IL-27 functions as a novel signal 3 cytokine and promotes cytotoxic T cell responses to tumors

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.