Durable outcomes in subsets of solid cancer patients treated with immune checkpoint inhibitors (ICI) or adoptive cell transfer (ACT) immunotherapy has driven interest in gaining a better understanding of resistance mechanisms that could identify novel druggable targets. Myeloid-derived suppressor cells (MDSC) have emerged as one such barrier based on their ability to inhibit innate and adaptive immunity. While elevated blood MDSC are recognized as a poor prognostic indicator in cancer patients, it is widely thought that the main effector site for MDSC is within the tumor microenvironment (TME). This is in line with the well-documented contact- dependent mechanisms involving short-lived intermediates that underlie known mechanisms of T cell suppression by MDSC. Our published and preliminary studies enlarge on this view, showing that MDSC also function outside the TME through an unprecedented mechanism of intravascular immune suppression. The proposed study builds on our discovery that circulating MDSC initiate contact-dependent cleavage of the L- selectin homing receptor on target T cells that substantially reduces antigen-driven expansion of cytotoxic T cells in lymph nodes. We further found that L-selectin loss coincides with the formation of stable MDSC clusters in the blood of murine tumor models and advanced cancer patients. We term these new structures circulating myeloid cell (CMC) clusters. These observations led us to hypothesize that CMC clusters are an unrecognized functional niche for systemic immune suppression in cancer. To test this hypothesis, we will first determine if blood-borne MDSC target not only naïve T cells, but more broadly attack stem cell memory and central memory T cells and natural killer cells that each require L-selectin for their antitumor activity. Secondly, we will determine if CMC clusters are the active site of L-selectin cleavage by using a multipronged genetic approach to examine L-selectin fate following disruption of MDSC-T cell conjugate formation in vivo. These mechanistic studies center on β2 integrins that are highly expressed by MDSC but are normally inactive on leukocytes in fast-flowing blood under non-pathological conditions. Thirdly, we will examine the translational relevance of CMC clusters during ICI or ACT therapy in a preclinical model in which blood is the primary effector site for MDSC due to their exclusion from the TME (by blocking chemokine-directed trafficking) and spleen (by splenectomy). We will deplete circulating MDSC in this model using antibodies or a clinically relevant liver-X-receptor agonist that induces MDSC-intrinsic apoptosis to establish if blood-borne MDSC contribute to therapeutic resistance. Complementary studies will test the hypothesis that combining the analysis of circulating MDSC with CMC clusters and/or T cell L-selectin will formulate an immunosuppressive signature that predicts response to first-line therapy in metastatic cancer patients. The proposed studies wil...