Transcriptional regulation of T cell exhaustion in immune checkpoint blockade resistance at single-cell resolution [in vivo]

Tumor-specific CD8+ T lymphocytes are crucial for anti-cancer immunity but can lose cytotoxic function in the immunosuppressive tumor microenvironment. Immune checkpoint blockade (ICB), like anti-PD-1 therapy, enhance and prolong anti-tumor T cell responses. Despite its widespread clinical use, resistance to ICB develops in some patients, characterized by the proliferation of exhausted T cell (Tex). Here, we establish two single-cell murine hepatocellular carcinoma (HCC) models to explore regulatory network in Tex with ICB resistance. We uncover distinct T cell compositions, including both early and terminal Tex subsets, following prolonged ICB treatment, and reveal the differentiation trajectory of Tex subsets. Finally, we not only identify transcription factor Runx2 and its downstream targets as contributors to T cell exhaustion in both models, but discover ICB response correlates to Runx2 level in human tumor-infiltrating lymphocytes. These findings elucidate Runx2 regulates T cell exhaustion in response to prolonged ICB treatment, influencing ICB effICBency, and suggest potential targets for combination therapy alongside ICB in HCC. Overall design: To uncover the crucial gene regulatory network that causes T cell exhaustion. We designed a mice model to examine the differential effects of anti-PD-1, an ICB targeting PD-1, on T cell. We inoculated C57BL/6 mice subcutaneously with 2 x 106 cells of hepatocellular carcinoma cell line, Hepa1-6. Mice were randomly assigned to two groups, anti-PD1 treatment or isotype, respectively, when tumors grew to reach an average volume of 200 mm3. For the short-term treatment experiment, either anti-mouse PD1 antibody (clone RMP1–14) or isotype control antibody (clone 2A3) was administered once per two days from day 15 to day 19, and the mice were sacrificed on day 20. Conversely, in the long-term treatment experiment, treatments were given every other day from day 15 to day 19 and then once per week starting from day 20, with the mice being sacrificed on day 57.