Abstract Monoclonal antibodies targeting CTLA-4, PD-1, and PD-L1 are widely used to promote anti-tumor immune responses in a range of human cancers, but can also lead to inflammatory toxicities, collectively referred to as immune-related adverse events (irAEs). Colitis is a common and severe irAE which can lead to treatment discontinuation, particularly in patients receiving anti-CTLA-4 antibodies alone or in combination with PD-1 blockade. Intestinal inflammation triggered by CTLA- 4 antibodies typically involves the colon and is characterized by the accumulation of CD4+ T lymphocytes and neutrophils in the intestinal tissue. Analyses of colitis-associated lymphocytes of melanoma patients receiving CTLA-4 and PD-1 inhibitors have further implicated the pathogenic contributions of tissue resident CD8+ T cells with cytotoxic features. However, the role of the microbiota and the immune pathways critical for immune checkpoint blockade (ICB) induced-colitis remain poorly understood. Our understanding of the immunological mechanisms of ICB induced-colitis have been impeded by the lack of robust animal models of colitis caused by immune checkpoint inhibitors. Laboratory mice are highly resistant to intestinal inflammation following treatment with antibodies targeting immune checkpoints. We found that this limitation can be overcome by using mice harboring the microbiota of wild-caught mice, referred to as wild mouse microbiome-reconstituted (WildR) mice, which develop overt colitis following treatment with anti-CTLA-4 antibodies. Intestinal inflammation is driven by unrestrained activation of IFN- producing CD4+ T cells and depletion of peripherally induced regulatory T cells through Fc receptor signaling. Accordingly, anti-CTLA-4 nanobodies that lack an Fc domain can promote anti-tumor responses without triggering colitis. We hypothesize that bacterial symbionts present in the WildR microbiota, but not conventionally raised specific pathogen-free (SPF) laboratory mice, are required to trigger over colitis after ICB. We further hypothesize that specific members of the WildR microbiota stimulate intestinal dendritic cells (DCs) to activate colitic antigen-specific CD4+ T cells in mice depleted of RORγt+ pTregs in the presence of CTLA-4 blockade. In this grant application, we propose three specific Aims to further understand how the WildR microbiota trigger colitis after ICB.