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

Tumor-specific CD8+ T lymphocytes are crucial for anti-cancer immunity but can lose cytotoxic function in the immunosuppressive tumor microenvironment. Immune checkpoint blockades (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 generate exhausted CD8+ T cells, we used P14 mice in which CD8+ T cells could specifically recognize gp33 peptide, repeatedly taking splenocytes with gp33 peptide to stimulate CD8+ T cell exhaustion. On different conditions, we generated four types of T cells: naïve, active, exhausted (72 h) and exhausted (96 h), respectively. CD8+ T cells from each group were collected by cell sorter (BD FACSAria III) and separated for single-cell multiome sequencing, including scRNA-seq and scATAC-seq. For sequencing, CD8+ T cells underwent nuclei isolation followed by the use of the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression kit (10x Genomics) according to the instruction provided by the manufacturer. Sequencing was then conducted on Illumina NovaSeq 6000 platform to generate the single-cell multiome dataset.