1. New York University -Derya Unutmaz, M.D. -Lina Kozaya Tempero Pharmaceuticals -Radha Ramesh -Alex Pellerin -Thaddeus Carlson, Ph.D. -Ivana Djuretic, Ph.D. University of Miami -Maria Abreu, M.D. -Jacob McCauley, Ph.D. -Maria Quintero Scripps Florida -Kelly McKevitt -Wei Cao, M.D., Ph.D. Funding -GlaxoSmithKline -Scripps – Florida New effector cytokine networks in IBD Mark Sundrud, Ph.D. The Scripps Research Institute msundrud@scripps.edu

2. New York University -Derya Unutmaz, M.D. -Lina Kozaya Tempero Pharmaceuticals -Radha Ramesh -Alex Pellerin -Thaddeus Carlson, Ph.D. -Ivana Djuretic, Ph.D. University of Miami -Maria Abreu, M.D. -Jacob McCauley, Ph.D. -Maria Quintero Scripps Florida -Kelly McKevitt -Wei Cao, M.D., Ph.D. Funding -GlaxoSmithKline -Scripps – Florida Unexpected features of pathogenic T cells associated with IBD Mark Sundrud, Ph.D. The Scripps Research Institute msundrud@scripps.edu

3. Disclosures Former employee of Tempero pharmaceuticals, Inc. No current relationship with Tempero or any other company

4. 1)T cell activation in regional lymph nodes 2) T cell differentiation into specialized “effector” subsets 3) Tissue infiltration 4) T cell effector function in tissue/pathogen clearance 5) Resolution of inflammation/memory Mechanisms of T cell-driven inflammation

5. Disease biology Models of T cell development Mechanisms of T cell-driven inflammation

6. “I go home today. They cured me using this new miracle drug. But I’m afraid it doesn’t work in humans.”

7. Mechanisms of T cell-driven inflammation What T cell subsets are in autoimmune target organs? What regulates their inflammatory function? Human Mouse

8. CD4 + T cell diversity Naïve CD4 + Th2iTregTh1Th17Th22T FH T-bet ROR  t Foxp3AhRBcl6 Gata3 CCR4 CRTH2 CXCR3CCR6CCR10CXCR5 IL-4 IL-5 IL-13 IL-21IL-22T cell activation IL-17A IL-17F IFN  IL-4 IL-21 IL-12 TNF  IL-6 TGF  IL-6 IL-2 IL-12 IL-1  IL-23

9. “Th17 cells are defined by and function through IL-17”

10. IL-17-independent functions of Th17 cells 1.IL-17A expression is instable; “Th17 cells are a transitional phenotype” 2.Th17 cells promote inflammation independent of IL- 17A 3. T cells that lack Il23r, Stat3, Rorc fail to induce experimental colitis upon transfer into lymphopenic mice Doodes et al., J Immunol 2008; Codarri et al., Nat Immunol 2010; Huber et al., Gut 2010; Huber et a., STM 2012

11. IL-17A Th17

12. What is a “Th17” cell in humans?

13. Stable features of Th17 cells Human memory T cells Wan et al., J Exp Med 2011 CCR6 expression defines effector/memory T cells with “Th17” features: –Express ROR  t –Capable of producing IL-17 cytokines

14. Stable features of Th17 cells Human memory T cells Wan et al., J Exp Med 2011 CCR6 expression defines effector/memory T cells with “Th17” features: –Express ROR  t –Capable of producing IL-17 cytokines IL-17A is transient; Th17 is stable IL-17A expression is maintained by IL-2 family cytokines (e.g., IL-2, IL-7, IL-15) - PI-3K/AKT signaling antagonizes FOXO1-mediated repression of IL17A

15. Stable features of Th17 cells Wan et al., J Exp Med 2011

16. Pathogenic/non-pathogenic Th17 cells Naïve T cells IL-23 Goreschi et al., Nature 2010 Hirota et al., NI 2011 Lee et al., NI 2012 Th17 TGF  1 IL-6 IL-17A Th17 IL-1  IL-17A TGF  3 IL-6 “Non-pathogenic” “Pathogenic” Homeostasis IL-10 Inflammation IFN  1.Express Il23r and induce EAE in an IL-23-dependent manner 2.Produce both Th17/Th1 cytokines (IL-17A + IFN  ) 3.Express a unique (“pathogenic”) transcriptional signature “Pathogenic” Th17 cells

17. Genetics of Th17 cells in autoimmune disease Jostens et al., Nature 2012 JAK2 IL-23R STAT3 RORC IL17A IBD-associated loci Locus not associated with IBD IFNG CSF2 (GM-CSF) STAT4 IL1R1 Pathogenic Th17- signature genes IL22 163 independent loci

18. What distinguishes pathogenic Th17 cells in humans?

19. MDR1 is selectively expressed by a subset of Th17.1 cells MDR1 Ramesh et al., J Exp Med 2014; Aller et al. Science 2009

20. Multi-drug resistance type 1 (MDR1) 1.1 of 50 human ATP-binding cassette (ABC) transporter genes 1.Expressed in chemoresistant tumors; transports chemotherapeutic agents (e.g., vinblastine, doxorubicin) 1.Expressed on epithelial cells (gut, lung, kidney, BBB) 1.Transports numerous structurally-unrelated molecules 1.Pharmaceutical drugs 2.xenobiotic compounds 1.Little is known about what it does in T cells

21. Assessing MDR1 expression/function by FACS CD4 + T cells 40C40C Rh123 MDR1 - 37 o C MDR1 + Ramesh et al., J Exp Med 2014

22. MDR1 + Th17 cells Ramesh et al., J Exp Med 2014 1.Restricted to the CCR6 + CXCR3 hi CCR4 lo (Th17.1) compartment 1.Enriched within CCR7 lo (T EM ) cells 1.Display “stem cell-like” characteristics (c-Kit, TCF7, LEF1, etc.)

23. MDR1 + Th17 cells Ramesh et al., J Exp Med 2014 T EM phenotypeSubset IL-17/ IFN  + IL23R (mRNA) P Th17- signature NP Th17- signature IL-23 response R6 - X3 hi R4 lo Rh123 hi MDR1 - Th1--+ R6 + X3 hi R4 lo Rh123 hi R6 + X3 lo R4 hi Rh123 hi -- + + + ++++-- + + R6 + X3 hi R4 lo Rh123 lo MDR1 - Th17 MDR1 - Th17.1 MDR1 + Th17.1

24. Ramesh et al., J Exp Med 2014 MDR1 + Th17 cells are enriched and activated in CD

25. Conclusions: MDR1 distinguishes pathogenic Th17 cells MDR1 is selectively expressed on IL-23-responsive pro- inflammatory Th17 cells MDR1 + Th17 cells are enriched and activated in involved areas of the gut in Crohn’s disease MDR1 + Th17 cells are refractory to glucocorticoids used to treat IBD Ramesh et al., J Exp Med 2014; Y. Bordon, Nat. Rev. Immunol. 2014

26. Mechanisms of T cell-driven inflammation What T cell subsets are in autoimmune target organs? What regulates their inflammatory function? Human Mouse

27. New York University -Derya Unutmaz, M.D. -Lina Kozaya Tempero Pharmaceuticals -Radha Ramesh -Alex Pellerin -Thaddeus Carlson, Ph.D. -Ivana Djuretic, Ph.D. University of Miami -Maria Abreu, M.D. -Jacob McCauley, Ph.D. -Maria Quintero Scripps Florida -Kelly McKevitt -Wei Cao, M.D., Ph.D. Funding -GlaxoSmithKline -Scripps – Florida Thank you! Mark Sundrud, Ph.D. The Scripps Research Institute msundrud@scripps.edu