854 Protein homeostasis enables T cell differentiation potential and sustains TIL function in cancer

Background CD8 + T cells can infiltrate tumor tissues and target tumor cells, but often fail to cure disease due to chronic activation in the immunosuppressive tumor microenvironment, resulting in differentiation into an exhausted T cell state. When acute infection is cleared in tissues, pathogen-specific T cells differentiate into tissue-resident memory (TRM) that remain to survey and protect from reinfection. And when TRM-like TIL are found in cancerous tissue, improved responses to immunotherapy and better patient outcomes are observed. Therefore, understanding the connection between exhausted TIL and TRM can inform efforts to manipulate T cell fates towards TRM-like TIL to benefit cancer immunotherapy.Methods To better understand the relationship among T RM-like TIL, exhaustion states, and TRM, we directly compared TRM from acute viral infection and exhausted TIL from tumors to find proteomic and transcriptional differences between these distinct T cell states.Results We focused on genes highly expressed by T RM that are shared by functional TIL but lost in terminally exhausted TIL as candidates that may mediate enhanced functions in tissues, and identified numerous genes related to protein homeostasis. This included multiple under-characterized E3 ubiquitin ligases, which when we enforced expression of these ligases in TIL, resulted in better tumor control and improved mouse survival. Protein regulation by the ubiquitin-proteosome system is an essential biological process, crucial for cell differentiation, and interestingly, by investigating protein homeostasis in TIL, we found that exhausted T cells have an excess of unfolded proteins accumulated in their cytosol. CRISPR knockout studies in cancer and acute infection showed that loss of our E3 ubiquitin ligases decreased T cell ‘stemness’ in tissues and accelerated differentiation to their terminal fate. The identified ligases were enriched in the nucleus, and proximity-labeling studies revealed the ligases can target transcriptional and translational machinery for degradation. When we enforced expression of these ligases in tumor-specific T cells in cancer, we found the transduced T cells showed a decrease in unfolded proteins and showed enhanced anti-tumor function: accumulation of stem-like T cells in the tumor-draining lymph node, accumulation of TIL in the tumor, and increased IFN-gamma production.Conclusions These data highlight an underappreciated relationship between protein homeostasis and T cell differentiation, and provide novel avenues of immunotherapeutic approaches for cancer.Ethics Approval The study was approved by UCSD’s Institutional Animal Care and Use Committee, protocol number S04105.