ME T H O D S I N MO L E C U L A R B I O L O G Y

Series EditorJohn M. Walker

School of Life and Medical SciencesUniversity of HertfordshireHatfield, Hertfordshire, UK

For further volumes:http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols andmethodologies in the critically acclaimedMethods in Molecular Biology series. The series wasthe first to introduce the step-by-step protocols approach that has become the standard in allbiomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagentsneeded to complete the experiment, and followed by a detailed procedure that is supportedwith a helpful notes section offering tips and tricks of the trade as well as troubleshootingadvice. These hallmark features were introduced by series editor Dr. John Walker andconstitute the key ingredient in each and every volume of the Methods in Molecular Biologyseries. Tested and trusted, comprehensive and reliable, all protocols from the series areindexed in PubMed.

Immune Mediators in Cancer

Methods and Protocols

Edited by

Ivana Vancurova and Yan Zhu

Department of Biological Sciences, St. John’s University, Queens, NY, USA

EditorsIvana VancurovaDepartment of Biological SciencesSt. John’s UniversityQueens, NY, USA

Yan ZhuDepartment of Biological SciencesSt. John’s UniversityQueens, NY, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)Methods in Molecular BiologyISBN 978-1-0716-0246-1 ISBN 978-1-0716-0247-8 (eBook)https://doi.org/10.1007/978-1-0716-0247-8

© Springer Science+Business Media, LLC, part of Springer Nature 2020This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material isconcerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproductionon microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,computer software, or by similar or dissimilar methodology now known or hereafter developed.The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulationsand therefore free for general use.The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed tobe true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Cover Illustration: Ovarian cancer cells grown in a 3D culture.

This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of SpringerNature.The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.

Preface

It is the expression of various immune mediators in tumor cells that dictates the crosstalkbetween tumor cells and immune cells and determines cancer cell survival, proliferation,invasion, and tumor progression. These immune mediators include a variety of cytokinesand chemokines, their receptors, as well as the recently identified immune checkpoints andtheir ligands. Knowledge of the quantitative and qualitative nature of these immune med-iators is critical for early cancer diagnosis, but also for our understanding of the mechanismsregulating cancer progression. However, analysis of these immune mediators in tumor cellsand tissues faces many challenges that include the low abundance, the need to distinguishbetween active and latent forms, and the need to measure multiple mediators in a singleassay. This volume provides a comprehensive collection of classic and cutting-edge meth-odologies, as well as bioinformatics and genome editing approaches that are used to quantifyimmune mediators and analyze their function and biological activity in cancer cells andtissues.

The chapters are divided into four main parts. The first part focuses on the detection ofimmune mediators in cancer cells and tissues, biopsies, and blood samples, using assays thatmeasure the total levels of mediators regardless of their biological activities. These assaysinclude immunoblotting, ELISA, flow cytometry, and immunohistochemistry. In addition,the complexity of immune responses has led to the development of multiplex technologiesthat allow for the simultaneous detection of large numbers of proteins in small volumes.Thus, the first part also includes the Luminex multiplex assays that allow the simultaneousmeasurement of multiple mediators in small sample volumes with time- and cost-savingadvantages.

However, since the aforementioned assays cannot distinguish between biologicallyactive and inactive molecules, bioassays are used to measure the different biological activitiesof various cytokines, chemokines, and immune checkpoints. These bioassays include analysisof cytokine-induced cancer cell invasion, migration, ability to grow and proliferate in 3Dculture spheroids, autophagy, ferroptosis, and isotype switching. Moreover, the second partincludes approaches used for blocking the cytokine biological activity by using DNAaptamers, as well as methods used for probing cytokine-inducedmetabolic changes in cancercells.

Often, there is a need to analyze the intracellular levels of various immune mediators orthe mechanisms regulating their expression and signaling in cancer cells and tumor micro-environment. The third part includes protocols for the analysis of cytokines and immunecheckpoints by immunoblotting and flow cytometry, as well as protocols for quantitativeanalysis of their promoter occupancy by using chromatin immunoprecipitation, and for theanalysis of gene polymorphisms and alternative splicing. In addition, the third part includeschapters focusing on the use of microscopic and optogenetic approaches to analyze inter-actions of cancer cells with tumor microenvironment and to manipulate signaling pathways.

Recent advances in DNA and RNA sequencing and proteomic technology have led tothe generation of a large number of datasets. Bioinformatical analyses of those datasets canprovide valuable information for studying the function and biological activities of immunemediators in cancer cells and tissues. The fourth part includes some of the bioinformaticaltools used for phylogenetic analyses and identification of long non-coding RNAs to expand

v

our knowledge of immune mediators’ genes and their regulation. This part also includesprotocols for gene knock-out and knock-in in cell-based models through genome editing.

There is no best method, since each has its own merits and limitations. Choosing theright method depends on the sample, purpose of the assay, and the available instrumenta-tion. Often, using combined information from several different approaches can yield themost accurate knowledge of the quantitative and qualitative nature of immune mediators’expression and regulation. The reliability of the protocols has been tested in laboratoriesaround the world. Each chapter is appended by notes that navigate through the protocoland serve as a troubleshooting guide. We hope that this book will be useful to not onlybiochemists, molecular biologists, cancer biologists, and immunologists but also physician-scientists working in the field of immunology and cancer research.

We would like to thank all the authors for their enthusiastic help and support inassembling this volume; we realize that in the highly competitive environment of academicresearch, many scientists are reluctant to commit their time to writing book chapters andmethod articles. We also would like to express our sincere gratitude to the series editor,Dr. John Walker, and the outstanding staff of Humana Press for their support, help, andencouragement.

Queens, NY, USA Ivana VancurovaYan Zhu

vi Preface

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vContributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

PART I DETECTION OF IMMUNE MEDIATORS IN CANCER

CELLS AND TISSUES

1 A Useful Guide for Analysis of Immune Mediators in Cancerby Fluorochrome (Luminex) Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Maria Faresjo

2 Analysis of Circulating HMGB1 in Human Serum . . . . . . . . . . . . . . . . . . . . . . . . . . 15Weiqiang Chen, Guoqiang Bao, Lin Zhao, and Haichao Wang

3 Analysis of IL-22 and Th22 Cells by Flow Cytometry in SystemicLupus Erythematosus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29Zhuang Ye, Ling Zhao, Qi Gao, Yanfang Jiang, Zhenyu Jiang,and Cong-Qiu Chu

4 Application of Immunohistochemistry in Basic and Clinical Studies . . . . . . . . . . . 43Aihua Li and Dong-Hua Yang

5 Interleukin-1 (IL-1) Immunohistochemistry Assay in Oral SquamousCell Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57Takaaki Kamatani

6 Luminex xMAP Assay to Quantify Cytokines in Cancer Patient Serum . . . . . . . . 65Helena Kupcova Skalnikova, Katerina Vodickova Kepkova,and Petr Vodicka

7 Detection of Cytokine Receptors Using Tyramide Signal Amplificationfor Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89Herui Wang, Ryan L. Pangilinan, and Yan Zhu

PART II CYTOKINE BIOASSAYS

8 Analysis of IFNγ-Induced Migration of Ovarian Cancer Cells . . . . . . . . . . . . . . . . 101Bijaya Gaire, Mohammad M. Uddin, Yue Zou, and Ivana Vancurova

9 Interleukin-8-Induced Invasion Assay in Triple-NegativeBreast Cancer Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107Mohammad M. Uddin, Bijaya Gaire, Betsy Deza, and Ivana Vancurova

10 Interleukin-8 Induces Proliferation of Ovarian Cancer Cellsin 3D Spheroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117Mohammad M. Uddin, Bijaya Gaire, and Ivana Vancurova

11 Detection of Ferroptosis by BODIPY™ 581/591 C11. . . . . . . . . . . . . . . . . . . . . . 125Alejandra M. Martinez, Ahryun Kim, and Wan Seok Yang

12 Methods for Studying TNFα-Induced Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . 131Sheyda Najafi, Ehab M. Abo-Ali, and Vikas V. Dukhande

vii

13 Isolation of Antibody Binders to MISIIR from a Phage DisplayLibrary by Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147Andy Qingan Yuan

14 Measuring Chimeric Antigen Receptor T Cells (CAR T Cells)Activation by Coupling Intracellular Cytokine Staining with Flow Cytometry . . 159Chong Xu and Yibo Yin

15 Analysis of Interleukin-4-Induced Class Switch Recombinationin Mouse Myeloma CH12F3-2 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167Wenjun Wu, Zhihui Xiao, Deon Buritis, and Vladimir Poltoratsky

16 Single-Stranded DNA Aptamers Against TNF and Their PotentialApplications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181Shao Tao, Pingfang Song, Xiaowei Zhang, Lingshu Zhang,and Cong-Qiu Chu

17 Probing Metabolic Changes in IFNγ-Treated Ovarian Cancer Cells . . . . . . . . . . . 197Pritpal Kaur, Shreya Nagar, Madhura Bhagwat,Mohammad M. Uddin, Yan Zhu, and Ales Vancura

PART III EXPRESSION AND REGULATION OF IMMUNE MEDIATORS

IN CANCER CELLS

18 Immunoblotting Analysis of Intracellular PD-L1 Levelsin Interferon-γ-Treated Ovarian Cancer Cells Stably Transfectedwith Bcl3 shRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211Sveta Padmanabhan, Yue Zou, and Ivana Vancurova

19 Flow Cytometry Analysis of Surface PD-L1 Expression Inducedby IFNγ and Romidepsin in Ovarian Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . 221Sveta Padmanabhan, Yue Zou, and Ivana Vancurova

20 Analysis of PD-L1 Transcriptional Regulation in Ovarian CancerCells by Chromatin Immunoprecipitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229Yue Zou, Sveta Padmanabhan, and Ivana Vancurova

21 Real-Time PCR Assay for the Analysis of Alternative Splicingof Immune Mediators in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241Ruizhi Wang, Md. Faruk Hossain, Jovan Mirkovic,Samuel Sabzanov, and Matteo Ruggiu

22 Combined Single-Cell Measurement of Cytokine mRNAand Protein in Immune Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259Julian J. Freen-van Heeren, Benoit P. Nicolet, and Monika C. Wolkers

23 Microscopic Methods for Analysis of Macrophage-Induced TunnelingNanotubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273Kiersten P. Carter, Jeffrey E. Segall, and Dianne Cox

24 Optogenetics: Rho GTPases Activated by Light in Living Macrophages . . . . . . . 281Maren Hulsemann, Polina V. Verkhusha, Peng Guo,Veronika Miskolci, Dianne Cox, and Louis Hodgson

viii Contents

PART IV BIOINFORMATICS AND GENOME EDITING APPROACHES

25 High-Throughput RNA Interference Screen TargetingSynthetic-Lethal Gain-of-Function of Oncogenic Mutant TP53in Triple-Negative Breast Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297Susumu Rokudai

26 Discovering Transcription Factor Noncoding RNA TargetsUsing ChIP-Seq Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305Vitalay Fomin and Carol Prives

27 Phylogenetic Analyses of Chemokine Receptors from SequenceRetrieval to Phylogenetic Trees . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313Juan C. Santos

28 Generation of IL17RB Knockout Cell Lines UsingCRISPR/Cas9-Based Genome Editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345Olivia Hu, Alessandro Provvido, and Yan Zhu

29 Engineering Mutation Clones in Mammalian Cells with CRISPR/Cas9 . . . . . . . 355Zijun Huo, Jian Tu, Dung-Fang Lee, and Ruiying Zhao

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371

Contents ix

Contributors

EHAB M. ABO-ALI • Department of Pharmaceutical Sciences, College of Pharmacy andHealth Sciences, St. John’s University, Queens, NY, USA

GUOQIANG BAO • Laboratory of Emergency Medicine, The Feinstein Institute for MedicalResearch, Manhasset, NY, USA

MADHURA BHAGWAT • Department of Biological Sciences, St. John’s University, Queens, NY,USA

DEON BURITIS • Department of Biological Sciences, St. John’s University, Queens, NY, USAKIERSTEN P. CARTER • Department of Anatomy and Structural Biology, Albert Einstein

College of Medicine, Bronx, NY, USAWEIQIANG CHEN • Laboratory of Emergency Medicine, The Feinstein Institute for Medical

Research, Manhasset, NY, USACONG-QIU CHU • Division of Arthritis and Rheumatic Diseases, Oregon Health & Science

University, Portland, OR, USA; Section of Rheumatology, VA Portland Health CareSystem, Portland, OR, USA

DIANNE COX • Department of Anatomy and Structural Biology, Albert Einstein College ofMedicine, Bronx, NY, USA; Gruss-Lipper Biophotonics Center, Albert Einstein College ofMedicine, Bronx, NY, USA; Department of Molecular and Developmental Biology, AlbertEinstein College of Medicine, Bronx, NY, USA

BETSY DEZA • Department of Biological Sciences, St. John’s University, Queens, NY, USAVIKAS V. DUKHANDE • Department of Pharmaceutical Sciences, College of Pharmacy and

Health Sciences, St. John’s University, Queens, NY, USAMARIA FARESJO • Department of Natural Science and Biomedicine, School of Health and

Welfare, Jonkoping University, Jonkoping, SwedenVITALAY FOMIN • Department of Biological Sciences, Columbia University, New York, NY,

USAJULIAN J. FREEN-VAN HEEREN • Department of Hematopoiesis, Sanquin Research-

Amsterdam UMC Landsteiner Laboratory and Oncode Institute, Amsterdam, TheNetherlands

BIJAYA GAIRE • Department of Biological Sciences, St. John’s University, Queens, NY, USAQI GAO • Department of Rheumatology and Immunology, The First Bethune Hospital, Jilin

University, Changchun, ChinaPENG GUO • Department of Anatomy and Structural Biology, Albert Einstein College of

Medicine, Bronx, NY, USA; Gruss-Lipper Biophotonics Center, Albert Einstein College ofMedicine, Bronx, NY, USA; Analytical Imaging Facility, Albert Einstein College ofMedicine, Bronx, NY, USA

MAREN HULSEMANN • Department of Anatomy and Structural Biology, Albert EinsteinCollege of Medicine, Bronx, NY, USA; Gruss-Lipper Biophotonics Center, Albert EinsteinCollege of Medicine, Bronx, NY, USA

LOUIS HODGSON • Department of Anatomy and Structural Biology, Albert Einstein Collegeof Medicine, Bronx, NY, USA; Gruss-Lipper Biophotonics Center, Albert Einstein College ofMedicine, Bronx, NY, USA

MD. FARUK HOSSAIN • Department of Biological Sciences, St. John’s University, Queens, NY,USA

xi

OLIVIA HU • Department of Biological Sciences, St. John’s University, Queens, NY, USAZIJUN HUO • Department of Integrative Biology and Pharmacology, McGovern Medical

School, The University of Texas Health Science Center at Houston, Houston, TX, USA;Department of Endocrinology, The First Affiliated Hospital of Sun Yat-sen University,Guangzhou, People’s Republic of China

YANFANG JIANG • Department of Central Laboratory, The First Bethune Hospital, JilinUniversity, Changchun, China

ZHENYU JIANG • Department of Rheumatology and Immunology, The First Bethune Hospital,Jilin University, Changchun, China

TAKAAKI KAMATANI • Department of Oral and Maxillofacial Surgery, Showa UniversitySchool of Dentistry, Ota-City, Tokyo, Japan

PRITPAL KAUR • Department of Biological Sciences, St. John’s University, Queens, NY, USAAHRYUN KIM • Department of Biological Sciences, St. John’s University, Queens, NY, USAHELENA KUPCOVA SKALNIKOVA • Laboratory of Applied Proteome Analyses, Institute of

Animal Physiology and Genetics of The Czech Academy of Sciences, Libechov, Czech RepublicDUNG-FANG LEE • Department of Integrative Biology and Pharmacology, McGovern

Medical School, The University of Texas Health Science Center at Houston, Houston, TX,USA

AIHUA LI • Epitomics—An Abcam Company, Burlingame, CA, USAALEJANDRA M. MARTINEZ • Department of Biological Sciences, St. John’s University, Queens,

NY, USAJOVAN MIRKOVIC • Department of Biological Sciences, St. John’s University, Queens, NY,

USAVERONIKA MISKOLCI • Department of Medical Microbiology and Immunology, University of

Wisconsin–Madison, Madison, WI, USASHREYA NAGAR • Department of Biological Sciences, St. John’s University, Queens, NY, USASHEYDA NAJAFI • Department of Pharmaceutical Sciences, College of Pharmacy and Health

Sciences, St. John’s University, Queens, NY, USABENOIT P. NICOLET • Department of Hematopoiesis, Sanquin Research-Amsterdam UMC

Landsteiner Laboratory and Oncode Institute, Amsterdam, The NetherlandsSVETA PADMANABHAN • Department of Biological Sciences, St. John’s University, Queens, NY,

USARYAN L. PANGILINAN • Department of Biological Sciences, St. John’s University, Jamaica, NY,

USAVLADIMIR POLTORATSKY • Department of Pharmaceutical Sciences, St. John’s University,

Queens, NY, USA; Department of Biological Sciences, St. John’s University, Queens, NY,USA

CAROL PRIVES • Department of Biological Sciences, Columbia University, New York, NY,USA

ALESSANDRO PROVVIDO • Department of Biological Sciences, St. John’s University, Queens,NY, USA

SUSUMU ROKUDAI • Department of Molecular Pharmacology and Oncology, GunmaUniversity Graduate School of Medicine, Maebashi, Gunma, Japan

MATTEO RUGGIU • Department of Biological Sciences, St. John’s University, Queens, NY,USA

SAMUEL SABZANOV • Department of Biological Sciences, St. John’s University, Queens, NY,USA

xii Contributors

JUAN C. SANTOS • Department of Biological Sciences, College of Liberal Arts and Sciences,St. John’s University, Queens, NY, USA

JEFFREY E. SEGALL • Department of Anatomy and Structural Biology, Albert Einstein Collegeof Medicine, Bronx, NY, USA; Gruss-Lipper Biophotonics Center, Albert Einstein College ofMedicine, Bronx, NY, USA

PINGFANG SONG • Division of Arthritis and Rheumatic Diseases, Oregon Health & ScienceUniversity, Portland, OR, USA; Rheumatology Section, VA Portland Health Care System,Portland, OR, USA

SHAO TAO • Division of Arthritis and Rheumatic Diseases, Oregon Health & ScienceUniversity, Portland, OR, USA; Rheumatology Section, VA Portland Health Care System,Portland, OR, USA; Division of Experimental Medicine, Faculty of Medicine, McGillUniversity, Montreal, QC, Canada

JIAN TU • Department of Integrative Biology and Pharmacology, McGovern Medical School,The University of Texas Health Science Center at Houston, Houston, TX, USA;Department of Musculoskeletal Oncology, The First Affiliated Hospital of Sun Yat-senUniversity, Guangzhou, People’s Republic of China

MOHAMMAD M. UDDIN • Department of Biological Sciences, St. John’s University, Queens,NY, USA

ALES VANCURA • Department of Biological Sciences, St. John’s University, Queens, NY, USAIVANA VANCUROVA • Department of Biological Sciences, St. John’s University, Queens, NY,

USAPOLINA V. VERKHUSHA • Department of Anatomy and Structural Biology, Albert Einstein

College of Medicine, Bronx, NY, USAPETR VODICKA • Laboratory of Applied Proteome Analyses, Institute of Animal Physiology

and Genetics of The Czech Academy of Sciences, Libechov, Czech RepublicKATERINA VODICKOVA KEPKOVA • Laboratory of Applied Proteome Analyses, Institute of

Animal Physiology and Genetics of The Czech Academy of Sciences, Libechov, Czech RepublicHAICHAO WANG • Laboratory of Emergency Medicine, The Feinstein Institute for Medical

Research, Manhasset, NY, USA; Donald and Barbara Zucker School of Medicine atHofstra/Northwell, Hempstead, NY, USA; The Feinstein Institute for Medical Research,Northwell Health System, Manhasset, NY, USA

HERUI WANG • Department of Biological Sciences, St. John’s University, Jamaica, NY, USARUIZHI WANG • Department of Biological Sciences, St. John’s University, Queens, NY, USAMONIKA C. WOLKERS • Department of Hematopoiesis, Sanquin Research-Amsterdam UMC

Landsteiner Laboratory and Oncode Institute, Amsterdam, The NetherlandsWENJUN WU • Department of Pharmaceutical Sciences, St. John’s University, Queens, NY,

USAZHIHUI XIAO • Department of Pharmaceutical Sciences, St. John’s University, Queens, NY,

USACHONG XU • Center for Cellular Immunotherapies, Perelman School of Medicine, University

of Pennsylvania, Philadelphia, PA, USA; The Parker Institute for Cancer Immunotherapy,Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA;Department of Pathology and Laboratory Medicine, Perelman School of Medicine,University of Pennsylvania, Philadelphia, PA, USA; Department of Medicine, PerelmanSchool of Medicine, University of Pennsylvania, Philadelphia, PA, USA

DONG-HUA YANG • Department of Pharmacology, College of Pharmacy and Health Sciences,St. John’s University, Queens, NY, USA

WAN SEOK YANG • Department of Biological Sciences, St. John’s University, Queens, NY, USA

Contributors xiii

ZHUANG YE • Department of Rheumatology and Immunology, The First Bethune Hospital,Jilin University, Changchun, China

YIBO YIN • Center for Cellular Immunotherapies, Perelman School of Medicine, University ofPennsylvania, Philadelphia, PA, USA; Department of Neurosurgery, Perelman School ofMedicine, University of Pennsylvania, Philadelphia, PA, USA; The Fourth Section ofDepartment of Neurosurgery, The First Affiliated Hospital of Harbin Medical University,Harbin, P.R. China

ANDY QINGAN YUAN • Department of Antibody Technology, EXCYTE LLC, Rockville, MD,USA

XIAOWEI ZHANG • Division of Arthritis and Rheumatic Diseases, Oregon Health & ScienceUniversity, Portland, OR, USA; Rheumatology Section, VA Portland Health Care System,Portland, OR, USA

LINGSHU ZHANG • Division of Arthritis and Rheumatic Diseases, Oregon Health & ScienceUniversity, Portland, OR, USA; Rheumatology Section, VA Portland Health Care System,Portland, OR, USA; Department of Rheumatology and Immunology, West ChinaHospital, Sichuan University, Chengdu, China

LIN ZHAO • Laboratory of Emergency Medicine, The Feinstein Institute for Medical Research,Manhasset, NY, USA

LING ZHAO • Department of Rheumatology and Immunology, The First Bethune Hospital,Jilin University, Changchun, China

RUIYING ZHAO • Department of Integrative Biology and Pharmacology, McGovern MedicalSchool, The University of Texas Health Science Center at Houston, Houston, TX, USA

YAN ZHU • Department of Biological Sciences, St. John’s University, Queens, NY, USAYUE ZOU • Department of Biological Sciences, St. John’s University, Queens, NY, USA

xiv Contributors