Fig. 3: T-bet⁺ T cells exhibit superior anti-tumor immunity.

a Flow cytometer analysis of expression of IFN-γ, GZMB, and CXCR3 in T-bet⁺ CD8⁺ (n = 1675) and T-bet^- CD8⁺ T cells (n = 12,852). b Flow cytometer analysis of expression of IFN-γ, GZMB, and CXCR3 in T-bet⁺ CD4⁺ (n = 1778) and T-bet^- CD4⁺ T cells (n = 19,704). c T-bet⁺ CD4⁺ T cells, T-bet⁺ CD8⁺ T cells, and T-bet⁺ CXCR3⁺ cells exist in the TME of GC tumors. MIHC images were taken from GC patients in the GC cohort. d Downstream genes of the transcription factor T-bet. In T cells, GZMB, PRF1, IFNG and CXCR3 are the direct targets of T-bet (Created in BioRender. Tang, Shiying. (2025): https://biorender.com/y38v503). e Line plots of average percentages of CD4⁺ T cells, CD8⁺ T cells, T-bet⁺ CD4⁺ T cells, and T-bet⁺ CD8⁺ T cells in the stroma compartments of normal tissue (N), normal-tumor margins (M), tumor edges (E), and tumor cores (C) (n = 19, 1 MSI-H GC, 4 EBV⁺ MSS GCs, 14 EBV- MSS GCs). Gray margins indicate standard errors. Original line plots were interpolated using interp1d function in SciPy (v 1.8.1). f Schematic graph of constructing tumor-bearing humanized mice model (Created in BioRender. Tang, Shiying. (2025): https://biorender.com/r39f251). g Line plot of tumor volumes over time (P = 2.197e-04, two-tailed two-way ANOVA test, using time and group as two independent variables). Tumor volumes were calculated as 1/2 × (Length × Width²). On the right, box plot of tumor weights at endpoints of mice transfused with T-bet^high or T-bet^low T cells (P = 0.0292, two-tailed paired-wise t-test. n = 5 biologically replicates from 5 different healthy PBMC donors). h mIHC of tumor-infiltrating T-bet⁺ CD4⁺ T cells and T-bet⁺ CD8⁺ T cells in humanized mice transfused with T-bet^high or T-bet^low T cells. i Box plots of percentages of T-bet⁺ CD8 T cells (P = 2.434e-03, two-tailed paired-wise t-test) and T-bet⁺ CD4⁺ T cells (P = 9.848e-03, two-tailed paired-wise t-test) in tumor-infiltrating immune cells, comparing mice transfused with T-bet^high T cells with mice transfused with T-bet^low T cells (n = 5, biologically replicates from 5 different healthy PBMC donors). Original data was generated by flow cytometer analysis. j Box plots of geometric mean fluorophore intensities (gMFIs) of IFN-γ (P = 7.685e-04, two-tailed Mann-Whitney U test) and GZMB (P = 2.461e-04, two-tailed Mann-Whitney U test) in T-bet⁺ CD8⁺ T cells and T-bet^- CD8⁺ T cells from 10 CDX GC tumors. k Kaplan-Meier curves of overall survival in patients in the GC cohort in relationship to T-bet⁺ CD8⁺ T cell abundance (on the left) and T-bet⁺ CD4⁺ T cell abundance (on the right). The percentages were calculated as the number of T-bet⁺ CD8⁺ or CD4⁺ T cells divided by the number of all cells in respective patient. A percentage of 0.2% was set as the cutoff to distinguish high or low infiltration of T-bet⁺ CD8⁺ and T-bet⁺ CD4⁺ T cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns is abbreviation for not significant. g, i, j 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.