Synergistic effects of indoleamine 2,3-dioxygenase (IDO) inhibitor drugs and chemotherapy in melanoma.

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1 Synergistic effects of indoleamine 2,3-dioxygenase (IDO) inhibitor drugs and chemotherapy in melanoma. Madhav D. Sharma Cancer Immunology, Inflammation, and Tolerance Program, Department of Pediatrics, Georgia Regents University, Augusta, Georgia, USA Abstract We have previously shown that large numbers of FoxP3 Treg cells undergo rapid reprogramming into activated T helper cells after vaccination with antigen plus Toll-like receptor 9 (TLR-9) ligand. Helper activity from converted Treg cells provided essential help during initial priming of CD8 T cells to a new cross-presented antigen. We now show that help from Treg cells is delivered via CD40L, and (unlike help from conventional non-treg CD4 cells) did not require preactivation or prior exposure to antigen. In hosts with established tumors, Treg reprogramming was suppressed by tumor-induced IDO. Therapeutically, there was fundamental synergy between blocking IDO-dependent suppression and driving CD40-mediated DC activation. When IDO was blocked, required CD40L signal could derive from pre-activated memory conventional CD4 helper cells (if available), reprogrammed Tregs, or exogenous anti CD40 agonist antibody. Agonist antibody and the IDO inhibitor drug 1-methyl-tryptophan showed potent synergy. Chemotherapy allowed extensive reprogramming of Tregs in tumor draining lymph nodes in response to anti-tumor vaccine, but only if IDO was blocked with 1MT. The effects of pharmacologic IDO inhibitor on Treg reprogramming was fully recapitulated by blocking the CTLA4 pathway. Thus reprogrammed Tregs participate as previously unrecognized contributors early CD8 T cell responses against tumors. Funding Support: "Supported by Grants from NIH (R01CA103320, R01CA096651, R01CA112431)" Introduction Hosts with established tumors can show markedly elevated levels of IDO in tumordraining lymph and tumor-induced IDO can directly activate Foxp3 Treg cells for enhanced suppressor activity. Conversely, if IDO is blocked (e.g., by the pharmacologic IDO-inhibitor 1- methyl-tryptophan), then the Treg cells in tumor-bearing hosts become unstable and can be driven by inflammation to undergo reprogramming into helper-like T cells, expressing IL-17 and other proinflammatory cytokines. We have shown that cells of the Foxp3 lineage can participate as an integral part of the CD4 T helper system. In certain settings, reprogrammed Treg cells were found to play an indispensable helper role, allowing innate inflammation to drive the early (priming) phase of CD8 Results T cell response to new antigen.

2 Figure1 D IDO in TDLN A TDLN for CD40L-mediated help is still needed B The source of CD40L is reprogrammed Tregs. C Helper Activity of Reprogrammed Treg Cellsin Tumor-Bearing Hosts Is Mediated viacd40l. (A) Tumor implanted C57BL/6 orcd40lg hosts, on day 7 received pmel-1 cellsand vaccine, with or without oral 1MT asindicated. 4 days later, DCs were analyzed intumor-draining LNs. (B) / C57BL/6 or Cd40lg mice Tregs were pretransferred in Tcra / mice before tumor implant. 4 days after pmel-1 cells and vaccine, TDLN analysis was performed, and tumor size was measured at necropsy. (C) C57BL/6 or Cd40lg / mice Tregs were pretransferred in Tcra / mice before tumor implant. 4 days after pmel-1 cells and vaccine, TDLN analysis was performed. One treatment group included activating anti- CD40 (clone FGK45). /

3 Figure 2 Blocking IDO pathway allows Tregs reprogramming. Treg Cell Reprogramming in Tumor-Bearing Hosts Is Antagonized by IDO.(A) GFP B16F10 tumor bearing Foxp3 mice received adoptive transfer of resting pmel-1 cells (sorted CD8 ), with or without vaccine. Groups were treated either with continuous 1MT in drinking water (beginning on day 6, 1 day prior to vaccination) or with vehicle control, as indicated. 4 days after vaccination, tumordraining LNs were harvested, treated with low-dose PMAionomycin, and stained. Figure 3 Agonist antibody and the IDO inhibitor drug 1-methyl-D-tryptophan showed potent synergy. B16F10 tumor bearing C57BL/6 or AHR d host mice received adoptive transfer of resting pmel-1 cells (sorted CD8 ), with or without vaccine. Groups were treated either with continuous 1MT in drinking water (beginning on day 6, 1 day prior to vaccination) or with vehicle control, as indicated 4 days after vaccination, tumor size were measured and photographs were Synergy taken. betweencd40lagonist C57BL/6 group of antibody and IDO-pathway inhibitor D-1MT.

4 d mice also received activating anti-cd40 (clone FGK45). Use of mice with deficient AhR signaling (AhR ) suggests that the AhR is a major pathway mediating effects of IDO. Figure 4 Synergy between IDO-pathway inhibitor and chemotherapy. CTLA4 blockade has similar effects on Tregs reprogramming as IDO-pathway inhibitor. 2 Chemotherapy allowed extensive reprogramming of Tregs in response to vaccine, but only if IDO was blocked blocked with 1MT. B16F10 tumor bearing C57BL/6 mice received adoptive transfer of resting pmel-1 cells (sorted CD8 ), with or without vaccine. Groups were treated either with continuous 1MT in drinking water (beginning on day 6, 1 day prior to vaccination) or with vehicle control, as indicated. Cyclophosphamide (Cy) was injected in chemotherapy group. At regular interval up to 14 days after vaccination, tumor size were measured,and mice sacrificed on day 21. Conclusion The effects of pharmacologic IDO inhibitor on Tregsreprogramming was fully recapitulated by blocking the CTLA-4 pathway.b16f10 tumor bearing C57BL/ mice received adoptive transfer of resting pmel-1 cells (sorted CD8 ), with or without vaccine. Groups were treated either with continuous 1MT in drinking water (beginning on day 6, 1 day prior to vaccination) or with vehicle control, or with anti CTLA-4 blocking antibody (clone 9H10). 4 days later, pmel-1 cells were analyzed in tumor-draining LNs. Two opposing loops in TDLN: tolerogenic and immunogenic loops.

5 References Sharma MD, Huang L, Shi H, Lee E, Wilson JM, Lemos H, Pan F, Blazer BR, Pardoll DM, Mellor AL, Munn DH. An inherently bi-functional subset of Foxp3 Treg/T-helper cells is controlled by the transcription factor Eos. Immunity, 38 (5) pp (2013) Sharma MD, Hou DY, Baban B, Koni PA, He Y, Blazer BR, Mellor AL, Munn DH. Reprogrammed Foxp3 Tregs provide essential help to support cross presentation CD8 T cell priming in naïve mice.immunity 33 (6) pp (2010) Sharma MD, Hou DY, Liu Y, Koni PA, Metz R, Chandler P, Mellor AL, He Y, Munn DH*. Indoleamine 2,3-dioxygenase controls conversion of Foxp3 Tregs to TH17-like cells in tumor-draining lymph nodes.blood 113: June (2009) Sharma MD, Baban B, Chandler P, Hou DY, Singh N, Yagita H, Azuma M, Blazar BR, Mellor AL, Munn DH. Plasmacytoid dendritic cells from mouse tumor-draining lymph nodes directly activate mature Tregs via indoleamine 2,3- dioxygenase. J Clin Invest 117: (2007) Munn DH, Sharma MD, Baban B, Harding HP, Zhang Y, Ron D, Mellor AL. GCN2 kinase in T cells mediates proliferative arrest and anergy induction in response to indoleamine 2,3-dioxygenase. Immunity 22: (2005) Munn DH, Sharma MD, Hou D, Baban B, Lee JR, Antonia SJ, Messina JL, Chandler P, Koni PA, Mellor AL. Expression of indoleamine 2,3-dioxygenase by plasmacytoid dendritic cells in tumor-draining lymph nodes. J Clin Invest 114: (2004) Munn DH, Sharma MD, Lee J, Jhaver KG, Johnson TS, Keskin DB, Marshall B, Chandler P, Antonia SJ, Burgess R, Slingluff CL Jr, Mellor AL. Potential regulatory function of human dendritic cells expressing indoleamine 2,3dioxygenase. Science 297: (2002)