Fig 2: NSCLC transfer CD39+ exosomes for instructing T cell mal-differentiation.A Healthy CD4+ T cells were pre-conditioned with conditioned media containing 10% and 20% A549 supernatant for 12 h, followed by activation and detection for T cell differentiation. Mean ± SEM from 5 individuals in each group. B Healthy CD4+ T cells were co-cultured with PKH67-labeled A549 cells for 12 h and detected for PKH67-labeled membrane vesicles. Mean ± SEM from 6 individuals in each group. C A549 cells were pre-treated with exosome inhibitor GW4869 (10 μM) for 24 h before pre-conditioning healthy CD4+ T cells for another 12 h. After that, CD4+ T cells were activated with anti-CD3/CD28 beads for 4 days and analyzed for T cell differentiation. Mean ± SEM from 5 individuals in each group. D CD39 protein in A549 cell-derived exosomes was detected by flow cytometry. Mean ± SEM from 6 individuals in each group. E–G Healthy CD4+ T cells were pre-treated with A549-derived exosomes for 12 h and activated with anti-CD3/CD28 beads. AMPK activation and mTOR activity were detected 3 days post-stimulation, while T-cell differentiation was determined on day 4. Mean ± SEM from 5–6 individuals in each group. H Healthy CD4+ T cells were pre-conditioned with exosomes from NSCLC patient-derived primary tumor cells for 12 h and activated with anti-CD3/CD28 beads for 4 days, and detected for T cell differentiation by flow cytometry. Mean ± SEM from 5 individuals in each group. I CD4+ T cells isolated from healthy donors, or NSCLC patients, were activated with anti-CD3/CD28 beads for 4 days, and detected for T cell differentiation by flow cytometry. Mean ± SEM from 5 individuals in each group. *p < 0.05, **p < 0.01, ***p < 0.001 with ANOVA plus Tukey method (A, C), paired student t-test (B, D–H) and unpaired student t-test (I).

Fig 3: NSCLC promotes AMPK activation for T cell mal-differentiation.A, B Healthy CD4+ T cells were pre-conditioned with A549 cells for 12 h, activated with anti-CD3/CD28 beads for 3 days, and tested for p-AMPKα and AMPKα by Western blot and flow cytometry. Mean ± SEM from 5 individuals in each group. C Co-localization of AMPK with LAMP1+ lysosomes detected by confocal microscopy. A representative from 3 individuals per group. Scale bar, 5 μm. Mean ± SEM from 30 individuals in each group. D, E Healthy CD4+ T cells were pre-conditioned with A549 cells for 12 h, activated with anti-CD3/CD28 beads for 3 days, and detected for p-S6 and p-AKT. Mean ± SEM from 5 individuals in each group. F, G A549 cell pre-conditioned CD4+ T cells were activated with anti-CD3/CD28 beads for 3 days in the presence or absence of AMPK inhibitor Compound C (10 μM), followed by detection of T cell differentiation. Mean ± SEM from 5 individuals in each group. *p < 0.05, **p < 0.01 with paired student t-test (A–E) and ANOVA plus Tukey method (F, G).