Fig. 4: Tumor-infiltrating T-bet⁺ CD8⁺ T cells are predictive of ICIs responses in GC patients.

a On the left, bar plots of CD8/CD4 ratio prior to transfusion (n = 5). The proportion of CD8⁺ T cells is 0.36 times that of CD4⁺ T cells (P = 0.008, two-tailed Mann-Whitney U test). On the right, bar plots of CD8/CD4 ratio in the TME of GCs (n = 5). The proportion of T-bet⁺ CD8⁺ T cells rose to 1.67 times that of T-bet⁺ CD4⁺ T cells (P = 0.016, two-tailed Mann-Whitney U test). Data were acquired by flow cytometry. Data are presented as mean ± standard error of the mean (s.e.m.). b UMAP plot of TBX21 (encoding T-bet) expression across cell types in GC tumors. c Box plots of numbers of T-bet⁺ cells in different cell types in GC tumors. In the TME of GC patients (n = 24), most T-bet⁺ cells are CD8⁺ T cells. d Process of tissue segmentation using inFrom software (v 2.3.0). Red areas represent the tumor compartments, and green areas represent the stroma compartments. Tissue segmentation was performed based on the expression pattern of pan-CK. e Scoring maps in the tumor and stroma compartments. Cells with green nucleus and yellow membrane were T-bet⁺ CD8⁺ T cells; cells with green nucleus and red membrane were T-bet⁺ CD4⁺ T cells. f Box plots of proportion of T-bet⁺ CD8⁺ T cells and T-bet⁺ CD4⁺ T cells in the stroma compartments and tumor compartments in the GC cohort (n = 120). The percentage was calculated by dividing the number of non-tumor cells in respective compartments. The triangles mark the mean value of each group. In the stroma compartments, T-bet⁺ CD8⁺ T cells were more abundant than T-bet⁺ CD4⁺ T cells (0.21% vs. 0.19%, P = 0.068, two-tailed Mann-Whitney U test). In the tumor compartments, T-bet⁺ CD8⁺ T cells were more abundant than T-bet⁺ CD4⁺ T cells (0.25% vs. 0.14%, P = 0.0018, two-tailed Mann-Whitney U test). g MIHC of TME of an ICI responder (ICI-12), an ICI non-responder (ICI-7), an MSS-PR patient (GC-97), and an MSS-NR patient (GC-24). T-bet⁺ CD8⁺ T cells were richly infiltrated into the tumor compartments of the ICI responder and the MSS-PR patient, as indicated by the white arrows. h The ROC curve for predicting of ICI responses in MSS patients based on cell fractions of tumor-infiltrating T-bet⁺ CD8⁺ T cells, estimated by CibersortX (AUC = 0.816). On the right, box plot of fractions of tumor-infiltrating T-bet⁺ CD8⁺ T cells in MSS responders (n = 7) and MSS non-responders (n = 31) in PRJEB25870 cohort (P = 0.010, two-tailed Mann-Whitney U test). The mean value of T-bet⁺ CD8⁺ T fraction in MSS responders is 0.090. i The ROC curve for predicting ICI responses in MSI-H patients based on cell fractions of tumor-infiltrating T-bet⁺ CD8⁺ T cells, estimated by CibersortX (AUC = 0.775). On the right, box plot of fractions of tumor-infiltrating T-bet⁺ CD8⁺ T cells in MSI responders (n = 12) and MSI non-responders (n = 10) in PRJEB25870 and PRJEB40416 cohorts. (P = 0. 032, two-tailed Mann-Whitney U test). The mean value of T-bet⁺ CD8⁺ T fraction in MSI-H responders is 0.069. R, responders; NR, non-responders. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns is abbreviation for not significant. c, f, h, i In the box plots, the bounds of the box span from 25% to 75% percentile, the line represents the median, and the whiskers show the 1.5 Interquartile range (IQR) of the data points.