Fig. 1

Functional and transcriptional profiles of Ag-specific CD8⁺PD1⁺ T-cell clones are distinctively related to CD28 expression. A Representative flow cytometry plots from two Ag-specific CD8⁺ T-cell clones isolated from peripheral blood of melanoma patients, with PD1⁺CD28⁻ (Melan-A-specific clone PT15.26, upper) or PD1⁺CD28⁺ (gp100-specific clone, PT08.24, lower) phenotype. Right, polyfunctionality of the same clones, as evaluated by intracellular staining of GrzB, IFN-γ, and TNF-α following activation with a Melan-A⁺/gp100⁺ melanoma cell-line for 4-5h in the presence of protein transport inhibitors. The percentage of positive expression is shown. B Pooled results from Ag-specific CD8⁺ T-cell clones isolated from 5 melanoma patients, grouped for PD1⁺CD28⁻ or PD1⁺CD28⁺ phenotype, in terms of anti-tumor cytotoxicity, as evaluated by Cr⁵¹ release (left, n = 26) and polyfunctionality (right, n = 24) against a Melan-A⁺/gp100⁺ melanoma cell-line. Each dot represents the mean value from two to five independent experiments performed on a single T-cell clone. P values were calculated by the Mann–Whitney unpaired two-sample test. ****P ≤ 0.0001. C-F RNAseq data from CD8⁺ PD1⁺CD28⁻ (n = 3) and PD1⁺CD28⁺ (n = 3) Ag-specific T-cell clones isolated from 4 melanoma patients. C Heatmap showing unsupervised clustering of the total 3700 genes (P_adj ≤ 0.05) in the two T-cell subsets. D Volcano plot analysis, based on differential expression of genes (DEG) between the two T-cell clone types. Blue dots indicate down-regulated genes (P_adj ≤ 0.05, FC < -0.25), and orange dots indicate up-regulated genes (P_adj ≤ 0.05, FC > 0.25). Selected genes are reported. The complete list of DEG is reported in Table S3. E Heatmap illustrating selected function-associated gene expression. In green, genes with P_adj ≤ 0.05; in blue, genes with P_adj ≤ 0.1; in black, genes not statistically significant. P adjusted values were calculated by the Benjamini & Hochberg adjustment method