Abstract The intestinal mucosa has a rich immune microenvironment capable of responding rapidly to invading pathogens while maintaining a regulated homeostatic state to commensal microbiota and dietary proteins. Tissue-resident memory T cells are critical for immune homeostasis at mucosal surfaces based on their ability to rapidly respond to invading pathogens. The CTLA-4 and PD-1 receptors inhibit T cell function and have become major therapeutic targets for boosting T cell-mediated immunity in cancer patients. However, targeting of these inhibitory receptors frequently induces inflammatory adverse events, and colitis is one of the most common and severe inflammatory adverse events induced by checkpoint inhibition (CPI). We recently reported an in-depth single cell analysis of immune cells in the mucosa of CPI colitis patients and healthy subjects. This analysis demonstrated dramatic accumulation of CD8 T cell populations with highly proliferative and cytotoxic states in all studied CPI colitis patients. We used the T cell receptor (TCR) sequences of CD8 T cells to investigate the origin of these clonally expanded T cells. Interestingly, we discovered that a large fraction of colitis-associated cytotoxic CD8 T cells had the same TCR sequences as tissue-resident memory CD8 T cells. We therefore hypothesize that the CTLA-4 and PD-1 inhibitory receptors hold tissue-resident memory T cells (Trm) in check, and that loss of these inhibitory signals can induce massive clonal expansion of Trm, a major step in the development of CPI colitis. Antibody blockade of CTLA-4 and PD-1 receptors in individuals with immunologically normal mucosa thus provides insight into the physiological function of these receptors in humans. In Aim 1, we will investigate how the CTLA-4 and PD-1 receptors regulate the function of Trm cells, both in humans and murine models. We will investigate whether targeting of inflammatory pathways may revert T cells from highly proliferative, cytotoxic states back into a Trm state by performing an in-depth single cell analyses on colon biopsies from CPI colitis patients enrolled in a separately funded randomized phase 2 clinical trial that evaluates TNFα blockade versus corticosteroids. Our single cell analysis demonstrated upregulated TNF gene expression signatures in CPI colitis, and retrospective clinical data indicate that TNFα blockade is efficacious in CPI colitis. We will also investigate this hypothesis in murine models by evaluating the impact of CTLA-4 and PD-1 antibodies on the activation and cytotoxic states of CD8 T cells in the gut mucosa. In Aim 2 we will study the spatial interactions of T cell populations with immune, epithelial and stromal cells in the intestinal mucosa using the CODEX technology that enables highly multiplexed immunofluorescence analysis of tissue sections using panels of DNA-barcoded mAbs. This high-dimensional imaging approach affords an opportunity to study the cell – cell interactions in the healthy ...