Fig. 4

Increased infiltration of exhausted PD1^Hi CD8⁺ T cells in HCC tumor tissues revealed by multiplex immunohistochemistry. a, Representative multiplex immunofluorescence images to show the distribution of CD8⁺ T cell subsets in peri-tumor and tumor: red arrows (CD8⁺TIM3⁺PD1^Hi), purple arrow (CD8⁺TIM3⁻PD1^Hi), green arrow (CD8⁺PD1^Int) and cyan arrow (CD8⁺PD1⁻). Tissue slides were stained by TSA method and scanned at 20x by Vectra3.0 Automated Imaging System. Scale bar, 50 μm. b, t-SNE analysis of CD8⁺ T cells from paired peri-tumor and tumor tissues validated the distinct classification of four CD8⁺ T cell subsets. c, Relative distribution analysis of total CD3⁺ T cells in paired peri-tumor and tumor tissues by dividing the total CD3⁺ T cells into CD8⁺ (cytotoxic T cells) and other cells (upper panel). CD8⁺ T cells were further split into CD8⁺PD1⁻, CD8⁺PD1^Int and CD8⁺PD1^Hi subpopulations (middle panel); and finally CD8⁺PD1⁺ T cells were separated into CD8⁺TIM3⁺PD1^Hi and CD8⁺TIM3⁻PD1^Int basing on the TIM3 expression (lower panel). d-h, Comparisons of the proportions of CD8⁺ among CD3⁺ T cells (d) and CD8⁺PD1^Hi (e), CD8⁺PD1^Int (f), CD8⁺TIM3⁺PD1^Hi (g) and CD8⁺TIM3⁻PD1^Hi (h) among CD8⁺PD1⁺ T cells between paired peri-tumor and tumor tissues in training cohort (n = 358). Error bars indicated median with interquartile range. Significance was assessed by Wilcoxon matched-pairs signed rank test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001