A systematic approach to uncover the basic mechanisms of checkpoint inhibitor immune related adverse events

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ABSTRACT Immune checkpoint inhibitors (ICIs) have profoundly altered cancer treatment. However, despite this technique’s success, there remain major challenges to ICIs use that must be met to best advance these therapies. These include increasing responsiveness to PD-1 and CTLA-4 blockade, uncovering new targets to optimize pathway blockade and addressing why more than one third of all patients that receive ICIs develop often devastating immune related adverse events (irAEs) syndromes characterized by various patterns of off-target organ inflammation. There is currently no clear understanding of the complexity and heterogeneity of irAEs and as ICIs approaches are expanded, understanding the basic mechanisms of irAEs stands to be a critical goal. We hypothesize that three distinguishable, but not mutually exclusive, pathogenic processes drive irAEs, and that the contribution of each varies according to the type of the irAEs. The first process is not tumor-related and is predetermined by known T cell autoimmune susceptibility genes, reflecting a failure of the negative selection mechanism. However, progression to an autoimmune disease is held in a latent preclinical stage by peripheral tolerance mechanisms until triggered by the inhibition of these mechanisms through ICIs. We hypothesize that the second process driving irAEs is an intrinsic consequence of the T cell immune response directed to tumor antigens. We envision that some of the TCR clonotypes responding directly to the tumor are specific for a peptide that is also expressed by target organ MHC molecules involved in the irAEs, and that activation of this expanded clone by ICIs initiates its attack on the tumor and on the target tissue, resulting in irAEs. Finally, since many of the tumor-infiltrating lymphocytes are not clonal, we hypothesize a third process of irAEs wherein the inflammatory response is mediated by promiscuous nonspecific TCR clones activated by ICIs or the tumor inflammatory milieu. Aim 1. To characterize the human T cell repertoire of irAEs patients. We will perform a gDNA repertoire analysis and single cell TCR seq of T cells isolated from the blood, synovial fluid, and synovial tissue of patients with irAEs, to uncover the physical-chemical properties of CDR3s in comparison to T cells that mediate other autoimmune diseases. We will take advantage of the gDNA to test the hypothesis that the entrance of these self-reactive TCRs in a patient’s repertoire is influenced by the variants of the same non-HLA alleles associated with other autoimmune disease susceptibility and the failure of negative selection and examine the role of the MHC allotype on repertoire selection and irAEs development. We will use humanized immune system mice to recreate, from hemopoietic stem cells, a replica of the irAEs patients’ pre-ICIs and pre-tumor TCR repertoire, to study repertoire evolution. Aim 2. To immunophenotype the irAEs mouse model. We developed a novel mouse model for ICIs-induced adverse events. We will perform CITEseq of T cells isolated from tumors and inflamed organs to uncover differential subsets of cells and their functions across different types of tumors. Through tetramer staining and TCRseq, we will uncover the contribution of tumor-specific T cells to the pathogenesis of irAEs. We will utilize multiplex IF studies to uncover the contribution of the tumor and the inflamed organ microenvironment to the function of the infiltrating cells.