Fig. 2

Tumor-derived EVs induce differentiation of T cells into Treg through TGF-β1 in Vitro. A Transmission electron microscopy (TEM) image of EVs secreted by 4T1 cells. Scale bar, 50 nm. B TGF-β1 concentration in total supernatant or EVs of 4T1 cells. C TGF-β1 concentration in total supernatant or EVs secreted by tumor pieces. 4T1 tumor model was established by injecting tumor cells subcutaneously into the fourth mammary pad of BALB/c mice. After 35 days, tumor tissues were collected, cut into pieces and cultured in vitro for 48 h. Culture medium was collected for EV isolation and ELISA was performed for analysis of TGF-β1. D Western blot analysis of TGF-β1 in EV or EV-depleted supernatant from 4T1 cells. E qPCR analysis of TGF-β1 mRNA in WT or TGFβ-1-ko 4T1 cell line. F ELISA analysis of TGF-β1 in supernatant from WT or TGFβ1-ko 4T1 cell line. G, H Naïve lymphocytes from the spleen of Foxp3-GFP transgenic mice were treated by anti-CD3/CD28 functional antibody. G The EVs secreted by WT or TGF-β1-ko 4T1 cells were added to the lymphocytes for 72 h and percentage of CD4⁺GFP⁺ cells (Tregs) were analyzed by flow cytometry. H Percentage of PHK⁺ cells in CD4⁺CD25⁺cells. EVs secreted by 4T1 cells were isolated and labeled with PHK. Naïve spleen cells from C57BL/6 mice were stimulated by anti-CD3/CD28 functional antibody and incubated with 500 µg/ml of PHK-labeled EVs for 72 h and percentage of PHK⁺ cells in CD4⁺CD25⁺cells were analyzed by flow cytometry. I The EVs secreted by WT or TGF-β1(5 ng/mL) with or without 1D11 (10 ng/mL) were added to the lymphocytes for 72 h and percentage of CD4⁺GFP⁺ cells (Tregs) were analyzed by flow cytometry. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. B, E, F were analyzed by T-test. G–I were analyzed by 2-way ANOVA