NFAT5 induction by the tumor microenvironment enforces CD8 T cell exhaustion (CUT&Tag)

Persistent exposure to antigen during chronic infection or cancer renders T cells dysfunctional. The molecular mechanisms regulating this state of exhaustion are thought to be common in infection and cancer, despite obvious differences in their microenvironments. We discovered that NFAT5, an NFAT family member lacking an AP-1 docking site, is highly expressed in exhausted T cells responding to chronic infection and tumors but is a central player selectively in tumor-induced exhaustion. While NFAT5 overexpression in CD8+ T cells reduced tumor control, NFAT5 deletion improved tumor control by promoting the accumulation of tumor-specific CD8 T cells that expressed less TOX and PD-1 and produced more cytokines specifically among precursor exhausted cells. Conversely, NFAT5 did not promote T cell exhaustion during chronic infection. While NFAT5 expression was induced by TCR triggering, its transcriptional activity was specific to the tumor microenvironment and required hyperosmolarity. NFAT5 thus promotes CD8 T cell exhaustion in a tumor-selective fashion. Overall design: NFAT5 KO cells were obtained from LN and spleen of CD4-Cre+/- NFAT5flox/flox mice. CD8 T cells were enriched by negative selection following the manufacturer's protocol (Stemcell) and plated at 0.4 106 cells /24-well with anti-CD3/28 antibodies and 20U/mL IL-2 24h pre-transduction. Viral supernatants were supplemented with 20U/mL IL-2, Polybrene at 8ug/mL, and 0.5mL were added to the CD8 T cells. Cells were spun for 60min at 1800RPM at 32°C and incubated for 24h followed by a resting period of 24h were cells when taken off stimulation. To induce exhausted cells were passed on a new anti-CD3 coated plate every 2 days. After a total of three rounds of stimulation, CD8 T cells were sorted and processed for CUT&RUNseq