MSI tumors upregulate TREX1 through cGAS–STING pathway to suppress CD8 ⁺ T cell–mediated antitumor immunity. (A) C57BL/6 mice (n = 5) were transplanted s.c. with 1 × 10⁷ MC38, MC38-Trex1^−/−, MC38-Cgas^−/−, and MC38-Trex1^−/−Cgas^−/− cells. 15 days later, tumor tissues were collected and analyzed by flow cytometry. The percentage of CD8⁺ T cell in CD45⁺ immune cells is shown. (B) Dot plot shows the significantly enriched hallmark pathways for highly expressed genes in MC38-Trex1^−/− vs. MC38-Trex1^−/−Cgas^−/− cells; the point color indicates the adjusted P value, and the point size indicates the upregulated gene count in each signal pathway. (C) UMAP plot of CD8⁺ T cell from the scRNA-seq data; the color of the dots is determined by the CD8⁺ T cell subtypes. (D) The proportion of each CD8⁺ T cell subtypes in four group samples. (E) Heatmap showing the expression of genes associated with different immune functions in each CD8⁺ T cell cluster identified from scRNA-seq data. (F–H) C57BL/6 mice (n = 5–10) were transplanted s.c. with 1 × 10⁷ MC38, MC38-Trex1^−/−, MC38-Cgas^−/−, and MC38-Trex1^−/−Cgas^−/− cells. 11 days later, tumor tissues were collected and analyzed by flow cytometry. The percentage of IFNγ⁺CD8⁺ T cell (F), TNFα⁺CD8⁺ T cell (G), and GZMB⁺CD8⁺ T cell (H) in CD8⁺ T cells is shown. (I and J) C57BL/6 mice (n = 10) were transplanted s.c. with 1 × 10⁷ MC38, MC38-Trex1^−/−, MC38-Cgas^−/−, and MC38-Trex1^−/−Cgas^−/− cells. 12 days later, tumor-bearing mice were injected with 100–200 μCi of ⁶⁸Ga-grazytracer. PET/CT imaging studies were performed at 0.5 h after injection. (I) One representative mouse from each group is shown, and all images of mice used for GZMB imaging have been placed in Fig. S5 G. Red circle indicates tumors. (J) Tumor (n = 10) uptake of ⁶⁸Ga-grazytracer was quantitatively analyzed. Data indicate mean ± SEM and are representative of two (F–J) and three (A) independent experiments. The statistical analysis was performed by unpaired Student’s two-tailed t test in A, F–H, and J. *P < 0.05, **P < 0.01, and ****P < 0.0001. UMAP, uniform manifold approximation and projection.