Cellular biomarkers of T lymphocyte func6on. Michael Be9s University of Pennsylvania Department of Microbiology

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1 Cellular biomarkers of T lymphocyte func6on Michael Be9s University of Pennsylvania Department of Microbiology

2 What T cell response parameters could be important for an effec6ve T cell based product? Specificity of responding cells Cytokine pa9ern of responding cells Phenotype of responding cells Homing capability Effector capability Homeostasis/maintenance Expression of inhibitory receptors

3 A short list of biomarkers useful in assessing T cell responses CD3, CD4, CD8, MHC tetramer: iden6fy T cells, subsets, an6gen specificity CD17a: iden6fy degranula6ng cells IFNγ, MIP1α/β, TNFα, IL2, IL4, IL17, etc: iden6fy cytokine produc6on Perforin, Granzyme A/B/K, Granulysin: iden6fy cytoly6c poten6al CD45RA/RO, CD27, CD57, CCR7, CD28, CD62L, etc: Iden6fy memory phenotype PD- 1, CD16, Lag- 3, 2B4, TIM3: Iden6fy exhaus6on status HLA- DR, Ki67, CD95, bcl2, CD38, CD69: Iden6fy ac6va6on status T- bet, Eomesodermin, GATA- 3, ROR- γt, BLIMP- 1: Iden6fy transcrip6onal programming pa9erns α4β7, CCR5, CCR9, CCR1, CD161, etc: Iden6fy 6ssue trafficking poten6al IL7R (CD127), IL15R (CD122): Assess homeosta6c poten6al CD25, CTLA- 4, FoxP3, GITR, TGFβ, etc: iden6fy poten6al Treg proper6es CFSE, Brdu: Assess prolifera6ve ability Main tool of the Be9s Lab: Polychroma6c Flow Cytometry

4 Focus of this presenta6on T cell polyfunc6onality- Different flavors, different viruses, different protec6ve capacity? T cell effector func6on- Modula6on and transcrip6onal control Disclaimer: All data shown is with ex vivo primary T cells, not a cellular product Use this informa6on as a guide- what do you want your T- cell based product to do?

5 What is T cell polyfunc6onality? Simultaneous expression of 3 or more measured T cell func6ons % CD8+ T cell response Do T cells against different viral pathogens always exhibit the same func;onal pa<erns?

6 Differen6al func6onal proper6es of human an6- viral CD8+ T cells Be9s, et al., Blood 26

7 Differen6al func6onal proper6es of HIV- specific CD8+ T cells by disease progression status Be9s, et al., Blood 26

8 Example of a panel used for tes6ng polyfunc6onality of an6viral CD8+ T cell effector responses FITC PerCP Cy5-5 MIP-1α CD4 Pac Blue Aqua Blue QD 565 QD 585 QD 65 QD 655 CD14/16/19 Viability CD57 CD3 CD8 CD27 APC Alexa 7 APC Alexa 75 IL-2 IFN-γ CCR7 PE TRPE PE Cy5-5 PE Cy5 PE Cy7 Perforin CD45RO Granzyme B CD17a TNF-α Note: Panel does work, but requires specialized properly validated flow cytometer

9 Differential CD8+ T cell effector properties between EBV, Flu, CMV, and Ad responses Func6ons measured: Perforin (P), IL- 2 (2), CD17a (7), IFN- γ (γ), TNFa (T) Makedonas, et al., PLoS Pathogens 21

10 Heterogeneity of IFN- γ producing CD8+ T cells: Differen6al IL- 2 and Perforin produc6on Makedonas, et al., PLoS Pathogens 21 An6viral CD8+ T cells tend to be maintained in either a memory- or an effector- like state that appear to be mutually exclusive

11 Differen6al effector capability of viral specific CD8+ T cell responses # of func6ons Purple Arc= perforin producing cells Makedonas, et al., PLoS Pathogens 21 Conclusions: 1. Polyfunc6onal T cell responses come in different flavors 2. Make sure that you know that your product can and does do what you want it to!

12 What drives differen6al effector capability of CD8+ T cells? Assessment of transcrip6on factors that control CD8+ effector func6on

13 T- bet Tbet- regulates lineage commitment in T cells and NK cells in murine models, but the role in human cells is less clear Promotes IFNg Represses IL- 2 T- box elements described on promoters for perforin, granzyme T- box Glimcher, et al. Nature Reviews Immunology. 24.

14 T- bet expression correlates with the presence of cytoly6c molecules in human CD8 + T cells <Pacific Blue-A>: Perforin 1 4 Perforin 1 3 <PE-Cy5-5-A>: Granzyme B 1 4 Granzyme B T- bet <FITC-A>: T-bet T- bet <FITC-A>: T-bet

15 Differen6al levels of T- bet within IL- 2 and perforin producing cells Superan?gen (SEB) s?mula?on; Naïve cells excluded Makedonas, et al., PLoS Pathogens 21

16 Rela6onship between T- bet expression and CD8+ T cell func6onality Unpublished data showing that perforin is primarily expressed in T- bet high cells, that IFNg is produced only in T- bet+ cells (high or low), and that TNF is produced irregardless of T- bet.

17 Modula6on of T- bet aoer s6mula6on and prolifera6on- baseline <Pacific Blue-A>: Perforin Perforin % of Max CD4+ T cells <Pacific Blue-A>: <APC-A>: RAK RAK tetramer Perforin Unpublished data showing rela6onship between EBV- specific tetramer+ Tetramer+ T cells and cells T- bet 1 expression NK cells <PE-A>: T-bet T- bet % of Max <APC-A>: RAK tetramer RAK tetramer <PE-A>: T-bet T- bet

18 Modula6on of T- bet aoer s6mula6on and prolifera6on- day <PE-A>: T-bet <Pacific Blue-A>: Perforin Unpublished data demonstra6ng increase in T bet expression upon prolifera6on in pep6de- s6mulated CD8+ T cells <CFSE-A>: Proliferation <CFSE-A>: Proliferation 1 1 CD4+ T cells Tetramer+ cells NK cells % of Max % of Max <PE-A>: T-bet T- bet <Pacific Blue-A>: Perforin Perforin

19 Prolifera6on of HIV- specific CD8+ T cells from chronic progressors does not recover effector poten6al <APC-A>: IFN-g hour re- s6m with op6mal HIV epitope pool on Day <CFSE-A>: Proliferation Unpublished data showing failure of prolifera6ng cells to upregulate T bet in chronic HIV progressors

20 Conclusions S6mula6on of T cells can generate a wide array of func6onal responses (cellular biomarkers) that can vary depending on specificity of the responding cells T- bet expression defines (in part) effector poten6al of CD8+ T cells Prolifera6on can alter func6onal and phenotypic proper6es of T cells, but may not overcome poten6al defec6ve responses T cell based cellular products may be inherently limited to in vivo pre- defined func6onal capabili6es (without addi6onal manipula6on)

21 Acknowledgements Be<s Lab Adam Hersperger George Makedonas Gabriela Cosma Korey Demers Jay Gardner Diane Carnathan Carolina Pombo- Mar6nez Danielle Haney Natalie Hutnick Sally Yuan UPenn CFAR Ian Frank Pablo Tebas Debbie Gudonis Duke University CFAR Guido Ferrari Kent Weinhold Penn John Wherry Steve Reiner FUNDING: NIAID R1 AI7666, HVDDT Harvard University CFAR Bruce Walker Florencia Pereyra University of Toronto Rupert Kaul Mario Ostrowski Prameet Sheth Lucy Shin Case Western University CFAR Michael Lederman Benigno Rodriguez Sco9 Sieg Leia Teixeira- Johnson University of Alabama Birmingham CFAR Paul Goepfert