Fig. 2
Frequency, differentiation stage and functionality of peripheral CD8+ T-cell subsets defined by PD1 and/or CD28 expression. A Representative flow cytometry plots showing PD1 vs CD28 staining, in ex vivo CD8+ T cells from PBMC of patients with three different tumors, as indicated. B Percentage of circulating PD1+CD28-, PD1+CD28+, PD1−CD28− and PD1−CD28+ subsets within CD8+ T cells from NSCLC (n = 48), melanoma (n = 10), PDAC (n = 10) and HDs (n = 25). No statistical difference was observed between the proportion of the four subsets in samples of different origins (Kruskal–Wallis). C Representative flow cytometry plots showing CCR7 vs CD45RA staining, in gated PD1/CD28 T-cell subsets, as indicated. D Percentage of CM (CCR7+CD45RA−), N (CCR7+CD45RA+), EM (CCR7−CD45RA−), and EMRA (CCR7−CD45RA+) within the gated PD1/CD28 T-cell subsets, in NSCLC patients (n = 21). E Representative flow cytometry plots showing intracellular GrzB, IFN-γ and TNF-α production, within the PD1/CD28 T-cell subsets, following anti-CD3 mAb activation (5-6h) in the presence of protein transport inhibitors. F Percentage of polyfunctional cells, defined as the ability to produce two or more lytic molecules simultaneously, within the PD1/CD28 T-cell subsets, in NSCLC (n = 35), melanoma (n = 10), and PDAC (n = 9) patients. G Polyfunctional increase following anti-CD3 mAb activation in the presence of anti-PD1 blockade with the specific mAb (n = 9), within the PD1/CD28 T-cell subsets (Wilcoxon rank test). Histograms show the polyfunctionality fold increase of each T-cell subset after PD1 blockade with respect to its own baseline functionality. H Expression of CD11ahigh (n = 22), CD137 (n = 21), ICOS (n = 31), and Ki67 (n = 10) was evaluated by flow cytometry, within ex vivo unstimulated T cells (CD11a, CD137, ICOS) or after 48h of stimulation with anti-CD3 mAb (Ki-67) in PBMC from NSCLC patients gated on PD1/CD28 T-cell subsets. P values were calculated using the Wilcoxon rank test, with Bonferroni correction for multiple comparisons. * P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Plots show median values with interquartile range. Expression percentages are shown within the flow cytometry dot plots