New insights into CD8+ T cell function and regulation. Pam Ohashi Princess Margaret Cancer Centre

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1 New insights into CD8+ T cell function and regulation Pam Ohashi Princess Margaret Cancer Centre

2 New insights into CD8+ T cell function and regulation Pam Ohashi Princess Margaret Cancer Centre No Disclosures

3 Role for NK cells in regulating T cell responses - Biron lab/bielekova lab NK cells inhibited T cell survival via contact dependent mechanisms - In lpr mice - disappearance of NK cells upon the development of autoimmunity (Takeda/Dennert JEM 1993) - decreased NK cell frequency/function associated with systemic juvenile RA (Villanueva J (2005) Arthritis Res Ther

4 Innate Immunity: NK regs? CPM MBP 1:4 1:8 1:16 1:32 1:64 1:128 Smeltz RB/ Swanborg R., J Immunol 163:1390 (1999)

5 NK cells limit CD8 immunity leading to chronic viral infection Virus Infection LCMV-Clone 13 Th Th NK NK NK X X CTL CTL CTL chronic Virus Infection LCMV-clone 13 Th Th Tn CTL CTL CTL CTL CTL CTL cleared Waggoner Nature (2012) 481:394 Lang et al PNAS (2012)109(4):1210

6 Presence of NK cells are associated with slow growth of tumor infiltrating lymphocytes percent Ovarian: % 25 CD3+ CD56- CD3- CD Rapid TIL growth Intermed/poor TIL growth

7 Do NK cells inhibit TIL growth? Sarah Crome Linh Nguyen

8 Slow growing cultures have increased % CD56+ cells CD8+ CD4+ CD3-CD56+ % of TIL cultures % of TIL cultures % of TIL cultures Fast slow TIL growth Fast slow TIL growth Fast slow TIL growth

9 NK cells inhibit the expansion of TILs

10 Regulatory NK cells suppress TIL expansion TIL (Cell trace) + anti-cd3 + anti-cd28 +/- NK (Slow growing) Proliferation % suppression % suppression + NK + NK

11 NK cells alter cytokine production by TILs TIL (Cell trace) TIL TIL + NK + anti-cd3 + anti-cd28 +/- NK (Slow growing) 3 days TNF-α CD4+ CD8+ cytokines IFN-γ

12 NK cells suppress IFN-γ production by TILs CD4+ CD8+ % IFN-γ % TNF-α % IFN-γ % TNF-α + NK + NK + NK + NK

13 TIL cultures with NKreg cells produce an altered cytokine profile IL-10 IL-6 IL-13 IL-4 Fast slow TIL growth Fast slow TIL growth Fast slow TIL growth Fast slow TIL growth

14 Does the presence of inhibitory NK cells correlate with disease progression? percent Ovarian: % 25 CD3+ CD56- CD3- CD Rapid TIL growth Intermed/poor TIL growth

15 Recurrence free survival correlates with presence of NKregs Recurrence Free Survival no NKreg (9) 12.4 months (n=6) +NKreg (6 patients) P = months (n=9) Time (months)

16 Summary: Subset of NK /ILCs inhibit T cell immunity - Increased proportion of NK cells in slow growing TIL cultures - NK cells limit TIL expansion - NK cells alter T cell cytokine production - Presence of NK regs in TIL cultures correlate with faster time to recurrence in ovarian cancer

17 Importance of NK cells in regulating Immunity CD4+ Helper T cell cytokines Treg DC NKreg CD8+ CTL: kills cytokines Tumor microenvironment

18 Role of novel T cell subsets in anti-tumor immunity Michael Saint Paul

19 Multiple different CD8+ T cell lineages TNF, IFN-γ Granzyme B Antiviral Antitumor IL-4, IL-5, IL-13 Granzyme B Parasites, Allergy, Autoimmunity Tc1 Tc2 IL-12 CD8+ IL-4, TGFb Tc9 IL-9 No Granzyme B Allergy? Tc17 Tc22 IL-22? Function? IL-17A, IL-17F, IL-22 No Granzyme B Infection, Autoimmunity (EAE)

20 Cytokine Production by different CD8+ T cell lineages IL-2 IL-10 IL-17 IL-22 TNF-a IFNg

21 Differential ability for Tc subsets to lyse target cells 60 % lysis :1 30:1 10:1 3:1 Effector: Target Ratio Granzme B

22 The Tc1 lineage can limit the growth of B16 melanoma cells 3 x 10 5 B16-gp33 sc days P14 polarized cells iv Monitor tumor growth Tumor area (mm 2 ) Day

23 The Tc22 subset can also limit tumor growth 3 x 10 5 B16-gp33 sc days P14 polarized cells iv Monitor tumor growth Tumor area (mm 2 ) No T cells Tc1 Tc Day

24 Summary: Tc lineages - can skew Tc 1, 2, 9,17, 22 in vitro - Tc22 make IL-2, TNFα, IL-22 but IFN-γ lo - Tc 1 and Tc22 show high cytotoxic activity in vitro - Tc1 and Tc22 can limit tumor growth

25 Sarah Crome Philipp Lang Marcus Bernardini Golnessa Mojtahedi Pat Shaw Anca Milea Michael St Paul Lewis Lanier Sandra Lopez-Verges Linh Nguyen Michael Pniak Jessica Nie Patty Yen

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27 Lab Focus: Basic mechanisms of immune regulation relevance/impact on immunotherapy Tumor infiltrating lymphocytes (TILs): 1) TIL therapy, melanoma, mesothelioma ovarian cancer 2) Biology of the Tumor Microenvironment

28 Adoptive T cell Therapy Test T cell specificity Expand Cells Culture T cells Tumor excision Dudley et al Science (2002) 298; 850 J Clin Oncol (2005) 22;2346 J Clin Oncol (2008) 26;5233 Nat. Rev. Immunol. 6:383. Transfer into treated patients ( cells) High-dose IL-2 Cyclophosphamide 60 mg/kg/day x2d Fludarabine 25 mg/m2/day x5d High dose IL2 = 720,000 IU/kg q8h to tolerance (max ~15 doses) 22% of patients durable CR of 93 patients 49% 59% 72% response rate

29 TIL from ovarian cancer specimens show variable rates of expansion 200 Total number of cells (millions) Fast Intermediate Slow/No

30 Tc1 cells are superior for limiting tumor growth

31 Multiple different CD4+ T cell lineages Intracellular pathogens IL-2, IFN-γ Th1 IL-12, IFN γ CD4+ IL-6, TNFα Th22 tissue remodelling, wound healing extracellular pathogens allergy IL-4, IL-5 IL-13 Th2 Th9 allergy autoimmunity IL-9 itreg TGF β suppression IL-10, TGF-β Tfh Th17 antibody production IL-21 IL-22 extracellular pathogens IL-17A, IL-17F, IL-22

32 Cytokine profile of Tc22s IL-22 IL-17 IFN-γ TNF-α IL-2

33 Cells of the Adaptive Immune System FEATURE: antigen specific receptors B Cells Antibody production T cells CD4+ cytokines regulatory Treg IL2 IL4 IL9 IL17 22 TFH Treg T cells CD8+ cytotoxic cytokines IL2 IL4 IL9 IL17 IL22

34 Innate Lymphoid Cells (ILCs) Group 1 Group 2 Group 3 NK IFNγ IL-5 IL-13 IL-17 IL-22 cytokines kill regulatory

35 Players in the Immune Response CD4+ Helper T cell cytokines DC Treg CD8+ CTL: kills cytokines Tumor

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37 Altered Cytokine-Profile of TIL With ILCregs

38 Innate Immunity: NK cells - regulated via a series of activating / inhibitory receptors - cytolytic function virus infected targets, tumor cells - important role for controlling virus infections and tumor growth - NK cell memory - have inhibitory function regulatory subpopulation -produce IL-10, limit CD8 responses (SH Lee JEM 206:2235 (2009)) - 2B4 regulates NK cell function: absence of 2B4, NK cells killed activated CD8 + T cells, perforin dependent (Waggoner SN JCI 120;1925 (2010)

39 Human NK Cells Crome et al, 2013

40 Mouse NK Cells Crome et al, 2013

41 CD16 expression distinguishes NK cells from fast versus slow growing TIL cultures CD16+ PBMC NK Fast growing Slow growing % CD16+ NK cells PBMC Fast Slow TIL expansion

42 Ovarian TIL Expansions Correlate with NK Ratio

43 NK cells inhibit adaptive immunity through a variety of mechanisms DC DC Limit # DCs to activate T cells perforin perforin Th DC NK NK IL-10 Th NK NK CTL IFN γ Anti-proliferative cytokines perforin CTL

44 NK cells inhibit adaptive immunity through a variety of mechanisms DC DC DC Limit # DCs to activate T cells perforin NK perforin NK Th NKG2D MICA/B ULB1/2/3 CD4+ T NKp46? NK NKG2A CD94 Qa-1 HLAE IL-10 Th NK NK CTL CD8+ T CTL IFN γ Anti-proliferative cytokines perforin NKG2D MICA/B ULB1/2/3 NK NKp46?

45 Players in the Immune Response Helper Helper T cell factors that enhance immune function IL-2 DC Treg Regulatory T cell Dendritic cell Killer cytotoxic T cell tumor kills tumor cells

46 TIL based adoptive T cell therapy Prior lymphodepletion 1. Tumor sample Expansion (IL-2) TIL infusion ~ cells i.v. Low dose IL- 2 therapy 4. Re-infuse TILs 2. TIL Evaluation 3. Rapid expansion of TILs (OKT3, feeder cells, lower-dose IL-2) Nguyen et al PLoS ONE 2010

47 Responses with TIL based adoptive T cell therapy Melanoma Maximum change in tumor size 0% -25% -50% -75% -100%

48 NK cells limit CD8 immunity WT mice NK depleted mice LCMV tetramer cytokine gp33 p < p < 0.01

49 Big questions: 1) How is the TME set up? Mutations, inherent properties of immune response in patients 2) Dissect cell types, function, markers 3) Predictive value: treatments, responses? 4) Need to include live cell analysis

50 Multiple different CD4+ T cell lineages Intracellular pathogens IL-2, IFN-γ Th1 IL-12, IFN γ CD4+ IL-6, TGF β Th17 extracellular pathogens IL-17A, IL-17F, IL-22 extracellular pathogens allergy IL-4, IL-5 IL-13 Th2 Th9 allergy autoimmunity IL-9 itreg TGF β suppression IL-10, TGF-β Tfh Th22 tissue remodelling, wound healing antibody production IL-21 IL-22

51 Multiple different CD8+ T cell lineages Intracellular pathogens IL-2, IFN-γ cytotoxic Tc1 IL-12, IFN γ CD8+ IL-6, TGF β Tc17 extracellular pathogens IL-17A, IL-17F, IL-22 Non Cytotoxic extracellular pathogens allergy IL-4, IL-5 IL-13 Weakly CTL Tc2 IL-4, TGFb Tc9 allergy autoimmunity IL-9, weakly cytotoxic Tc22 tissue remodelling, wound healing IL-22 cytotoxic

52 Team TILs

53 NK cells limit T cell proliferation in vitro NK cells from naïve mice T cells NK cells from infected mice T cells NK cells from naive mice + IFNα T cells Anti-CD3/28 - proliferation

54 CD16 expression distinguishes NK cells from fast versus slow growing TIL cultures CD16+ PBMC NK Fast growing Slow growing

55 NK cells limit T cell function via perforin Control αnk1.1 Perforin -/- αnk1.1 Perforin -/- αnk1.1 Control ank1.1 Perforin -/- Perforin -/- ank1.1 * * * P14 LCMV αnk1.1 IFN-gamma production

56 Points for discussion: 1) Define markers/signature for this NKreg/ILC population. 2) What triggers the development of NKregs in vivo, in the tumor setting? 3) Do they have prognostic value? 4) Do they play a role in other cancers?