Figure 3: Donor-derived exosomes induce donor antigen specific Tregs.

CD4⁺ T cells were isolated by MACS from the wild hearts and allograft hearts and analyzed by flow cytometry. (A) the CD4⁺ T cells were gated. (B–F) the gated dot plots indicate the frequency of Tregs in the gated CD4⁺ T cells of (A). PAR2-n: PAR2-deficient mice. (G) the bars indicate the summarized data of (B–F). (H–J), Tregs (CD4⁺ CD25⁺ CD127^-) were isolated from the heart-isolated T cells, labeled with CFSE, and cultured in the presence of exosomes and DCs (T cell:DC = 5:1) for 3 days. The gated histograms indicate the frequency of the proliferating Tregs. (K) samples were collected from the culture medium of (G–I) and analyzed by ELISA. The bars indicate the TGF-β levels in the medium. (L–O) Tregs were isolated from the allograft hearts (the recipient mice were treated with donor-derived exosomes) and cultured with polarized Th2 cells at a ratio of 1:1 in the presence of DCs and exosomes for 48 h. The cells were stained with Annexin V reagents and propidium iodide (PI), and analyzed by flow cytometry. (L) the CD4⁺ T cells were gated first. (M–O) the gated dot plots indicate the frequency of apoptotic cells. (P) the bars indicate the summarized data of M-O. #, the exosomes were obtained from non-donor mice. The data of bars are presented as mean ± SD. *p < 0.01, compared to group B (G), or group G (K), or group L (P). Each group consists of 10 mice. Samples from individual mice were processed separately.