4DN Interrogation of T Cell Exhaustion in Cancer

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PROJECT ABSTRACT/SUMMARY Immunotherapies that enhance the ability of T cells to recognize and kill tumor cells have been transformational in the treatment of human cancer, but immunotherapy is not effective in all patients or cancers, and therefore studies interrogating the molecular basis for durable T cell responses to cancer are needed. A critical barrier for the sustained activation of tumor-infiltrating T cells is the development of T cell ‘exhaustion,’ which leads to the stable expression of inhibitory surface receptors, poor response to tumor antigens, and low cell proliferation and persistence of T cells in vivo. However, to date, it has been difficult to study the gene regulatory mechanisms that control the development of T cell exhaustion in humans, due to a lack of sensitive genomic tools to study primary immune cells from patients. We recently developed a suite of high-throughput epigenomic technologies that enable the measurement of three-dimensional (3D) genome conformation and single-cell chromatin accessibility in primary T cells from human tumors. In the proposed research, we aim to utilize these methods to identify changes in 4D nucleome (4DN) organization and accessibility that underlie the development of human T cell exhaustion. In Aim 1, we will define 3D genome interactions that occur in human T cell exhaustion in patients with advanced skin cancer. Exhaustion-associated genome conformation will be compared across several cancer types to identify a consensus exhaustion profile, and these findings will be integrated with chromatin accessibility and gene expression data to identify transcriptional effects of 3D changes. In Aim 2, we will determine the dynamics and reversibility of regulatory 3D interactions in exhaustion using a novel chimeric antigen-receptor (CAR)-T cell model. In Aim 3, we will perturb these interactions using CRISPR/Cas9 genome editing in primary T cells, coupled with single-cell epigenomic read-outs, to engineer improved, durable, next- generation immunotherapies. If successful, these findings will have a direct impact on the future design of immunotherapy strategies, which will have a significant impact on the clinical care of cancer patients. Finally, we will facilitate the dissemination of these findings by freely distributing protocols and data and releasing custom software tools, and we will use these studies as a collaborative launch point in the 4DN network. We anticipate that these results will lead to novel insights into the molecular regulation of T cell exhaustion and serve as an effective research program for Dr. Satpathy to establish his independent laboratory at the interface of immunology and genome science.