Investigation of Novel Regulators of CD8+ T Cell Function in Cancer

Abstract

CD8+ T cells can recognize and kill cancer cells, yet their chronic exposure to tumor antigens and immunosuppressive signals in the tumor microenvironment initiates an altered differentiation trajectory. This trajectory culminates in dysfunctional CD8+ T cells that are ineffective at mediating tumor control. To date, several T cell states have been identified along this trajectory, each defined by its own transcriptional profile and functional capacity. A deep understanding of this trajectory and the gene programs that control the cell state transitions within it has great potential to inform the next generation of immunotherapies. The tumor microenvironment varies widely by cancer type and is known to influence this differentiation trajectory. To identify potential T cell regulators active across multiple tumor microenvironments, we analyzed a compendium of human single-cell RNA sequencing data from CD8+ tumor-infiltrating lymphocytes. This meta-single-cell analysis uncovered a pan-cancer T cell dysfunction gene program that highlighted CXCR6 as a pan-cancer marker of chronically activated T cells. In mouse models, CXCR6 expression increased with tumor progression and upon checkpoint blockade, with highest expression observed on dysfunctional PD1+Tim3+ cells. Investigation of CXCR6 transcriptional regulation revealed active repression by TCF1, a key transcriptional regulator of the stem-like CD8+ T cell state, thus identifying a regulatory axis spanning stem-like to dysfunctional cell states. Deletion of CXCR6 in CD8+ T cells uncovered its role in sustaining tumor growth control by promoting pro-survival signals in T cells via enhanced CD28 expression, thus highlighting a role for CXCR6 in counterbalancing PD1-mediated suppression of CD28 signaling. In addition, we identified the transcription factor KLF2 as a potential regulator of effector CD8+ T cells in cancer. In subcutaneous tumor models, KLF2 marked a small population of CD8+ T cells that expressed high levels of the effector markers CX3CR1 and KLRG1 as well as multiple cytokines. Transfer of genetically modified T cells that either lacked or overexpressed KLF2 resulted in diminished or heightened tumor control, respectively. Further investigation revealed that KLF2 not only controlled trafficking to the tumor site but also increased CD8+ T cell cytotoxicity. We also confirmed the impact of KLF2 on trafficking to tumor sites and cytotoxicity in a B cell leukemia model. Overall, KLF2 overexpression increased T cell tumor trafficking, antigen-specific tumor cell killing, and tumor control, making it an attractive target for adoptive cell therapies. In summary, this work uncovered CXCR6 and KLF2 as novel regulators of CD8+ T cell function in cancer. This knowledge can inform future research to fine tune T cell infiltration, effector function, and spatial organization and persistence in the tumor microenvironment. These concepts can then be leveraged to improve cancer therapeutics.