Fig. 6

Activin-A-treated human lung tumor-infiltrating CD4⁺ T cells exhibit a less exhausted profile. A Primary lung tumor-infiltrating leukocytes were isolated from NSCLC patients and stimulated with antibodies against CD3/CD28 in the presence of activin-A or PBS. Cytokine levels in culture supernatants of PBS- or activin-A-treated lung tumor-infiltrating leukocytes (n = 20–25 donors). Data are mean ± SEM of triplicate wells. B Cumulative percentages of CD69, PD-1, CTLA-4 or LAG-3-expressing cells among human lung tumor-infiltrating CD3⁺CD4⁺ T cells cultured as in Fig. 6A (n = 12 donors). C Representative flow cytometry plots (left) and cumulative percentages (right) of Foxp3-expressing cells among human lung tumor-infiltrating CD3⁺CD4⁺ T cells cultured as in Fig. 6A (n = 9 donors). D Activin-A- or PBS-treated human lung tumor-infiltrating CD4⁺ T cells, generated as in Fig. 6A, were CellTracker Red-labelled and co-cultured with CellTrace-CFSE-labelled autologous peripheral blood (PB) CD4⁺ T responders. Representative flow cytometry plots showing T responder cell proliferation (left) and cumulative data (right) (n = 9 donors). E Cytokine levels in culture supernatants of lung tumor-infiltrating act-A- or PBS-CD4⁺ T cells (n = 8 donors). Data are mean ± SEM of triplicate wells. F CD45⁻ lung cancer cells were co-cultured with autologous PB CD8⁺ T cells pre-treated with CM from act-A- or PBS-CD4⁺ T cell cultures. Cumulative data of cytotoxicity, assessed by LDH measurement in cell culture supernatants (n = 6). Statistical significance was obtained by Wilcoxon matched-pairs signed rank test; *P < 0.05, **P < 0.01 and ****P < 0.0001