Fig. 5

PD1, TIGIT, TIM-3, CTLA-4, and LAG-3 co-expression differently impacts the functionality of CD8⁺ T cells depending on CD28 expression. A Representative flow cytometry plots showing TIGIT, CTLA-4, TIM-3, and LAG-3 expression in gated CD8⁺ PD1⁺CD28⁻ or PD1⁺CD28⁺ T-cell subsets, in ex vivo unstimulated cells from PBMC (P), adjacent non-tumor tissue (NT) and tumor site (T) of one NSCLC patient. The percentage of positive expression is shown. B Analysis of single TIGIT, CTLA-4, TIM-3, and LAG-3 IR expression in ex vivo unstimulated CD8⁺ PD1⁺CD28⁻ or PD1⁺CD28⁺ T-cell subsets from matched PBMC, NT, and tumor site from 19 NSCLC patients. P values were calculated using the Wilcoxon rank test, with Bonferroni correction for multiple comparisons. C Quantification of the 16 possible combinations of IRs co-expression, in gated PD1⁺CD28⁻ or PD1⁺CD28⁺ T cells from PBMC, NT, and tumor site from 12 NSCLC patients. Small grey dots show the absence while large black dots indicate the presence of the corresponding inhibitory receptors. P values were calculated using the Friedman test between the three districts. D Polyfunctionality, evaluated after stimulation with anti-CD3 mAb (5-6h), of the 16 IR subgroups identified within PD1⁺CD28⁻ or PD1⁺CD28⁺ subsets, in cells from peripheral blood and tumor site from 10 NSCLC patients. P values were calculated by comparing the polyfunctionality of each different subgroup (IR2-IR16) vs the quadruple-negative (IR1) group (orange, lower functionality; green, higher functionality) (Wilcoxon rank test). * P ≤ 0.05, **P ≤ 0.01. NS, not significant. Plots show median values with interquartile range