Fig. 3

PD1⁺CD28⁻ and PD⁺CD28⁺ T-cell subsets show different functionality in peripheral blood and tumor site. A Representative flow cytometry plots showing PD1 vs CD28 staining, in ex vivo CD8⁺ T cells isolated from PBMC (P), adjacent non-tumor tissue (NT), and tumor site (T), in two NSCLC patients. B Percentage of total CD8⁺, gated according to their dimensions (FSC, forward-scatter and SSC, side-scatter parameters), and PD1/CD28 T-cell subsets, from the periphery to the tumor site, in 68 NSCLC patients (PBMC, n = 48; NT, n = 30; and Tumor, n = 43). P values were calculated by unpaired Mann–Whitney test, with Bonferroni correction for multiple comparisons. C Comparison between the frequencies of CD8⁺ PD1⁺CD28⁻ and PD1⁺CD28⁺ T cells within the same district (Wilcoxon rank test). D Percentage of CM, N, EM, and EMRA, evaluated by CCR7 and CD45RA expression, within CD8⁺ PD1⁺CD28⁻ and PD1⁺CD28⁺ T-cell subsets, in ex vivo unstimulated PBMC, NT and tumor site in NSCLC patients (n = 21) (Mann–Whitney test, with Bonferroni correction). E Intra-cellular GrzB, IFN-γ, and TNF-α expression following anti-CD3 mAb activation (5-6h) in the presence of protein transport inhibitors, in CD8⁺ PD1⁺CD28⁻ or PD1⁺CD28⁺ T-cell subsets, in ex vivo T cells from PBMC, NT and tumor site, in one NSCLC patient. F-G Quantification of single or simultaneous lytic molecule production, in CD8⁺ PD1⁺CD28⁻ or PD1⁺CD28⁺ T-cell subsets, from the periphery to the tumor site (F), or in the comparison between the two subsets within the same district (G), in 35 NSCLC patients (PBMC, n = 27; NT, n = 17; T, n = 24). F, Mann–Whitney test, with Bonferroni correction. G, Wilcoxon rank test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. NS, not significant. Plots show median values with interquartile range. Expression percentages are shown within the flow cytometry dot plots