Genetic and immunological dissection of coinhibitory crosstalks between human T cells and cancer cells

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PROJECT SUMMARY / ABSTRACT The goal of this proposal is to identify novel coinhibitory checkpoint molecules operating orthogonally to currently targeted checkpoints in cancer patients, such as PD-1. Despite the unprecedented success of checkpoint blockade immunotherapy as a therapeutic approach against multiple types of cancer, significant inter-individual variability remains, ranging from no response to complete remission of systemically metastasized lesions. Various tumor-intrinsic parameters, such as tumor mutational burden, neoantigen burden, and mismatch repair pathway deficiencies, only show limited predictive power for favorable clinical outcomes. Therefore, complementary to such tumor-intrinsic factors, it is plausible to hypothesize that humans have hitherto unknown coinhibitory mechanisms orthogonal to currently known immune checkpoints and that a minor fraction of humans with germline or somatic mutations in such pathways could benefit from exceptionally potent antitumor immunity triggered by checkpoint blockade immunotherapy. Identification of such orthogonal pathways would significantly enhance the health of patients with multiple types of cancer; for instance, the 15 types of cancer for which pembrolizumab has been indicated. The current project aims at dissecting the human genetic and immunological basis of coinhibitory crosstalk between human T cells and cancer cells by tackling the two specific aims. Specific Aim 1 is to delineate the molecular mechanisms of PD-L1/L2-mediated inhibition of T cell activation independent from PD-1. This aim is supported by preliminary observations that both primary and immortalized T cells from a patient with inherited complete PD-1 deficiency, as well as PD-1-negative human T cell lines Jurkat and HuT78, all responded to co-inhibition by bead- immobilized PD-L1/L2. CD83 was identified as a candidate for PD-L1-mediated immunosuppression. The proposed work includes searching for additional candidates through RNA sequencing and surface receptor profiling and functionally validating the candidates via RNA silencing and CRISPR-guided knockout. Specific Aim 2 is to characterize the immunoregulatory function of CD83 in human T cells. Previous studies suggest that CD83 can serve as both an immunosuppressive receptor and ligand for human T cells, although its counterreceptor(s), downstream signaling, and relevance to antitumor immunity remain mostly elusive. The proposed work includes biochemical characterization of CD83 signaling in human T cells with or without cancer cell coculture utilizing genetic knockout and lentiviral rescue of CD83 gene. The proposed project will help untangle the biochemical and immunological basis of human T cell coinhibitory circuits in the crosstalk between cancer cells and also substantially bolster the applicant's scientific portfolio and expedite the growth toward an independent investigator pioneering human genetics of cancer immunology and immunotherapy.