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