Chicago, Chicago, IL, USA; 2Department of Medicine, The University of Chicago, Chicago, IL, USA; 3Department of Microbiology/Immunology, Northwestern University, Chicago, IL, USA.Transplantation tolerance has been achieved in experimental models and in limited clinical settings. Infections and TLR signals at the time of transplantation are known to prevent the induction of tolerance, but their effect on established tolerance is less clear. We here report that infection with Listeria monocytogenes abrogated cardiac transplantation tolerance in mice, resulting in T cell-dependent acute rejection. Reversal of established tolerance required both IL-6 and IFNß, and was consistent with their playing non-redundant roles in enhancing T cell proliferation and IFNg production, respectively. Both cytokines were transiently produced during Listeria infection and IFNg-producing cells were detected in the spleen and lymph nodes at 7 but not at 14 days post-Listeria infection. A second donor heart, transplanted 14 days after Listeria inoculation and 7 days after rejection of the fi rst, was spontaneously accepted. The unexpected lack of sensitization following rejection of an established allograft establishes the resilience of the tolerant state, that cytokine perturbations may only transiently override this state, and that interventions directed at Type I IFNs or IL-6 might maintain tolerance under conditions of innate activation. Finally, that tolerance spontaneously returns with resolution of infl ammation or infection might bode well for similar tolerance strategies in the clinic.

Abstract# 79 Poster Board #-Session: P4-IICardiac Allograft Survival Is Prolonged by the Induction of Mixed Hematopoietic Chimerism Two Months after Heart Transplant. Timothy M. Millington,1 Akihiro Aoyama,1 Svjetlan Boskovic,1 James S. Allan,1 Gilles Benichou,1 Tatsuo Kawai,1 Benedict Cosimi,1 David H. Sachs,1 Joren C. Madsen.1 1Department of Surgery, Massachusetts General Hospital, Boston, MA, USA.Immunologic tolerance to renal allografts has been achieved in non-human primates by the induction of transient mixed hematopoietic chimerism at the time of transplant. Although this technique prolongs the survival of cardiac allografts, tolerance is not achieved. We tested whether the recipient of a previously transplanted heart could be made tolerant through mixed chimerism.

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kidney transplantation or modifi ed osteomyocutaneous hindlimb transplantation from ACI rats. Recipients were treated with MMF (15 mg/kg PO) and FK506 (1 mg/kg IP) daily starting 3 days prior to hindlimb transplantation until rejection.Project number Graft suvival (days) Means (days)unsensitized, FK+MMF 5 8, 55*, 55*, 90*, 90* 59.6sensitized, no treatment 4 3, 4, 5, 5 4.3sensitized, FK+MMF 4 3, 4, 6, 6 4.8sensitized, no treatment, kidney Tx 3 15, 20, 25 (minutes) 0* stop treatmentResults: The sensitized recipients of osteomyocutaneous fl aps did not demonstrate hyperacute rejection but slightly accelerated rejection of allografts. Sensitized WF rats treated with immunosuppressants rejected allografts between 3-6 days. Sensitized rats who did not receive immunosuppression rejected CTA allografts between 3-5 days. In striking contrast, renal allografts were rejected immediately upon revascularization in sensitized WF rats.

Conclusions: Presensitization by skin transplant prior to CTA did not result in hyperacute rejection but instead accelerated rejection. While alloantibody may play a role, the mechanism of this rejection is likely mediated by activated memory T cells.

ORAL POSTER EXCHANGE 2 - T CELLS AND T REGS I

Abstract# 82 Poster Board #-Session: P7-IIExpansion of CD4+CD25+FoxP3+ Regulatory T Lymphocytes with Low-Dose Anti-Thymocyte Globulin Leads to Prolonged Survival of Cardiac Allografts but Not Tolerance. Timothy M. Millington,1 Olaf Boenisch,2 Svjetlan Boskovic,1 Nader Najafi an,2 Mohamed Sayegh,2 Joren C. Madsen.1 1Massachusetts General Hospital, Boston, MA, USA; 2Brigham and Women’s Hospital, Boston, MA, USA.Purpose: Studies in liver and kidney transplant recipients suggest that patients successfully withdrawn from immunosuppression exhibit increased Tregs in peripheral blood. The generation and propagation of Tregs in heart transplant recipients may allow development of new strategies for inducing immunologic tolerance. Culture of PBMCs with a low dose of rabbit-derived anti-thymocyte globulin leads to expansion of Treg. We have recently demonstrated in non-human primates that infusion of rATG leads to a similar expansion in vivo. We hypothesized that using rATG to expand Treg in heart transplant recipients would lead to prolonged graft survival.Procedures: Heterotopic heart transplants were performed on fully MHC-mismatched pairs of cynomolgus macaques. rATG (2mg/kg, one tenth the dose used for lymphodepletion) was administered on days 0, 1, 2, 7, 14, 21 and 28. Regimens included no treatment, sirolimus alone, rATG alone and sirolimus plus rATG. Graft status was assessed by manual palpation and confi rmed by biopsy. Numbers of CD4+CD25+FoxP3+ lymphocytes were determined by FACS.Results: Heart transplantation without immunosuppression was associated with rejection within 7 days of transplant. Transplantation with low-dose rATG alone was associated with a modest prolongation of survival (14 days) and rATG/sirolimus combination therapy led to survival of 31-54 days. The combination of low-dose rATG and sirolimus led to an expansion in CD25+FoxP3+ Tregs from 1-2% of CD4+ cells pretreatment to a peak of 5-7% two weeks post-transplant.Conclusion: Treatment of a primate cardiac allograft recipient with low-dose rATG either as monotherapy or in combination with sirolimus led to expansion of Tregs. Although graft survival was prolonged, acute cellular rejection occured within 5-7 weeks of transplantation. A 3-4 fold expansion of Tregs is not suffi cient to achieve tolerance to a cardiac allograft but may be useful in combination with other strategies.

Abstract# 83 Poster Board #-Session: P8-IIEasiness To Generate Is Compensated with Limited Therapeutic Applicability. Arthur Andakyan,1 Xiu-Da Shen,1 Sigrid Burruss,1 Sergei Romanov,1 Natalya V. Semiletova.1 1Surgery, UCLA School of Medicine, Los Angeles, CA.The understanding of chronic rejection (CR) mechanisms is the principal goal of transplantation. In this study we tested cardiac transplant model for CR development in connection with emerging T-regs. Three regimens to generate T-regs were tested. Methods: Transplanted recipients were treated either with 7 days CsA (10 mg/kg) or with 1 mg donor-like MHC class I protein, or with 40 mer peptides derived from donor MHC class I a1-helix of a1 domain. 120 days post-Tx spleens were harvested from recipients with active grafts. Different sub-types of T cells were purifi ed and i.v. injected into new cohorts of lightly irradiated syngeneic recipients. Results: Cardiac recipients treated with total T cells (107) from 7d CsA or MHC protein or peptide groups survived their cardiac transplant >120 d (n=8, 17 & 3, resp.). Hosts transferred with CD4+ (106) cells either from 7d CsA or MHC class I protein reated groups demonstrated graft survival >100 days (n=5 &12, resp.). Control hosts treated with naïve syngeneic CD4+ acutely rejected their grafts (3x12d & 13d; n=4). CD4CD25 cells from MHC protein treated group were effective at dose 3x105 cells per host (n=3). Hosts transferred with syngeneic CD8+ cells from 7d CsA or MHC protein treated groups survived their cardiac grafts indefi nitely (n=5 & 5, resp.). Naïve CD4+ and CD4 from tolerant hosts were i.v. injected into syngeneic cardiac recipients that were undergoing thymectomy 2 weeks before transplantation. All hosts demonstrated delayed type of graft rejection (naive CD4+ , MST = 44+20; n=3 & tolerant CD4+, MST= 30+10; n=3). Histology analysis of long-term surviving cariac grafts after T-regs therapy demonstrated development of TVS and chronic rejection (CR) in all treated groups. Recipients transferred with CD4+ cells obtained from hosts after MHC class I protein therapy had lowest TVS and CR levels. Those grafts had minimal intragraft infi ltration with CD4, CD25 or FoxP3 positive cells but level of infi ltrating CD8+ cells was between low to medium abundance (n=5, grafts analyzed). Conclusion: T-regs ability to transfer specifi c immunosuppression does not depend on the type of therapy that was used to induce tolerance. Syngeneic CD4, CD8 and CD4CD25 as well as total splenic T cells can be used to regulate recipient immune response and protect allogeneic graft from acute rejection but T-regs fail to prevent the development of TVS and CR in long-term surviving grafts and T-regs require functional thymus to be effective.

Abstract# 84 Poster Board #-Session: P9-IIGeneration of Human Tregulatory Cells with the Capacity To Suppress Islet Allograft Rejection in a Xenogeneic Mouse Model. Alicia N. McMurchy,1 Derek L. Dai,2 Jana K. Gillies,1 C. Bruce Verchere,2 Megan K. Levings.1 1Surgery, Vancouver Coastal Health Research Institute and UBC, Vancouver, BC, Canada; 2Pathology and Laboratory Medicine, Child and Family Research Institute and UBC, Vancouver, BC, Canada.The potential use of Tregulatory cells (Tregs) as a cellular therapy to prevent graft rejection has been demonstrated in many mouse models. The major barriers preventing the clinical translation of this approach are limiting cell numbers and a lack of pre-clinical models to evaluate the effi cacy of human Tregs in vivo. We developed a method to generate human Tregs by transducing conventional T cells with lentivirus encoding the transcription factor FOXP3. FOXP3-transduced T cells recapitulate the expected phenotype and in vitro function of ex vivo Treg cells, are a stable and homogeneous population of cells, and can be generated in suffi cient numbers for use as a cellular therapy for transplant patients. The purpose of the present study is to test the in vivo suppressive capacity of FOXP3-transduced T cells in a xenogeneic mouse model of human islet allograft rejection. Clinical grade human islets are transplanted into NOD/SCID mice previously treated with streptozotocin to induce diabetes. Once blood glucose levels have normalized, indicating a functional graft, mice are injected with allogeneic PBMCs to initiate rejection, which is monitored on the basis of blood glucose. The objective is to test the relative ability of co-injected FOXP3-transduced T cells versus ex vivo Tregs to suppress allograft rejection. Initial experiments focused on optimization of methods to expand ex vivo Tregs while preserving FOXP3 expression. We found that sorting naive Tregs (CD4+CD25hiCD45RA+) resulted in the most homogeneous population of FOXP3+ cells. To obtain suffi cient Tregs for injection into mice, different expansion conditions were tested including different media and stimuli. Over 100-fold expansion of sorted Tregs could be achieved after 14 days with 90% of cells remaining FOXP3+ when cells were stimulated with artifi cial antigen presenting cells in OpTmizer T cell Expansion Medium in the presence of 100ng/mL rapamycin for the fi rst 7 days. Ongoing experiments aim to determine the optimal ratio of expanded ex vivo Tregs:PBMCs necessary to prevent allograft rejection so that we can evaluate the relative effi cacy of FOXP3-transduced T cells. Evidence that human Tregs can prevent rejection of human islets will provide a strong rationale for clinical translation of cellular therapy to prevent allograft rejection.

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Abstracts

Abstract# 85 Poster Board #-Session: P10-IIEffect of FTY720 Treatment on CD4+ T Cell-Endothelial Cell Interactions in Ischemia/Reperfusion Injury of the Pancreas. Dirk Uhlmann,1 Andrea Prescher,1 Christina Mohry,1 Matthias Martin.1 12nd Department of Surgery, University of Leipzig, Leipzig, Germany.Aim: CD4+ T cells contribute to disturbances of pancreatic microcirculation after cold and even after warm ischemia/reperfusion (I/R). The aim of this study was to investigate a possible protective role of FTY720 in this setting.Material and Methods: In an in vivo model (42 Wistar rats), ischemia of the pancreas was induced for 60 min under anesthesia with xylazin/ketanest. Sham operated (s.o.) (I), untreated ischemic (II) and treatment (III) group with FTY720 (1 mg/kg bw i.v.) were investigated. The effect of FTY on I/R injury was assessed by in vivo microscopy 30-90 min after reperfusion (capillary diameter, functional capillary density, leukocyte-endothelium interactions, lymphocyte-endothelium interactions), by measurement of lipase/amylase, RT-PCR of IL-2, IL-6, IL-10, TNF-α, toll-like-receptor 4 (TLR-4), and by histological investigation.Results: In the untreated ischemic group (II), capillary constriction to 79.3 ± 6.3 % of s.o. diameters and a reduction of functional capillary density to 58.6 ± 8.7 % of the s.o. group (356.8 ± 8.3 1/cm) was measured after reperfusion. After 30 min of reperfusion, the number of T cells in capillaries was increased (203.1 ± 16.4 % of s.o., p< 0.05 vs I). FTY treatment reduced this number to 124.1 ± 8.9 % of s.o. (p<0.05 vs II). Likewise, the number of adherent leukocytes in capillaries (145.4 ± 11.2 % of s.o.) was reduced in group III (109.3 ± 11.4 %; p<0.05 vs II), leading to an improvement in functional capillary density in the treatment group (79.3 ± 8.5 % of s.o.; p<0.05 vs II). In the treatment group, vascular constriction was attenuated (88.6 ± 5.8 % of s.o.). According to improved microcirculation, lipase/amylase values and histological tissue damage were reduced in the therapy group (p<0.05). RT-PCR revealed an increased expression of IL-2, TNF-α and TLR-4 in the non-treated ischemic group. This expression was clearly reduced in the treatment group (p<0.05). However, an elevated expression of IL-6 and IL-10 mRNA was found in this group.Conclusion: In conclusion, FTY720 ameliorates the microcirculatory, biochemical and histological manifestations of pancreatic I/R injury by preventing T cell infi ltration. These results indicate that T cells are pivotal mediators in the I/R of the pancreas and may have important implications in pancreas transplantation.


Abstract# 87 Poster Board #-Session: P12-IIEx-Vivo Expanded CD4+CD25+ Treg Cells Suppress T and B Immune Response. Avneesh K. Singh, Caleb N. Seavey, Keith A. Horvath, Muhammad M. Mohiuddin. Cardiothoracic Surgery Research Program, NHLBI, NIH, Bethesda, MD, USA.The CD4+CD25+FoxP3+ regulatory T (Treg) cells play an important role in regulating immune response. A very small number of Treg cells are present in peripheral blood and lymphoid organs and have a high potential for immunotherapy in clinics. Due to their low number, these cells have been expanded in-vitro by different methods using IL-2, TCR and co-stimulation. We have purifi ed and successfully expanded naturally occurring baboon CD4+CD25+ Treg (nTregs) cells that exhibit similar characteristics to the natural occurring Tregs and demonstrated that ex-vivo expanded baboon Tregs (iTregs) suppress anti-porcine xenogeneic immune responses effectively. We have also expanded and enriched CD4+CD25+FoxP3+ Treg from CD4+ T cells of baboon in the presence of rapamycin (0.1-10nM) using irradiated pig PBMCs and IL-2. CD4+CD25+FoxP3+ iTreg cells were increased up to two times in presence of rapamycin as compared without rapamycin in cultures. Purifi ed CD4+CD25+ Treg cells enriched from CD4+ cells in presence of rapamycin were able to suppress the baboon anti-porcine xenogeneic immune responses in vitro up to 90% at 1:1 ratio (T regulatory Cells: T effector cells) and suppression ability exists even at 1:256 ratio whereas freshly isolated natural Treg cells suppress only 70% at 1:1 and loose their suppressive ability (>50%) at 1:16. To investigate the other function of iTreg cells we have co cultured the ex-vivo expanded CD4+CD25+ iTreg cells with B cell in the presence of polyclonal B-cell activators. We found that the proliferation of B cells was suppressed. This proliferation was reduced to only 40-50% when ex-vivo expanded CD4+CD25Hi iTreg cells were added to the culture at a 1:1 ratio whereas, the addition of CD4+CD25neg cells induced vigorous proliferation. Further, mechanism for the inhibition of B cell proliferation is being investigated in our laboratory. These results suggest that the ex-vivo expanded CD4+CD25+ iTreg cells can be used to effi ciently inhibit T and B cells proliferation and may be the hyperacute xenograft rejection.

ORAL POSTER EXCHANGE 3 - T CELLS AND T REGS II

Abstract# 88 Poster Board #-Session: P13-IIBoth Rejection and Tolerance of Allogeneic Transplants Can Occur in the Absence of Secondary Lymphoid Organs. Cavit D. Kant,1 Katsunori Tanaka,1 Yoshinobu Akiyama,1 Yoshiko Iwamoto,1 Susan Shea,1 Sarah E. Connolly,1 Gilles Benichou.1 1Surgery, M, Boston, MA, USA.We investigated the roles of secondary lymphoid organs in the alloantigen recognition and alloresponse and allograft rejection by T cells in skin- and heart-transplanted mice. Aly/Aly mice which are lacking lymph nodes and peyer patches rejected skin and heart fully allogeneic BALB/c transplants acutely (10 ± 2 days). However, unlike normal wt B6 mice, they failed to mount a Th1 indirect alloresponse after skin allografting. Aly/Aly mice which had been splenectomized (Aly/Aly SPL-) also rejected BALB/c skin grafts acutely, although in a slightly delayed fashion (14 ± 3 days), but accepted permanently BALB/c cardiac allotransplants. Aly/Aly SPL- which had accepted a BALB/c heart subsequently accepted a BALB/c skin allograft but not a third-party C3H skin allograft. Interestingly, while these mice had clearly developed donor-specifi c tolerance, they developed chronic rejection (CAV) detectable 50 days post-cardiac transplantation. In contrast, the placement of BALB/c skin grafts in Aly/Aly SPL- mice either prior to or at the time of heart transplantation resulted in the development of a memory T cell alloresponse and the acute rejection of both skin and heart allografts. Finally, we observed that thymectomized Aly/Aly SPL- mice failed to develop tolerance in the same model. Therefore, both rejection and tolerance can be acheived in mice lacking secondary lymphoid organs. We have accumulated some preliminary data showing that T cell priming in Aly/Aly SPL- rejecting skin allografts can occur in the bone marrow. It is also possible that T cells may get sensitized in the skin graft itself. The mechanisms underlying donor-specifi c tolerance induction in these mice are being explored.

Abstract# 89 Poster Board #-Session: P14-IISuppressing Antigen-Bearing Dendritic Cells by Double Negative Regulatory T Cells. Julia Fang Gao,1,2 Megan S. Ford McIntyre,2 Alexandra Kowalczyk,2 Ramesh B. Vanama,2 Stephen C. Juvet,2 Jun Diao,2 Bardia M. Naeini,2 Mark Cattral,2 Li Zhang.1,2 1Department of Immunology, University of Toronto, Toronto, ON, Canada; 2Multi-Organ Transplantation Program, Toronto General Research Institute, University Health Network, Toronto, ON, Canada.Regulatory T cells (Tregs) play an important role in controlling the development of a variety of immune pathologies. As a subset of Tregs, peripheral αβ-TCR+CD3+CD4-

CD8-NK1.1- double negative (DN) Tregs, found in both mice and humans, comprise 1-3% of peripheral T lymphocytes. We and others have demonstrated that DN Tregs can suppress syngeneic CD8+ and CD4+ T cells in various disease models. However, whether DN Tregs can exert regulatory effects directly on dendritic cells (DCs) remains unknown. To investigate whether DN Tregs are able to suppress syngeneic and/or allogeneic mature and immature DCs, we used a single MHC class I locus (Ld) mismatched transgenic mouse model. Activated 2C×dm2 F1 (Ld-) DN Tregs were cocultured with syngeneic B6×dm2 F1 (Ld-) or allogeneic B6 ×BALB/c F1 (Ld+) bone marrow derived DCs. We demonstrate that DN Tregs can kill alloantigen-expressing DCs in an antigen-specifi c manner, mainly through the Fas-FasL pathway. Furthermore, DN Tregs were also able to downregulate costimulatory molecules expressed on mature DCs. Mature DCs coincubated with DN Tregs were impaired in their ability to stimulate CD8+ T cell proliferation, and cytotoxic lymphocyte antigen 4 (CTLA-4) is involved in DN Treg-mediated suppression of mature DCs. In conclusion, these data demonstrate that DN Tregs are not only able to kill activated antigen-specifi c T effector cells, but are also able to prevent naïve donor-specifi c T cell activation by suppressing both the direct and indirect alloantigen presentation pathways. Thus, DN Tregs have a great potential to be developed as a novel treatment for graft rejection.

Abstract# 90 Poster Board #-Session: P15-IIMechanisms of Immunological Tolerance Induced by Double Negative Regulatory T Cells in a Semiallogeneic Model of Graft-Versus-Host Disease. Stephen C. Juvet,1,2,5 Mei Han,4,5 Edward Y. Kim,3,5 Oyedele Adeyi,4,5 Ramesh Vanama,4,5 Li Zhang.1,3,4,5 1Institute of Medical Science, University of Toronto; 2Clinician-Scientist Training Program and Division of Respirology, Department of Medicine, ; 3Dept. of Immunology, ; 4Dept. of Laboratory Medicine and Pathobiology, ; 5Toronto General Hospital Research Institute, University Health Network, University of Toronto, Toronto, ON, Canada.Double negative regulatory T cells (DN Tregs) express the αβ TCR but lack CD4, CD8, and NK cell markers. They suppress allo- and xenograft rejection in mice, and DN Treg cell number is inversely correlated with graft-versus-host disease (GVHD) severity in patients after bone marrow transplantation (BMT), but the mechanisms involved therein are incompletely understood. To explore how DN Tregs inhibit

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alloreactive CD4+ T cells during GVHD, we developed a semiallogeneic BMT model in which lethally irradiated C57BL/6×BALB/c F1 (CB6F1, H-2b/d) mice were reconstituted with C57BL/6 (B6, H-2b) T cell depleted bone marrow. They also received either B6.Thy1.1+ CD4+ T cells (H-2b) (BM+CD4), B6.Thy1.1+ CD4+ T cells and DN Tregs from B6.lymphoproliferative (B6.Faslpr) mice (H-2b) (BM+CD4+DN), or no additional treatment (BM only). We found that DN Tregs produced IFNγ, reduced CD4+ T cell expansion and infi ltration of GVHD target organs, and prolonged survival. In contrast, Fas ligand-defi cient DN Tregs from B6.FasLgld mice did not suppress GVHD.

Suppression of CD4+ T cell proliferation by DN Tregs in vitro depended on IFNγ. Interestingly the target of IFNγ was not CD4+ T cells since IFNγ receptor defi cient (IFNγR-/-) CD4+ T cells were also suppressed. Instead, IFNγ may act on DN Tregs since suppression of IFNγR-/- CD4+ T cells was also reduced when IFNγ was neutralized. These data demonstrate that DN Tregs suppress CD4+ T cell-mediated GVHD in a semiallogeneic model of BMT, and both FasL and IFNγ are involved in suppression of alloreactive CD4+ T cells. They also shed light on our understanding of DN Treg function and may contribute to the development of novel therapies for GVHD and graft rejection.

Abstract# 91 Poster Board #-Session: P16-IISynergistic Effect of CsA and Tregs in Preventing Memory T Cell Induced Tolerance Abrogation. Anja Siepert,1 Birgit Sawitzki,2 Holger M. Reichardt,3 Jens van den Brandt,3 Horst Nizze,4 Markus Tiedge,1 Manfred Lehmann,1 Hans D. Volk,2 Petra Reinke.5 1Inst.of Med.Biochem.and Mol.Biology, Univ.of Rostock, Rostock, Germany; 2Inst.of Med.Immunol., Charité Univ.Medicine Berlin, Berlin, Germany; 3Cell.and Mol.Immunol., Univ. of Göttingen, Göttingen, Germany; 4Inst.of Pathol., Univ. of Rostock, Rostock, Germany; 5Med.Clinic of Nephrol.and Internal Medicine, Charité Univ.Medicine Berlin, Berlin, Germany.INTRODUCTION: Transfer of regulatory T cells (Tregs) is increasingly recognized for clinical tolerance induction in transplant recipients. Preexisting donor-reactive memory T cells (Tmem) are known to be a barrier for adoptive transfer of Tregs. To prevent reactivation of Tmem and to avoid graft rejection before tolerance is established, conventional immunosuppressive drugs are given in conjunction with Tregs early pTx. It is therefore essential that these drugs do not block Treg generation. Our aim was to develop a transplant model which simulates the clinical situation of an increased Tmem pool and Treg function.METHODS: LEW rats received GFP+ transgeneic DA-specifi c Tmem 7 or 100 days before transplantation. The LEW rats of DA renal allografts were T-cell depleted and treated either with short term or permanent low dose CsA. A third group received short term CsA therapy plus Tregs at day 3 pTx. Serum creatinine and survival time were used as read-out parameters.RESULTS: T cell depletion resulted in long term survival (MST >150d). Additional application of Tmem 7 or even 100 days before Tx resulted in allograft rejection (MST 13.7±14.0d vs 51.5±55.4d) and was associated with increased frequency of Tmem escaping T cell depletion and a dramatic B cell infi ltration (161.0±8.3 vs 15.8±8.1). Short term CsA application led to delayed rejection with MST of 51.3±24.8d. Therapy of Tregs plus short term CsA resulted in 79% long-term survival within the group with decreased frequency of Tmem starting at day 5 pTx. Although permanent CsA therapy induced 100% long-term survival, the treatment could not reduce Tmemaccumulation (0.25±0.28 % vs 0.07±0.04%).CONCLUSION: Our data show that Tmem even when aquired long term prior to transplantation escape T cell depletion leading to increased B cell infi ltration and tolerance abrogation. Only T cell depletion combined with Tregs and short term CsA therapy controls Tmem accumulation. Our model represents an attractive approach to avoid lifelong immunosuppressive therapy in pre-sensitized transplant recipients.*Supported by Else Kröner Fresenius Foundation.

Abstract# 92 Poster Board #-Session: P17-IICD200 Immunomodulation Increases Tregs and Prolongs CTA Survival. Rishi Jindal,1 R. Sucher,1 Y. Wang,1 D. Zhang,1 J. V. Unadkat,1 B. J. Pulikkottil,1 R. Zanoun,1 G. Brandacher,1 X. X. Zheng,1 W. P. A. Lee,1 V. S. Gorantla.1 1Plastic Surgery, University of Pittsburgh, Pittsburgh, PA, USA.Donor antigen-specifi c tolerance without the need for long-term immunosuppression remains the goal in composite tissue allotransplantation (CTA). Regulation of immune responses through immunomodulation rather than immunosuppression

may be the key to tolerance. CD200 fusion protein (CD200) is a novel agent shown to enhance regulatory T-cell (Treg) proliferation and thereby counteracts anti-tolerogenic effects of cyclosporine-A (CsA). We hypothesize that Treg upregulation by CD200 will create a tolerogenic cellular and cytokine milieu that will prolong graft survival under a short-term immunosuppression regimen. Lewis rats received hind-limb transplants from Brown-Norways (groups in Figure 1). Endpoints were graft survival >150 days or limb necrosis.

Mean graft survival for groups 1, 2, and 3 were 39.8±2.8, 35.8±4.0, and 43.3±4.2 days. In group 4, 4 recipients rejected grafts by day 43.5±4.7, while 4 went on to long-term survival (p<0.05; ongoing). FACS at day 10 showed increased CD4+CD25+FoxP3+ Tregs in CD200 animals vs. control (p<0.05). By day 21, CD200-stimulated Tregs had decreased, though animals that would eventually go on to long-term survival had signifi cantly more Tregs than rejecting CD200 animals or controls.

At day 100, H&E showed minimal infi ltrate in long-term survivors’ grafts, and at day 150 they received same-donor and 3rd party skin grafts. 3rd party rejected while same-donor skin survived >30 days (p<0.05). We have shown that short-course CD200 therapy with ALS+CsA prolongs allograft survival in a highly immunogenic transplant model without long-term immunosuppression. Increased Tregs and polarization towards pro-regulatory milieu are likely responsible for this prolongation. Further studies are ongoing to address the difference in CD200 recipients’ outcomes.

Abstract# 93 Poster Board #-Session: P18-IIA Novel Strategy and Unique Model for Composite Tissue Allotransplantation Tolerance. Rishi Jindal,1 J. V. Unadkat,1 D. Zhang,1 T. Ng,1 Y. Wang,1 B. J. Pulikkottil,1 R. Zanoun,1 R. Sucher,1 P. N. Afrooz,1 W. P. A. Lee,1 X. X. Zheng.1 1Plastic Surgery, University of Pittsburgh, Pittsburgh, PA, USA.Purpose: Recently, we developed a novel strategy to induce composite tissue allotransplantation (CTA) survival and tolerance that combines short-course clinical immunosuppression, consisting of anti-lymphocyte serum and cyclosporine A, with an immunomodulatory agent, interleukin-2 fusion protein (IL-2/Fc, a long-lasting form of IL-2). As CsA blocks IL-2 transcription and Tregs require it, adding IL-2/Fc will enhance Tregs and consequent CTA survival.

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Abstracts

Methods: Hind-limb transplant was performed in BN to Lew rats.Group Treatment Regimen Size (n)Control Untreated 41 ALS 42 CsA 53 ALS+CsA 64 CsA+IL-2/Fc 45 ALS+CsA+IL-2/Fc 11Table 1. Experimental groups and treatment protocols. ALS=Anti-lymphocyte serum, 0.5mL ip day -4 & +1; CsA=CyclosporineA, 10mg/kg ip x21days; IL-2/Fc=Interleukin-2 fusion protein, 15µg/kg ip x21daysBlood, lymph nodes, skin and muscle were harvested at various points during and after treatment. T-cells (CD3,CD4,CD8,CD45R,FoxP3) were analyzed by FACS. Ifn-γ,Prf1,GzmB,FoxP3&IL-2 expression were measured by PCR.Results: ALS+CsA+IL-2/Fc induced long-term graft survival (>150days) in 6/11 animals. Interestingly, all long-term survivors rejected at day 46.7+3.8, then spontaneously recovered with no intervention; therefore, we have established a CTA model with two types of rejection, benign vs progressive. FoxP3 to Prf1, GzmB, or Ifn-γ ratio in skin biopsied at fi rst signs of acute rejection (day 41.9+4.2) was 2-5x higher in IL-2/Fc-treated benign (6/11 animals) vs. progressive rejectors (5/11), indicating biopsies correlated with outcome (p<0.05).

In 4 long-term survivors, we tested tolerance with donor vascularized osteocutaneous grafts. Preliminary results indicate 1 rat accepted the graft (robust tolerance), 2 rejected skin only (split tolerance), and 1 rejected both grafts (ignorance). Further transplants are ongoing.Conclusions: This model provides a unique opportunity to study immunological mechanisms of rejection and tolerance, and to develop individualized diagnostic and therapeutic protocols to predict, prevent rejection, and induce CTA tolerance.

ORAL POSTER EXCHANGE 4 - EXPERIMENTAL TOLERANCE,

INNATE IMMUNITY, TOLERANCE PROTOCOLS

Abstract# 94 Poster Board #-Session: P19-IIStudies on Some Immune Properties of the Pancreatic Progenitor Cells Derived from Human Fetal Pancreas. Man Ting Ma,1 Po Sing Leung.1 1School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China.The potential use of human fetal pancreas as an alternative source for transplantable beta-cells to treating type 1 diabetes lies in its immune privilege as well as high capacity to proliferate and differentiate. This notion has been substantiated by a recent study showing a reduced immunogenicity of fi rst-trimester human fetal tissue. Meanwhile, we have recently reported the isolation, culture and characterization of a population of pancreatic progenitor cells (PPCs) derived from human fetal pancreas that is amenable to growth and differentiation into insulin-producing islet-like cell clusters (ICCs); however, the immunogenicity of these cells has yet to be characterized. Thus, we hypothesized that the PPCs and ICCs derived from fi rst-trimester possess lower immunogenicity than that from second-trimester of human fetal pancreas and we tested this hypothesis by characterizing and comparing the immune properties of these cells. Firstly, the expression pattern of selected immune-related genes was examined by real-time PCR. Expression of some of the major histocompatibility complex class I (MHC-I) molecules upon stimulation of interferon-gamma (IFN-γ) was further examined by fl ow cytometry. Results showed positive gene expression of a selection of immune-related genes including MHC molecules, complement components (C3), apoptotic markers (TNFSF10), chemokine ligands (CCL19), co-stimulatory molecules (CD80) and innate molecules (B7H4). Another co-stimulatory molecule CD86 was found to be negative in both PPCs and ICCs. Upon stimulation of IFN-γ, MHC-I expression on both PPCs and ICCs were upregulated in a dose-dependent manner. Comparison of these

immunological parameters in PPCs from different trimesters was also undertaken. Differential gene expression of MHC-I and MHC-II were observed in PPCs from 9 to 15 gestation weeks, being with an enhanced expression from second trimester. Expression of the immune-related genes was signifi cantly elevated in PPCs from second trimester, indicating an immuno-privilege of PPCs from fi rst trimester. In addition, we also confi rmed a higher inducibility of MHC-I expression in PPC and ICCs from second-trimester by IFN-γ. These data conclude that PPCs of fi rst-trimester has immune privilege over second-trimester derived from human fetal pancreas, thus indicating its potential use as a source of transplantable islets with enhanced survival of islet graft.

Abstract# 95 Poster Board #-Session: P20-IIHMGB1 Regulates Tubular Epithelial Cell (TEC) Expression of MCP-1 in Ischemic Kidney Injury. Arthur Lau,1 Zhu-Xu Zhang,1,2,3,4 Shuang Wang,2,4 Anthony M. Jevnikar.2,3,4 1Pathology, Univerity of Western Ontario, London, ON, Canada; 2Multi-Organ Transplant Program, London Health Sciences Centre, London, ON, Canada; 3Medicine, University of Western Ontario, London, ON, Canada; 4London Health Research Institute, London, ON, Canada.Ischemia reperfusion injury (IRI) occurs in all kidney transplants as a consequence of donor organ procurement and contributes to graft dysfunction and rejection in recipients. The initial ischemic insult induces widespread necro-apoptotic death of kidney parenchymal cells such as TEC, which results in organ dysfunction and the release of damage associated molecular pattern (DAMP) proteins into the extracellular space. High Mobility Group Box-1 (HMGB1) and other DAMP proteins may further contribute to infl ammatory kidney injury. However their effect on survival or pro-infl ammatory function of TEC remains unknown. We studied the expression and effect of HMGB1 in kidney ischemic injury. Uni-nephrectomized C57BL/6J mice were subjected to clamping of the remaining renal pedicle for 45 min to induce IRI. In vitro, the effect of hypoxia was tested in TEC (NG1.1) subjected to 5-10% oxygen for 20 min. In vivo, HMGB1 protein gradually increased in ischemic kidney samples over 24 hours. TLR4, a major receptor for HMGB1, had increased levels of mRNA at 24 hours. In contrast, HMGB1 mRNA did not increase, suggesting the increase in protein was post transcriptionally regulated or released from necrotic cells. Hypoxia induced TEC expression of the chemokine MCP-1 at 24 h. The addition of glycyrrhizic acid (GZA), a functional inhibitor of HMGB1, to hypoxic NG1.1 TEC prevented this increase in a dose dependent manner, to levels observed in naive TEC. This was observed in TEC without an effect on cell death. We have shown that MCP-1 has a chemotactic effect on NK cells, an important effector cell in IRI. In conclusion, HMGB1 is released following hypoxic kidney injury but importantly may have a previously unrecognized effect in promoting renal infl ammation by increased TEC expression of MCP-1. In addition to a potential role as an early renal injury biomarker, HMGB1 inhibitors may provide novel therapeutic strategies to attenuate IRI in kidney transplants.

Abstract# 96 Poster Board #-Session: P21-IISerum Anti-MHC Immunoglobulins Ineffective in Prevention of Acute Graft Rejection. Arthur Andakyan,1 Sigrid Burruss,1 Sergei Romanov,1 Xiu-Da Shen,1 Natalya Semiletova.1 1Surgery, UCLA School of Medicine, Los Angeles, CA.MHC class I therapy with short course of CsA prolonged cardiac graft survival and averted development of chronic rejection. Emergence of protective donor allo-Abs was tested in this study. Methods: One ml of whole serum or IgG fraction (0.1-1 mg/rat) from long-term (120d) surviving ACI recipients of WF hearts were i.v. injected into ACI rats at the day of cardiac transplantation. Results: Cardiac recipients treated with syngeneic serum that has high-titer of allo-IgG demonstrated 40% rate of graft survival beyond 100 days ( 2x12 d, 2x15 d, 43 d, 55 d, 4x110 d; n=10) unlike hosts that were administered naïve serum (2x26 d, 34 d; n=3). Allo-Abs high-titer serum failed to prolong graft life in syngeneic recipients transplanted with third-party cardiac grafts (BN-ACI, MST=11+1, n=3 vs WF-ACI, MST 59+45, n=10). Immunohistology of long-term surviving grafts revealed IgG deposits on graft vessels. ELISA analysis demonstrated that total IgG1 and IgG2c levels in the serum with high-titers of allo-Abs was similar to control, naïve serum. In contrary, titers for allo-IgG showed statistically signifi cant increase for IgG1 and IgG2c sub-types. Allo-IgG high-titer serum directly inhibited metabolic activity of IFN-g activated donor WF ECs in RNA turnover assay and signifi cantly blocked recipient T cell preliferative responses toward donor antigen stimulation in MLR test. ELISA for IgG subtypes binding to resting or IFN-g activated ECs was performed with 1:4, 1:8 & 1:16 dilutions. Marked increase in IgG2c binding to activated donor ECs was detected through several serial dilutions. IgG fractions were prepared with Melon Gel IgG spin purifi cation system and purity assessed on SDS-PAGE gel (>95%). ACI recipient were i.v. injected with 0.1 mg or 1 mg of pure IgG fractions in PBS at day of cardiac transplantation. Control groups were treated with IgG purifi ed from naïve syngeneic serum. All transplants were acutely rejected in both groups (MST=13+1.5 & 12+2 d, n=4 & 4). Hosts treated with IgG fractions purifi ed from serum with high-titer of allo-IgG failed to prolong graft survival time and all recipients rejected their grafts in acute manner (MST=12.5+2 & 13.5+1.2; n=6 & 4). Conclusion: Serum IgG fractions enriched with anti-MHC Abs can not prevent allograft rejection in the

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model of heterotopic cardiac transplantation. However, therapy with the serum that has high-titer of allo-Abs clearly demonstrated 40% rate of graft survival.


Abstract# 98 Poster Board #-Session: P23-IILentiviral Transduction of Face and Hand Allografts: Implications for Immunomodulation of Composite Tissue. Angelo A. Leto Barone,1,2,3 Zhao Y. Zhou,1 Ruth M. Schulman,1 Steven Lee,1 Francesco Moschella,3 Erin L. Weber,1 Curtis L. Cetrulo.1,2 1Laboratory for Stem Cell-Based Microsurgical Tissue Engineering, Division of Plastic and Reconstructive Surgery, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA, USA; 2Division of Plastic and Reconstructive Surgery, Harvard Medical School, Boston, MA, USA; 3Dipartimento di Discipline Chirurgiche ed Oncologiche, Sezione di Chirurgia Plastica e Ricostruttiva, Universita’ degli Studi di Palermo, Palermo, PA, Italy.INTRODUCTION: Ex vivo introduction of an immunomodulatory transgene into a face or hand allograft may improve the risk/benefi t ratio in composite tissue allograft (CTA) transplantation. In particular, abrogation of the immunogenicity of the skin component of a face or hand allograft may decrease the risk of rejection and/or permitting immunologic tolerance. Proof-of-principle demonstrations of transduction of composite tissue have been established using adenoviral vectors, producing only transient gene expression. We hypothesized that transduction, integration, and long-term expression of a transgene in a CTA could be achieved using lentiviral vectors, which permanently integrate into the genome.METHODS: Ex vivo transduction of heterogeneous primary rat cell lines representative of a composite tissue fl aps cellular architecture (keratinocytes, dermal fi broblasts and endothelial cells) was performed using a luc-eGFP HIV-1-based lentiviral vector. In addition, we investigated ex vivo intravascular vs. intradermal injection of free rat SIEA fl aps.RESULTS: Quantifiable reporter expression by FACS analysis and in vitro bioluminescence was observed. The luc-eGFP vector exhibits broad tropism, integrates into the genome, and allows permanent, constitutive transgene expression in relevant cell lines, as well as throughout the SIEA fl ap through ex vivo intradermal transfection, resulting in long-term (> 6 mo), constitutive transgene expression and genomic integration (as demonstrated by RT-PCR). Similarly, effi cient intradermal transfection of face and hand fl aps in a rat model corroborated this approach. Ex vivo intravascular perfusion of the vector proved inferior to intradermal injection.CONCLUSIONS: Intradermal delivery of the transgenes proved superior to intravascular perfusion. Optimization of this gene-delivery approach may allow long-term, constitutive expression of immunomodulatory proteins in face and hand allografts. Future goals include replacement of the luciferase and eGFP reporter genes with key immunomodulatory proteins.

Abstract# 99 Poster Board #-Session: P24-IIAllogeneic Porcine CD4+CD25+ T Cells Regulate the Development of Transplant Arteriosclerosis in Porcinized Mice. Gregor Warnecke,1 Nodir Madrahimov,1 Knoefel Ann-Kathrin,1 Avsar Murat,1 Dreckmann Karla,1 Thissen Stefanie,1 Kruse Bianca,1 Haverich Axel,1 Strueber Martin.1 1Division of Thoracic and Cardiovascular Surgery, Hannover Medical School, Hannover, Germany.Introduction: We have shown previously that long term graft acceptance correlated with the frequency of CD4+CD25+ regulatory T cells in a porcine allogeneic lung transplantation model. However, it is unknown, if this type of T cell regulation is the cause for or merely an epiphenomenon of long term allograft survival. Thus, we implanted porcine arteries and adoptively transferred porcine PBMC into NODrag-/-gammac-/- mice to study T cell regulation in vivo.Methods:Porcine intercostal arteries were implanted into the murine abdominal aorta. Animals received either 10x106 autologous (Group A), or allogeneic PBMC (Group B, graft and cells were obtained from two MHC mismatched pigs), group C received allogeneic PBMC depleted of CD4+CD25+ cells, whereas group D recipients received allogeneic PBMC augmented with additional CD4+CD25+ T cells. Porcine CD45+ cell engraftment was monitored by FACS postoperatively. Transplant arteriosclerosis was histologically assessed on postoperative day (POD) 28. Quantifi cation was done by measuring intima to media ratio.Results: All groups showed good porcine cell engraftment as monitored by FACS on POD 14 and 28. Transplant arteriosclerosis was different among groups (p<0.05) and severely pronounced in group C. Mean intima to media ratio was higher (p<0,05) in group C as compared to groups A and B, respectively (80.3+/-11.6% vs 23.8+/-5.2% and 47.7+/-13.6%). Addition of CD4+CD25+ T cells in group D resulted in a marked reduction of the mean intima to media ratio towards the level of the control group A (23.0+/-7.0%).

Conclusion: Firstly, regulatory T cells control the development of transplant arteriosclerosis in vivo. Secondly, our model enables the functional study of allogeneic T cell regulation in large animals and humans.

ORAL POSTER EXCHANGE 5 - TOLERANCE/IMMUNOLOGICAL MONITORING,

AND BIOMARKERS

Abstract# 100 Poster Board #-Session: P1-IIIEstablishment of Local Tolerance for Cell Transplants, toward Clinical Application. Lina Lu,1,2 Ching-Chuan Hsieh,1 Hong-Shiue Chou,1 John J. Fung,2 Shiguang Qian.1,2 1Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA; 2General Surgery, Transplantation Center, Cleveland Clinic, Cleveland, OH, USA.Organ transplantation has been applied for decades, but outcomes for cell transplants remain disappointing. This is true in animal models. Liver allograft transplants in mice are spontaneously accepted, but hepatocyte transplants are acutely rejected, suggesting a crucial role of non-parenchymal cells in immune regulation. We have demonstrated that hepatic stellate cells (HpSC), liver stromal cells, have potent immunosuppressive activity, and co-transplantation with HpSC achieved long-term survival in 66% of islet allografts without requirement of immunosuppression via induction of local immune hyporesponsiveness. This has great potential in clinical application. However, a hurdle is that the co-transplanted HpSC have to be syngeneic to patient himself, since allogeneic HpSC are not ptotective. Obviously, obtaining suffi cient HpSC from the liver adds risk to patients. To test HpSC being replaced by other stromal cells, BM-derived stromal cells, mesenchymal stem cells (MSC), were co-transplanted, showing very mild effect. None islet grafts survived >28d, indicating that only specifi c stromal cells, such as HpSC, possess potent immuosuppressive activity. We demonstrated, in this study, that HpSC were potent inducers of myeloid-derived suppressor cells (MDSC). Co-transplantation with HpSC mainly induced CD11b+CD11c- infi ltrating mononuceocytes (89%), while 90% being CD11b+CD11c+ in islet alone grafts. Addition of HpSC into the culture of bone marrow precursors at a 1:80 ratio in the presence of GM-CSF effectively shifted generation of CD11b+CD11c-, instead of CD11c+cells. They shared many properties with MDSC (immature phenotype, resistance to maturation, powerful T cell inhibition). Co-transplantation with HpSC-induced MDSC effectively protect islet allografts from rejection similar to co-transplantation with HpSC. Induction of MDSC by HpSC was mediated by soluble factor (s) by using transwell plate culture, and confi rmed by use of HpSC culture supernatant. Identifi cation of responsible soluble factor (s) has been narrowed down to complement component 3 (C3). HpSC have shown extraordinary capacity of binding complement Factor H (FH) which facilitates generation of inactive C3b (iC3b). iC3b is a ligand for CD11b. Addition of iC3b leads to generation of MDSC from BM precursors.Induction of local tolerance by MDSC will facilitate cell transplantation in clinic.

Abstract# 101 Poster Board #-Session: P2-IIIHigh Levels of IDO-Expressing CD16+ Peripheral Cells, and Tregs in Graft Biopsies from Kidney Transplant Recipients under Belatacept Treatment. Janette Furuzawa-Carballeda,1 Guadalupe Lima,1 Norma Uribe,2 Carmen Avila-Casado,3 Eduardo Mancilla,4 Luis E. Morales-Buenrostro,5 Jesús Pérez-Garrido,6 María Pérez,4 Guillermo Cárdenas,6 Luis Llorente,1 Josefi na Alberú.6 1Immunology and Rheumatology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico, DF, Mexico; 2Pathology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico, DF, Mexico; 3Pathology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico, DF, Mexico; 4Nephrology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico, DF, Mexico; 5Nephrology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico, DF, Mexico; 6Transplants, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico, DF, Mexico.Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme which suppresses T lymphocyte activity. Costimulation blockade through CTLA4Ig increase IDO in antigen presenting cells. The suppressive effect of IDO is mediated by Foxp3+CD4+CD25+ regulatory T cells (Tregs).The percentage of IDO-expressing peripheral cell subpopulations as well as Tregs, was evaluated in 27 stable kidney transplant recipients (KTR) receiving either Belatacept (LEA29Y) (n=19) or Cyclosporine (n=9). Blood samples were obtained at 23±6 months (Belatacept) and 24±2 months (Cyclosporine) post-KT. Intracellular IDO was analyzed by fl ow cytometry in CD14+, CD11c+, CD16+, CD56+ and CD8+ cell subpopulations. Tregs were assessed by intracellular Foxp3 detection. CD3+, CD4+, CD8+, CD20+, CD68+, IDO+, and Foxp3+ cells were evaluated by immunohistochemistry on graft biopsies obtained at pre-implantation, at 12 months post-KT, and in those with acute rejection/dysfunction (R/D) during the fi rst 12 months.

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Abstracts

Percentages of IDO-expressing peripheral cells were comparable in both groups except for a subpopulation of CD16+ monocytes which was signifi cantly increased in the group receiving Belatacept (47±25% vs. 4.6±2.0%, P = 0.009). No differences were found in peripheral Tregs between groups. In contrast, higher percentages of Tregs were observed in R/D and 12 months graft biopsies compared with baseline in KTR receiving Belatacept, and in R/D compared with Cyclosporine. Tissular CD3+, CD4+, CD8+, and CD68+ was higher in R/D group compared with baseline in KTR receiving Belatacept.These observations show that KTR receiving Belatacept have higher amounts of peripheral blood CD16+/IDO+ cells and Tregs on graft biopsies than those under Cyclosporine treatment.

Abstract# 102 Poster Board #-Session: P3-IIIEndothelial Cells Co-Cultured with Embryoid Bodies Enhance Insulin-Producing beta-Cell Differentiation through BMP-2 Signaling. Dodanim Talavera-Adame,1 Gordon Wu,1 Jae Y. Hwang,1 Daniel L. Farkas,1 Donald C. Dafoe.1 1Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA.Pancreatic islet cell transplantation is one of the effective approaches to cure type 1 diabetes mellitus. However, the main limitation is the severe shortage of transplantable donor islets. One attractive approach is the generation of functional insulin-producing beta cells from embryonic stem cells (ES). In vivo studies have shown that a close interaction between endothelial cells (EC) and embryonic endoderm is crucial to develop well-differentiated insulin-producing cells. However, it is not known if endothelial cells exert the same effects in vitro. We investigated whether the differentiation of mouse embryoid bodies (EBs) to insulin-producing cells could be enhanced by interaction with cultured endothelial cells. We established a co-culture system. Embryoid body cells were either in direct contact with human microvascular endothelial cells (HMEC) or without cell-cell contact. We also tested the effects of HMEC culture medium. Embryoid bodies co-cultured or treated with HMEC culture medium were analyzed at different time points. The beta cell marker expression was quantifi ed by immunocytochemistry and qRT-PCR. By Immunocytochemistry, a signifi cant increase in the percentage of proinsulin (60.08 ± 1.71 vs 29.65 ± 3.16), GLUT2 (60.22 ± 3.86 vs 48.038 ± 3.19), and PDX-1 (68.38 ± 4.59 vs 17.59 ± 2.46) positive cells was found in co-cultured EBs versus controls. Quantifi cation of mRNA expression of selected genes for islet cells (insulin 1, insulin 2, KIR-6.2, SUR1, glucokinase, PDX-1, Glut-2, glucagon, somatostatin, amylin, amylase, MafA, PAX-4, PAX-6, Nkx6.1, neurod1, and NGN3) corroborated the immunocytochemical results. Bone morphogenic protein-2 (BMP-2) was found in the HMEC culture medium. Activation of BMP-2 pathway with Smad1/5/8 phosphorilation was observed in treated EB cells that expressed proinsulin. Human recombinant BMP-2 (hrBMP-2) exerted similar effects on EB cells. Noggin partially inhibited these effects. These results indicate that differentiation of embryonic stem cells to insulin-producing cells can be promoted by endothelial cells in vitro and that BMP-2 is one of the factors involved in the process. Future studies will be directed to the isolation, functional characterization, and transplantation of these cells generated from EC-EB co-cultures.

Abstract# 103 Poster Board #-Session: P4-IIIEffect of Rituximab on the Regulation of Sphingomyelinase-Like Phosphodiesterase 3b-Precursor To Prevent FSGS Recurrence after Renal Transplantation. Junichiro Sagheshima, Alessia Fornoni, Changli Wei, Maria Saenz, Jing Li, Adela Mattiazzi, Marco Ladino, Prabakar Kamalaveni, Camillo Ricordi, Maria Pia Rastaldi, Peter Mundel, Jochen Reiser, George W. Burke III. Surgery and Medicine, University of Miami Miller School of Medicine, Miami, FL, USA.Background: Focal segmental glomerulosclerosis (FSGS) is a considerable cause of end stage renal disease, and recurrent FSGS after transplantation occurs in up to 70% of affected patients. Rituximab, an antibody directed against CD20, has been shown to have non-traditional functions, such as the ability to bind sphingomyelinase-like-phosphodiesterase-3b-precursor (SMLPD-3b) in podocytes, and may control recurrent FSGS. We hypothesize that podocyte SMLPD-3b expression is down regulated in recurrent FSGS, and that rituximab can prevent this phenomenon.Methods: We used rituximab induction in 29 renal recipients (4-24 y.o.) with FSGS (rituximab group) and compared the incidence of FSGS recurrence with 11 FSGS recipients without rituximab treatment (historical control). We evaluated CD20 and SMLPD-3b expression in post transplant kidney biopsies (n=14). We also exposed normal human podocytes to the sera collected pre and post transplantation from patients with (n=12) or without (N=21) recurrent disease, with/without rituximab.Results: The incidence of recurrent FSGS (urine protein/creatinine > 3.5) posttranspalnt was signifi cantly low in the rituximab group as compared with the control group (27.6% vs. 63.6%, p=0.04). Human podocytes expressed SMLPD-3b in vitro and in vivo. CD20 was not expressed. The number of SMLPD-3b positive podocytes correlated with posttransplant proteinuria (p=0.018, r2=0.52), rituximab prevented the down-regulation of SMLPD-3b (p=0.05) when podocytes were exposed to the sera of patients with recurrent disease.Conclusions: Rituximab induction seems to reduce the incidence of recurrent FSGS after transplantation. Rituximab may affect podocyte function in a CD20 independent

manner through modulation of SMLPD-3b. Our fi ndings suggest novel mechanisms for proteinuria in recurrent FSGS and may lead to new therapeutic indications for rituximab treatment of proteinuric kidney diseases.

Abstract# 104 Poster Board #-Session: P5-IIIIndoleamine 2,3-Dioxygenase (IDO) Immune Status Monitoring Post-Transplantation. Vikas Dharnidharka,1 Sushil Gupta,1 Timothy Garrett.1 1University of Florida.Introduction: Under- or over-immunosuppression increases rejection or infection risk, respectively, both detrimental to graft survival. Indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme in immune cell catabolism of tryptophan (trp) to kynurenines (kyn) and determines immune activation or anergy to alloantigens. Brandacher et al demonstrated raised blood and urinary krp/trp levels with acute rejection. We hypothesized that blood and urine kyn/trp levels would provide good discrimination of both extremes of immunosuppression.Methods: In this study, we prospectively and serially tested blood and urine IDO kyn/trp levels in children at months 1-12 post-kidney transplant at our center. We compared levels in stable patients to levels obtained in the 30-day period prior to biopsy-proven acute rejection episode or viral PCR proven infection with BK, CM or EB virus. Concentrations of kyn and trp in serum and urine were measured by mass spectrometry in blinded fashion.Results: Analysis of 39 blood and urine samples from 9 transplant recipients revealed higher kyn/trp levels in urine prior to 6 infection events , with smaller elevation during the solitary acute rejection episode (panel A, p = 0.0642 by 3-way ANOVA) . In contrast, blood levels of kyn/trp were higher in the acute rejection episode (panel B, p = 0.0506 by 3-way ANOVA). Trough tacrolimus levels were not signifi cantly higher in the infection group compared to the stable group (p = 0.14).Conclusions: Early results suggest that kyn/trp levels may be able to discriminate both extremes of immunosuppression. Infection is a more common event these days than acute rejection.

Abstract# 105 Poster Board #-Session: P6-IIILiver Transplant Recipients Successful ly Weaned Off Immunosuppression Lack Donor-Specifi c Anti-HLA Antibodies. Alin Girnita,2,3 George V. Mazariegos,1,2 Antonino Castellaneta,1,2 Jorge Reyes,2,3 Lynn Ostrowski,2,3 Carol Bentlejewski,2,3 Natalie Ku,2 Angus W. Thomson,2 Adriana Zeevi.2,3 1Children’s Hospital of Pittsburgh, University of Pittsburgh; 2Starzl Transplantation Institute, University of Pittsburgh; 3Department of Pathology, University of Pittsburgh.Background and Aim: Although liver allografts are regarded as less susceptible than other organs to damage induced by HLA antibodies (Abs), there have been reports of increased episodes of acute rejection in presensitized recipients. Thus, the impact of donor-specifi c Ab (DSA) on liver transplant outcome is still controversial. The aim of this study was to evaluate the role of anti-HLA Abs after liver transplantation in three clinically stable recipient groups defi ned as patients who were operationally tolerant (TOL; n=19); undergoing prospective drug weaning (PW; n=34) or who required maintenance immunosuppression (MI; n=20).Patients and methods: Seventy three recipients (children and adults) were eligible for study. Cross-sectional screening for anti-HLA Abs was performed by ELISA utilizing commercial LAT-M™ and LAT-1288™ ELISA kits (One Lambda; Canoga Park, CA).Results: Six out of 19 (31%) patients in the TOL group had anti-HLA Abs, compared to 23/34 (96%) in the PW group (p=0.02) and 9/20 (45%) in the MI group. None of the 6 ELISA-positive patients in the TOL group had DSA. By contrast, we identifi ed DSA in 14/19 patients (74%) tested for DSA in the PW and MI groups (9/12, p=0.01 and 6/7, p=0.01 respectively). Five years after the initial evaluation, the majority (18/19; 95%) of TOL patients continued to be off drug. Only one patient, positive for ELISA Ab required reinstitution of drug therapy following an episode of rejection. Furthermore, among 7 TOL patients available for re-testing, only one patient exhibited DSA. In the PW group, 4/34 patients (12%) were successfully weaned

T CELLS AND T REGS III

26

and 4 patients experienced rejection. Two of the 4 successfully weaned recipients were HLA Ab negative and remained negative upon retesting. All four patients that rejected exhibited anti-HLA Ab before and upon retesting.Conclusion: These fi ndings show that the majority of liver TOL recipients lack DSA and maintain this status for a prolonged period. By contrast, >75% of MI and/or PW patients who were tested for anti-HLA Abs exhibited DSA. These observations suggest a possible role for DSA as a biomarker of liver graft immunereactivity and in helping to drive clinical decision-making in relation to the management of immunosuppression minimization.

ORAL POSTER EXCHANGE 6 - T CELLS AND T REGS III

Abstract# 106 Poster Board #-Session: P7-IIIPro-Infl ammatory Cytokines Modulate the Ability of Regulatory T Cells To Control Alloimmunity. Giorgio Raimondi, Mahesh Pillai, Yoram Vodovotz, Angus W. Thomson. Surgery, University of Pittsburgh, Pittsburgh, PA, USA.Multiple studies have demonstrated the potential of naturally-occurring regulatory T cells (Treg) to control allo-responses. However, recent data raise concern that Treg suppressive function may be compromised in a pro-infl ammatory environment. We recently demonstrated that alloantigen-specifi c Treg (AAsTreg) lose their suppressive ability when alloreactive T cells are stimulated by maturing dendritic cells (DC). Identifying the molecules responsible for this effect and delineating their mechanism of action are essential for the design of successful tolerance-inducing strategies.Methods. Mouse LPS-treated DC were used to generate a maturation supernatant (MATSup) containing the cocktail of cytokines (R-cocktail) that liberates T cells from Treg suppression. The composition of the MATSup was investigated by Luminex™ assay. Microarray analysis of maturing DC provided additional insights into the composition of the R-cocktail. An in vitro DC-free CFSE-based T cell suppression assay (CFSE-assay) was devised to test the infl uence of specifi c molecules on the proliferation of CD4 T cells.Results. Microarray analysis identifi ed IL-12, TNF-α, and IL-27 as possible components of the R-cocktail in addition to IL-6 (a known R-cocktail component). The Luminex™ assay confi rmed the presence of these molecules in the MATSup. As expected, addition of MATSup in the CFSE-assay caused T cells to resist Treg-mediated suppression. IL-6 alone provided a moderate degree of resistance that was, however, inferior to that obtained with MATSup. IL-6 along with either IL-12 or TNF-α further increased T cell resistance. IL-12 and TNF-α were interchangeable, suggesting redundant activity. The infl uence of the R-cocktail was exerted on effector T cells and not on Treg, since a short pre-exposure of CD4 T cells was suffi cient to release T cells from suppression. Differently, pre-exposure of Treg to MATSup increased their suppressive capacity. Thus, differential effects are exerted by infl ammatory cytokines on T cells vs. Treg, with the effect on the former being dominant over the latter. Unexpectedly, when IL-27 was utilized in the CFSE-assay, we observed increased Treg suppressive function.Conclusions. Pro-infl ammatory cytokines exert distinct effects on effector T cells and Treg, promoting counter-balancing activities perhaps used by the immune system to fi nely tune its reactivity. Interference with this process may then represent an innovative approach to modulation of allo-reactivity.

Abstract# 107 Poster Board #-Session: P8-IIITransplantation Tolerance to Non-Inherited MHC Class I Maternal Antigens: New Insights Using TCR Transgenic Models. Gilles Benichou,1 Stephane Caucheteux,2 Yoshinobu Akiyama,1 Katsunori Tanaka,1 Cecile Vernochet,2 Colette Kanellopoulos-Langevin.2 1S, MGH/Harvard Medical School, Boston, MA, USA; 2IGA Laboratory, Institut J.Monod, CNRS & U. Paris-Diderot, Paris, France.The elucidation of the mechanisms underlying the infl uence of NIMA (non-inherited maternal antigens) on the offspring immune system has been diffi cult owing to the fact that the precise nature of the NIMA and the T cell clones recognizing these maternal antigens are unknown. To overcome this, we designed a double transgenic model in which the mother’s NIMA and offspring’s anti-NIMA T cells were well defi ned. In this model, all the offspring’s CD8+ T cells corresponded to a single clone recognizing the Kb MHC class I protein. On the other hand, the mother and the father of the offspring differed by the expression of a single antigen, Kb that served as NIMA. This allowed us to study the infl uence of NIMA exposure on the offspring T cell repertoire selection during ontogeny and on its T cell response during adulthood. First, we showed that the NIMA mice were tolerant to Kb as they accepted Kb+ heart allotransplants permanently. Functional analysis of anti-Kb alloreactive T cells in NIMA mice revealed the absence of pro-infl ammatory T cells producing IL-2 and gIFN. In turn, while these mice were tolerant to CBK allografts, they displayed some T cells producing IL-4 and high numbers of IL-10-producing T cells when challenged with Kb+ allostimulators i.e. through the direct allorecognition pathway. Therefore, exposure of fetuses and/or neonates to NIMA resulted in the selection of T cells producing type 2 cytokines, IL-4 and IL-10. we showed that we showed that tolerance to Kb could be transferred to control non-exposed mice by injection of

CD4+CD25+FoxP3+ T cells collected from the spleen of NIMA mice. The majority of these tolerogenic CD4+ T cells secreted IL-10 and was donor-specifi c in that they did not suppress the rejection of third-party allografts.

Abstract# 108 Poster Board #-Session: P9-IIIPregnant Level Estrogen and Progestone Affect the Regulatory T Cells in Mouse. Xing-Guang Lin,1 Sheng Chang,1 Li Wang,2 Hong-Min Zhou,1 Wen-Tao He,1 Zhen-Long Luo,1 Zhong-Hua Klaus Chen.1 1Institute of Organ Transplantation, Tongji Hospital, Huazhong Univerisity of Science and Techology, Wuhan, Hubei, China; 2Xiehe Hospital, Huazhong Univerisity of Science and Techology, Wuhan, Hubei, China.Objective To investigate whether the estrogen(E2) ,progesterone(P4) within the physiological concentration range of pregnancy can affect the expansion of CD4+CD25+ Foxp3+ regulatory T cell. Methods Using the ovariectomized and castrated mouse models, the models were injected subcutaneously with 100 µl of saline or with E2, and/or P4. After rebuilding the sex hormone to the physiological concentration range of pregnancy for 2 weeks, we sacrifi ced the models, and obtained lymphocytes from thymus, spleen, iliac lymph nodes and blood of the mice, tested the CD4+CD25+ Foxp3+ regulate T cell of total CD4+ T cell by fl ow cytometry and Immunohistochemistry. Results The group with E2 alone show a notable increase in the proportion of the Tregs that marked CD4+CD25+ Foxp3+ in spleen, iliac lymph nodes and blood. CD4+CD25+ Foxp3+ cells constituted 10.5% of all CD4+ cells in spleen, 7.63% in iliac lymph nodes and 6.13% in blood. While the proportion in the thymus can’t be enhanced compared to the vehicle groups. The expansion of the CD4+CD25+ Foxp3+cell pool was consistent in all mice analyzed, with only minor variation.The P4 can improve the proportion of Tregs lightly, without statistically signifi cant. The group of E2 combined with P4, each tissue shown a same statistically signifi cant trend in the increase of Tregs with E2 alone. Furthermore, we found that there is no difference in all groups between the males or females. Although the expression of male minor histocompatibility antigens such as H-Y may affect the treatment. The similar results can be found in Immunohistochemistry. Conclusions In vivo, E2 can drive the expansion of the Tregs in the secondary lymphoid compartments, while P4 doesn’t have the effect. Neither of them can improve the proportion of Tregs in thymus. As we know that E2 may directly act on CD4+CD25-T cells via ER-α, and convert to CD4+CD25+ Treg. We found no differece of the ER-α distribution between male or female groups, although the male has the male-specifi c minor histocompatibility antigens: H-Y.

Abstract# 109 Poster Board #-Session: P10-IIIWithdrawn

Abstract# 110 Poster Board #-Session: P11-IIIIndoleamine 2,3-Dioxygenase (IDO) and Treg Support Are Critical for CTLA4Ig Mediated Tolerance Induction to Solid Organ Allografts. Robert Sucher,1,2 Nikolaus Fischler,1 Rupert Oberhuber,1 Irmgard Kronberger,1 Dietmar Fuchs,1 Ernst Werner,1 Bettina Zelger,1 George Tellides,3 Nina Pilat,4 Thomas Wekerle,4 Raimund Margreiter,1 W. P. A. Lee,2 Gerald Brandacher.1,2 1Dept. of Visceral-, Transplant- and Thoracic Surgery, Innsbruck Medical University, Austria; 2Dept. of Surgery, Division of Plastic and Reconstructive Surgery, UPMC, USA; 3Dept. of Surgery, Yale University, USA; 4Dept. of Surgery, Division of Transplantation, Medical University Vienna, Austria.Purpose: Costimulatory blockade of CD28-B7-interaction with CTLA4Ig is a well-established tolerance induction strategy. Although previous in vitro studies confi rm that CTLA4Ig up-regulates IDO expression in DCs, the precise mechanisms of CTLA4Ig and IDO interaction remain unclear. Here we studied if concerted immunomodulation in vivo by CTLA4Ig, IDO and Tregs accounts for indefi nite survival of murine cardiac allografts.Methods: C57BL/6 IDO(WT/knock outs) mice received BALB/c-hearts. Group1[No treatment], Group2[Donor-specific transfusion (DST)], Group3[CTLA4Ig], Group4[CTLA4Ig+DST], Group5[CTLA4Ig+DST+ IDO inhibitor 1-methyl-tryptophan (1-MT)] and Group6[CTLA4-Ig+DST+ αCD25 mAb]. 1-MT was delivered in slow-release-pellets (at surgery or POD50). Serum enzyme-activity of IDO (kyn/trp) was analyzed by HPLC. Quantitative PCR was used for mRNA expression of IDO1/IDO2, Foxp3 and granzyme B. Anti-donor Abs were screened by FACS. Histopathology (H&E) and immunohistochemistry (for IDO,Foxp3,CD4,CD8,CD20,CD68 and C4d) of tissues was performed.Results: Graft survival: Group1[7.7±1.9 d], Group2[10.7±1.3 d], and Group3[47.7±29.8 d]. Group4:Indefi nite graft survival [>100 d] and tolerance without chronic rejection in IDO WT but acute rejection [16.5±5.9 d] in IDO knock out recipients. Group5:IDO inhibition with 1-MT, either at transplant or at POD 50, abrogated CTLA4Ig+DST tolerance induction. Group6:αCD25 mAb depletion of Tregs prevented CTLA4Ig+DST tolerance induction. Tolerant recipients had signifi cantly higher IDO activity as compared to non-tolerant animals, which markedly correlated with intragraft IDO and Foxp3 levels on immunostaining. IDO1/IDO2 mRNA-expression was similar in tolerant and non-tolerant recipients. Anti-donor-Abs were absent in all long-term-survivors.

MINIMIZATION STRATEGIES AND CHRONIC REJECTION

27

Abstracts

Conclusion: This study provides the fi rst direct in vivo evidence that CTLA4Ig induced tolerance to murine cardiac allografts is critically dependent on synergistic cross-linked interplay of IDO and Tregs.

Abstract# 111 Poster Board #-Session: P12-IIIAnti-HLA Abs along with Defensins Contribute to BOS Pathogenesis. Deepti Saini,1 Venkataswarup Tiriveedhi,1 Nataraju Angaswamy,1 Sabarinathan Ramachandran,1 Aviva Aloush,1 Ramsey R. Hachem,3 Elbert P. Trulock,1 Alexander Patterson,1 T. Mohanakumar.1,2 1Surgery, Washington University School of Medicine, Saint Louis, MO, USA; 2Pathology and Immunology, Washington University School of Medicine; 3Internal Medicine, Washington University School of Medicine.This study is towards determining the role of anti-HLA class I antibodies and defensins in the pathogenesis of chronic rejection, Bronchiolitis Obliterans Syndrome (BOS), following lung transplantation. Bronchoalveolar Lavage and serum collected from lung transplant recipients (21 BOS+ve, 20 BOS-ve) and 12 normals were analyzed by ELISA for α-defensins (HNP1-3) and human β-defensin2 (HBD2). BAL and serum from BOS+ patients showing higher levels of HNP1-3 and HBD2 compared to BOS- recipients or normal sera (p<0.05). Higher HNP1-3 and HBD2 levels also correlated with the presence of circulating Anti-HLA Abs. Airway Epithelial Cells (AEC) treated with anti-HLA Abs, HNP-1 or 2, produced HBD2 following stimulation with either of them and with synergistic effects on treatment with both. In a mouse model of Obliterative Airway Disease wherein intrabronchial administration of anti-MHC class I Abs resulting in immune responses to self antigens and lesions similar to BOS, a twofold increase in α-defensin and β-defensin levels was observed in the BAL at day 5, 9, 11, 13 and 15 post Ab administration compared to control. Neutrophil activity, measured by Myeloperoxidase (MPO) assay was also 3 fold higher in the lungs of mice treated with Anti-MHC Abs compared to isotype control. We conclude that Anti-HLAclass I Abs and α-defensins stimulate β-defensin production by epithelial cells leading to increased cellular infi ltration and infl ammation. Therefore, chronic stimulation of epithelial cells both by defensins and Anti-HLA Abs can lead to increased growth factor production and epithelial proliferation contributing towards the pathogenesis.

ORAL POSTER EXCHANGE 7 - MINIMIZATION STRATEGIES AND CHRONIC REJECTION

Abstract# 112 Poster Board #-Session: P13-IIIMinimization of Immunosuppression in Primary Adult Liver Transplantation with Steroid-Free Regimen Using Low Dose of Tacrolimus with Mycophenolate Mofetil after Daclizumab Induction. Olivier Boillot, gabriella pittau, Thomas Gelas, Catherine Boucaud. Liver Transplant Unit, Edouard Herriot Hospital, Lyon, France.In the aim to minimize side-effects of steroids and calcineurin inhibitors in liver transplant (LT) patients, we conducted a pilot study comprising a steroid-free regimen using low dose of tacrolimus (Tac) with mycophenolate mofetil (MMF) after daclizumab induction.Patients and methods. From september 2005 to december 2007, 64 primary LT adult patients were enrolled in this prospective one-year pilot study. Two doses of daclizumab were given at post-operative day (POD) 0 and 7, a 500 mg intra-operative bolus of solumedrol was administrated and thereafter stopped. Tac was started at POD 1 in order to reach trough levels below 8ng/mL and 2 g per day of MMF were given according to white blood cell count. All alive patients were followed for one year.Results. Mean Tac trough levels at POD 7, 3 months and 1 year were 7.5, 6.7 and 5.9nl/ml respectively. Mean serum creatinine levels prior LT, at 3 months and 1 year were 81, 102 and 94 mol/L respectively. Ten (15.6%) patients were withdrawn from MMF secondary to side effects. Acute rejection was histologically diagnosed in 18 (28%) patients. Six were mild and observed on the one-year protocol biopsy with normal LTs, 10 were moderate and 1 severe. All rejection episodes resolved with no treatment (n=5), increased tacrolimus dose (n=11) and solumedrol boluses (n=2). In univariate analysis, risk factors for rejection were initial MMF doses and donor BMI. Twelve patients were switched to everolimus or cyclosporine and 6 (10%) patients developed de novo diabetes. One year patient and graft survival was 96.8%.Conclusions. The one-year results of this dual immunosuppressive steroid-free regimen comprising Tac low dose and MMF suggest that side-effects can be minimized without jeopardizing effi cacy.

Abstract# 113 Poster Board #-Session: P14-IIIFeasibilty of Long-Term Sirolimus Monotherapy after Liver Transplantation. Dirk Uhlmann,1 Tonja Weber,1 Michael Bartels,1 Sven Jonas,1 Helmut Witzigmann.2 12nd Department of Surgery, University of Leipzig, Leipzig, Germany; 2Department of Surgery, Hospital Dresden-Friedrichstadt, Dresden, Germany.Aims: Sirolimus (SIR) is an alternative immunosuppressive agent in liver transplant patients who developed renal insuffi ciency caused by calcineurin inhibitors (CNI). Furthermore, SIR has been proven to have antitumoral activity. The aim of the study was to determine the effi ciency and the safety of long-term SIR monotherapy. A further aim was to examine if there is an improvement in renal function in liver transplant recipients after switching to SIR.Methods: Twenty four patients (14 with deterioration of renal function, 6 tumor patients, 4 pat. with the intention to reduce CNI-based immunosuppression after OLT) were included. Inclusion criteria were stable liver function and in the patients with nephrotoxicity (n=14) a serum creatinine between 130 and 250 µmol/l. Before OLT and every 3 months thereafter, liver and kidney function, blood pressure, lipids and side effects were assessed. The mean follow-up is 38.4 months.Results: Mean interval between OLT and initiation of SIR monotherapy was 51.7 months (6 – 102 months). The mean SIR whole blood throught level was 9.0 ± 2.8 ng/ml after 6 months and 6.0 ± 1.8 ng/ml after 18 months. No rejection episode occurred.In 12 patients with CNI-induced nephrotoxicity, before conversion the mean GFR measured by 99mTc-DTPA–clearance was 27.4 ± 6,8 mL/min/1.73m2 and mean serum creatinine 156.1 ± 54.9 µmol/l. Six months after conversion, GFR increased to 40.9 ± 6.3 mL/min/1.73m2 (p<0.05) and mean serum creatinine decrease slightly to 129.1 ± 34.7 µmol/l. At last follow-up (at an average of 3 years after conversion), serum creatinine was 112.4 ± 17.9 µmol/l (p<0.05 vs levels before SIR). In 2 further patients with serum creatinine > 200 µmol/l, no signifi cant improvement of kidney function was found after conversion. Cholesterol levels were 5.5 ± 0.8; 5.7 ± 1.6 und 5.2 ± 1.1 mmol/l after 0, 6 and 12 months; triglyceride levels 3.0 ± 2.6; 3.5 ± 2.2 and 2.2 ± 1.4 mmol/l (p<0.05). The values remained stable up to the last update. The systolic blood pressure decreased from 143.5 ± 15.6 to 127.0 ± 15.2 mmHg and the diastolic from 87.0 ± 11.6 to 79.0 ± 5.5 mmHg. Antihypertensive medication could be reduced after conversion in 12 pat.Conclusions: The conversion to SIR monotherapy after OLT results in an amelioration of renal function and in a reduced blood pressure. It reveals no increased risk of rejection and minimal side effects during long-term follow-up.

Abstract# 114 Poster Board #-Session: P15-IIIExcellent Outcomes with Alemtuzumab Induction and Steroid-Free Maintenance Compared to Interleukin-2 Receptor Antagonist or Thymoglobulin with Prednisone Maintenance. Joseph B. Africa,1 Marvin J. Tucker,1 Michael Orquiza,1 Alejandro O. Aquino,1 Seyed R. Ghasemian,1 Jimmy A. Light.1 1Surgery, Transplant Services, Washington Hospital Center, Washington, DC, USA.We compared Alemtuzumab induction with rapid steroid withdrawal to interleukin-2 receptor antagonist (IL2RA) and Thymoglobulin both with prednisone maintenance. All received FK/MMF maintenance.METHODS: Retrospective, cohort study of 603 kidney transplant recipients January 1, 2001 - December 31, 2006. Alemtuzumab: FK trough targeted at 4-8ng/dl; MMF was started at 1 gm BID, decreased to 500 mg BID by the 2nd week; Prednisone taper only for DD/DGF, but stopped by 2nd week. Thymo/ILRA (Daclizumab 91%): FK trough 6-10ng/dl; MMF 1gm BID; Prednisone tapered down to 5mg/d by 4th wk. All had intraop SM 500mg, then 250mg,125mg at POD#1,#2. Exclusion: SPK/PAK transplants, maintenance other than FK/MMF, combination or OKT3 induction regimens, Alemtuzumab patients on steroids prior to transplant, CIT > 36 hrs, desenstized pts, and HIV recipients. Outcomes: Patient/graft survival; acute rejection rate; serum creatinine; infections requiring hospitalization; BK shedding/nephritis.Demographics

ALEMTUZUMAB THYMO IL2RATOTAL 281 65 257Follow-Up (Median) 36 months 56 months 60 monthsAGE 50 52 52AA 68%* 58% 57% p=0.036DD 53% 35% 21%* p<0.01PRA > 20% 16% 20% 7%* p=0.001REPEAT Tx 2% 21%* 6% p<0.001

ALEMTUZUMAB THYMO IL2RAACUTE REJECTION (0-6 months, 6-12 months, > 12 months)

27% (10%, 4%, 12%)

34% (13%, 3%, 18%)

25% (5%, 4%, 16%) ns

CREATININE mg/dl 1 wk; 3, 6, 12 months

1.99, 1.45, 1.50, 1.52, 1.48

3.30*, 2.45*, 2.02*, 2.14*, 2.44*

1.94, 1.49, 1.50, 1.35, 1.34 * p<0.05

GRAFT SURVIVAL 89% 74%* 85% p=0.005PATIENT SURVIVAL 94% 86% 93% nsINFECTIONS 24% 17% 21% nsBK urine/biopsy 28%/ 1% 29%/ 1% 23%/ 2% ns

CLINICAL SCIENCE, CHRONIC AND ANTIBODY-MEDIATED REJECTION

28

RESULTS: More retransplant/sensitized patients received Thymoglobulin. More AA in the Alemtuzumab group. Eighty percent of the Alemtuzumab patients remained steroid-free at the end of 3 years.CONCLUSION: Alemtuzumab induction, FK/MMF without maintenance steroids produced superior serum creatinine and graft survival compared to Thymoglobulin. Despite IL2RA’s more favorable pateint characteristics, when compared with Alemtuzumab, no difference was seen in patient/graft survival, acute rejection rate, and serum creatinine. The incidence of infections and BK viruria/nephritis were the same for the three groups.

Abstract# 115 Poster Board #-Session: P16-IIIA l e m t u z u m a b I n d u c t i o n , S t e ro i d - F re e M a i n t e n a n c e Immunosuppression in African-Americans vs Non-African Americans. Joseph B. Africa,1 Marvin J. Tucker,1 Alejandro O. Aquino,1 Seyed R. Ghasemain,1 Jimmy A. Light.1 1Surgery-Transplant Services, Washington Hospital Center, Washington, DC, USA.African-Americans (AA) have been excluded from steroid-free studies due to dismal early experience outcomes. Recent reports using antibody induction have been encouraging, with the best results using lymphocyte depletion. We report our experience with Alemtuzumab induction/steroid-free maintenance regimen in AA compared to non-African-Americans (NAA).METHODS: Retrospective cohort study of kidney transplant recipients from January 1, 2004 - December 31, 2006. Excluded were: SPK/PAK transplants; induction other than Alemtuzumab; cold ischemia time > 36 hours; those on steroids prior to transplant; maintenance other than FK/MMF. Tacrolimus trough was targeted at 4-8ng/dl; MMF was started at 1gm BID and decreased to 500 mg BID by the 2nd week and further dose adjustments made as clinically indicated. Outcomes: patient/graft survival; acute rejection rate; serum creatinine; infections requiring hospitalization; BK shedding/nephritis, tacrolimus trough levels, patients remaining steroid-free. Median follow-up 36 months.Characteristics

AA NAAAGE 49 51TOTAL 190 91DD 59% 42% p=0.005PRA>20% 17% 14%REPEAT Tx 3% 2%

ResultsINFECTION 25% 21% ns

BK URINE / BIOPSY 24% / 0% 35% / 2% ns / p=0.062

ACUTE REJECTION (DD/LD) 28% (35% / 19%) 23% (26% / 21%) nsGRAFT SURVIVAL (DD/LD) 87% (87% / 89%) 93% (89% / 96%) nsPATIENT SURVIVAL (DD/LD) 92% (88%* / 97%) 98% (95% / 100%) *p<0.0001STEROID FREE (DD/LD) 78% (71% / 84%) 84% (78% / 87%) ns

RESULTS: More AA received DD. Tacrolimus trough levels at 1 wk, 3, 6, and 12 months were the same (AA 3.1 nd/dl vs NAA 3.8 ng/dl; 6.1 vs 7.04; 6.06 vs 6.2; 5.5 vs 6.3). Serum creatinine was better for NAA LD at 1 week (AA 1.54 mg/dl vs NAA 1.36 mg/dl) and NAA DD at 3 months (AA 1.65 mg/dl vs NAA 1.21 mg/dl); however, no signifi cant difference was noted at 6, 12 and 24 months (AA DD/LD vs NAA DD/LD: 1.6/1.49 vs 1.29/1.44; 1.73/1.46 vs 1.31/1.38; 1.59/1.6 vs 1.39/1.24). Mycophenolate doses were the same in both groups. There was no signifi cant difference in acute rejection rates, infections and proportion of patients remaining steroid free. NAA had better DD patient survival and a trend towards more BK infection.CONCLUSION: Steroid-free immunosuppression using Alemtuzumab may be successfully done in AA, producing the similar outcomes compared to NAA. This protocol also allowed minimal Tacrolimus and Mycophenolate dosing.

Abstract# 116 Poster Board #-Session: P17-IIIThe Significance of Immune Cell Function Testing in Renal Transplantation. Vaughn E. Whittaker,1 Geo Serban,2 Jianshe Fan,2 David J. Cohen,1 Lloyd E. Ratner,1 Nicole Suciu-Foca,2 Adriana I. Colovai.2 1Transplant Surgery and Nephology, Columbia University Medical Center, New York, NY, USA; 2Immunocytogenics, Columbia University Medical Center, New York, NY, USA.The Cylex Immune Function Test is currently used to assess cell-mediated immunity in transplant recipients. This test was designed to measure the amount of ATP released by PHA-activated CD4+ T cells from whole blood, and to assist in adjusting immunosuppression. Low ATP activity has been associated with increased risk of infections, while high ATP has been reported to predict rejection. Since the effect of lymphocyte depletion agents on immune cell function, as measured with the Cylex test, has not been carefully investigated, we studied a population of 58 renal transplant recipients following Thymoglobulin induction and initiation of maintenance immunosuppression. In parallel, we analyzed by fl ow cytometry the phenotype and the proliferation capacity of CD4+ T cells. Overall, the incidence of infections during the fi rst year post-transplantation was signifi cantly higher in patients with low ATP activity (<225 ng/ml) compared to patients with moderate activity (225-525 ng/ml; p=0.03). However, there was no association between ATP activity and rejection.

ATP levels did not correlate with CD4+ T cell counts. Notably, ATP activity varied over a wide range (35-843 ng/ml) in samples displaying low CD4+ T cell counts (<100/ul). These samples accounted for 88% of the specimens obtained during the fi rst two months post-transplantation. No phenotypic features were identifi ed which distinguished CD4+ T cells from samples with low ATP activity compared to samples with moderate/high ATP level. Post-depletion CD4+ T cells were predominantly CD25+CD45RO+CD45RA-, consistent with an activated/memory phenotype, and produced Th1 cytokines, such as IL2, IFN-gamma and TNF-alpha. There was no association between ATP activity and the ability of CD4+ T cells to proliferate in response to PHA, as measured by CFSE labeling. ATP activity measured within the fi rst two months post-transplantation was not associated with infection or rejection. Our fi ndings indicate that cell populations other than CD4+ T cells, possibly myeloid progenitors, contribute to the signal measured by the Cylex assay. Therefore, Cylex results should be interpreted with caution. High ATP activity does not justify increased immunosuppression, particularly in lymphopenic patients.

Abstract# 117 Poster Board #-Session: P18-IIIBronchoalveolar Lavage CD4+ Fox P3+ T Cells Are Decreased in Lung Transplant Recipients with Bronchiolitis Obliterans Syndrome. Sangeeta M. Bhorade,1 Hong Chen,1 Luciana Molinero,1 Chuanhong Liao,1 Rebecca Shilling,1 Anne Sperling,1 Anita Chong,2 Maria Luisa Alegre.1 1Medicine, University of Chicago Medical Center, Chicago, IL, USA; 2Surgery, University of Chicago Medical Center, Chicago, IL, USA.Lung transplantation is severely limited by chronic rejection which affects lung allografts with an incidence and severity exceeding most other transplanted organs. Alloimmune responses play an important role in progression to chronic rejection that manifests as bronchiolitis obliterans syndrome (BOS), but no biomarker is currently available that can predict the progression to BOS. Studies in animal models suggest that intra-graft T regulatory cells are important to maintain transplantation tolerance and FoxP3 is the protoypic marker for these cells. In patients, however, FoxP3+ T cells have been reported to accumulate during episodes of acute rejection in different organ transplant settings. To gain insight into potential role of Tregs in lung allografts, we assessed the percentage of Fox P3+ T cells among CD4+ T cells in BAL and peripheral blood (PBMC) of stable lung transplant recipients compared to those with BOS. 20 lung transplant recipients (14 who remained stable and 6 who developed BOS) underwent 63 bronchoscopies with BAL and simultaneous collection of PBMC. Stable patients had a signifi cantly increased percentage of FoxP3+ cells among CD4+ cells in BAL (10% vs 3%; p, 0.001) and greater levels of the Treg-attracting chemokine CCL22, than patients who developed BOS. The difference between the two groups remained signifi cant when single BAL samples obtained at 1 year post-transplant were compared between the two groups. There was also a signifi cant increase in the percentage of FoxP3+CD4+ cells during acute rejection in patients whose lung function stabilized compared to those who developed BOS (p = 0.01). There was no difference in the percentage of FoxP3+CD4+ cells in the PBMCs between the two groups. In this cohort of clinical lung transplant recipients, BAL fl uid CD4+ T cells were enriched in FoxP3+ cells in stable recipients compared to those developing BOS. Our results indicate that BAL may be more refl ective of local immune responses than peripheral blood and suggest that increased percentages of FoxP3+ cells correlate with improved allograft fate. Further longitudinal data will determine if ratio of BAL FoxP3+ T cells to effector T cells will predict lung transplant outcomes.

ORAL POSTER EXCHANGE 8 - CLINICAL SCIENCE, CHRONIC

AND ANTIBODY-MEDIATED REJECTION

Abstract# 118 Poster Board #-Session: P19-IIILiver Function Tests Do Not Correlate with Liver Biopsy Results in Pediatric Patients Who Have Undergone Liver Transplantation. Jason D. Fraser,1 Vicki Fioravanti,1 Pablo Aguayo,1 Robert E. Kane,1 James F. Daniel,1 Walter S. Andrews.1 1Departent of Transplantation, Children’s Mercy Hospital, Kansas City, MO, USA.IntroductionProtocol-based annual liver biopsies for pediatric patients who have had a liver transplant are standard in some centers. However, disagreement exists whether such surveillance is necessary, especially when liver function tests are normal. Therefore, we reviewed our experience in patients who have undergone annual liver biopsies to determine if there was correlation between liver function tests and biopsy results.MethodsWe retrospectively reviewed all pediatric patients with liver transplants who underwent annual biopsies from 10/1994 to 10/2008. Biopsy results were classifi ed as normal (normal, no rejection, nonspecifi c, or minimal fi brosis), fi brosis (mild or severe), and rejection (all forms). Variables were analyzed using ANOVA, Student’s T-test, and Chi-squared test where appropriate. Pearson’s correlation was used to evaluate biopsy results and liver function tests. Two-tailed P value was determined from the correlation coeffi cient and signifi cance was defi ned as P ≤ 0.05.

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ResultsA total of 259 biopsies were done in 62 patients, 159 as part of annual surveillance. The average time from transplant to biopsy was 5.1+/-3.5 years. There was no signifi cant differences among the average liver function tests in each of the groups. There were 78 (49%)normal biopsies, 41 (26%) with fi brosis, and 40 (25%) with rejection. There were 81 (52%) abnormal biopsies. Liver function test did not signifi cantly correlate with biopsy results. Serial biopsies were completed in 47 of the 62 patients. Twenty-six (55%) showed no progression of disease. Fourteen (30%) demonstrated serial regression in level of fi brosis or rejection while 7 (15%) demonstrated progression of fi brosis or rejection. Patients who were on monotherapy were more likely to have an abnormal biopsy compared to those who were on a multi-drug therapy at the time of biopsy (P = 0.02). There was no difference in time from time to biopsy between patients who had normal versus abnormal biopsies (P = 0.60).ConclusionsOur data demonstrate that there was no signifi cant correlation between liver function tests and biopsy results in pediatric liver transplant patients who underwent surveillance biopsies. Despite the presence of normal liver function tests, 52% of this population had abnormal hepatic histology, including 25% with rejection. Therefore, to forgo biopsy based solely on normal liver function test may not be prudent.

Abstract# 119 Poster Board #-Session: P20-IIIAntibody Mediated Rejection Secondary to Anti-DP Antibodies in a Kidney Transplant Recipient. Maria P. Martinez Cantarin,1 Pooja Singh,1 Beth Colombe,2 George Francos.1 1Nephrology, Thomas Jefferson University; 2Hystocompatibility Laboratory, Thomas Jefferson University.Introduction: Solid phase platforms are able to distinguish DP antibodies. The role of DP antibodies in allograft rejection is unclear. We present the case of a 54 year old transplant recipient that was highly sensitized and had antibody mediated rejection from a DP donor specifi c antibody (DSA).Case reports and results: A 54 year old African American male with past medical history of end-stage renal disease secondary to hypertension of 24 years duration that was status post 3 prior cadaveric kidney transplants presented for deceased donor transplantation at our institution. The patient was anuric on hemodialysis. He received a zero mismatch kidney from a brain death donor that was hepatitis C positive, as was the recipient. Initial B and T cell crosmatches were slightly positive by only 3 to 4 channel shifts, so it was decided to treat the patients with 100 grams of IVIG. The patient did well and never required hemodialysis. His creatinine at the time of discharge was 6.5 mg/dl.and was 2.9 mg/dl at follow-up. Two weeks after his transplant his creatinine increased to 4.0 mg/dl. He was admitted for steroid pulse and kidney biopsy was performed showing only borderline cellular rejection but the immunofl uorescence showed diffuse C4d deposition in the peritubular capillaries. HLA typing of the donor and the recipient showed a strong antidonor DPA1*0103. We obtained confi rmation that the antidonor antibody was complement fi xing. The antibody titer at that time was 1/1024. The patient underwent treatment with plasmapheresis and low dose IVIG, requiring 9 sessions to decrease his titers to 1/64, where he has remained stable. His creatinine started to decrease maintaining good urine output. He also received a dose of rituximab, 1 gram. His last creatinine was 1.6 mg/dl.Conclusions: We presented the case of a patient that suffers from antibody mediated rejection from DPA1 antidonor antibody. Prior publications have shown how DP antibodies can cause a positive crossmatch but the cases of acute antibody mediated rejection secondary to DP DSA are scarce. With the actual tissue typing techniques including high resolution antigen typing and antibody detection with solid phase beads we are staring to see how our standard six antigen typing may not be enough to avoid complications especially in highly sensitized patients.

Abstract# 120 Poster Board #-Session: P21-IIIMachine Perfusion in Kidney Transplantation: Lower Rate of Acute Rejection in the Presence of Longer Pump Time, Regardless of Immunosuppression. Gaetano Ciancio,1 Jeffrey L. Gaynor,1 Junichiro Sageshima,1 Linda Chen,1 David Roth,2 Warren Kupin,2 Giselle Guerra,2 Lissett Tueros,1 Alberto Zarak,1 Lois Hanson,1 Susan Ganz,1 Phillip Ruiz,1 William W. O’Neill,3 Alan S. Livingstone,4 George W. Burke III.1 1Division of Transplant Surgery, University of Miami Miller School of Medicine, Miami, FL, USA; 2Division of Nephrology, University of Miami Miller School of Medicine, Miami, FL, USA; 3Division of Cardiology, University of Miami Miller School of Medicine, Miami, FL, USA; 4Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, USA.Machine perfusion(MP) preservation has been used for all deceased donor(DD) kidney transplants(KT’s) at our center. Kidneys are placed in cold storage(CS) at retrieval, then transferred to MP upon arrival at our center. We compared our delayed graft function(DGF) incidence with other centers predominantly using cold storage(CS), and evaluated the prognostic impact of pump time.Three-hundred-and-ninety(339/390 DD) KT recipients from 3 randomized,

immunosuppression trials at our center were analyzed. Categorical data analysis compared our DGF incidence vs. a pooled cohort of 8,023 patients from 23 published, randomized trials. Stepwise logistic and Cox regression along with a matched pairs analysis were used in determining the prognostic value of pump time.Our mean CS and Pump times were 6.6hr and 26.7hr, respectively. Our DGF percentage was signifi cantly lower than that reported by other centers: 3.8%(15/390) vs. 23.5%(585/2488) in study arms with >75%DD (P<.000001). While no signifi cant association of pump time with DGF incidence existed, a signifi cantly lower fi rst biopsy-proven acute rejection(BPAR) rate was observed for longer pump time (e.g., >24hr) among more immunologically active(Non-caucasion, age<50yr) recipients (univariable P=.002;multivariable P=.02), which was confi rmed in the matched pairs analysis. Immunologic graft failure for pump time <24 and >24hr was 13.8%(16/116) vs. 7.6%(17/223) (P=.10). Finally, our 9.0%(35/390) fi rst BPAR rate at 12mo contributed to our 1 and 5 year graft survivals being 94±1% and 77±3%, respectively, approaching published living donor graft survival rates.With relatively short CS times, MP with prolonged pump times favorably reduced both DGF and fi rst BPAR, translating into improved long-term graft survival.

Abstract# 121 Poster Board #-Session: P22-IIISocioeconomic Status and Acute Rejection in Kidney Transplant Recipients. S. Lodhi,1 K. Lamb,1 J. Gregg,1 P. Patton,1 P. Minnick,1 D. Tomlin,1 H. U. Meier-Kriesche.1 1Division of Nephrology, University of Florida, Gainesville, FL, USA.Socioeconomics are implicated in poor graft survival in kidney transplant recipients. Many patients are experiencing a loss of monetary assistance across the nation. A major concern is if low-income patients are noted to have worse outcomes, they may be screened out by centers because of tightened CMS oversight. Our center serves a large population of low-income recipients and we are able to track them for a lifetime. In order to elucidate a correlation between fi nancial status and kidney transplant outcome, we studied the incidence and severity of acute rejection (AR) in recipients stratifi ed by income level.We analyzed patients by estimated income, and stratifi ed them into 4 groups by cluster analysis. The primary outcomes were time to fi rst acute rejection and a composite endpoint of time to graft loss, death, or AR. Outcomes were described by KM analysis confi rmed by Cox proportional hazard models.Our data indicate a signifi cantly higher risk of ARs in recipients from a lower income group as compared to the high income group [AHR=1.71(1.18-2.48)]. Many of these ARs are vascular and occur within the fi rst 3 years, with signifi cantly more among recipients with an income of <$28K as compared to incomes >$52K followed over 9 years (p=.03). The composite endpoints KM analysis showed a clear stratifi cation of event risk by income.

African-American race was corrected for with multivariate analysis and did not add to the risk. Additionally, the low income group encountered more rejections not returning to baseline after treatment. We submitted an IRB to merge our social work with our transplant database, and will present correlations between AR and insurance status.Our fi ndings corroborate the link between economic status and transplant outcomes. This correlation is seen prior to the 3-year point when Medicare support ends. We see patients return with devastating rejections after stopping medications due to fi nancial woes, despite an aggressive effort to solve these problems before transplantation. Further research is needed to determine if diverting federal resources to enhance medical compliance will avoid future cost-burdens of graft failure.

Abstract# 122 Poster Board #-Session: P23-IIIThe Effect of Change or Modulation of Maintain Immunosuppressive Regimen on Long-Term Transplant Result. Jung Jun Lee,1 Myoung Soo Kim,1 Kyu Ha Huh,1 Soon Il Kim,1 Yu Seun Kim.1 1Departments of Surgery, Yonsei University College of Medicine, Seoul, Korea.Type of maintenance immunosuppressive regimen (ISR) is a major prognostic factor that determines graft survival. Therefore, a change or modulation of the ISR may be a potential determinant that affect the transplant results. This study evaluates the effects of change or modulation of ISR on long-term transplant results.A total of 1,164 patients that underwent kidney transplantation from January, 1997 to December, 2008 in Yonsei University Health System were enrolled in this study. The change mode of immunosuppressant, the reason for change, and the transplant results were retrospectively collected.

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Among 1,164 kidney transplant recipients, 201 recipients (17.3%) were started under the double regimen (DR) (calcineurin inhibitor (CNI) and steroid) and 963 recipients (82.7%) were under triple regimen (TR) (CNI+steroid+antimetabolite). Seventy-seven recipient (38.3%) with DR and 271 recipients (28.1%) with TR were changed ISR during post-transplant period. Among the recipient with DR, the most frequent reason for regimen change was acute rejection in early post-transplant period and chronic allograft dysfunction in late period. Conversion to TR (69/201, 34.3%) was predominant change mode of recipient with initial DR. Unlike recipient with DR, major causes of regimen change in recipients with TR were complications related with over-immunosuppression and drug toxicity regardless of post-transplant period. Conversion to DR and change of antimetabolite were performed in 127 recipients (127/ 963, 12.6%) and 98 recipient (98/963, 10.2%) respectively. The change group of initial DR showed statistically superior graft survival rate (GSR) (p=0.032) compared with maintain group of initial DR. On the other hand, the change group of initial TR showed inferior GSR (p<0.001).

The cause and mode of regimen change was clearly different by type of initial regimen. And effect of regimen change on transplant result was also different. The type of regimen after post-transplant change or modulation affects the long-term transplant result. The triple maintain regimen with antimetabolite showed a superior GSR.

Abstract# 123 Poster Board #-Session: P24-IIIUnusual Lymphoma and Concurrent Presentation of Graft Arteriopathy in a Hand Transplant Recipient Three Years Post Transplant. Christina L. Kaufman,1 Kadiyala V. Ravindra,2 Brenda Blair,1 Warren Breidenbach.1 1Christine M. Kleinert Institute for Hand and Microsurgery Inc, Louisville, KY, USA; 2Transplant Center, Jewish Hospital and St Mary’s Healthcare, Louisville, KY, USA.Objective: We evaluated monoclonal T and B cell populations found in the PB and marrow in a hand transplant patient. Immunosuppression was drastically reduced, restarted, and then further increased in response to evidence of initimal hyperplasia in deep vessels. The effects of changes in immunusuppression on these unusual populations are presented.Methods: Patient 3 recieved a hand tx in 2006. Recipient and donor were EBV positive. He recieved Campath induction and Prograf/MMF maintainence. At 22 months post transplant routine blood work indicated an excessive amount of lymphocytes in the peripheral blood. Flow cytometry revealed monoclonal B and T cell populations. An intial diagnosis of PTLD was made, and MMF was immediately discontinued and tacrolimus was lowered to 5ng/ml.Results: The patient was asymptomatic at time of diagnosis. PET/CT scans revealed no adenopathy. The clonal B cell population (kappa restricted) was composed of small cells (CD19, CD20, CD25, CD5-, CD10-, CD23-, bcl-1-, bcl-6- ). The T cell clone (Vb restricted) had cytologic and phenotypic features consistent with large granular lymphocytes (CD8+, TCR, dim CD3+, dim CD5+). In situ hybridization and quantitative PCR were EBER- and EBV-. Eight weeks after reduction of immunosuppression, The B and T cell clones persisted unchanged. The graft showed signs of swelling and grade II/III rejection and the patient decided to resume immunosuppression. Analysis of frozen peripheral blood obtained six months PRIOR to transplant revealed the presence of the B cell clone, but not the T cell clone. Recent deep tissue biopsies revealed intimal hyperplasia and prednisone was added to the immunosuppression regimen in September 09. Evaluation of the the unusual circulating clones three weeks after intitiation of steroid therapy demonstrated minimal changes.Conclusion: The phenotype of the B cell clone is consistent with a marginal zone lymphomas (MZL) per WHO. The T cells could represent clonal expansion against the B cell clone or an unknown antigen. The failure of the clones to respond to signfi cant changes in immunosuppression, combined with the presence of the B cell clone prior to transplant suggest this lymphoma is not PTLD. We will continue to monitor both the B cell and T cell clone, but at this time, immunosuppression will be maintained.

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Abstract# 124 Poster Board #-Session: P1-IComparison of Standard and High Dose Rabbit Antithymocyte Globulin Induction on Renal Allograft Outcomes in High Risk Recipients with Slow Graft Function. Winston A. Ally,1,2 Benjamin Duhart,1,2 Amy G. Krauss,1,2 Samir J. Patel,3 Maung Mya,1,2 James Eason.1,2 1Methodist University Hospital Transplant Institute, Memphis, TN, USA; 2University of Tennessee, Memphis, TN, USA; 3Methodist Hospital, Houston, TX, USA.Slow graft function (SGF) is associated with poor long-term graft outcomes in renal allograft recipients with increased acute rejection risk and decreased graft survival. Previous studies have not addressed the optimal dosing for antithymocyte globulin (ATG) induction for recipients with SGF. We defi ned SGF as a decline in serum creatinine less than 25% by POD 2 without the need for dialysis. At our center, patients with immediate graft function receive a standard total dose of 4.5 mg/kg over 3 days, whereas patients with slow or delayed graft function often receive > 4.5 mg/kg of ATG.The purpose of this study was to determine the effect of standard and high dose ATG induction on 1 year graft outcomes in cadaveric kidney transplant recipients with SGF.We retrospectively reviewed renal allograft recipients transplanted from January 2000 through April 2005. Patients were stratifi ed according to the total dose of ATG induction (standard dose: ≤ 4.5 mg/kg vs. high dose: > 4.5 mg/kg). Of 97 recipients identifi ed with SGF, 66% were African American, 66% were male, mean age at transplant was 46.3 years, 10.3% were re-transplants, mean number of HLA mismatches was 4.0, and 15.5% had peak panel-reactive antibody >30%.Baseline demographics were compared between those with standard (n=41) and high dose induction (n=56) with no detected differences. The total ATG induction dose between the standard and high dose groups was 3.9 vs. 6.3 mg/kg (p<0.0001). De novo maintenance immunosuppression was either tacrolimus (43.9 vs. 53.6%, p=0.41) or sirolimus based (56.1 vs. 46.4%, p=0.41). There was no difference in the 1 year incidence of acute rejection (12.2 vs. 5.4%, p=0.28), graft loss (7.3 vs. 5.4%, p=0.69), death censored graft loss (4.9 vs. 3.6%, p=1.00), and overall mortality (2.4 vs. 1.8%, p=1.00) between standard and high dose groups respectively. There was also no signifi cant difference in serum creatinine or glomerular fi ltration rate at 1, 3, 6, and 12 months. There was no difference in the incidence of neutropenia and thrombocytopenia.Our data suggest that a total dose of ≤ 4.5 mg/kg of ATG for induction may be suffi cient for cadaveric renal allograft recipients with SGF as similar graft and patient outcomes were achieved.

Abstract# 125 Poster Board #-Session: P2-IPharmacodynamic Monitoring of IMPDH Activity To Optimize MMF Therapy after Renal Transplantation. Ferdi Sombogaard,1 Ron Mathot,1 Willem Weimar,1 Teun van Gelder.1 1Erasmus University Medical Center, Rotterdam, Netherlands.Background: Mycophenolic acid (MPA) –the active moiety of MMF– exerts its immunosuppressive effect by inhibiting inosine monophosphate dehydrogenase (IMPDH). There is increasing interest for patient tailor optimize dosing instead of fi xed dosing to further improve the balance between effi cacy and toxicology of MPA therapy. IMPDH activity can be used for that aim. This study developed a pharmacokinetic-pharmacodynamic (PK-PD) model to characterize this relationship and to explore the predictive value of IMPDH activity for BPAR and adverse events (AE).Methods: In a prospective study 101 de novo renal transplant patients were followed. Time profi les of MPA plasma concentration and IMPDH activity were assessed at day 6, 21, 49 and 140 post-transplantation and one sample was taken to determine IMPDH mRNA levels and pharmacogenetic polymorphisms. The PK-PD relationship was assessed using nonlinear mixed effects modeling.Results: MPA PK was described using a 2-compartment model. An inhibitory Emax model adequately described the relationship between MPA concentration and IMPDH activity. Maximal inhibition of IMPDH activity (Emax) was 89%. Baseline IMPDH activity (E0) increased from 56.1±11.2 (day 6) to 92.0±22.1µmol/s/mol AMP (day 140), while the IC50 was decreased from 0.75±0.45 (day 6) to 0.17±0.10mg/L (day 140). The inter-patient variability for E0 and IC50 was 9.0±5.5% and 99.0±27% respectively. The inter-patient variability was partly explained by race (p<0.05) and the IMPDH type II 3757T>C polymorphism (p<0.05). E0 was correlated to leukocytes count (p<0.01) and IC50 was correlated to albumin (p<0.05).E0 was 12% higher in BPAR patients (p=0.16) and the IMPDH activity exposure was also slightly higher in BPAR patients compared to non-BPAR patients (246 vs 208h·µmol/s/mol AMP; p=0.15). IMPDH mRNA type I and type II levels pre-dose were signifi cantly lower in BPAR patients compared to non-BPAR patients (type I p<0.05; type II p<0.01). No correlations were found between any determinant and MMF-related AE.Conclusion: A PK-PD model has been developed describing the relationship between MPA concentration and IMPDH activity. Using this model, intra-patient variability over time and inter-patient variability could partly been explained. PD parameters are

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correlated to BPAR –but not to AE– and could have a predictive value. This model may be used to study the usefulness and feasibility of combined PK-PD monitoring to optimize and individualize MMF therapy after renal transplantation.

Abstract# 126 Poster Board #-Session: P3-IThe Value of Chronic Renal Allograft Injury Score for the Conversion from Cycspoline to Tacrolimus or Sirolimus in Chronic Allograft Nephropathy. Jiqiu Wen,1 Leishi Li,1 Jisong Chen,1 Shuming Ji,1 Huiping Chen,1 Qiquan Sun,1 Dongrui Cheng,1 Zhihong Liu.1 1Department of Nephrology, Jinling Hospital, Research Institute of Nephrology, Nanjing, Jiangsu, China.Objective:[/bold] This study want to explore the indication of the conversion of immunosuppressive in chronic renal allograft nephropathy(CAN). Methodology: CAN diagnosied by biopsy received cyclosporin A were selected from June 2003 to June 2008. Each patient was evaluated by the immunological injury score(IIS) and non immunological injury score(NIIS) with semiquantitative method according to the histological changes(Banff 97).The IIS and NIIS in different subgroups

Effective group Ineffective group P valueTacrolimus IIS 12.15±4.24* 10.6±4.30** 0.396Tacrolimus N IIS 4.85±1.72# 7.0±1.89## 0.010Sirolimus IIS 6.36±2.25* 14.90±3.24** 0.000Sirolimus N IIS 5.18±1.17# 4.09±2.88## 0.258* P=0.001 # P=0.059 ** P=0.017 ##P=0.003The IIS includes allograft glomerulitis, mononuclear cell interstitial infl ammation, tubulitis,intimal arteritis(light microscope),C4d(immunofluorescence), CD4,CD8,CD68,HLA-DRexpression (immunohistochemistry). NIIS includes arteriolar hyaline thickening, fibrous intimal thickening, mesangial matrix increase, interstitial fi brosis, tubular atrophy. The patients had conversion from CsA to tacrolimus and sirolimus and were followed for 12 months. Each group was separated into effective and ineffective subgroup according to the serum creatinine on the 12th month contrast to the baseline. The IIS and NIIS were analyzed between these subgroups and try to fi nd the indications for the conversion. Result: There were 13 effective patients and 10 ineffective patients in tacrolimus group. IIS has no difference between two subgroups, The NIIS in ineffective group was higher than effective group There were 11 effective patients and 11 ineffective patients in sirolimus group. IIS in ineffective group was higher than effective group, and the NIIS has no difference. In all effective group, the IIS of tacrolimus was higher than sirolimus group, and the NIIS has no difference; In all ineffective group, the IIS in sirolimus was higher than tacrolimus and the NIIS in tacrolimus was higher than sirolimus groupConclusion: Our primary analysis disclosed that the predominant of immunological injury was unsuitable for sirolimus, and the predominant of non immunological injury was unsuitable for tacrolimus in CAN based of CsA.

Abstract# 127 Poster Board #-Session: P4-IEffi cacy of Biopsy as a Method of Detecting Early Kidney Transplant Rejections. Mohammed Shenaq,2 Jason Denny,1 Dean Kim,1 Aamir Memom,1 Vrishali Patil,1 Marwan Abouljoud,1 Robert Slater,1 Ahmad Abou Abbass,1 Anita Patel,1 Lauren Malinzak.1 1Transplant Institute, Henry Ford Hospital, Detroit, MI, USA; 2Wayne State University School of Medicine, Detroit, MI, USA.We reviewed kidney biopsies done within 30 days of transplant for presence of rejection. 1045 patients received kidney transplant at a single center from 1998 - 2008. 264 patients (25%) underwent biopsy for graft dysfunction between 1 and 30 days after transplant. Biopsy results and induction data were available for 200 patients (76%). Biopsy results alone were available for 64 patients (24%).Kidney Biopsy Results

Induction Number (%)

Mean Days to Biopsy

Mean Recipient Agewith rejection

Living/Deceased Donor with rejection

Mean Peak PRA with rejection

Rejection on Biopsy (%)

Thymoglobulin 90 (34) 10 47 4/6 21 10 (11)Unknown 64 (24) 9 42 6/7 39 13 (20)Anti IL-2 58 (22) 9 50 5/7 23 12 (20)None 36 (14) 9 30 9/2 3 11 (30)OKT3 11 (4) 10 37 4/1 12 5 (45)ATGAM 5 (2) 12 49 0/1 100 1 (20)Overall 264 28/24 52 (20)

Overall, allograft rejection within the fi rst 30 days after transplant was found in 20% of biopsies done for early kidney dysfunction. Patients receiving thymoglobulin had the lowest rate of rejection compared to patients without thymoglobulin (p<0.05). Despite having the lowest PRA at time of transplant, patients with no induction had a higher rate of rejection on biopsy.Diagnosis of rejection with biopsy for kidney dysfunction after kidney transplantation yields acceptable results and hence, should remain the gold standard test. One cannot rely heavily on the absence of pre-transplant immune risk factors to guide induction or need for biopsy. In the future, correlation with novel non-invasive immune monitoring may help delineate the need for post-transplant biopsies and guide the degree of immunosuppression.

Abstract# 128 Poster Board #-Session: P5-IWhat Is Optimal Induction Therapy for Expanded Criteria Donor Kidneys? Paul L. Tso,1 Nicole A. Turgeon,1 Kenneth A. Newell,1 Thomas C. Pearson,1 Christian P. Larsen,1 Allan D. Kirk.1 1Department of Surgery, Emory University, Atlanta, GA, USA.The optimal immunosuppressive strategy for expanded criteria donor (ECD) kidneys including induction regimen remains to be defi ned. A retrospective review of a single center experience with 48 consecutive ECD kidney transplants looked at delayed graft function (DGF), rejection at 1 and 6 months, BK viremia, death-censored graft loss, and mortality at 6 months by induction agent: rabbit anti-human thymocyte globulin (ATG), basiliximab, or none. Maintenance therapy consisted of tacrolimus, mycophenolate mofetil, and steroids.ECD kidney transplants by induction agent: 48 patients

ATG basiliximab none# of patients 22(46%) 19(40%) 7(15%)DGF 10/22(45%) 13/19(68%) 2/7(29%)Rejection<1month 3/22(14%)* 8/19(42%) 4/7(57%)Rejection<6months 7/22(32%) 8/19(42%) 5/7(71%)Death<6months 0/22(0%) 3/19(16%) 0/7(0%)Graft loss<6months (censored for death with functioning graft) 0/22(0%) 2/16(12%) 0/7(0%)

DGF+rejection<1month 0/22(0%)** 7/19(37%) 2/7(29%)BKviremia<6months 8/22(37%) 4/19(21%) 3/7(43%)*p<0.05 ATG vs none, ** p<0.05 ATG vs basiliximabThe rate of DGF was not signifi cantly infl uenced by induction agent. ATG was more effective in preventing rejection within the fi rst month than no induction, but by 6 months there were no signifi cant differences in the incidence of rejection between groups. Six month death-censored graft loss and mortality were not signifi cantly affected by induction strategy. No patient receiving ATG induction experienced both DGF and rejection within the fi rst month. Of note, 2 of 3 patients who died within 6 months had both DGF and rejection within the fi rst month.Therapy for rejection in fi rst 6 months by induction agent

ATG basiliximab none

Rejection therapy ATG - 1 patient, steroids - 5 patients, IVIg - 1 patient

ATG - 7 patients, IVIg - 1 patient*, steroids - 1 patient

steroids - 5 patients

*after receiving ATG for rejectionIncidence of BK viremia within the fi rst 6 months was not different between the 3 groups, possibly due to intensifi ed immunosuppression necessitated by rejection episodes. Short term (6 month) follow-up does not show a superior strategy for induction therapy in ECD kidneys. Induction with ATG was very effective in preventing rejection within the fi rst month and no patient in that group experienced both DGF and rejection within the fi rst month. Well designed prospective studies or analyses of registry data will provide more insight to this important topic.

Abstract# 129 Poster Board #-Session: P6-IWithdrawn




Abstract# 133 Poster Board #-Session: P10-ISingle Dose Alemtuzumab Induction Followed by Steroid Free Immunosuppression in Filipino Cohort. Beverly C. Aguila, Stephanie C. Andres, Joseph Bernard B. Africa. Medicine-Nephrology, University of Santo Tomas Hospital, Espana , Manila, Philippines.Purpose: This aims to report the one-year experience using single dose alemtuzumab followed by a steroid free regimen in standard risk primary kidney transplant patients.Methods: This is a retrospective cohort of 24 Filipino renal transplant patients who were given a single dose of alemtuzumab(30mg/IIV)as induction.A total of 1 gram of methylprednisolone/IV was given over four days and discontinued thereafter.Postoperatively, tacrolimus 0.075 mg/kg/day (target trough level of 4-7 ng/ml) and mycophenolate mofetil 2 g/day were given.Serum creatinine (SCr)) levels, estimated glomerular fi ltration rate (eGFR), and acute cellular / humoral rejection (ACR / AHR) episodes were noted at 1, 3, 6 months and 1 year. The incidence of infection and leukopenia were noted within 6 months and > 6 months post-transplant.Results: The mean SCr (mg/dl) and eGFR (ml/min/1.73m2) using the MDRD equation was 1.14 ± 0.26 and 66 ± 16 at 1 month, 1.14± 0.4 and 70± 21 at 3 months ,0.81 ± 0.41 and 51± 19 at 6 months and 0.98± 0.22 and 74± 14 at 1 year. The cumulative incidence of ACR was 0% at 1 and 3 months, 4% at 6 months and 0% at 1 year . AHR occurred in 0% at 1 month, 8% at 3 months and 0% at 6 months and 1 year. Graft survival rate at 1 year was 100%. The incidence of infections within 6 months were: 7 urinary tract

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infection, 3 CMV infection, 3 bacterial pneumonia ,2 pulmonary tuberculosis and 3 fungal infection. Three patients had UTI > 6 months post transplant. Leukopenia occurred in 12 patients, 4 of whom required granulocyte colony stimulating factor, within 6 months. None of them developed leukopenia >6months post transplant.Conclusion: Alemtuzumab is a safe and effective induction agent for steroid free maintenance immunosuppression in standard risk Filipino renal transplant patients. The incidence of infection and leukopenia was higher during the fi rst 6 months post-transplant. Its advantages are: single dose induction, use of low doses of calcineurin inhibitor and low acute rejection rate at one year.

Abstract# 134 Poster Board #-Session: P11-ISuccessful Immunosuppression Minimization in Pediatric Liver Transplantation. Udeme D. Ekong,1 Hardik Bhagat,1 Estella M. Alonso.1 1Pediatrics, Children’s Memorial Hospital, Northwestern University, Chicago, IL, USA.This report describes a group of pediatric liver transplant recipients who have undergone immunosuppression minimization at Children’s Memorial Hospital, Chicago, between January 1, 2001 and November 30, 2008. We defi ned successful minimization as normal liver enzymes at 1 year after minimization, with normal enzymes throughout all follow-up.To qualify for minimization, the patient had to meet strict, set criteria. Patients who did not meet the set criteria or had lost an organ to chronic rejection were not considered for minimization. There were 147 patients in our organ transplant tracking record (OTTR) who were ≥ 5 years post liver transplant. Of these, 56 underwent IS minimization. Patients who met the set criteria were placed on once daily Calcineurin Inhibitor at half their previous dose.50 patients successfully achieved IS minimization, while 6 patients failed at a mean of 3.7±3.2 months following the dosing change. The mean interval from transplant was signifi cantly longer in those patients who achieved successful minimization compared to those who failed IS minimization (P=0.05). Importantly, there have been no graft losses.Immunosuppression minimization is safe in carefully selected recipients, with a longer interval post liver transplantation increasing the likelihood of success.

Abstract# 135 Poster Board #-Session: P12-ITransplant Arteriopathy and Immunosuppression Minimization in Hand Transplant Recipients. Warren C. Breidenbach,1 Kadiyala V. Ravindra,2 Brenda Blair,1 Carolyn Burns,3 Robert Zaring,3 Christina L. Kaufman.1 1Christine M. Kleinert Institute, Louisville, KY, USA; 2Transplant Center, Jewish Hospital and St Mary’s Healthcare, Louisville, KY, USA; 3Pathology, Jewish Hospital and St. Mary’s Healthcare, Louisville, KY, USA.Purpose: Chronic rejection has not been defi ned in a medically compliant hand transplant recipient. Our fourth hand transplant recipient recently lost his graft at 9 months post transplant due to severe irreversible ischemia. We present data on this graft loss as well as the status of transplant arteriopathy in our four other CTA recipients. We sought to determine if this arteriopathy was associated with reduced immunosuppression regimens.Methods: We reviewed the pathology the explanted CTA graft. This led us to perform deep tissue biopsies in the four current hand transplant recipients. The biopsies included a 3mm 6 mm skin biopsy, biopsy of muscle, artery and vein from the forearm.Results: 5 hand transplants have been performed in Louisville since 1999. In 2005 we initiated a plan to reduce immunosuppression in these patients. Patients 3. 4 & 5 received Campath 1H induction were on tacrolimus and MMF maintenance with topical use of clobetasol and Prograf. Patient 4 developed ischemia of the graft and had the transplant removed at 9 months. Study of the tissues from the explant showed major changes in the arterial system. Both the major and small arteries had extensive intimal hyperplasia causing near total occlusion. Features consistent with acute cellular and humoral rejection in the other tissues were minimal. There was no evidence of donor specifi c antibodies and C4d staining was non-specifi c. Analysis of tissues from patients 1,2,3 and 5 showed evidence of chronic vascular changes some intimal hyperplasia. In all cases vascular imaging studies did not refl ect the vascular changes we saw upon deep biopsy.Conclusions: Vascular changes such as hypertrophy of the media and intimal hyperplasia can occur in CTA grafts without changes in appearance of the graft or a detectable loss of function. Additionally these changes may occur in the face of normal vascular fl ow studies. This arteriopathy was more apparent in the last three recipients who were on lower overall immunosuppression than the fi rst two patients. We have implemented research protocols to monitor arterial wall thickness. We strongly encourage programs implementing immunosuppression minimization to carefully monitor arteriopathy as well as skin rejection in CTA recipients.

Abstract# 136 Poster Board #-Session: P13-IAfrican-American Patients on a Steroid-Free Immunosuppression Regimen Using Alemtuzumab Have Excellent Outcomes Compared to Steroid-Containing Immunosuppressive Regimens Using Interleukin-2 Receptor Antagonist or Thymoglobulin Based Inductions. Elliot J. Zahtz,1 Alex O. Aquino,2 Reza Ghasemian,2 Marvin J. Tucker,2 Joseph B. Africa,2 Jimmy A. Light.2 1Surgery, Washington Hospital Center, Washington, DC, USA; 2Transplantation Services, Washington Hospital Center, Washington, DC, USA.Steroid-free immunosuppression in African-Americans (AA) has generally resulted in poor outcomes. We report our 5 year experience in AA patients with alemtuzumab induction with a steroid-free maintenance regimen, & thymoglobulin/interleukin-2 receptor antagonist (IL2RA) induction regimens with maintenance steroids.PURPOSE: To demonstrate similar outcomes in AA using alemtuzumab induction & steroid-free immunosuppression compared to steroid containing regimens using thymoglobulin/IL2RA for induction.METHODS: Single center retrospective study of AA kidney transplant recipients from 2001-06. Groupings: alemtuzumab, steroid-free; thymoglobulin/IL2RA, with steroid maintenance. All were placed on tacrolimus & MMF maintenance regimens. Excluded were patients with SPK/PAK transplants; alternate induction; & alternate maintenance regimens.RESULTS: 387 transplants were performed in patients identifi ed as AA. 196 underwent alemtuzumab induction alemtuzumab & were maintained without steroids. The remaining 191 were induced with thymoglobulin/IL2RA & maintained with steroid containing regimens. Acute rejection and mortality rates were similar in all 3 groups. A signifi cantly higher rate of graft loss was noted in the thymoglobulin based group. 202 organs were from living donors. 185 were from deceased donors. The IL2RA group had more living donors. Acute rejection, graft loss, and mortality rates were similar between alemtuzumab and thymoglobulin with a signifi cantly increased rate of acute rejection & graft loss in the living donor IL2RA group. Mean followup was 36, 56, & 60 months respectively for the alemtuzumab, thymoglobulin, & IL2RA groups.

Alemtuzumab Thymoglobulin ILR2A P-valuesPatients 196 41 150Deceased Donors (DD) 116 28 41 <0.0001Living Donors (LD) 80 13 109* <0.0001Graft Loss 24 (12.2%) 13 (31.7%)* 28 (18.7%) 0.015Acute Rejection (LD) 18 (22.5%) 8 (61.5%) 35 (32.1%)* 0.014Graft Loss (LD) 9 (11.3%) 6 (46.2%) 18 (16.5%)* 0.016(“*” indicates a statistically signifi cant result) CONCLUSIONS: Alemtuzumab based induction protocols can maintain appropriate immunosuppression in the AA patient without maintenance steroids.

Abstract# 137 Poster Board #-Session: P14-IThe Indication of Conversion from Cyclosporine to Sirolimus in Chronic Allograft Nephropathy. Jiqiu Wen,1 Zhihong Liu,1 Jinsong Chen,1 Dongrui Cheng,1 Shuming Ji,1 Qiquan Sun,1 Huiping Chen,1 Leishi Li.1 1Department of Nephrology, Jinling Hospital, Research Institute of Nephrology, Nanjing, Jiangsu, China.Objective: Chronic allograft nephropathy (CAN) was the main cause of renal graft loss and the immunosuppressive conversion from cyclosporin A (CsA) to sirolimus was an effective therapy in partial patients. This study want to analyze the indication of this conversion. Methodology: This is a retrospective study. Total 22 patients diagnosed CAN by biopsy received CsA. Each patient was evaluated by the immunological injury score(IIS) and non immunological injury score(NIIS) with semiquantitative method according to the histological changes. The IIS includes allograft glomerulitis, mononuclear cell interstitial infl ammation, tubulitis,intimal arteritis(light microscope),C4d(immunofluorescence),CD4,CD8,CD68,HLA-DRexpression (immunohistochemistry). NIIS includes arteriolar hyaline thickening, fi brous intimal thickening, mesangial matrix increase, interstitial fi brosis, tubular atrophy. The patients received the conversion from CsA to sirolimus and they were followed for 12 months. They were separated into effective and ineffective subgroup according to the serum creatinine on the 12th month contrast to the baseline. The IIS and NIIS were analyzed between two subgroups and try to fi nd the indications for the conversion. Result: There were 11 effective patients and 11 ineffective patients. IIS in ineffective subgroup was higher than effective subgroup(14.90±3.24 vs 6.36±2.25, P<0.01), and the Mononuclear cell interstitial Infl ammation, tubulitis, intimal arteritis, C4d, CD4, CD8, CD68, HLA-DR expression were higher than effective group. Although the NIIS has no difference between two subgroups(5.18±1.17 vs 4.09±2.88,P=0.258) , but the arteriolar hyaline thickening fi brous(p<0.01) and intimal

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Abstracts

thickening(p<0.01) in effective group was higher than the ineffective group. The further analysis show the achievement ratio of conversion was 100% in the patients with IIS≤10 and the NIIS ≥4.

Conclusion: Our analysis disclosed that the predominant of nonimmunological injury with low immunological injury was suitable for conversion from CsA to sirolimus in CAN patients.

Abstract# 138 Poster Board #-Session: P15-IBortezomib for Treatment of Antibody Mediated Rejection (AMR) in Intestinal Transplantation. Kyle Soltys,1 John Lunz,2 Rakesh Sindhi,1 Geoffrey Bond,1 George Mazariegos,1 Adriana Zeevi.2 1Pediatric Transplantation, Children’s Hospital of Pittsburgh, Pittsburgh, PA, USA; 2Tissue-Typing Lab/Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA.A highly-sensitized 15-year old patient presented for re-transplantation of a combined intestine and colon allograft 2 years after his primary intestinal allograft failed from complications of acute cellular rejection (ACR). At the time of retransplant, PRA was 68% with donor specifi c antibodies (DSAs) on Luminex assay. Alemtuzumab (0.4mg.kg) preconditioning was utilized with steroids and tacrolimus. As B and T cell crossmatches were positive,14 days of alternating day plasmapheresis (1.5 volume), IVIG (1g/kg) and a single dose of rituximab (375mg/m2) was utilized. Despite this, DSAs (A23,DR13 & DR52) began to rise on Luminex assay, reaching a peak during the second post-operative week. These fi ndings correlated with class I and II ELISA titers. Cylex assays (54ngATP/mL) and lymphocyte subsets showed dramatic supression. On day 12, combined ACR and AMR was diagnosed with diffuse capillary c4d staining and crypt loss due to apoptosis. Bortezomib (1.3mg/m2) was given, followed by two days of anti-thymocyte globulin (total 4 mg/kg). Follow-up biopsy demonstrated resolved ACR and minimal c4d staining. Despite resolution of ACR, disappearance of c4d staining and reduction in ELISA titers (fi g 1), DSAs persisted by Luminex analysis. Three additional doses of bortezomib were given on days 25, 34 and 38. No adverse effects of bortezomib were encountered; however there has been a persistence of class II DSAs with strong mean fl ourecence indices (MFIs >2000). There has been no recurrence of AMR or c4d depostion, however a mild, asymptomatic steroid sensitive ACR was treated on POD 46 with resolution. The patient remains on tacrolimus, steroids and mycophenolate mofetil and will continue to have weekly surveillance biopsies and quantitative follow-up of DSAs.

Conclusions. Bortezomib treatment may represent a useful adjunct in the treatment or prevention of AMR in intestinal transplantion. Our case demonstrates its potential utility and need for further investigation in this population.



Abstract# 141 Poster Board #-Session: P18-IComplication Rates after Cardiac Catheterization in Patients with Cirrhosis. Tarun K. Narang,1 Anna Montegudo,1 Mark W. Russo,1 Sanjeev Gulati.2 1Center for Digestive Diseases and Liver Transplantation, Department of Medicine, Carolinas Medical Center, Charlotte, NC, USA; 2Sanger Heart and Vascular Institute, Department of Medicine, Carolinas Medical Center, Charlotte, NC, USA.Background: Cardiac catheterization is performed in select liver transplant candidates to identify patients with signifi cant coronary artery disease that precludes transplantation.Aim: We sought to determine the complication rate related to cardiac cath in patients with cirrhosis undergoing evaluation for liver transplantation.Methods: Medical records of cirrhotic patients who underwent pre-liver transplant cardiac cath at our center from January 2005 to June 2009 were reviewed for demographic and clinical factors and complications within 30 days.Results: Of 106 patients with cirrhosis, 75.5% were males and the mean age was 59 +/- 7.5 years. Etiologies of cirrhosis included hepatitis C (24%), cryptogenic cirrhosis (21%), alcoholic liver disease (18%) and non-alcoholic fatty liver disease (10%). 9 (8.5%) patients underwent placement of a cardiac stent. The mean MELD pre cath was 15.6. 55 (51.8%) patients subsequently underwent transplantation. 8 (7.5%) patients developed a complication within 30 days of cardiac cath.Patient Complication Interval Intervention OutcomeA Retroperitoneal hematoma 0 day RBC transfusion, surgical repair AliveB Cath site hematoma 0 day Pressure, femoral stop Alive

C Complete heart block, hypotension 0 day IV atropine, IV fl uids Alive

D Afi b 3 weeks Cardizem, anticoagulation AliveE Afi b 2 weeks B blockers, anticoagulation AliveF ALF, renal failure 1 day CPR Dead

G SBP and sepsis 3 weeks IV antibiotics, cardiorespiratory support Dead

H Encephalopathy and respiratory failure 2 days IV antibiotics, cardiorespiratory

support Dead

No complications were seen in those who underwent stent placement.Conclusion: The overall complication rate was 7.5% and 30 day mortality 2.8%. This is higher than that reported for non-cirrhotic patients (overall complication rate 0.5-1%, mortality rate 0.1%). Deaths occurred in patients with advanced cirrhosis and may have been due to complications from portal hypertension and not directly related to cardiac cath. Prior to proceeding with cardiac cath, there should be compelling reasons for not initially utilizing noninvasive cardiac stress testing modalities.

Abstract# 142 Poster Board #-Session: P19-IPost-Transplant Lymphoproliferative Disorders in Children: Recent Outcomes and Response to Dual Rituximab/Chemotherapy Combination. Vikas Dharnidharka,1 Sushil Gupta,1 William Slayton,1 Regino Gonzalez-Peralta,1 Jay Fricker,1 Pamela Schuler.1 1Pediatrics, University of Florida.Post-transplant lymphoproliferative disorder (PTLD) is a neoplastic complication of over-immunosuppresion after organ transplantation. Treatment options, not standardized, include reduction of immunosuppression, rituximab and chemotherapy. Reduction of immunosuppression in the absence of immune tolerance may lead to acute rejection, graft and patient loss. At our center, we have recently used a combination dual Rituximab/Chemotherapy (R/C) with reduced dose Cyclophosphamide (600 mg/m2) for PTLD treatment. In this analysis we assessed outcomes of PTLD after chemotherapy.We retrospectively identifi ed 30 PTLD cases at our center across four solid organ systems in children between January, 1995 and July, 2008. Event-free survival (EFS) was defi ned as a period with no recurrence of PTLD or graft failure or death.Histologically most PTLDs were polyclonal (71%), polymorphic (75%), B cell type (86%) and Epstein-Barr viral RNA (EBER) positive (79%). Overall median time to PTLD was 34 months (range 4-156 months). Median follow-up time was 45 months (range 3.5-128 months). Overall four (13%) cases entered remission with reduction in immunosuppression. Eleven patients received R/C combination treatment. Four of the eleven patients received treatment as a part of the COG ANHL0221 study and were excluded from this analysis. With limiting our analysis to patients receiving any chemotherapy, 2-year EFS was 80% with the dual R/C combination and 60% with other regimens. Infection events and treatment-related morbidity were not worse with R/C.

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The R/C combination may allow more intense immunosuppression to be maintained and prevent immune reconstitution.Clinical-Pathological Characteristics and Outcomes

Organ Type No. Median time to

PTLD (months)Tumor EBER + n/total (%)

Tumor CD20 + n/total (%)

B cell type n/total (%)

2-year EFS (%)

R/C used %

Kidney 8 41 7/8 (87) 7/8 (87) 8/8 (100) 100 25Liver 9 36 7/8 (87) 8/8 (100) 7/8 (87) 86 44Heart 10 24 7/10 (70) 6/8 (75) 7/9 (78) 90 20Lung 3 32 2/3 (67) 1/2 (50) 2/3 (67) 67 0




Abstract# 146 Poster Board #-Session: P23-IEffect of Intrahepatic Stainable Iron on Cardiac Dysfunction Post Orthotopic Liver Transplantation. Tarun K. Narang,1 Khanh Le,1 Herbert B. Bonkovsky,1 Mark W. Russo,1 Ahrens A. William,2 Steven Zacks.1 1Center for Digestive Diseases and Liver Transplantation/Department of Medicine, Carolinas Medical Center, Charolotte, NC, USA; 2Department of Pathology, Carolinas Medical Center, Charlotte, NC, USA.Background/Aim:It remains unclear whether patients with increased iron burdens are predisposed to cardiac dysfunction post orthotopic liver transplantation (OLTX). We sought to determine the association between intrahepatic iron and cardiac dysfunction post OLTX in patients with end-stage liver disease (ESLD).Methods:Medical records of patients with ESLD who underwent OLTX at our center from January ‘05 to June ‘09 were reviewed. Data regarding demographics, clinical factors, and cardiac dysfunction within 30 days post OLTX were recorded. Cardiac dysfunction was defi ned by presence of any of the following: left ventricular ejection fraction (LVEF) <50% on echocardiography, radiological evidence of cardiomegaly, pleural effusion and/or pulmonary vascular congestion. Iron staining of explanted livers was graded on a scale of 0-4 (Scheuer-Lefkowitch). Those with 0/1+ staining were considered ‘group 1’ and those with 2+ or greater, ‘group 2’.Results:173 patients underwent OLTX during the study period of which 132 explants were stained for iron. Of these, 71% were males and mean age (+ SD) was 54 + 8.5 years. Etiologies of ESLD included hepatitis C (41%), NAFLD (22%), alcoholic liver disease (11%), autoimmune hepatitis (7.5%), and PSC (5%). 6 patients had hemochromatosis of which 5 were heterozygotes and 1 was a homozygote. Clinical characteristics of the patients were: 3.7%- coronary artery disease, 32.6%- diabetes mellitus, 25%- systemic arterial hypertension, 9.8%- obesity/ overweight, and 56%- alcohol abuse and/or smoking. 65% had stainable iron present in the explant. Mean Iron score was 1.17.In both groups, cardiac dysfunction was observed commonly, especially post OLTX (group 1 pre OLTX 33%, post OLTX 69%; and group 2 pre OLTX 35%, post OLTX 77%). LVEF decreased post OLTX in both groups but, interestingly, did not signifi cantly differ across the 2 groups. 58 of 84 patients (69%) in group 1 developed post OLTX cardiac dysfunction compared to 37 of 48 (77%) belonging to group 2. This difference in prevalence was not statistically signifi cant (p=0.32)Conclusion:Cardiac dysfunction is frequent among US adults with ESLD, and is present in more than two-thirds of such subjects post-OLT. Stainable iron in the explanted liver was not associated with cardiac dysfunction post OLTX.



Abstract# 149 Poster Board #-Session: P26-IChronic Rejection Results in Different Changes of Urine Metabolite and Protein Patterns Than Immunosuppressant Toxicity in the Rat. Uwe Christians,1 Wenzel Schoening,1 Volker Schmitz,1 Ryan Lawrence,1 Jelena Klawitter,1 Jost Klawitter.1 1Anesthesiology, Clinical Research and Development, University of Colorado Denver, Aurora, CO, USA.We studied the changes of urinary metabolite patterns caused by chronic rejection in the Fisher-to-Lewis kidney transplantation model and whether these were different from changes caused by immunosuppressant toxicity.Eight to 10 week old male inbred Lewis rats served as recipients in a life-supporting kidney transplantation of Fischer 344 kidneys (n=12). Prior to transplantation, on post-operative day 1, and followed by two week intervals, we collected 24-hour urine of recipient Lewis rats and carried out 1H-MRS urine metabolite profi le analysis. Urine kidney dysfunction markers including the FDA/EMEA protein panel and rat cytokines/ chemokines were analyzed using commercial multiplexing assays.The results were:- Histology showing that the animals developed chronic rejection of the transplant kidney within 30 weeks. This was not, however, refl ected by a signifi cant change in creatinine concentrations in serum- Microalbumin in urine being a more sensitive indicator which started to increase between 12-16 weeks- A principal component analysis of 1H-MRS showing metabolite patterns in individual rats beginning to separate after 12 weeks, and after 30 weeks being rendered completely different to those 4 weeks after transplantation- These metabolite patterns, however, were different from those observed during treatment of non-transplanted animals with cyclosporine, which showed metabolites patterns typically associated with immunosuppressant-induced proximal tubulus injury- The detection of several urine metabolites that seemed specifi cally associated with the kidney injury caused by the renal alloimmune processes in our rat model- Analysis of protein kidney dysfunction markers in urine showed that, as indicated by a the lack of change in markers typically associated with proximal tubulus injury such as KIM-1 and clusterin, the proximal tubulus was not affected. Interestingly, we observed marked changes in other protein markers such as EGF, a marker for distal tubulus injury.Our results suggested that, unlike immunosuppressant nephrotoxicity, the proximal tubulus is not an early target during chronic rejection. Changes in metabolite patterns were detected earlier than changes of markers currently used in the clinical management of transplant patients (creatinine concentrations in serum, creatinine clearance).

Abstract# 150 Poster Board #-Session: P27-ISirolimus, but Not Everolimus, Enhances Tacrolimus Nephrotoxicity in the Rat. Uwe Christians,1 Rahul Bohra,1 Wenzel Schoening,1 Touraj Shokati,1 Nina Brunner,1 Lawrence Ryan,1 Schniedewind Bjoern,1 Jost Klawitter,1 Jelena Klawitter.1 1Anesthesiology, University of Denver, Aurora, CO, USA.The goal of this study was to assess potential interactions between tacrolimus and the proliferation signal inhibitors sirolimus and everolimus in Lewis rats.Dosing followed a checkerboard format (0, 0.5, 1.5 and 3 mg/kg tacrolimus and 0, 0.5, 1.5 and 3 mg/kg sirolimus or everolimus, all possible combinations, n=4/ dose combination). Animals were euthanized after 28 days and blood, urine and tissues were collected. A combined 1H-MRS/ GC-MS metabolic profi ling strategy was used to identify the molecular mechanisms responsible for changes in urinary metabolites.Our results showed that tacrolimus alone and in combination with sirolimus had very similar effects on urine metabolite patterns as those observed previously for cyclosporine and its combination with sirolimus. As with cyclosporine, the kidney histologies showed specifi c tubular damage with no effect on the glomeruli or vasculature. Sirolimus clearly enhanced the negative effects of tacrolimus. To compensate for a decrease in the generation of intermediates succinate and citrate by the mitochondrial Krebs cycle, these substrates are increasingly taken up from urine via the NaDC1 and NaDC3 transporters in proximal tubulus cells. A decrease in urine Krebs cycle intermediate concentrations is a surrogate marker for mitochondrial dysfunction. Combination of sirolimus decreased succinate and citrate concentrations, suggesting inhibition of mitochondrial metabolism. As observed in the case of cyclosporine, tacrolimus and sirolimus in combination increased kidney aminoacylase activity, resulting in lower hippurate concentrations in urine. Combination of tacrolimus and everolimus resulted in very different urine metabolite patterns suggesting a lower negative impact on the kidney than the combination of tacrolimus and sirolimus: the urine hippurate, creatinine, succinate

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Abstracts

and citrate concentrations were no different than those of the controls. This was confi rmed with GC-MS analysis, in which a principal components analysis showed a distinct separation of the urine metabolite spectra of rats treated with a combination of tacrolimus and sirolimus from those treated with tacrolimus and everolimus or the controls.Our study showed that everolimus is different than sirolimus. While sirolimus enhances the negative effects of tacrolimus on the rat kidney everolimus seems to have no or a signifi cantly less negative effect.

Abstract# 151 Poster Board #-Session: P28-IIL-21 and IL-21 Receptor (IL-21R): Potential Targets for Immunotherapy in Highly Sensitized Kidney Transplant Patients. Reza Tavana, Shuqing Liu, Jean Tchervenkov. Department of Surgery, McGill University, Montreal, QC, Canada.Background: highly sensitized patients are at high risk of transplanted organ rejection since they carry high levels of anti-HLA antibodies in their serum. Interleukin-21 is a type I cytokine that acts as a regulator of B-cell anti-body production and differentiation into plasma cells as well as immunoglobulin class switching. We hypothesized IL-21 could be related with degree of sensitization. Purpose: to 1) study the effect of IL-21 on the anti-body production capacity of B-cells, 2) evaluate the expression of IL-21R on the isolated human peripheral blood B-cells and T-cells after inducing polyclonal and allogenic type stimulation in-vitro. 3) to compare the expression of IL-21R on the isolated peripheral B-cells of highly sensitized and non-sensitized patients awaiting kidney transplantation.Methods: 1) isolated B-cells were stimulated with anti-CD40 monoclonal antibody, recom binant IL-21 or combination of both and the produced IgG and IgM levels were measured by Elisa. 2)IL-21 receptor expression was compared on B-cells stimulated by SAC vs AntiCD-40 mab and also on T-cells stimulated by PHA vs anti-CD3 antibody. 3) The expression of IL-21R was studied on the peripheral blood mononuclear cells and B-cells obtained from non-sensitized (PRA<5%) and highly sensitized (PRA>50%) patients selected from the transplant waiting list. Results: (1) In-vitro Co-stimulation of B-cells with IL-21 and anti-CD40 results in higher antibody production compared with isolated anti-CD-40 or IL-21 stimulation. (2) Allogenic stimulation of human B-cells and T-cells increases the expression of IL-21receptor on these cells. (3) The expression of IL-21R is signifi cantly higher on the peripheral B-cells in highly sensitized patients compared to non-sensitized patients. (4) A signifi cant correlation exists between PRA levels and the percentage of peripheral B-cells expressing IL21 receptor.

Conclusion: IL-21 and IL-21R may be an important cytokine in B and T-cell interactions and production of antibody in highly sensitized renal transplant recipients. These molecules may represent new targets for immunotherapy and be aimed in desensitization protocols.

Abstract# 152 Poster Board #-Session: P29-IChronic Humoral Rejection of Human Kidney Allografts Is Associated with Matrix Metalloproteinase-2 Accumulation in Glomeruli and Its Release in the Urine. Waichi Wong,1 Julie Devito,1 Hoa Nguyen,1 David Sarracino,1 Fabrice Porcheray,1 Ian Dargon,1 Patricia Della Pelle,1 A. Bernard Collins,1 Rex Neal Smith,1 Nina Tolkoff-Rubin,1 Robert Colvin,1 Emmanuel Zorn.1 1Transplant Center, Massachusetts General Hospital, Boston, MA, USA.Chronic humoral rejection (CHR) accounts for a large number of graft failures following kidney transplantation. Clinical detection of CHR relies on serum creatinine, circulating donor specifi c antibodies and confi rmation with kidney biopsy. Yet, CHR is often diagnosed at a time when irreversible tubular injury has already occurred. Early non-invasive markers for CHR are crucially needed. Overall, mechanisms of renal tissue destruction associated with CHR are poorly understood. A hallmark of CHR is the interstitial fi brosis and accumulation of extra cellular matrix (ECM) in the allograft. Matrix metalloproteinase-2 (MMP-2), a 72kDA type IV collagenase, is a key regulator of ECM formation. We examined the presence of MMP-2 in allograft biopsies and in the urine of kidney transplant recipients with CHR to assess its potential contribution to kidney damage in this complication. Accumulation of MMP-2 in the glomeruli was detected by immunohistochemistry for most patients experiencing CHR but not patients with other kidney complications. The staining of the mesangium and the glomerular basement membrane (GBM) appeared uniform. Using a multiplex strategy, we found that urinary levels of MMP-2 were also signifi cantly higher in CHR patients (median 4942 pg/ml, N=27)

compared to non-CHR patients (median 598 pg/ml, N=65; p < 0.001). Elevated urinary MMP-2 correlated with higher levels of proteinuria in both patient groups. Longitudinal analysis of 4 CHR patients indicated that increase in urine MMP-2 coincided with diagnosis of humoral rejection as documented by the biopsies. Lastly, the proteolytic activity of MMP-2 in the urine was revealed with an enzymatic assay, demonstrating the presence of the active form of this enzyme. Our fi ndings together with the known function and substrates of MMP-2 suggest an active role for this enzyme in glomerular modifi cation and GBM disruption associated with CHR. Additionally, detection of these MMPs in the urine of transplant recipients may prove a dependable and non-invasive marker for CHR and potentially other types of immune rejection.


Abstract# 154 Poster Board #-Session: P31-IIn Vitro Assessment of Alloreactivity in Pediatric Living Related Liver Transplantation. Udeme D. Ekong,1 Xunrong Luo,2 Stephen D. Miller,3 Maurice R. G. O’Gorman.4 1Pediatrics, Northwestern University, Children’s Memorial Hospital, Chicago, IL, USA; 2Medicine, Northwestern University, Chicago, IL, USA; 3Microbiology-Immunology, Northwestern University, Chicago, IL, USA; 4Pathology, Northwestern University, Children’s Memorial Hospital, Chicago, IL, USA.Background: [1]identification of liver transplant recipients who demonstrate immunological quiescence vs. immune reactivity to their donor requires simple, robust, ideally noninvasive surrogate biomarkers. Objective: we sought to demonstrate donor specifi c alloreactivity in a group of pediatric living related liver transplant recipients (LRLT); and show that immune quiescence to the graft was a result of active regulation by T-regulatory cells. Design: pediatric LRLT recipients, on a single agent, specifi cally a CNI, followed at The Siragusa Transplant Center at Children’s Memorial Hospital, Chicago, were enrolled in our study. Participants: 17 recipients, their respective donors, the non-donor parent, and unrelated 3rd party individuals. Method: CD69, 71 and 25 expression following donor and non-donor stimulation was measured, along with cytokine production. Suppression of proliferation by recipient T-regulatory cells following donor and non-donor stimulation was also assessed. Results: within our cohort of recipients studied thus far, we identifi ed a group whose CD69, 71 and IFN-γ production was decreased by more than 50%, following donor vs. non-donor stimulation. This difference was signifi cant for the activation markers, CD69 and 71, (p=0.05 & 0.01) and IFN-γ (p=0.04). Additionally, lymphoproliferation in a mixed lymphocyte reaction was suppressed in the presence of 3rd component regulatory T-cells, following donor stimulation vs. non-donor stimulation, in a manner suggestive of active regulation. Conclusion: the ratio between donor vs. non-donor stimulation of lymphocyte activation markers, as well as proinfl ammatory cytokines may identify transplant recipients who demonstrate immune reactivity to their graft vs. those in immune quiescence, and active regulation is one of the mechanisms underlying this difference in ratios.

Abstract# 155 Poster Board #-Session: P32-IATP in CD4+ T Cells as an Independent Predictor for Infection and Rejection: Preliminary Results from a Pediatric Liver Transplant Study. Gunnar Brandhorst,1 Sebastian Schulz-Jürgensen,2 Victor W. Armstrong,1 Martin Burdelski,2 Michael Oellerich.1 1Department of Clinical Chemistry, University Hospital Goettingen, Goettingen, Germany; 2Department of Pediatrics, University Hospital Schleswig Holstein, Campus Kiel, Kiel, Germany.Independent biomarkers are needed for monitoring the minimization of immunosuppressive therapy. Within the scope of our study we investigated ATP in CD4+ T cells as a pharmacodynamic marker for cell-mediated immune response in stable pediatric liver transplant recipients. Twenty nine transplant patients (median age 58 months, range 11-141) and 11 outpatients without liver disease (median age 93 months, range 6-133) were included (10 patients on CSA monotherapy, 1 on CSA/MMF, 12 on TCL monotherapy, 1 on TCL/MMF, 2 on TCL/prednisolone, 2 on MMF monotherapy, 1 on everolimus/MMF). The reactivity of the immune system was measured as rise of intracellular ATP (iATP) in CD4+ T-lymphocytes following PHA-stimulation (Cylex® ImmuKnow®). A wide range of iATP levels was observed in both liver transplant recipients (359 ng/mL, range 17-828) and pediatric controls (median 394 ng/mL, range 225-801). There was no signifi cant difference in iATP levels between the two groups (p=0.276). No signifi cant correlation between drug trough concentrations and iATP levels was found, neither for CSA (r=0.037, p=0.905) nor for TCL (r=0.152, p=0.575). Two patients displayed particularly informative courses during treatment. A 2 year old boy developed EBV infection during TCL treatment (trough level 19.8 ng/mL, EBV copies 1.4x10^5). Cell-mediated immune response was low (181 iATP ng/mL, EBV copies <10^3) but recovered after TCL withdrawal and acyclovir therapy (279 iATP ng/mL). Furthermore, a 15 year old girl on MMF therapy displayed a dramatic increase in cell-mediated immune response (from 325 to >1000 iATP ng/mL) accompanied by a modest increase in liver enzymes. Thirty days later an acute rejection was clinically documented, while

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the high immune response persisted and liver enzymes increased substantially (AST 257 U/L, ALT 379 U/L, gGT 690 U/L). These preliminary results indicate that the ImmuKnow® assay might provide independent additional information to identify either over- or underimmunosuppression in pediatric liver transplant patients. Further investigations are necessary to validate and extend these fi ndings.

Abstract# 156 Poster Board #-Session: P33-ISignatures of Cytomegalovirus Protective Immunity in the Setting of Immunosuppression. Aneesh K. Mehta,1,3 Megan McCausland,2 Prasad Mulupuri,2 Jennifer Cheeseman,3 Christine Martens,3 Rafi Ahmed,2 Christian P. Larsen.3,4 1Division of Infectious Diseases, Department of Medicine, Emory University, Atlanta, GA, USA; 2Emory Vaccine Center, Emory University, Atlanta, GA, USA; 3Emory Transpant Center, Emory University, Atlanta, GA, USA; 4Department of Surgery, Emory University, Atlanta, GA, USA.Immunosuppressive medications have distinct effects on the human immune system. Most research in organ transplantation has focused on these changes effect allograft function and rejection, with relatively little data put forth into the understanding of interactions between these altered immune systems and specifi c pathogens until recently. Human Cytomegalovirus (CMV) is a latent pathogen in most humans that contributes signifi cant morbidity in immunosuppressed populations. However, little is known about how specifi c changes in immunologic memory from immunosuppressive medication relate to viral reactivation and disease. Here we have examined peripheral blood mononuclear cells (PBMCs) by fl ow cytometry to assess how immunosuppression and viral infection alters the phenotypes of T lymphocytes and correlation of these changes to disease in the patient. CMV viremia has been found in a signifi cant portion of our cohort. CMV viremia leads to proliferation CMV specifi c T cells and skews T cell memory toward the Effector Memory (CCR-, CD45R-) and Terminal Effector (CCR7-, CD45RA+) phenotype. Persistent CMV viremia also leads to increased PD1 expression on CMV specifi c memory cells and on bulk cytotoxic T cells. These changes remain after viremia has cleared. Eventually clearance of viremia is associated with the maintenance of polyfunctional (TNF+, IFN-γ+, IL-2+) CMV specifi c T cells. Thus, lack of true viral protective immunity may be predicted by ex vivo examination of patients’ immune response and might be used to develop risk algorithms and treatment protocols.



Abstract# 159 Poster Board #-Session: P36-ITOL101; a Novel αβ TCR Targeting Monoclonal Antibody. Maria Siemenow, Stephen A. Brown, John S. Thompson, Stephen D. Miller, Daniel R. Getts. Transplant, Cleveland Clinic Foundation, Cleveland, OH, USA; Research Service, University of Kentucky, Lexington, KY, USA; Research Service, University of Kentucky, Lexington, KY, USA; Department of Microbiology-Immunology, Northwestern University, Chicago, IL, USA; Science/Technology and Department of Microbiology-Immunology, Tolera Therapeutics and Northwestern University, Kalamazoo, MI, USA.Depletion of allo-reactive T cells at the induction phase after kidney transplantation promotes engraftment and donor organ survival. However, current therapies utilized to induce this depletion including Rabbit Anti-Thymocyte globulin (RIgG; ThymoglobulinTM) and Alemtuzamab (Campath-1HTM) deplete numerous cell subsets and are associated with a cytokine release syndrome, enhanced susceptibility to opportunistic infection and malignancy. Therefore there is a clear clinical requirement for a highly specifi c therapy that depletes T cells without altering other elements of the immune system or causing cytokine storm events. One such therapy may include TOL101, a monoclonal antibody specifi cally designed to deplete ab-T cells.TOL101, is a murine IgM monoclonal antibody, which we have shown by fl ow cytometry to be highly specifi c for human ab-T cells. Functionally, TOL101 triggers a number of events when it binds to its cognate target. Firstly, binding of TOL101 to the αβ T cell receptor (TCR) results in down regulation/internalization and/or shedding of not only the αβ heterodimer and through this interaction triggers modulation of CD3ε also. Importantly this activity occurs in a non-mitogenic fashion, as peripheral blood monocytes (PBMC) treated with soluble TOL101 failed to proliferate or secrete large amounts of IL2. Furthermore, TOL101 stimulated PBMC fail to produce signifi cant amounts of IFN-g, TNF or IL6 compared to OKT3 or PMA/ionomysin treated PBMC. Finally, TOL101, being an IgM has a high affi nity for complement, and thereby may also trigger specifi c lysis of ab-T cells.Taken together the data suggests TOL101 may provide a unique highly specifi c and non-mitogenic tool for depleting ab-T cells without increasing susceptibility to opportunistic infections. Clinical trials with TOL101 are planned to begin in December 2009.

DISCLOSURE: Siemenow, Maria: Grant/Research Support, tolera therapeutics; Brown, Stephen A.: Grant/Research Support, tolera therapeutics; Thompson, John S.: Grant/Research Support, tolera therapeutics; Miller, Stephen D.: Grant/Research Support, tolera therapeutics; Getts, Daniel R.: Employee, tolera therapeutics.

Abstract# 160 Poster Board #-Session: P37-IEffect of AEB-071 on Prevention of Acute Heart Allograft Rejection in the Rat. Jung Jun Lee,1,3 Yu Hui Fang,1 Dong Jin Joo,1,3 Beom Jin Lim,2 Joon Ye Kim,1 Myoung Soo Kim,1,3 Hyeon Joo Jeong,2 Yu Seun Kim.1,3 1The Research Institute for Transplantation, Yonsei University Health System, Seoul, Korea; 2Department of Pathology, Yonsei University College of Medicine, Seoul, Korea; 3Departments of Surgery, Yonsei University College of Medicine, Seoul, Jordan.Inhibition of T-cell activation is the most effi cient way to prevent transplant rejection. Protein kinase C (PKC) is an important signaling enzyme to activate and regulate T lymphocytes. AEB071 (AEB) is a low-molecular-weight compound effectively blocking early T-cell activation by selective inhibition of PKC, which is a different mechanism from that of calcineurin inhibitors. This study is aimed to reveal the effect of AEB-tacrolimus (Tac) combined therapy to prevent acute rejection in heart-transplanted rats.Brown Norway-Lewis rat strain combinations were used to investigate graft survival on 30-day, post-transplant oral therapy with AEB (15, 30, or 60 mg/kg/bid), Tac (0.3, 0.6, or 1.2 mg/kg/d), or combination regimens at varying doses (60+0.3 mg/kg, 60+0.6 mg/kg, or 60+1.2 mg/kg). Grafts were monitored by daily palpation, and cessation of palpable ventricular contraction was considered as rejection. Some cases were histologically examined 7 days after transplantation.In untreated recipients, the mean survival time (MST) of allograft was 6.83±0.41 days. AEB at 15, 30 or 60 mg/kg and Tac at 1.2 mg/kg signifi cantly prolonged graft survival to MST of 12.33±1.21, 16.67±1.21, 19.33±3.83, and 17.00±6.90 days, respectively. Combination therapy with three different doses led to extensively longer MST than monotherapy (29.5±5.74, 39.67±11.68, and >50 days, respectively). Histological analysis revealed that combination therapy notably decreased acute rejection rate. Histopathological analysis showed myocardium structure was similar to control groups despite slight change of interstitial cellular infi ltration.In conclusion, AEB, a PKC inhibitor, can successfully prevent allograft rejection with additive or synergistic effects on graft survival when combined with Tac.

Abstract# 161 Poster Board #-Session: P38-IPeripheral Tolerance in Porcine Lung Transplantation Induced by Splenocyte Infusion and Low Dose Irradiation – Modifi cations of the Protocol for Improved Clinical Feasibility. Karla Dreckmann,1 Warnecke Gregor,1 Avsar Murat,1 Knoefel Ann-Kathrin,1 Madrahimov Nodir,1 Thissen Stefanie,1 Kruse Bianca,1 Ziehme Petra,1 Sommer Wiebke,1 Gottlieb Jens,1 Simon R. Andre,1 Karstens H. Johann,1 Haverich Axel,1 Strueber Martin.1 1Hannover Medical School, Hannover, Germany.Purpose: Low dose irradiation and donor splenocyte infusion led to the maintenance of peripheral tolerance in a porcine lung transplantation model. Here, we wished to study modifi cations of the protocol for improved clinical feasibility.Methods: Left-sided lung transplantation from MHC mismatched donors was performed in 40 adult minipigs. Intravenous pharmacologic immunosuppression was withdrawn after 28 postoperative days. The animals received a splenocyte infusion on the day of lung transplantation, either from the lung donor, or from a MHC mismatched third party donor. In one group splenocytes were processed to subcellular antigen. Non-myeloablative irradiation was administered within 12 hours before transplantation. Allograft survival was monitored by chest radiographs and bronchoscopy.Results: Transplant survival was prolonged beyond 200 days in the group receiving donor splenocytes (n=5). Upon infusion of third party splenocytes, 2 out of 6 animals rejected before day 200, but the remainder developed stable tolerance. Substitution of intact splenocytes with donor-specifi c subcellular antigen led to long term survival in only 2 out of 6 animals. High levels of circulating CD4+CD25high+ regulatory T cells were present in eventual long term survivors. Furthermore preliminary data suggests that shielding of the bone marrow from irradiation, but not applying whole body irradiation at a reduced dose, signifi cantly reduced side effects while staying effective for tolerance induction.Conclusion: This data shows that the infusion of easily available non-specifi c cellular alloantigen is nearly as effi cient as donor-specifi c alloantigen. Additionally, the toxicity of the irradiation regime may be further reduced, enhancing feasibility in human lung transplantation.

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Abstracts

Abstract# 162 Poster Board #-Session: P39-INovel Transplantation Approach for Correction of Type 1 Diabetes without Insulin or Immunosuppression. Subhadra C. Gunawardana.1

1Molecular Physiology & Biophysics, Vanderbilt University, Nashville, TN, USA.Based on intriguing observations made during correction of type 1 diabetes (T1D) through subcutaneous transplantation of embryonic pancreas, this study explores a revolutionary new approach for T1D requiring no insulin replacement and little or no immunosuppression.Literature and preliminary data indicate adipose tissue hormones can both compensate for and complement insulin in maintaining glucose homeostasis. This study was performed to determine whether replenishment of healthy adipose tissue alone can correct T1D independent of insulin. Adipose tissue of T1D recipients was replenished by subcutaneous transplantation embryonic brown adipose tissue or neonatal white adipose tissue from same strain donors.Results: In 5 streptozotocin-diabetic mice including 2 immune-competent C57BL/6 mice, average basal blood glucose decreased from 374 ±60 to 165±24 mg/dl (p<0.005) in 3-6 weeks after adipose tissue replacement. Fig. 1 shows signi? cantly improved glucose tolerance in C57BL/6 mice 7 weeks after embryonic brown adipose tissue transplants. While complete normalization of glucose tolerance is not reached yet, our previous data indicates subcutaneous transplants may take up-to 12 weeks to achieve euglycemia, a delayed response compared to deeper sites. From 9 mice receiving transplants, 4 were unsuccessful and did not improve diabetes. Histological examination of tissue post-mortem demonstrated that adipose tissue in successful transplants had less in? ammation. Histology was also used to verify that no false positive results occurred through regeneration of pancreatic beta cells.

Conclusions: These data demonstrate the ability of adipose tissue to compensate for insulin under speci? c conditions. Subcutaneous transplantation of embryonic brown adipose tissue led to considerable regeneration of recipients’ adipose tissue followed by marked improvement of glucose tolerance, provided the replenished adipose tissue remained healthy and un-in? amed. As these outcomes occurred with no immunosuppression, these data show great promise for a safe, convenient and non-invasive transplantation approach for T1D without the complications of insulin replacement therapy.

Abstract# 163 Poster Board #-Session: P40-ITreatment with ILT3-Fc Improves Allogeneic Pancreatic Islet Graft Survival in hu-NOD/SCID Mice. George Vlad,1 Zhuoru Liu,1 Qinying Zhang,1 Raffaello Cortesini,1 Nicole Suciu-Foca.1 1Pathology, Columbia University, New York, NY, USA.Diabetic NOD/SCID mice were transplanted with human pancreatic islet cells. Mice in which euglycemia normalized following transplantation were “humanized” by injection of allogeneic peripheral blood mononuclear cells and treated with ILT3-Fc or control human IgG. Rejection (diagnosed after two consecutive blood glucose readings >350 mg/dl) became apparent in all control animals within 49 days. In contrast, ILT3-Fc-treated mice retained functional, insulin-producing islets for up to 91 days. Human CD4 and CD8 T-cells recovered from the spleens of these mice revealed that ILT3-Fc treatment suppressed the expression of cytokines and CD40L and induced the differentiation of human CD8(+) T suppressor cells that inhibited Th alloreactivity against graft HLA antigens. T-cells allostimulated in vitro in the presence of ILT3-Fc inhibited pro-in? ammatory changes in human pancreatic islet cells. Finally, ILT3-Fc treatment was also shown to be effective in preventing ongoing rejection by initiating the treatment only after detection of hyperglycemia in some animals. The data indicates that ILT3-Fc is a potent immunoregulatory agent that suppressed islet allograft rejection in humanized NOD/SCID mice.


Withdrawn

Abstract# 165 Poster Board #-Session: P42-IImmune Active Effect of Chemokine RANTES on Human Peripheral Mononuclear Cells. Xiao Gu,1 Jin Yang,2 Tao Gu,4 Hong Zaho.3

1Department of Urology, University of Oklahoma, Oklahoma City, OK, USA; 2Department of Urology, Af? liated Hospital of Yangzhou University, Yangzhou, Jiangsu, China; 3Institute for Cellular Therapeutics, University of Louisville, Louisville, KY, USA; 4Department of Immunology, State University of New York at Buffalo, Buffalo, NY, USA.BACKGROUND: Chemotactic signals not only induce oriented movement, but also function on immune activation. RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) is one of the important chemokines in the family. We observed the proliferation of peripheral mononuclear cells (PMNCs), phenotype of lymphocytes and in? uence of pyrrolidine dithiocarbamate (PDTC) and CTLA4Ig on proliferation after treatment of recombinant human RANTES using PMNCs as subjects, including lymphocytes and monocytes, and investigated the involved mechanisms by which RANTES works.METHODS: PMNCs from healthy donors were isolated by density gradient centrifugation, stimulated by different concentrations of recombinant human RANTES and/or anti-CD3 monoclonal antibody. Cells with signi? cantly proliferative response were intervened by pyrrolidine dithiocarbamate (PDTC) or CTLA4Ig.3H-thymidine incorporation assay was used to detect the proliferation of mononuclear cells. Flow cytometry was applied to test the phenotypes of lymphocytes.RESULTS: Proliferation of PMNCs reached two peaks with recombinant human RANTES concentrations of 100 µg/L and 5000 µg/L respectively. The 100 µg/L induced proliferation was signi? cantly higher than that in the presence of 50 µg/L anti-CD3 monoclonal antibody alone(P < 0.05). There were no rivalry or synergistic effect between them. The immune active effects of recombinant human RANTES could be inhibited by PDTC or CTLA4Ig in a dose dependent manner. After RANTES treatment, CD25 expression upregulated (P < 0.05) and the CCR5 expression decreased (P < 0.05), but there were no signi? cant differences in CD28 expression and the ratio of CD4/CD8 (P > 0.05).Effects of recombinant human (rh) RANTES on expression of CD25, CCR5 and CD28 of lymphocytesPhenotype of lymphocytes Before stimulation After stimulation PCD25 2.48±0.13 3.92±0.17 0.011CCR5 1.08±0.09 0.49±0.13 0.036CD28 6.14±0.95 6.41±0.16 0.670

CONCLUSION: RANTES is able to induce the immune activation of mononuclear cells, which depends on the activation of interleukin-2 signal pathway, CD28 co-stimulatory pathway and nuclear factor-κB, but independent of CD3 activation.

Abstract# 166 Poster Board #-Session: P43-I

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