cord blood-derived regulatory t cells: a novel cure for graft-versus-host-disease siddharth damania...

Cord Blood-Derived Regulatory T Cells:A Novel Cure for Graft-Versus-Host-

Disease

Siddharth Damania2007

University of MinnesotaCancer Center

Bone-Marrow and Stem-Cell Transplants

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6,000 Americans

Bone-Marrow and Stem-Cell Transplants

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Bone-Marrow and Stem-Cell Transplants

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Over half develop GVHD

Graft-Versus-Host-Disease (GVHD)

• GVHD occurs when transplanted cells reject host cells in the patient

Patient CellTransplanted

Cell

GVHD

GVHD

GVHD

GVHD

GVHD

Purpose

• To use T-reg cells to cure mice with human GVHD

First Goal

• To devise a new protocol to derive and purify CD4+25+ T-reg cells using human umbilical cord blood

Types of T cells

• Cytotoxic T cells• Helper T cells• T-reg cells

http://www.britannica.com/eb/art/print?id=85110&articleTypeId=0

CD4+25+ T-reg Cells

• Sakaguchi (2005)– Increased capability to suppress

immune responses– Low numbers increase risk of

autoimmune diseases

CD4+25+ T-reg Cells

• Sakaguchi (2005)– Increased capability to suppress

immune responses– Low numbers increase risk of

autoimmune diseases

• Suri-Payer et al (2006)– Potential to suppress multiple types

of autoimmune diseases

Umbilical Cord Blood

• Godfrey et al (2005)– T-reg cells isolated from peripheral

blood are often impure and require multiple purification processes

Umbilical Cord Blood

• Godfrey et al (2005)– T-reg cells isolated from peripheral

blood are often impure and require multiple purification processes

– Umbilical cord blood facilitates easier purification and presents purer quantities of T-reg cells

Procedure

Procedure

Procedure

Procedure

Procedure

CD4+25+ T-reg Cells

3/28 KT32

KT32 -41 BBL KT32 -OX40

FACS T-reg Cell Purity (CD4/CD25)

86.2% 95.4%

91.5% 81.0%

Second Goal

• To culture derived CD4+25+ T-reg cells

Second Goal

• To culture derived CD4+25+ T-reg cells

CD4+25+ T-reg Cells

Second Goal

• To culture derived CD4+25+ T-reg cells

CD4+25+ T-reg Cells

Second Goal

• To culture derived CD4+25+ T-reg cells

CD4+25+ T-reg Cells

CD4+25+ T-reg Cells

3/28 Cell Line

KT32 Cell Line

KT32-41BBL Cell Line

KT32-OX40L Cell Line

Culture Expansion of T-reg Cells

KT-32-OX40:y =

504.24e0.7776x

R2 = 0.7983

3/28:y =

10715e0.5056x

R2 = 0.8031

KT32-41BBL:y =

6.4462x5.0346

R2 = 0.8129

KT32:y =

8077.2x1.5986

R2 = 0.40660.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

6.00E+06

0 2 4 6 8 10 12 14

Day (After Umbilical Cord Blood Derivation)

Number of Cells

3/28'KT32KT32-OX40KT32-41BBLExpon. (KT32-OX40)Expon. (3/28')Power (KT32-41BBL)Power (KT32)

Culture Decline of T-reg Cells

3/28:

y = 3E+16x-8.5021

R2 = 0.6171

KT32:y = -14345x + 304762

R2 = 0.9924

KT32-OX40:y = -58036x + 2E+06

R2 = 0.3654

KT32-41BBL:y = -30548x +

719881

R2 = 0.5407

0.00E+00

1.00E+06

2.00E+06

3.00E+06

4.00E+06

5.00E+06

6.00E+06

14 15 16 17 18 19 20 21 22

Days (After Umbilical Cord Blood Derivation)

Number of Cells

3/28'

KT32

KT32-OX40

KT32-41BBL

Power (3/28')

Linear (KT32)

Linear (KT32-OX40)Linear (KT32-41BBL)

Third Goal

• To analyze the histological effect of CD4+25+ T-reg cells in GVHD mice

Procedure

Procedure

Procedure

GVHD

GVHD

GVHD

GVHD

GVHD

Procedure

GVHD

GVHD

GVHD

GVHD

GVHD

Procedure

GVHD

GVHD

GVHD

GVHD

GVHD

Healthy Liver

Diseased Liver

Diseased Liver

Diseased Liver

Diseased Liver

Diseased Liver

GVHD Reduction with T-reg Cells

Liver

GVHD Lung

GVHD Spleen GVHD

Colon GVHD

GVHD Mice 3.8 3.4 3.0 2.0 T-reg Treated Mice 1.9 1.9 3.6 1.1

GVHD Reduction with T-reg Cells

Liver

GVHD Lung

GVHD Spleen GVHD

Colon GVHD

GVHD Mice 3.8 3.4 3.0 2.0 T-reg Treated Mice 1.9 1.9 3.6 1.1

GVHD Reduction with T-reg Cells

Liver

GVHD Lung

GVHD Spleen GVHD

Colon GVHD

GVHD Mice 3.8 3.4 3.0 2.0 T-reg Treated Mice 1.9 1.9 3.6 1.1

GVHD Reduction with T-reg Cells

Liver

GVHD Lung

GVHD Spleen GVHD

Colon GVHD

GVHD Mice 3.8 3.4 3.0 2.0 T-reg Treated Mice 1.9 1.9 3.6 1.1

50%

GVHD Reduction with T-reg Cells

Liver

GVHD Lung

GVHD Spleen GVHD

Colon GVHD

GVHD Mice 3.8 3.4 3.0 2.0 T-reg Treated Mice 1.9 1.9 3.6 1.1

44%

GVHD Reduction with T-reg Cells

Liver

GVHD Lung

GVHD Spleen GVHD

Colon GVHD

GVHD Mice 3.8 3.4 3.0 2.0 T-reg Treated Mice 1.9 1.9 3.6 1.1

20%

GVHD Reduction with T-reg Cells

Liver

GVHD Lung

GVHD Spleen GVHD

Colon GVHD

GVHD Mice 3.8 3.4 3.0 2.0 T-reg Treated Mice 1.9 1.9 3.6 1.1

45%

Future Work

• Optimize culture expansion

Future Work

• Optimize culture expansion • Increase rate of cure in mice

Future Work

• Optimize culture expansion • Increase rate of cure in mice• Test T-reg cells as a therapeutic

cure for GVHD in humans

Acknowledgements• Dr. Keli Hippen• Dr. Paul Harker-Murray• The University of Minnesota• Lois Fruen• Advanced Science Research

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Cord Blood-Derived Regulatory T Cells:A Novel Cure for Graft-Versus-Host-

Disease

Siddharth Damania2007