Determination of chromatin protein dynamics in CD8 T cells

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Determination of chromatin protein dynamics in CD8 T cells CD8 T cells are crucial for antiviral and antitumor immunity. How these cells respond to stimulations and how to manipulate their effector function for therapeutic purposes have been under intensively investigations. It is widely accepted that T cell antigen receptor (TCR) and co-receptor signaling drives almost all the key processes of CD8 T cell differentiation and function via transcriptional and epigenetic regulation of gene expression. Factors modulating TCR signal transduction or target gene expression presumably play important roles in dictating CD8 T cell differentiation and immunological functions. Although proteins directly mediating TCR and co-receptor signal transduction have been well defined, the nuclear proteins modulating target gene expression remain to be fully investigated. Unbiased approaches are required to comprehensively reveal the nuclear proteins regulating TCR and co-receptor dependent gene expression, potentially leading to the identification of novel factors governing CD8 T cell differentiation and function that determine antiviral and antitumor immune response. Recently CRISPR screening offers an unprecedented efficiency to systematically delineate the regulators of CD8 T cell differentiation and function. However, this method does not uncover the mechanistic linkage between candidate factors and signaling pathways and is also severely constrained by available experimental readouts. We reasoned that defining the dynamic proteins of the active gene promoters and enhancers in CD8 T cells upon TCR and co-receptor stimulation would be a solution, because proteins recruited to or depleted at these regions during this process would reveal novel key regulators. To determine the dynamic chromatin proteins at genomic regions of interest, we recently developed a comparative proteomics method using antibody-guided proximity biotinylation in a separate study. In preliminary experiments, we applied this method to mouse primary CD8 T cells and profiled the dynamic proteins at active enhancers and promoters marked by histone H3 lysine 27 acetylation (H3K27ac) upon TCR and co-stimulation, because this signaling appears to act on these genetic elements to control important functional genes in CD8 T cells (such as cytokines and Myc) by coordinating with the histone acetylation pathway. This experiment revealed known TCR signal transducers and a number of novel factors overrepresented or underrepresented. We then performed an in vivo pooled CRISPR screening by knocking out these dynamic proteins and discovered several positive and negative regulators of tumor infiltrating CD8 T cells. On the basis of these results, we propose to further define the chromatin protein dynamics in CD8 T cells and determine their roles in modulating inducible gene expression and CD8 T cell antitumor activity. Overall, we will employ a new proteomics method, developed by us, to determine signal dependent chromatin protein dynamics to unbiasedly identify novel regulators of CD8 T cell function. These factors may serve as new targets to improve CD8 T cell based immunotherapies.