Regulation of T cell exhaustion via 3D chromatin architecture

PROJECT SUMMARY CD8+ T cells are essential players in mounting protective cellular immune responses against pathogens and cancers. A productive CD8+ T cell response clears pathogens that cause acute infections and forms protective memory T cells; in contrast, CD8+ T cells enter an exhausted/hypofunctional state when exposed to persisting antigens due to chronic viral infection or tumor microenvironment. Promoting memory formation and reversing exhaustion are sought-after goals. Advances in these areas have important bearings on the rational design of prophylactic vaccines and immunotherapy to treat viral infections and cancers. The exhausted T (Tex) cells have been extensively studied for their transcriptome, epigenome, and chromatin accessibility dynamics on bulk and single cell levels. Dozens of Tex regulators are identified, but it is less understood how the actions of regulators on one-dimensional (1D) genomic elements are integrated to drive various Tex cell fates. Further, little is known about how Tex programs are controlled under the framework of three-dimensional (3D) genome topology. In our preliminary studies, we performed Hi-C on Tex-stem and Tex-term cells at the effector phases, and discovered that the early Tex cells acquired de novo chromatin interaction (ChrInt) hubs compared with Tn and acute infection-elicited Teff cells. We thus hypothesize that persistent antigen stimulation mobilizes CTCF and reshapes 3D genome topology in Tex-specific manner to program Tex fate decision, self- renewal, and effector differentiation. We will test this in two specific aims: Aim 1. To construct 3D genome topology-based, integrative gene regulatory modules that programs Tex differentiation. We will longitudinally map ChrInt hub dynamics in Tex cells with distinct fate trajectories and predict novel regulators that control stemness, effector and exhaustion programs in Tex cells. We will characterize the candidate factors and their architectural connectivity-based enhancers, and construct a searchable Tex gene regulatory module database for the community use. Aim 2. To dissect how CTCF differentially utilizes its insulator and coactivator dual functions to direct Tex fate decision. CTCF has dual functions: as an insulator to establish boundaries and as a transcription factor/cofactor to mediate enhancer-promoter interaction. We will discern the contribution of each function, by specific perturbation of insulation and dissection of its interplay with partner factors utilized by Tex cells. Overall, this proposal will delve into a less-charted research area, higher order genome reorganization in Tex cell biology. These efforts may lead to paradigm-shifting advances in scientific knowledge and help devise more effective vaccines and therapeutics for infectious diseases and cancers.