Bcell–Derived IL35Drives STAT3-DependentCD8þT-cell Exclusion in Pancreatic Cancer A C

◥ Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy characterized by a paucity of tumor-proximal CD8þ T cells and resistance to immunotherapeutic interventions. Cancer-associated mechanisms that elicit CD8þ T-cell exclusion and resistance to immunotherapy are not well-known. Here, using a Krasand p53-driven model of PDA, we describe a mechanism of action for the protumorigenic cytokine IL35 through STAT3 activation in CD8þ T cells. Distinct from its action on CD4þ T cells, IL35 signaling in gp130þCD8þ T cells activated the transcription factor STAT3, which antagonized intratumoral infiltration and effector function of CD8þ T cells via suppression of CXCR3, CCR5, and IFNg expression. Inhibition of STAT3 signaling in tumor-educated CD8þ T cells improved PDA growth control upon adoptive transfer to tumor-bearing mice. We showed that activation of STAT3 in CD8þ T cells was driven by B cell– but not regulatory T cell–specific production of IL35. We also demonstrated that B cell–specific deletion of IL35 facilitated CD8þ T-cell activation independently of effector or regulatory CD4þ T cells and was sufficient to phenocopy therapeutic anti-IL35 blockade in overcoming resistance to anti–PD1 immunotherapy. Finally, we identified a circulating IL35þ B-cell subset in patients with PDA and demonstrated that the presence of IL35þ cells predicted increased occurrence of phosphorylated (p)Stat3þCXCR3 CD8þ T cells in tumors and inversely correlated with a cytotoxic T-cell signature in patients. Together, these data identified B cell–mediated IL35/gp130/ STAT3 signaling as an important direct link to CD8þ T-cell exclusion and immunotherapy resistance in PDA. Introduction Infiltration of cytotoxic T cells into the tumor parenchyma correlates with better outcomes in a variety of tumor types, especially in the context of immunotherapy (1). Thus, better understanding of the mechanisms that modulate T-cell trafficking and function in tumors is necessary to overcome inefficient immune responses. This is particularly relevant in pancreatic ductal adenocarcinoma (PDA), an aggressive and deadly disease often characterized by lack of infiltration and/ or dampened functionality ofCD8þTcells (2, 3). In the setting of PDA, immunotherapy has been unsuccessful (4, 5). The mechanisms that restrict tumor-directed CD8þT-cell function in PDA are thought to be linked to immunosuppression (6–8). Significant research efforts have described a variety of tumor cell-intrinsic or extrinsicmechanisms that may act to restrict CD8þ T-cell activity in the PDA tumor microenvironment (TME), among which are myeloid cell recruitment and polarization, expansion of regulatory and gd T cells, as well as modulation of T-cell infiltration by tumor cells themselves (9–17). These studies suggest that reversing immunosuppression in pancreatic cancer could improve endogenous T-cell activity. Although it is becoming clear that contribution from both tumor cell-intrinsic and extrinsic mechanisms may dictate the type of immunosuppression present in the TME, the mechanisms that these various immunosuppressive arms utilize to directly control T-cell function in PDA remain poorly defined. We previously demonstrated that the cytokine IL35 promotes pancreatic tumorigenesis (18, 19). IL35 is a member of the IL12 family of cytokines, forms via heterodimerization of p35 and Ebi3, and is thought to signal in na€ ve CD4þ T cells through the IL35 receptor (IL35R, consisting of IL12Rb2 and gp130 subunits). This activates STAT1 and STAT4 signaling pathways (20). Elevated IL35 can be detected in lymphoma cells and lung cancer, and predicts poor outcome in cases of leukemia, colorectal, and pancreatic cancer (20). Regulatory T cells (Treg), na€ ve CD4þ T cells (iTr35), dendritic cells (DC), and B cells are known to produce IL35 (11, 19, 21–25), whose expression has been linked to its ability to modulate immune Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina. Department of Cell Biology, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina. Department of Pathology and LaboratoryMedicine, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina. Department of Radiation Oncology, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina. Department of Surgery, Barnes-Jewish Hospital and the Alvin J. Siteman Comprehensive Cancer Center, Washington University School of Medicine, St. Louis, Missouri. Department ofMedicine, Barnes-JewishHospital and theAlvin J. Siteman Comprehensive Cancer Center, Washington University School of Medicine, St. Louis, Missouri. Department of Biostatistics, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina. Department of Surgery, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina. Department of Medicine, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina. Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina. Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. Department of Genetics, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina. Note: Supplementary data for this article are available at Cancer Immunology Research Online (http://cancerimmunolres.aacrjournals.org/). CorrespondingAuthor:Yuliya Pylayeva-Gupta, TheUniversity ofNorth Carolina at Chapel Hill, 450 West Drive, Chapel Hill, NC 27599. Phone: 919-962-8296; Fax: 919-966-8212; E-mail: yuliyap1@email.unc.edu Cancer Immunol Res 2020;8:292–308 doi: 10.1158/2326-6066.CIR-19-0349 2020 American Association for Cancer Research. AACRJournals.org | 292 on January 3, 2021. © 2020 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from Published OnlineFirst February 5, 2020; DOI: 10.1158/2326-6066.CIR-19-0349