1. Dynamic Programming of CD8 + T Cell Effector and Memory Development by Innate Cytokines David Farrar Department of Immunology UT Southwestern Medical Center Dallas, TX

2. Differentiation versus acute response Proliferation/expansion Antigen recognition Costimulation Cytokines Acute effector response Antigen recognition Costimulation Cytokines Cytokine secretion Lytic activity DC

3. CD8 + Cytotoxic T Lymphocytes (CTL) 1.Home to sites of infection 2.Secrete inflammatory cytokines: IFN-  and TNF-  3.Kill infected cells via perforin/granzyme-mediated lysis.

4. Innate cytokines regulate adaptive immune responses IL-1 IL-6 IL-12 IL-23 TNF-  TGF-  IFN-  DC PAMPs TLRs, NODs, RIG-I/MAVS, etc.

5. IL-12R IL-12 STAT4 T-bet Th1/Tc1 IFN-  IFNAR Proliferation, etc.Antiviral, etc. Are IL-12 and IFN-  redundant signals regulating Th1 development? &

6. P-Y STAT 1 STAT 2 P-Y IFN-  Signaling STAT 4 Y-P Antiviral Activity Our Previous Studies: 1. IFN-  does not promote Th1 development 2. Enhances IL-2 secretion and development of T CM cells 3. Inhibits Th2 development

7. IL-12, but not IFN- , regulates IFN-  and TNF-  expression in human CD8 + T cells

8. CCR7 and CXCR3 direct T cell traffic in opposite directions Inflammed tissues: IP-10, MIG Lymph nodes: CCL19, CCL21 Naïve: CCR7+ CXCR3- Effector: CCR7- CXCR3+ Central Memory: CCR7+ CXCR3-

9. CXCR3+ Effector/E M : -Preferentially traffic to periphery -High cytolytic activity -High effector cytokine expression -Terminally differentiated/poor expansion to recall CCR7+ Memory/C M : -Preferential lymph node traffic -Low cytolytic activity -Low effector cytokine, high IL-2 -Multi-potent/strong expansion to recall Induction of effector and memory CTL development by IL-12 and IFN- 

10. Chowdhury, et. al. Blood 2011 CD8+ effector functions are driven by IL-12 and acquired as a function of proliferation

11. CXCR3+ cells preferentially secrete IFN-  Ramos, et. al. Blood 2009

12. IL-12 + IFN-   -CD3  -CD28 Sort: CD8+ CD45RA+ Sort: Day7 CXCR3+/CCR7- CXCR3-/CCR7+ Examination of recall responses Ramos, et. al. Blood 2009

13. Chowdhury, et. al. Blood 2011 Cytokine and lytic activity of T EM cells ex vivo

14. Effector Memory: CXCR3+/CCR7- IFN-  /TNF-  Pfp/GrzB Low proliferative capacity Terminally differentiated IL-12 IFN-  Cytokine-regulated central and effector memory CD8 + T cell development Central Memory: CXCR3-/CCR7+ Low cytokine secretion GrzB hi, Pfp lo High proliferative capacity Able to generate secondary effectors What is the mechanism?

15. How do IL-12 and IFN-  co-regulate the development of both T EM and T CM ? Differential cytokine responsiveness?

16. IL-12 and IFN-  promote expression of their own receptors Ramos, et. al. Blood 2009

17. Cytokine responsiveness as a function of cell division Ramos, et. al. Blood 2009

18. Cytokine responsiveness as a function of cell division

19. Chowdhury, et. al. Blood 2011 Distinct gene expression patterns are acquired as a function of cytokine responsiveness and proliferation

20. Chowdhury, et. al. Blood 2011 Unique gene signatures of T EM and T CM

21. Chowdhury, et. al. Blood 2011 Identification of IL-12-induced genes that are stably expressed in T EM in vivo

22. Dynamic programming of T EM and T CM cells IL-12 Signal Strength IFNAR2 IL-12R  2 T CM T EM Effector Memory IFN-  Central Memory Recall

23. Jon Huber, Navin Chowdhury, Didem Agac, Leo Estrada, Ann-Marie Schaefer, Sarah Gonzales, Hilario Ramos, Ann Davis Jim Forman, Ph.D. Becky Gruchalla, M.D., Ph.D. Michelle Gill, M.D., Ph.D. Amnis/Millipore: Tad George, Ph.D. Flow Cytometry Core, UTSW: Angela Mobley, M.S. Christina Nguyen, B.S. Funding: NIH, NIAID Crystal Charity Ball Acknowledgements