Decoding the regulation and function of GZMK within tumor-infiltrating CD8+ T cells

Project Summary/Abstract The emerging roles of granzyme K (GZMK)+ CD8+ T cells in conditions associated with defects in immune regulation, from rheumatoid arthritis to inflammaging, have garnered significant interest. The infiltration of GZMK+ CD8+ T cells is a prominent feature of the tumor microenvironment (TME) across several human cancers, and yet their role and regulation remain undefined. Recent studies indicate that tumor-infiltrating GZMK+ CD8+ T cells display distinct spatial and functional characteristics compared to other CD8+ T cells within tumors. Notably, high levels of GZMK+ CD8+ T cell infiltration are associated with poorer prognosis in certain cancers. Unlike other well- studied members of the granzyme family such as granzyme B (GZMB), the mechanisms driving GZMK expression in CD8+ T cells within the TME and the effects of CD8+ T cell-derived GZMK on antitumor immunity remain unclear. CD8+ T cell differentiation in the TME is shaped by the strength of T cell receptor (TCR) signaling upon interaction with tumor-specific antigen. Studies have shown that persistent and overly-strong TCR interactions drive T cell dysfunction, while insufficient TCR activation leads to functional inertness. Surprisingly, my preliminary data suggests that both these extremes of TCR signaling strength predispose CD8+ T cells to express GZMK. Furthermore, our group has demonstrated that GZMK can act as a novel initiator of the complement cascade, a known impediment to antitumor immunity through its ability to recruit suppressive myeloid cells to the TME. Building on these findings, I propose to investigate the mechanisms underlying GZMK expression in CD8+ T cells within the TME and its impact on antitumor immunity. I hypothesize that GZMK expression is driven in a subset of tumor-infiltrating CD8+ T cells that experience distinct TCR signaling, leading to the upregulation of context-specific transcription factors (TFs), and that eliminating GZMK from the TME will enhance antitumor immunity. To address my central hypothesis, in Aim 1, I will examine the impact of TCR signaling strength and cell-extrinsic signals on Gzmk expression in tumor-specific CD8+ T cells. First, I will assess the endogenous polyclonal CD8+ T cell response to cancer using Nur77 TCR-signaling reporter mice. Then, using OT-I CD8+ T cells and tumors with OVA-SIINFEKL variations, I will assess how controlled variation of TCR signaling strength impacts Gzmk expression. In Aim 2, I will identify the cis-regulatory elements and TFs that modulate Gzmk levels in tumor-infiltrating CD8+ T cells by perturbing key epigenetic and transcriptional regulators in vivo. In Aim 3, I will use Gzmk-deficient mice and CD8+ T cell-specific Gzmk-deficient mice to explore its impact on overall antitumor immunity. Together, these studies aim to provide critical insights into the regulation and function of GZMK in tumor-infiltrating CD8+ T cells and explore the potential of GZMK as a novel target for therapeutic intervention in cancer immunotherapy.