Modulation of CD8+ T cell differentiation and anti-tumor immunity by metabolic manipulation

PROJECT SUMMARY / ABSTRACT CD8+ T cells are critical determinants of the anti-tumor immune response. The TCF1+ progenitor-exhausted subset of CD8+ T cells is critical to their therapeutic efficacy, providing a more durable response than their TCF1- more effector-like counterparts. However, the mechanisms underlying the generation and maintenance of this TCF1+ differentiation state remain unclear. T cell metabolism has been intimately linked to differentiation, and recent work from our group and others has begun to interrogate the potential of manipulation of metabolic pathways to alter differentiation. This R03 proposal aims more comprehensively to identify metabolic enzymes controlling the generation of TCF1+ progenitor cells for further functional and mechanistic studies, and characterize key transcriptomic and metabolic characteristics of TCF1+ cells induced under different conditions. The first aim, Identifying metabolic enzymes whose loss results in an altered T cell differentiation state, will interrogate the effect of loss of specific metabolic enzymes on TCF1+ progenitor cell generation across multiple TCR affinities. A CRISPR/Cas9 screen in vitro will target enzymes from pathways upregulated upon T cell activation. Candidate genes regulating TCF1 expression and T cell differentiation will be identified by sequencing and functionally validated by flow cytometric and metabolic analyses. Finally, effects of target manipulation on anti-tumor efficacy will be evaluated in vivo in mice and in an immunocompetent human patient- derived organoid system. The second aim, Identifying transcriptional and metabolic characteristics of TCF1+ progenitor-like cells induced by different metabolic alterations, will determine core properties of these cells. Using drugs and cytokine cocktails known to induce this TCF1+ population through differing mechanisms, activated T cells will be manipulated in vitro to identify transcriptomic and metabolomic similarities and differences. Pharmacological agents targeting hits identified in Aim 1 will also be tested. Data from these aims will allow me to identify both therapeutically targetable nodes for TCF1+ progenitor state induction, and key transcriptional and metabolic characteristics of TCF1+ progenitor cells, including their core characteristics and intrinsic heterogeneity. These data will be leveraged to identify high-quality candidates for future mechanistic studies, and provide a basis for a future R01 submission and career transition from a KL2- supported scholar to an independent investigator.