Project Summary Despite the clinical success of immune checkpoint blockade (ICB), most cancer patients do not respond. Cytotoxic CD8+ T cells are critical mediators of tumor growth control following ICB and depend on 3 signals: signal 1 (TCR), signal 2 (costimulation), and signal 3 (cytokines). While many immunotherapies target signals 1 and 2, we do not know the necessary signal 3 cytokines and mechanisms regulating them to induce potent anti- tumor cytotoxic CD8+ T cells. To identify regulators of CD8+ T cell responses to tumors, including signal 3 cytokine receptors, we developed a CRISPR-based screening platform for assessing gene knockouts (KOs) in naive CD8+ T cells differentiating in response to a tumor. We identified IL-27 receptor subunits IL27Ra and IL6st, IL- 2R subunits, and Stat3 (downstream transcription factor from IL27Ra) as the most important cytokine pathway members needed to promote CD8+ T cell abundance in tumors. KO of IL27Ra on CD8+ T cells or neutralization of IL-27 significantly decreased CD8+ T cell infiltration, cytotoxic marker expression, and tumor growth control. Strikingly, we found that IL-27 supplementation during CD8+ T cell priming was sufficient to promote cytotoxic CD8+ T cell responses to tumors and potently controlled tumor growth. Based on these data, our overarching hypothesis is that IL-27 is the signal 3 cytokine that promotes differentiation of naive CD8+ T cells into anti-tumor cytotoxic CD8+ T cells. Our overall goals are to (1) define mechanisms by which IL-27 drives cytotoxic CD8+ T cell responses to tumors and (2) determine mechanisms that regulate IL-27 production. Aim 1 will determine mechanisms by which IL-27 promotes CD8+ T cell cytotoxicity. We will compare cytotoxic functions of mouse WT and IL27Ra KO CD8+ T cells in tumors by intravital microscopy and human WT and Stat3 KO CD8+ T cells treated with IL-27 by live cell imaging. We will also assess the similarity of IL27Ra KO and Stat3 KO CD8+ T cells by flow cytometry and RNA-sequencing. We will use Stat3 ChIP-qPCR in mouse and human CD8+ T cells to determine if IL-27 activates Stat3 to bind to cytotoxic gene loci. Aim 2 will determine mechanisms regulating IL-27 production and whether therapeutic IL-27 improves tumor immunity. We will identify cell types that produce IL-27 by imaging mouse and human melanoma clinical samples. We will determine if IFNg controls IL-27 expression in tdLNs by treating tumor-bearing IL-27 reporter mice with IFNg neutralizing antibodies. To determine the therapeutic potential of IL-27 administration, we will treat mice with recombinant IL-27 with or without PD-1 blockade and assess tumor growth control. Lastly, we will analyze transcriptional data from human cancers to determine if IL-27/IL27Ra levels correlate with cytotoxic CD8+ T cell responses. Together, the proposed project will improve our conceptual understanding of the signal 3 cytokines that positively regulate cytotoxic CD8+ T cell responses to tumors. If IL-27 is the primary signal 3 cytokine for CD8+ T cell responses to tumors, this may lead to new approaches for treating cancer patients by modulating the IL-27 pathway.

Project Narrative Despite the success of immune checkpoint blockade (ICB) in diverse cancer types, the majority of patients do not respond to ICB and the determinants of successful ICB responses are not yet clear. This project will test the hypothesis that the cytokine IL-27 functions as the primary signal 3 cytokine in tumor immunity and will elucidate (1) how IL-27 mediates CD8+ T cell cytotoxicity in cancer and (2) how IL-27 production is regulated. These findings will inform new strategies for improving ICB responses by modulating the IL-27 pathway.