Natural CD8+25+ regulatory T cell-secreted exosomes capable of suppressing cytotoxic T lymphocyte-mediated immunity against B16 melanoma

Natural CD4+25+ and CD8+25+ regulatory T (Tr) cells have been shown to inhibit autoimmune diseases. Immune cells secrete exosomes (EXOs), which are crucial for immune regulation. However, immunomodulatory effect of natural Tr cell-secreted EXOs is unknown. In this study, we purified natural CD8+25+ Tr cells from C57BL/6 mouse naive CD8+ T cells, and in vitro amplified them with CD3/CD28 beads. EXOs (EXOTr) were purified from Tr cell's culture supernatants by differential ultracentrifugation and analyzed by electron microscopy, Western blot and flow cytometry. Our data showed that EXOTr had a “saucer” or round shape with 50-100 nm in diameter, contained EXO-associated markers LAMP-1 and CD9, and expressed natural Tr cell markers CD25 and GITR. To assess immunomodulatory effect, we i.v. immunized C57BL/6 mice with ovalbumin (OVA)-pulsed DCs (DCOVA) plus Tr cells or EXOTr, and then assessed OVA-specific CD8+ T cell responses using PE-H-2Kb/OVA tetramer and FITC-anti-CD8 antibody staining by flow cytometry and antitumor immunity in immunized mice with challenge of OVA-expressing BL6-10OVA melanoma cells. We demonstrated that DCOVA-stimulated CD8+ T cell responses and protective antitumor immunity significantly dropped from 2.52% to 1.08% and 1.81% (p < 0.05), and from 8/8 to 2/8 and 5/8 mice DCOVA (p < 0.05) in immunized mice with co-injection of Tr cells and EXOTr, respectively. Our results indicate that natural CD8+25+ Tr cell-released EXOs, alike CD8+25+ Tr cells, can inhibit CD8+ T cell responses and antitumor immunity. Therefore, EXOs derived from natural CD4+25+ and CD8+25+ Tr cells may become an alternative for immunotherapy of autoimmune diseases.