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

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

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

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

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

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

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

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

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

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

IL-17A Th17

What is a “Th17” cell in humans?

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

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

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

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

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 IL independent loci

What distinguishes pathogenic Th17 cells in humans?

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

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

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

MDR1 + Th17 cells Ramesh et al., J Exp Med 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.)

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

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

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

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

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