1. 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 21 st, 2016. Berlin, Germany 1

2. Hypothesis T cells isolation Patient With advanced cancer/chronic infection Effectors T cells Ex vivo specific activation and differentiation Diminished antitumor/viral activity T cell growth factors (such as IL-2)

3. 3 Strength of APC signal Proliferative capacity Self renewal ability Long-term persistence Therapeutic efficacy 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). 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.

4. Hypothesis Exogenous TGF-β ex vivo specific activation and differentiation T cells isolation Patient Memory T cells T cells isolation Patient With advanced cancer/chronic infection Effectors T cells Ex vivo specific activation and differentiation Enhanced antitumor/viral activity Diminished antitumor/viral activity + T cell growth factors (such as IL-2) 4

5. PBMC T cells aCD3 coating/CD28 Blood samples from healthy donors T-cell enrichment Ficoll Polyclonal stimulation TGF-β (5ng/ml) GW788388 (2.5µM) ø Methodology Flow cytometry qPCR Day 14 Day 7 - Monitoring: -Proliferation - Survival - Phenotype: differentiation, activation, exhaustion - Functionality - Transcriptional signature 5

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7. Activation, expansion and ex- vivo culture Effect of TGF-β modulation on T-cell differentiation 7 Modified from Gattinoni, Klebanoff and Restifo. Nature. 2012.

8. TGF-β promotes the accumulation of central memory T cells Effect of TGF-β modulation on T cell differentiation CD45RO CD62L nT: naive T cells. Tcm: central memory T cells Tem: effector memory T cells Teff: effector T cells Tcm Tem CD4 + T cells 8 Tn Teff CD8 + T cells N=10; *P ˂ 0.05; **P ˂ 0.01; ***P ˂ 0.001 by one tailed paired T test.

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10. What is the transcriptional signature of TGF-β-induced memory cells TGF-β exposure is associated with ID3 up regulation CD4 + A B 10 PRDM1: encode for BLIMP1 N=4 for ID3, N=3 for BLIMP1 N=4 - CD8 + Memory associated genes Effector associated genes

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12. CD8 + CD4 + CD45RO CD62L siRNA-CTRLsiRNA-ID3 siRNA-CTRL siRNA-ID3 ID3 ID3 Knock Down Effect of TGF-β on T- cell differentiation in ID3 Knock Down TGF-β favors central memory differentiation through the induction of ID3 IS the effect of TGF-β on memory differentiation ID3- dependent? aCD3/CD28 + TGF-β 12 Tcm Tem Tcm Tem Tcm Tem Tcm Tem

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14. hCD8 UntreatedTGF-β-treated Peripheral blood Spleen hCD4 TGF-β treated-T cells show enhanced expansion, persistence and alloreactivity in vivo 0 CTRL or TGF-β-T cells 71421 28 NSG Analysis Day 2.25 Gy 14 N=8 TGF-β treated-T cells induce GVHD

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16. PBMC Blood samples 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 + EBNA1-specific T cell line “Clinical-like” protocol for the generation of EBNA1-specific T-cell line (Epstein-Barr virus antigen) 21-28 days Irradiation 16

17. 17 A B C D Effect of TGF-β on EBNA1 T-cell line differentiation, function and expansion (EBV antigen) TGF-β favors memory differentiation of CD4 + EBNA1-T-cell line without affecting their expansion or capacity to release IFN-γ upon antigen recognition. IFN-γ ELISPOT assay N=5; *P˂0.05; **P˂0.01; ***P˂0.001 by one tailed paired T test. No peptide EBNA1 library ø TGF-β

18. ID3 BLIMP1 X + LCK P P P Memory T cell In vivo: - Persistence - Proliferation - Reactivity In vivo: - Persistence - Proliferation - Reactivity Polyfunctionality ? ? 18 Clinical pertinence: Memory differentiation of CD4+ EBNA1-T-cell line Clinical pertinence: Memory differentiation of CD4+ EBNA1-T-cell line

19. In this study: – We have identified a new role of TGF-β in human T cell biology: memory differentiation – We provide a rationale for clinical use of TGF-β for the preparation of improved T cells for adoptive immunotherapy 19 Take home message

20. Supervisor: Dr Jean-Sébastien Delisle Lab members: Cédric Carli Manon Richaud Valérie Janelle Julie Orio Eustache Ouassa 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 Collaborators: Dr Denis-Claude Roy lab’s Dr Claude Perault Dr Heather Melichar Dr Nathalie Labreque Acknowledgement Thank you for your attention

21. Supplementary data 21

22. Effect of TGF-β modulation on T-cell phenotype TGF-β promotes the expression of CD62L and CCR7 in ex vivo stimulated hT cells CD62L CD8 + CD4 + A CCR7 B ø TGF-β N=10 for CD62L; N=9 for CCR7; *P ˂ 0.05; **P ˂ 0.01; ***P ˂ 0.001 by one tailed paired T test. 22

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24. TNF-α IL-2 IFN-γ CD4 CD8 Effect of TGF-β signalling modulation on polyfunctionality N=4 ; **P˂0.01 TGF-β does not impede the acquisition of polyfunctionality of T cells 24 CD4 + CD8 +

25. Ratio of cytokine secreting T cells (normalized to untreated condition) CD4 + CD8 +

26. A B C ø TGF-βGW Day 3 Day 7 Day 14 CTV ø TGF-βGW C CD8 + CD4 +

27. CD3 CD45RO CD45RA CCR7 CD3 + CD45RO + T cells CD3 + CD45RA + CCR7 + T cells A B CD3 + CD45RO + T cells CD3 + CD45RA + CCR7 + T cells CD4 + T cellsCD8 + T cells

28. TGF-β does not induce Treg or Th17 CD4 + CD8 + AB C Unstimulated ø + TGF- β TReg 1:8 T cells/Treg To responder ratio CTV dilution aCD3/CD28 D 28

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30. siRNA-ID3 siRNA-CTRL aCD3/CD28 aCD3/CD28 + TGF-β CD4 + T cells ID3 expression(d14)

31. aCD3/CD28 aCD3/CD28 + TGF-β CD4 + T cells Does ID3 down regulation affect differentiation of TGF-β-treated T cells? CD45RO CD62L siRNA-ID3 siRNA-CTRL

32. siRNA-ID3 siRNA-CTRL aCD3/CD28 aCD3/CD28 + TGF-β CD8 + T cells ID3 expression(d14)

33. aCD3/CD28 aCD3/CD28 + TGF-β CD8 + T cells Does ID3 down regulation affect differentiation of TGF-β-treated T cells? CD45RO CD62L siRNA-ID3 siRNA-CTRL

34. hCD4 hCD8 ø TGF-β Day 7Day 14Day 21Day 28

35. aCD3/CD28 aCD3/CD28 + TGF-βaCD3/CD28 + GW CD8 + CD4 + Day 3 Day 7 Day 14 CTV Results are representative of one of 3 independent experiments. Effect of TGF-β modulation on T-cell proliferation N=6 donor; Error bars represent SEM variation. CD8 + CD4 + CD8 + CD4 + 35

36. TGF-β modulation does not affect CD4 + and CD8 + T-cell proportion. Effect of TGF-β modulation on CD4 + and CD8 + T-cell proportion N=6 donor; Error bars represent SEM variation. 36

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38. TGF-β signaling inhibition affects slightly T-cell viability on day 7. Effect of TGF-β modulation on T-cell viability CD8 + T cells N=3; Error bars represent SEM variation. *P˂0.05. * Annexin V PI * 38 CD4 + T cells

39. TGF- β modulation does not affect T cells’ viability Effect of TGF-β modulation on T cells viability N=6 donor; Error bars represent SEM variation. 39

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41. PBMC T cells aCD3 coating/CD28 T cells enrichment FACS Day 7 Day 14 TGF-β IL-7 + TGF-β GWIL-7IL-15 IL-7 + GW IL-15 + TGF-β IL-15 + GW IL-7 + IL-15 + TGF-β Cytokines IL-7 + IL-15 + GW ø 41

42. N=3; Error bars represent SEM variation. * *** * * CD8 + T cells CD4 + T cells 42

43. A B C Effect of TGF-β on WT1-T-cell line differentiation, function and expansion (Tumor associated antigen)

44. Déterminer la concentration optimale in vitro de GW chez l’humain 44

45. Conclusion: - La concentration optimale de GW semble être 2.5µM (comme chez la souris). Effet du GW788388 au jour 7 45

46. Effet du GW788388 au jour 14 Conclusion: - Au jour 14, l’effet dose dépendent du GW n’est plus appréciable. 46

47. Gattinoni, Klebanoff et Restifo. Nat Rev Cancer, 2012 Strategies that might be used to preserve or to confer stemness to T cells 47

48. IL-7Rα CCR7 BCL2 E2A CXCR5 KLF2 SOCS3 ID3 X X X X X X X X BLIMP1 X + + ID3 X CD-122 LCK P P P Cell cycle arrest Differentiation

49. IL-7Rα CCR7 BCL2 E2A CD-122 CXCR5 KLF2 SOCS3 ID3 BLIMP1 MC2,3,10 Topa2 KIF2c, KIF4a,… Cdac5, cdac8 FOXM1 NeK2 STK39 + + + + ID3 + + + + + + + X + Differentiation X X INK4A + miR-23a? Cell cycle arrest Differentiation X LCK P P P X X + +

50. E2A ID3 BLIMP1 Maintenance of self-renewal Multipotency Maintenance of genome integrity + + + ID3 X + Replicative senescence Differentiation X X Survival Homeostasis + + Cell cycle arrest Differentiation X X miR-23a? X LCK P P P

51. ID3 BLIMP1 X + LCK P P P Memory T cell - Persistence - Proliferation - Reactivity - Persistence - Proliferation - Reactivity Polyfunctionality E2A X X Transcription al activity 51