This document discusses research on using regulatory T cells (Tregs) for graft-versus-host disease (GVHD) prevention after allogeneic hematopoietic cell transplantation (HCT). Key points include: 1) Tregs show promise in controlling GVHD while retaining the graft-versus-leukemia effect in mouse models of allogeneic HCT. 2) Studies demonstrate that higher levels of Tregs early after HCT in patients correlate with less severe acute GVHD. 3) Researchers have developed methods to successfully expand Tregs from umbilical cord blood (CB) through CD25 selection and CD3/CD28 bead stimulation while maintaining a functional

1. Adoptive Therapy with
Regulatory T-Cells for
Prevention of
Graft versus Host Disease

2. Regulatory T cell
CD127lo

3. Busy Life
of Tregs

5. G
V
H
D

6. 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 eGFPbAct
luc+ reporter mouse

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

8. 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

9. Tregs and GVHD
• 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.

10. Magenau et al. BBMT. 2010.
38%
63%
Circulating Tregs predict OS
Non Relapse Mortality Overall Survival

13. NO POST-SCT IST

14. Outcomes – U. of Perugia
• Deaths: 12/26
• Regimen Related Toxicities:
– Veno-occlusive disease (3)
– Multi-organ failure (1)
• Acute GVHD grade III-IV (2)
• Serious infections (7)
• Relapse (AML 1)
D’Ianni et al. Blood 2011

15. • N=43
• High Risk AML =33; ALL = 10
• Conditioning: TBI based regimen
• Grafts included
- CD34+ cells= 9.7 × 106/kg
- Tregs= 2.5 × 106/kg)
- Tcons=1.1 × 106/kg
• NO post-transplant immunosuppression was given
• Full-donor type engraftment = 95%
• ≥grade 2 acute GVHD =15%
• DFS @ median FU 46 mos = 56%
• Cumulative incidence of relapse = 5% Blood 2014 Jul 24;124(4):638-44

16. MDACC EXPERIENCE

19. 4. Magnetic
Activated Cell
Sorting,
MidiMACS
3. Anti-CD25
microbeads
(CD25 Reagent,
MiltenyiBiotec)
1. CB unit
5a. CD25 positive cells “Treg”.
Cultured at 1x106 cell/ml
in T-cell expansion medium.
IL-2 @ 200 u/ml
Initiated with *CD3/28 beads at
3 beads: 1 cell ratio.
+
-
6. Cellularity monitored
every
2 days& re-adjusted to
~1x106 cell/ml
(as required)
IL-2 added to maintain
200 u/ml.
(No new beads added)
7. Flow cytometry: to
reveal CD3/4/8/25
(19/56)
and
FoxP3 and
CTLA4
5b. CD25 negative cells “Non-Treg”.
Cultured at 1x106CD3+cell/mlin T-cell
expansion medium.
IL-2 @ 200 u/ml
Initiated with *CD3/28 beads at
3 beads: 1 cell ratio.
Current CB “Treg” ex vivo expansion technique
2. Ficoll to isolate
CB MNC
8. Functional in
vitro assay: Mixed
Lymphocyte
Reaction (MLR)
Notes:
(a) IL-2: Proleukin®, Chiron Corp., Emeryville, CA.
(b) *CD3/28 beads:
(i) Carl H. June MD (University of Pennsylvania), or
(ii) invitrogen Dynabeads® ClinExVivoTM CD3/CD28
(c) T-cell expansion medium: RPMI, 5% heat inactivated human
serum, antibiotics and L-glutamine (2mM)

20. Day 7
Day 0
Day 0
Day 7
CD25 Positive Fraction CD25 Negative Fraction

21. Expanded CB Tregs are
Functional
Vβ 1.0 Vβ 2.0 Vβ 3.0 Vβ 4.0 Vβ 5.1 Vβ 5.2 Vβ 6.0 Vβ 7.0
Vβ 8.0 Vβ 9.0 Vβ 11.0 Vβ 12.0 Vβ 13.0 Vβ 14.0 Vβ 15.0
Vβ 16.0 Vβ 17.0 Vβ 18.0 Vβ 20.0 Vβ 21.0 Vβ 22.0 Vβ 23.0 Vβ 24.0
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
Countsperminute
CD25
FOXP3
CD4
CD127
CD25
94.9%
96.79%
97.59%
95.49%
CD4

22. Successful ex vivo expansion of
UCB Tregs

23. Ex vivo Expanded CB Tregs
Maintain
Polyclonal Vβ Repertoire

24. BASELINE FLOW (WHOLE CB)

25. Expanded CB Tregs Phenotype:
CD4+CD25+FOXP3+CD127lo

26. Expanded CB Tregs:
CD4+CD25+FOXP3+CD127lo

27. DeMethylation status of FOXP3
gene regions represents
expanded CB Treg
Baron et al, EMBO

28. Expanded CB Tregs show
FOXP3 Demethylation

29. FOXP3 demethylation correlates with CB
Treg Phenotype and Function
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
percentgatedlymphocytes
0
5000
10000
15000
20000
25000
D1+D2 D1+D2+TREG(1:1)
CPM
0.00
20.00
40.00
60.00
80.00
100.00
120.00
FOXP3 (7-1)
2CpG
FOXP3 (9-1)
2CpG
FOXP3 (10-1)
2CpG
FOXP3 (11-1)
2CpG
Tregs
Baseline
Methylation%
N=3

30. 0
5000
10000
15000
20000
25000
30000
35000
40000
45000
DONOR 1 DONOR 2 TREG D1+D2 D1+TREG D2+TREG 10:1 1:1 1:10
CB Tregs significantly suppresses
2-way Allogeneic MLR
cpm

31. 0
5000
10000
15000
20000
25000
30000
35000
40000
45000
DONOR 1 DONOR 2 TREG D1+D2 D1+TREG D2+TREG 10:1 1:1 1:10
cpm
CB Tregs significantly suppresses
2-way Allogeneic MLR

32. 0
5000
10000
15000
20000
25000
30000
35000
40000
45000
DONOR 1 DONOR 2 TREG D1+D2 D1+TREG D2+TREG 10:1 1:1 1:10
cpm
CB Tregs significantly suppresses
2-way Allogeneic MLR

33. 0
5000
10000
15000
20000
25000
30000
35000
40000
45000
DONOR 1 DONOR 2 TREG D1+D2 D1+TREG D2+TREG 10:1 1:1 1:10
cpm
Treg:MNC ratio
CB Tregs significantly suppresses
2-way Allogeneic MLR

34. Expanded CB Tregs Suppress
Two-Way Allogeneic MLR

35. Xenogenic GVHD Mouse Model

37. Xenogenic GVHD Mouse Model
Lymphoid
follicle
Extra-
medullary
hematopoeisis
mixed with
histiocytes
Spleen
Small Intestine 400x
(Apoptosis with Enteritis)
Apoptotic bodies diagnostic for GvHD
(arrows)
Apoptotic
bodies
Interstitial
edema
Skin
vein B.D.
A
B.D.
Yellow arrows represent dead hepatocytes.
Outlined area is a combination of necrotic
hepatocytes, fibrosis, and some lymphocytes.
Small Intestine
Liver

38. PBMC + Tregs PBMC ONLY
EDEMA LYMPHOCYTES
APOPTOTIC BODIES DIAGNOSTIC FOR GVHD (arrows)
Small Intestine 400x (Apoptosis with Enteritis)

39. 3rd Party CB Tregs injection on
day -1 at 1:1 ratio
Day -1
CB Tregs 1x107

40. 3rd Party CB Tregs Prevent GVHD
when infused on day -1 at 1:1 ratio
NO Treg WITH Treg
NO Treg WITH Treg
LUNGLIVER
PBMC
1 x107
PBMC+
Treg 1 x107

43. CHALLENGE!
• Limitation in generating clinically relevant
doses of Tregs from a single CB unit

44. CB Hematopoieitic Cells
have Decreased Ability
to bind Selectins
• P- and E-selectin are expressed by the
microvasculature of the hematopoietic
microenvironment and mediate hematopoietic cell
homing and adhesion
Katayama et al. Blood. 2003;102:2060-7

45. Fucosylation can Restore
Selectin Binding
Xia et al. Blood. 2004 ;104:3091-6
Hidalgo and Frenette Blood. 2005;105:567-75

46. Fucosyltransferase (FT) VI
CB Hematopoieitic
Cell
GDP-Fucose
Glycoprotein
Fucosylated
glycoprotein
√
X
P- & E-Selectin
BM
endothelium
Impact on
homing &
engraftment?Bone marrow
microvasculature
Enhancing Selectin Binding
through Ex Vivo Fucosylation

47. Fucosylation of CB CD34+ cells
enhances engraftment in NSG mice
4.5x104 CD34+ cells/mouse
Fucosylated
Untreated
100 101 102 103 104
CLA FITC
CB FTVI 40.008100 101 102 103 104
CLA FITC
washed cb.008
CLA
Untreated
Fucosylated
CD34

48. Preparative Therapy
Days -8 to -2
0
Thaw
and
infuse
CB#1
Thaw, treat
with FT-VI and
GDP-fucose
(30 minutes),
wash and infuse.
CB#2
Preparative Therapy
Days -8 to -2
0
Thaw
and
infuse
CB#1
Thaw
and
infuse
CB#1
Thaw, treat
with FT-VI and
GDP-fucose
(30 minutes),
wash and infuse.
CB#2
Thaw, treat
with FT-VI and
GDP-fucose
(30 minutes),
wash and infuse.
CB#2
CB Fucosylation Study
MDACC #2010-0658, IND#14897
High-Risk Hematologic Malignancies

49. MDACC CB Fucosylation Trial
N=23
FT-VI treated CB
Time to Neutrophil
Engraftment
(ANC ≥ 500)
Median: 17 Days
Range: 12 – 34
Time to
Platelet Engraftment
(plts ≥ 20,000)
Median: 33 Days
Range: 18 -100
Popat et al., ASH 2013

50. Is there Impact of Fucosylation
on CB Treg Homing?

51. Fucosyltransferase-VI-GDP Fucose
(FT-VI) Treatment Increases CB Treg
Fucosylation
CD4
Fucosylation (CLA/sLex)
Control + FT-VI

52. Fucosylated CB Tregs show Increased
E-selectin Ligand Binding Ability
Fig 1C
Fig 1D
Fig 1E
Control + FT-VI
0
10
20
30
40
50
60
70
80
90
100
CLA (Fuco) E-Selectin P-Selectin L-Selectin
percentbinding
untreated
FTVI treated
P=0.008
P=0.0001
P=0.004
P=0.8

53. Suppression of in vitro Allogeneic MLR by
Fucosylated CB Tregs is comparable to
Untreated Ex vivo Expanded CB Tregs
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
Countsperminute

54. 4. Magnetic
Activated Cell
Sorting,
MidiMACS
3. Anti-CD25
microbeads
(CD25 Reagent,
MiltenyiBiotec)
1. CB unit
5a. CD25 positive cells “Treg”.
Cultured at 1x106 cell/ml
in T-cell expansion medium.
IL-2 @ 200 u/ml
Initiated with *CD3/28 beads at
3 beads: 1 cell ratio.
+
2. Ficoll to isolate
CB MNC
Experimental Procedure:
Imaging Tregs
Day 3: eGFP-FFluc retro-viral transduction
Continually culture for 11 additional days
30 min incubation w FTVI enzyme
Treg FTVI-Treg

55. Longer in-vivo Persistence of
Fucosylated CB Tregs
Day 7
Day 10
Fig 2F
T-Tre
Day -1
Day 0
FT-Treg + PBMC Treg+PBMC
Fig 2A Fig 2B
Fig 2D
FT-Treg 1 x107
+ PBMC
Treg 1 x107
+ PBMC

56. Are Fucosylated Tregs more
Potent?
PBMC: 1x107
Group 1
PBMC: 1x107
Treg: 1x106
Group 2
PBMC: 1x107
Fucosylated Treg: 1x106
Group 3

57. Fucosylated CB Tregs Preserve
Recipient Weight at 1-log less Dose
P =0.03
70
75
80
85
90
95
100
105
110
Day
0
Day
1
Day
3
Day
7
Day
10
Day
13
Day
14
Day
17
Day
18
Day
19
Day
20
Day
21
Day
26
Day
31
Actualweight/baselineweightx
100
Treg + PBMC
FT-Treg + PBMC
PBMC alone
PBMC+ Treg: 1x106
PBMC + FT-Treg: 1x106
PBMC : 1x107

58. Fucosylated CB Tregs Render
Lower GVHD Score at 1-log less
Dose
P 0.009
0
1
2
3
4
5
6
7
Day
0
Day
1
Day
2
Day
3
Day
4
Day
5
Day
6
Day
7
Day
8
Day
9
Day
10
Day
11
Day
12
Day
13
Day
14
Day
17
Day
18
Day
19
Day
20
Day
21
Day
26
Day
31
GVHDSCORE
Treg + PBMC
FT-Treg+ PBMC
PBMC alone
PBMC+ Treg: 1x106
PBMC + FT-Treg: 1x106
PBMC : 1x107

59. Fucosylated CB Tregs Improve
Survival at 1-log less Dose
P<0.0001
PBMC +
FT-Treg x106
PBMC +
Treg x106
PBMC
1 x107
FTVI-Treg 106 cell dose

60. Are Fucosylated Tregs more
Potent?
PBMC: 1x107
Group 1
PBMC: 1x107
Treg: 1x105
Group 2
PBMC: 1x107
Fucosylated Treg: 1x105
Group 3

61. Fucosylated CB Tregs Improve
Survival at 2-log less Dose
PBMC +
FT-Treg x105
PBMC +
Treg x105
PBMC
1 x107
P=0.01
FTVI-Treg 105 cell dose

62. PILOT STUDY OF INFUSION OF
FUCOSYLATED REGULATORY T CELLS
TO PREVENT
GRAFT VS. HOST DISEASE
Protocol Number: 2014-0150
Principal Investigator: Simrit Parmar, MD

64. Pilot study of CB Fucosylated
Tregs to prevent GVHD
Day -8 Day -7 Day -6 Day -5 Day -4 Day -3 Day -1 Day 0
Flu
40mg/m2
Flu
40mg/m2
Flu
40mg/m2
Flu
40mg/m2
TBI FTVI-
Treg
infusion
SCT
Cytoxan
50mg/kg +
MESNA
IST:
Sirolimus
+ MMF
‘Off the Shelf’ CB Fucosylated Tregs

65. CB Treg Dose Levels
Dose Cohort Treg Dose
Dose Level 1 1 × 106 FTVI-Tregs/kg
Dose Level 2 1 × 107 FTVI-Tregs/kg

66. Thank You!!