Fig 2: Expression of GITR protein among T cell subsets from different tissues of GC patients(A) Schematic gating of GITR on Treg cells (left), Tconv cells (middle), and CD8 T cell (right) from tumor tissues of GC patients.(B–E) Expression of GITR among T cell subsets from (B) tumor tissues, (C) peritumor tissues, (D) TDLN, and (E) PBMC.(F) Schematic gating of GITR on Treg cells from different tissues of GC patients.(G–I) Expression of GITR on (G) Treg cells, (H) Tconv cells, and (I) CD8 T cells from different tissues. The schematic gating strategy for analysis of human T cells from GC or other tissues used in Figure 2 is shown in Figure S1A. Data are represented as mean +/− SEM and were analyzed by unpaired Student’s t test in (B–E and G–I). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig 3: Distribution of GITR protein in T cells from different tissues of GC patients(A) Total GITR (GITRTOT) expression in Treg cells from different tissues.(B) Comparison of surface GITR (GITRSUR) and GITRTOT expression in Treg cells among different tissues.(C) Composition ratio of GITRSUR and intracellular GITR (GITRIC) in Treg cells from different tissues.(D) Confocal images of GITR (clone: D919D) distributed on the surface membrane of Treg cells from GC tissues.(E) Confocal images of GITR (clone: D919D) distributed intracellularly in Treg cells from PBMC. The images showed in (D and E) are stained with DAPI (blue), CD4 (green), FOXP3 (magenta), and GITR (red). The rightmost panel is merged view of the four left images. The scale bar (10 μm) is placed at the left corner of each image.(F) Right: confocal image of GITR (clone: ERP20566) distributed intracellularly in iTreg cells and stained with DAPI (blue), CD4 (green), and GITR (red). Left: confocal image of isotype control in iTreg cells and stained with DAPI (blue), CD4 (green), and IgG (red). The scale bar (10 μm) is placed at the left corner of each image.(G) GITRTOT expression in Tconv cells from different tissues.(H) Comparison of GITRSUR and GITRTOT expression in Tconv cells among different tissues.(I) Composition ratio of GITRSUR and GITRIC in Tconv cells from different tissues.(J) GITRTOT expression in CD8 T cells from different tissues.(K) Comparison of GITRSUR and GITRTOT expression in CD8 T cells among different tissues.(L) Composition ratio of GITRSUR and GITRIC in CD8 T cells from different tissues.The clone number of GITR antibody used in (A–C and G–L) is 108–17. Data are represented as mean +/− SEM and were analyzed by unpaired Student’s t test in (A–C and G–L). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig 4: Intratumoral GITR+ Treg cells exhibit stronger suppressive capacity(A) FOXP3 expression of GITR+ Treg cells and GITR- Treg cells from GC tissues.(B) CD25 expression of GITR+ Treg cells and GITR- Treg cells from GC tissues.(C–G) The immune checkpoints expression, including (C) CTLA-4, (D) PD-1, (E) OX40, (F) LAG3, and (G) CD39, of GITR+ Treg cells and GITR- Treg cells from GC tissues.(H) Suppressive capacity of GITR+ Treg cells and GITR- Treg cells isolated from GC tissues. CD8 T cells were co-stimulated with anti-CD3/CD28 beads. Left: histograms showing CTV dilution peaks indicating effector T (Teff) cell proliferation. Right: percentages of proliferative CD8 Teff cells co-cultured with GITR+ or GITR- Treg cells at ratios of 2:1, 4:1, 8:1, or no Treg. The schematic gating strategy for flow sorting of GITR+ Treg cells and GITR- Treg cells is shown in Figure S2A. Positive percentage is used to evaluate the level of CD39. MFI are used to evaluate the levels of FOXP3, CD25, CTLA-4, PD-1, OX40, and LAG3. Data are represented as mean +/- SEM and were analyzed by unpaired Student’s t test. *p < 0.05, **p < 0.01, ****p < 0.0001; NS: not significant.

Fig 5: Single-cell Atlas of human GC(A) Bubble plot depicting the expression of canonical marker genes in tumor-infiltrating immune cell clusters. The size of the circle represents the percentage of cells expressing the gene in that specific immune cell cluster, whereas the color represents the average expression of the gene.(B) UMAP plot representation of 13 unique cell clusters color coded by their corresponding immune cell subtype. Each dot in the UMAP represents a single cell.(C) Comparison of the infiltration levels of major cell types in 26 tumor tissues and 11 peritumor tissues from GC patients. Box plot center, box, and whiskers correspond to the median, IQR, and 1.5 × IQR, respectively.(D and E) Volcano plot shows differentially expressed genes between GITR+ (red dots) and GITR− (green dots) CD4 T cells, including (D) Treg cells and (E) Tconv cells. The names of the partial significant genes are indicated in the plots. See also Tables S1 and S2.(F) Pairwise Spearman’s correlations of immune cell subclusters are performed, using the average expression of marker genes. The color gradient shows the range of correlation coefficients from −1 (blue) to 1 (red). Data are represented as mean +/− SEM and were analyzed by unpaired Student’s t test in (C). ∗p < 0.05, ∗∗p < 0.01, ns: not significant.