1. New Strategies for the
Prevention and Treatment of
Graft vs. Host Disease (GVHD)
Simrit Parmar, MD
Stem Cell Transplant & Cellular Therapy
BTG2013, Hong Kong

2. Risk Factors for Acute GVHD
• HLA disparity
• Increasing age
• Donor and recipient gender disparity
• Type and status of underlying disease
• Amount of radiation and intensity of the
transplant conditioning regimen
• Doses of methotrexate and cyclosporine or
tacrolimus

3. Acute GVHD: Pathophysiology
1. Recipient conditioning
2. Donor
T cell activation
3. Cellular and
Inflammatory Effectors

4. Acute GVHD
• Acute GVHD
– Typically occurs around the time of engraftment.
– Previously mis-defined as GVHD which occurs prior to day
100 post-transplant.
– Three main organs involved:
• Skin: macularpapular rash
• GI system: Nausea / Vomiting and Diarrhea
• Liver Abnormalities: typically cholestatic (jaundice).
– Incidence of 9-50% of sib transplants.
Vigorito et al. Blood 2009

7. Acute GVHD: Survival and Relapse
• Grade 0 acute GVHD — hazard ratio (HR) for TRM: 1.0
• Grade I — HR 1.5 (95% CI 1.2-2.0)
• Grade II — HR 2.5 (95% CI 2.0-3.1)
• Grade III — HR 5.8 (95% CI 4.4-7.5)
• Grade IV — HR 14.7 (95% CI 11-20)
• Grade 0 acute GVHD — hazard ratio (HR) for relapse 1.0
• Grade I — HR 0.94 (95% CI 0.8-1.2)
• Grade II — HR 0.60 (95% CI 0.5-0.8)
• Grade III — HR 0.48 (95% CI 0.3-0.8)
• Grade IV — HR 0.14 (95% CI 0.02-0.99)
D
E
A
T
H
R
E
L
A
P
S
E

8. “Be good or I’ll send you to
transplant”
“”I am telling you, by the time
they get done with you, you’ll be
wearing diapers”
“Do you want a
little vidaza or
total body skin
sloughing?”

9. GVHD Prophylaxis

10. “No Free Lunch” Principle
GVHD
• Relapse
• Rejection
• Delayed Immune
Reconstitution
GVHD

11. Immune Function in HCT
• Dysfunctional immune responses are common in
clinical medicine
• Major mechanism of disease control due to GVT
reactions, yet major limitation of allogeneic HCT is
GVHD
• Controlling GVHD could lead to use of allogeneic
HCT in other clinical settings such as treatment of
autoimmune diseases and tolerance induction for
organ transplantation

12. Risk of GVHD in Two Eras
Gooley et al. N. Engl. J Med 363:2091, 2010

13. In vivo tracking of
light emitting donor cells
Allogeneic HCT
B
T
M
BM BM
BM
B
T
Bone Marrow
Splenocytes
FVB/N
WT
luc+
Balb/c
H-2q/Thy1.1H-2d/Thy1.2
CD4+
CD8+
B220+
NK1.1+
Gr-1/Mac-1+
2x105 cells/well Absolute light
emission
0.00 0.05 0.10 0.15
Luciferase 2A eGFPAct
luc+ reporter mouse

14. Acute Graft-vs-Host Disease Development
Beilhack, A. et al. Blood. 2005. 106:1113

15. The Evolution of acute GVHD

16. Approaches to the Prevention of GVHD
• Pharmacologic
– CNI/MTX
– CNI/MTX vs Rapa/MTX
• Graft source
– BM vs PBPC
– MRD vs URD vs UCB
• T Cell depletion
– CD34 Selection
– ATG, Campath
• Immune regulation

17. Regulation of Immune Function
• Critically important in health and disease
• Compartmentalization of immune
responses
• Cytokines
• Regulatory T cells (Treg, NK-T, iTreg, others)
RegulationReactivity
T regulatory cellT effector cell
CD4+ T Cell Subsets

18. CD4+CD25+ Regulatory T Cells
• Major population of cells which regulate immune
reactions
• Express transcription factor FoxP3
• Deficiency or mutation of FoxP3 has autoimmune
consequences in animal models and humans
• Cell contact-dependent suppression of alloreactive
responses in mixed lymphocyte reactions (MLR)
• Prevent organ specific autoimmune diseases in animal
models (e.g. IBD, diabetes)
• IL-10 and TGF- implicated in mediating suppressive
effect in vivo

19. Regulatory T-cells
• Allogeneic HCT recipients with aGVHD had Treg
frequencies 40% less than those without aGVHD.
• Treg frequencies decreased linearly with acute
GVHD severity.
• The frequency of Tregs at acute GVHD onset
predicted response to therapy.
Magenau et al. BBMT. 2010.

20. Magenau et al. BBMT. 2010.
38%
63%
Circulating Tregs predict OS

21. d15 Death from
GVHD
100
5000
1000
20000
1
10
100
1000
10000
0 20 40 60 0 20 40 600 20 40 60
Time [d] post BMT
RelativeSignalIntensity
0
25
50
75
100
0 20 40 60
Time [d] post BMT
Survival[%]
TCD BM only, n = 14
TCD BM + Tcon, n = 15
TCD BM + Tcon + Treg n = 9
Control of GVHD with Retention of GVL
TconBM only Tcon + Treg
500
5000
d5
Edinger et al. Nature Medicine 9:1144, 2003

22. Challenges for Clinical Translation of Treg
• Treg are rare cell populations
• Paucity of unique markers for isolation and
availability of clinical grade reagents
• Marginal functional assays in humans
• Regulatory requirements

23. Expanded CB Tregs show FOXP3
demethylation and suppress alloMLR

24. 3rd Party CB Tregs Prevent GVHD

25. In vivo tracking of Treg transduced
with GFP and Firefly Luciferase

26. Treg Treg+PBPC
Day -1
Day 0
Day 3
dorsal

27. Treg Treg+PBPC
Day -1
Day 0
Day 3
dorsal

28. Treg Treg+PBPC
Day -1
Day 0
Day 3
dorsal

29. Treg Treg+PBPC
Day -1
Day 0
Day 3
dorsal

30. Treg Treg+PBPC
Day 3
Day 10
ventral

31. Proposed phase I Clinical Trial
Treg Doses to be Studied
Dose Cohort Treg Dose
Dose Level 1 1 × 105 Tregs/kg
Dose Level 2 5 × 105 Tregs/kg
Dose Level 3 1 × 106 Tregs/kg
Dose Level 4 5 × 106 Tregs/kg
Dose Level 5 1 × 107 Tregs/kg

32. Next Step: Adoptive Therapy with Treg
Day
-8
Day
-7
Day
-6
Day
-5
Day
-4
Day
-3
Day
-2
Day-
1 0 +1 +2
Day
+3
Day
+4
Day
+6
BU
Test
Dose
32mg/m2
Rest BU BU BU BU BMT Infusion
of
Ex-vivo
Expanded
Tregs
FLU
40
mg/m
2
FLU
40
mg/
m2
FLU
40
mg/
m2
FLU
40
mg/
m2
CY**
50
mg/k
g
CY**
50
mg/k
g
Day
-6
Day
-5
Day
-4
Day
-3
Day
-2
Day-1
0
MEL BU BU BU Infusion of
Ex-vivo
Expanded
Tregs
BMT
FLU
40
mg/m2
FLU
40
mg/m2
FLU
40
mg/m2
FLU
40
mg/m2
MMF+Sirolimus

34. Individual clinical outcome of patients
who received a Treg dose > 30x105/kg

36. Haploidentical Transplant Schema
(Stanford)
Mel, TT, Flu +
Thymoglobulin@
0 +14 +16Day -10
CD34+ cell
selected
graft
CD4+CD25+
Treg
CD4+/CD8+
Tcon
Cell
Dose
5-10 x
106/kg
105/kg
3x105/kg
106/kg
Endpoints:
Chimerism
Immune reconstitution
Acute and chronic
GVHD
EFS, OS
BB IND13923

38. Selection of CD4+CD25+ Tregs (U. Perugia)
Cells (x109) 1060 (540-1370) 280 (202- 390)
%CD4CD25 3.0 (1.5-7.45) 92.4 (90-97.1)
N° cells (x 106) 330 (221-1020) 256 (185.6-365.4)
%CD4CD25high 0.3 (0.12- 0.89) 33.6 (14.4-39.6)
N° cells (x 106) 36.12 (19.98 - 84) 68.6 (20.9-143)
Starting fraction Final fraction
CD25
CD127
CD4
FoxP3
Gate on CD4CD25+high
Gate on CD4CD25+
Fox P3+ cells
71.9 ± 15 %
Immunomagnetic
Selection of
CD4+CD25+Cells
1st step:
Depletion of
CD8+/CD19+cells
2ndstep:
Enrichment of
CD25+ cells

39. >50 >100 >200
0
50
100
150
200
CD4/ l
DayspostBMT
>50 >100 >200
0
20
40
60
80
100
CD8/ l
DayspostBMT
Recovery of CD4+ and CD8+ T cell subpopulations
0
50
100
150
200
250
1 2 3 4 5 6 7 8 9 10 11 12
SpectratypecompexityScore
Donors
Months after transplant
Complexityscore
Spectratyping
Pattern of immunoreconstitution

40. Evaluable Patients
Patients with CMV reactivation
0
10
20
30
40
50
60
70
80
90
100
0-30 31-60 61-90 91-120 121-150 151-180 181-365 >365
100
96
82
75
67
56
48
2928
50
34
22
9 9
1 1
0
5
10
15
20
25
30
0-30 31-60 61-90 91-120 121-150 151-180 181-365 >365
27
21
16
10
9
5
2
12
5
1
0 0 0 0 0
Days after transplant
Days after transplant
CMV reactivation episodes
Tregs Group
Control Group
p<0.05

41. Outcomes – U. of Perugia
Event-Free Survival
12/26 (46%)
• Regimen Related Toxicities:
– Veno-occlusive disease (3)
– Multi-organ failure (1)
• Acute GVHD grade III-IV (2)
• Serious infections (7)
• Relapse (AML 1)
Median follow-up 18.5 months
(range 16.1-27.6)
D’Ianni et al. Blood 2011

42. Conclusions
• GVHD remains the most significant
complication following allogeneic HCT
• Murine studies have demonstrated that
immune regulatory mechanisms play a
significant role in controlling dysfunctional
immune responses including GVHD
• Clinical translation is ongoing with promising
early results