Fig. 5
Protein cargos in M1-EVs induced M1 polarization and stimulated T lymphocytes. A GSEA showed that M1-EVs were significantly enriched with proteins related to M1 protein signature. B GSEA suggested that M1-EV proteins positively correlated with lymphocyte activation. C Expression of M1 marker genes in naïve macrophages treated with 1.5 × 109/mL of M0- or M1-EVs for 8 h. D Expression of M1 marker genes in naïve macrophages treated with 1.5 × 109/mL of regular or heated M1-EVs for 8 h. EVs were heated at 95 °C for 10 min to denature the proteins. E, F Flow cytometry analysis showing the protein levels of iNOS and Arg1 in BMDMs treated with 6 × 109/mL of regular (E) or heated (F) M0- and M1-EVs for 8 h. G–J Murine splenocytes were primed with anti-CD3 antibody (1 µg/mL) in the absence or presence of 1 × 1010/mL M1-EVs for 72 h, followed by flow cytometry analysis and cytokine measurement. G–H Zombie Violet staining to assess viability of total splenocytes (G), T cells (CD4+ and/or CD8+) and non-T cells (CD4 and CD8 negative, H). I Proliferation of CD4+ and CD8+ lymphocytes measured using CellTrace CFSE dye. J IFN-γ release from anti-CD3-primed splenocytes treated with regular or heated M1-EVs. Data presented as mean ± STD (N = 3). **P < 0.01