Cord Blood-Derived Regulatory T Cells:
A Novel Cure for Graft-Versus-Host-Disease
Siddharth Damania
2007
University of Minnesota
Cancer Center
Bone-Marrow and Stem-Cell Transplants
6,000 Americans
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Bone-Marrow and Stem-Cell Transplants
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Bone-Marrow and Stem-Cell Transplants
Over half develop GVHD
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Graft-Versus-Host-Disease (GVHD)
• GVHD occurs when transplanted cells
reject host cells in the patient
Transplanted
Cell
Patient Cell
Purpose
• To use T-reg cells to cure mice with
human GVHD
GVHD
GVHD
GVHD
GVHD
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
FACS T-reg Cell Purity (CD4/CD25)
3/28
KT32
86.2%
KT32-41BBL
91.5%
95.4%
KT32-OX40
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
6.00E+06
KT-32-OX40:
y=
504.24e0 .7 7 7 6 x
R2 = 0.7983
Number of Cells
5.00E+06
4.00E+06
3/28'
KT32
KT32-OX40
KT32-41BBL
Expon. (KT32-OX40)
Expon. (3/28')
Power (KT32-41BBL)
Power (KT32)
3.00E+06
2.00E+06
KT32-41BBL:
y=
3/28:
y=
6.4462x5 .0 3 4 6
R2 = 0.8129
10715e0 .5 0 5 6 x
R2 = 0.8031
1.00E+06
KT32:
y=
8077.2x1 .5 9 8 6
R2 = 0.4066
0.00E+00
0
2
4
6
8
10
12
Day (After Umbilical Cord Blood Derivation)
14
Culture Decline of T-reg Cells
6.00E+06
Number of Cells
5.00E+06
3/28'
4.00E+06
KT32
KT32-OX40
3.00E+06
KT32-41BBL
3/28:
y = 3E+16x
Power (3/28')
- 8 .5 0 2 1
R2 = 0.6171
2.00E+06
Linear (KT32)
KT32-OX40:
y = -58036x + 2E+06
1.00E+06
Linear (KT32OX40)
Linear (KT3241BBL)
KT32-41BBL:
y = -30548x +
719881
2
R = 0.5407
2
R = 0.3654
KT32:
y = -14345x + 304762
2
R = 0.9924
0.00E+00
14
15
16
17
18
19
20
21
Days (After Umbilical Cord Blood Derivation)
22
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
GVHD Mice
T-reg Treated Mice
Liver Lung Spleen Colon
GVHD GVHD GVHD GVHD
3.8
3.4
3.0
2.0
1.9
1.9
3.6
1.1
GVHD Reduction with T-reg Cells
GVHD Mice
T-reg Treated Mice
Liver Lung Spleen Colon
GVHD GVHD GVHD GVHD
3.8
3.4
3.0
2.0
1.9
1.9
3.6
1.1
GVHD Reduction with T-reg Cells
GVHD Mice
T-reg Treated Mice
Liver Lung Spleen Colon
GVHD GVHD GVHD GVHD
3.8
3.4
3.0
2.0
1.9
1.9
3.6
1.1
GVHD Reduction with T-reg Cells
GVHD Mice
T-reg Treated Mice
Liver Lung Spleen Colon
GVHD GVHD GVHD GVHD
3.8
3.4
3.0
2.0
1.9
1.9
3.6
1.1
50%
GVHD Reduction with T-reg Cells
GVHD Mice
T-reg Treated Mice
Liver Lung Spleen Colon
GVHD GVHD GVHD GVHD
3.8
3.4
3.0
2.0
1.9
1.9
3.6
1.1
44%
GVHD Reduction with T-reg Cells
GVHD Mice
T-reg Treated Mice
Liver Lung Spleen Colon
GVHD GVHD GVHD GVHD
3.8
3.4
3.0
2.0
1.9
1.9
3.6
1.1
20%
GVHD Reduction with T-reg Cells
GVHD Mice
T-reg Treated Mice
Liver Lung Spleen Colon
GVHD GVHD GVHD GVHD
3.8
3.4
3.0
2.0
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 Damania
2007