Post on 30-Jan-2021
TRANSFORMING GROWTH FACTOR-β PROGRAMS CENTRAL-MEMORY DIFFERENTIATION IN EX-VIVO STIMULATED HUMAN T
CELLS BY MODULATING ID3 EXPRESSION
Amina Dahmani
Dr Jean-Sébastien Delisle lab’s
Université de Montréal
Hôpital Maisonneuve-Rosemont Research Center
5th European Immunology & Innate Immunity
July 21st, 2016. Berlin, Germany
1
HypothesisHypothesis
Patient
With advanced
Effectors T cells
Ex vivo specific
activation and
differentiation
T cell growth
as IL-2)
T cell growth
factors (such
as IL-2)
T cells isolation
With advanced
cancer/chronic
infection
differentiation
Diminished antitumor/viral activity
Strength of
The adoptive transfer of T cells with early memory features may improve the therapeutic potential of AI
Transforming Growth Factor β (TGF-β)
• TGF-β is a quiescence
factor that promotes
hematopoietic stem cell
maintenance (Blank and
Karlsson, Blood. 2015).
3
Strength of
APC signal
Proliferative
capacity
Self renewal
ability
Long-term
persistence
Therapeutic
efficacy
Karlsson, Blood. 2015).
• A recent study showed that
continuous transforming
growth factor-β signaling is
required to maintain the
identity of memory T cells in
mice (Ma et Zhang, PNAS.
2015).
Modified from Gattinoni, Klebanoff and Restifo . Nature. 2012.
HypothesisHypothesis
Exogenous
TGF-β
Exogenous
TGF-β
ex vivo specific
activation and
differentiation Patient
Memory T cells
Patient
With advanced
Effectors T cells
Ex vivo specific
activation and
differentiation
+
T cell growth
as IL-2)
T cell growth
factors (such
as IL-2)
differentiation
T cells isolationT cells isolation
With advanced
cancer/chronic
infection
differentiation
Enhanced antitumor/viral activityDiminished antitumor/viral activity 4
PBMC T cells aCD3 coating/CD28
Blood samples
from healthy
donors
T-cell enrichment
FicollPolyclonal
stimulation
TGF-β GW788388ø
Methodology
TGF-β
(5ng/ml)
GW788388
(2.5µM)ø
Flow cytometry
qPCR
Day 14Day 7
- Monitoring:
-Proliferation
- Survival
- Phenotype: differentiation, activation,
exhaustion
- Functionality
- Transcriptional signature
5
6
Activation,
expansion and ex-
vivo culture
Effect of TGF-β modulation on T-cell differentiation
7Modified from Gattinoni, Klebanoff and Restifo . Nature. 2012.
Effect of TGF-β modulation on T cell differentiation
CD45RO
CD
62
L
nT: naive T cells.
Tcm: central memory T cells
Tem: effector memory T cells
Tcm
Tem
CD4+ T cells
Tn
Teff
CD8+ T cells
TGF-β promotes the accumulation of central memory T cells
Tem: effector memory T cells
Teff: effector T cells
8
N=10; *P˂0.05; **P˂0.01; ***P˂0.001 by one tailed paired T
test.
9
What is the transcriptional signature of TGF-β-induced memory cells
CD4+A
B
CD8+
TGF-β exposure is associated with ID3 up regulation10PRDM1: encode
for BLIMP1
N=4 for ID3, N=3 for BLIMP1
N=4
CD8
Memory associatedgenes
Effectorassociated
genes
11
CD
4+
siRNA-CTRL siRNA-ID3 siRNA-CTRL siRNA-ID3
ID3 Knock Down
Effect of TGF-β on T-
cell differentiation in
ID3 Knock Down
IS the effect of TGF-β on memory differentiation ID3-dependent?
aCD3/CD28 + TGF-β aCD3/CD28 + TGF-β
Tcm Tcm
CD
8+
CD
4
CD45RO
CD
62
L
ID3
TGF-β favors central memory differentiation through the induction of ID312
Tem Tem
Tcm
Tem
Tcm
Tem
13
hC
D8
Untreated TGF-β-treated
Peri
ph
era
l b
loo
dS
ple
en
TGF-β treated-T cells show enhancedexpansion, persistence and alloreactivity in vivo
0
CTRL or TGF-β-T cells
7 14 21 28
NSG
AnalysisDay -1
2.25 Gy
N=8
TGF-β treated-T cells induce GVHDhCD4
14
TGF-β treated-T cells induce GVHD
15
PBMC
Blood samples
from healthy
EBNA1-specific T cell line
“Clinical-like” protocol for the generation of EBNA1-specific T-cell line (Epstein-Barr virus antigen)
21-28 days
from healthy
donors
IL-7 + IL-15
+ TGF-β
- Monitoring:
- CM differentiation (FACS).
- Functionality (IFN-γ ELISPOT asssay)
- Expansion
EBNA1- pulsed
Dendrtitic cells
IL-7 + IL-15
EBNA1 peptide
library
+
Irradiation
16
A B
C
Effect of TGF-β on EBNA1 T-cell line differentiation, function and expansion (EBV antigen)
IFN-γ ELISPOT assay
No
peptide
EBNA1
library
ø
17
D
TGF-β favors memory differentiation of CD4+ EBNA1-T-cell line without affecting
their expansion or capacity to release IFN-γ upon antigen recognition.
N=5; *P˂0.05; **P˂0.01;
***P˂0.001 by one tailed
paired T test.
TG
F-β
BLIMP1X
LCKP
P
P
?Clinical pertinence:
Memory
differentiation of
CD4+ EBNA1-T-cell
line
ID3
X+
Memory T cell
In vivo:
- Persistence
- Proliferation
- Reactivity
Polyfunctionality 18
• In this study:
– We have identified a new role of TGF-β in human
T cell biology: memory differentiation
Take home message
– We provide a rationale for clinical use of TGF-β for the preparation of improved T cells for
adoptive immunotherapy
19
Supervisor: Dr Jean-Sébastien Delisle
Lab members:
Cédric Carli
Manon Richaud
Valérie Janelle
Julie Orio
Eustache Ouassa
Caroline Lamarche
Collaborators:
Dr Denis-Claude Roy lab’s
Dr Claude Perault
Dr Heather Melichar
Dr Nathalie Labreque
Acknowledgement
Caroline Lamarche
Sheena Blaise
Cecile Grange
Shirin Lak
Thomas Pincez
Victoria Georgest
Guillaume Brodeur
Mathieu Goupil
Myriam Khalili
Cell sorting – Martine Dupuis
Animal care staff
Healthy donor volunteers
Thank you for your attention
Supplementary data
21
Effect of TGF-β modulation on T-cell phenotype
CD62L
CD8+CD4+
A B
ø
TGF-β
TGF-β promotes the expression of CD62L and CCR7 in ex vivo stimulated hT cells
CCR7
N=10 for CD62L; N=9 for CCR7; *P˂0.05; **P˂0.01; ***P˂0.001 by one tailed paired T test.
22
23
CD
8
Effect of TGF-β signalling modulation on polyfunctionality
CD4+
CD8+
TNF-α
IL-2
IFN-γ
CD4
CD
8
N=4 ; **P˂0.01
TGF-β does not impede the acquisition of polyfunctionality of T cells24
CD4
se
cre
tin
gT
ce
llsu
ntr
ea
ted
co
nd
itio
n)
CD4+ CD8+
Ra
tio
of cyto
kin
e s
ecre
tin
g(n
orm
aliz
ed
to u
ntr
ea
ted
A
B
C
ø TGF-β GW
Da
y 3
Da
y 7
ø TGF-β GWCD8+ CD4+
Da
y 7
Da
y 1
4
CTV
C
CD3 CD45RO
CD
45R
A
CCR7
CD3+CD45RO+ T cells
CD3+CD45RA+
CCR7+T cells
A
B
CD
45
RA
+
cell
s
CD4+ T cells CD8+ T cells
CD
3+C
D4
5R
O+
T c
ell
s
CD
3+C
D4
5R
A
CC
R7
+T
ce
lls
TGF-β does not induce Treg or Th17
CD4+
CD8+
A B D
CUnstimulated
ø + TGF-β TReg
1:8
T c
ells/T
reg
To
re
sp
on
der
rati
o
CTV dilution
aCD3/CD28
28
29
siR
NA
-CT
RL
aCD3/CD28aCD3/CD28
+ TGF-β
CD4+ T cells
ID3 expression(d14)
siR
NA
-ID
3
aCD3/CD28 aCD3/CD28 + TGF-β
CD4+ T cells
Does ID3 down regulation affect differentiation of TGF-β-treated T cells?
siR
NA
-CT
RL
CD45RO
CD
62Ls
iRN
A-I
D3
siR
NA
-CT
RL
aCD3/CD28aCD3/CD28
+ TGF-β
CD8+ T cells
ID3 expression(d14)
siR
NA
-ID
3
aCD3/CD28 aCD3/CD28 + TGF-β
CD8+ T cells
Does ID3 down regulation affect differentiation of TGF-β-treated T cells?
siR
NA
-CT
RL
CD45RO
CD
62Ls
iRN
A-I
D3
ø
Day 7 Day 14 Day 21 Day 28
hCD4
hC
D8T
GF
-β
aCD3/CD28 aCD3/CD28 + TGF-β aCD3/CD28 + GW
CD8+ CD4+
Da
y 3
Da
y 7
Da
y 1
4
Effect of TGF-β modulation on T-cell proliferation
CD8+ CD4+ CD8+ CD4+
Da
y 1
4
CTVResults are representative of one of 3 independent experiments.
N=6 donor; Error bars represent SEM variation.35
Effect of TGF-β modulation on CD4+ and CD8+ T-cell proportion
TGF-β modulation does not affect CD4+ and CD8+ T-cell
proportion.
N=6 donor; Error bars represent SEM
variation.
36
37
Effect of TGF-β modulation on T-cell viability
CD8+ T cells
*
AnnexinV
PI
*
CD4+ T cells
TGF-β signaling inhibition affects slightly T-cell viability on day 7.
N=3; Error bars represent SEM variation. *P˂0.05.
*
38
Effect of TGF-β modulation on T cells viability
TGF-β modulation does not affect T cells’ viability
N=6 donor; Error bars represent SEM
variation.
39
40
PBMC T cells aCD3 coating/CD28
T cells
enrichment
FACS
Day 7
Day 14
Cytokines
TGF-β
IL-7
+
TGF-βGW IL-7 IL-15
IL-7
+
GW
IL-15
+
TGF-β
IL-15
+
GW
IL-7
+
IL-15
+
TGF-β
IL-7
+
IL-15
+
GWø
41
* * * *
CD8+ T cells
CD4+ T cells
N=3; Error bars represent SEM variation.
**
CD8+ T cells
42
AB
Effect of TGF-β on WT1-T-cell line differentiation, function and expansion (Tumor associated antigen)
C
Déterminer la concentration Déterminer la concentration
optimale in vitro de GW chez
l’humain
44
Conclusion:
- La concentration
optimale de GW semble
être 2.5µM (comme chez
la souris).
Effet du GW788388 au jour 7
45
Effet du GW788388 au jour 14
Conclusion:
- Au jour 14, l’effet dose
dépendent du GW n’est
plus appréciable.plus appréciable.
46
Strategies that might be used to preserve or to
confer stemness to T cells
Gattinoni, Klebanoff et Restifo. Nat Rev Cancer, 201247
IL-7Rα
CCR7
BCL2
X
X
X
X
BLIMP1
X ID3
X CD-122
LCKP
P
P
E2A
CXCR5
KLF2
SOCS3
ID3
X
X
X
X
+
+
X
Cell cycle arrest
Differentiation
IL-7Rα
CCR7
BCL2ID3
BLIMP1
+
+
+
X+
LCKP
P
P
E2A
CD-122
CXCR5
KLF2
SOCS3
ID3
MC2,3,10 Topa2
KIF2c, KIF4a,…
Cdac5, cdac8
FOXM1 NeK2
STK39
+
+
+
+
ID3
+
+
+
+
Differentiation
X
X
INK4A +
Cell cycle arrest
DifferentiationX
X
X
+
+
ID3
BLIMP1
X+
LCKP
P
P
E2A
Maintenance of
self-renewal
Maintenance of
self-renewal
Multipotency
Maintenance of
genome integrity
+
+
+
ID3
Replicative senescence
Differentiation
X
X
Survival
Homeostasis
+
+
Cell cycle arrest
Differentiation
X
X
X
ID3
BLIMP1X
+
LCKP
P
P
Memory T cell
- Persistence
- Proliferation
- Reactivity
Polyfunctionality
E2A
XX
Transcriptional activity
51