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 (EXO(Tr)) 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 EXO(Tr) 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 (DC(OVA)) plus Tr cells or EXO(Tr), and then assessed OVA-specific CD8(+) T cell responses using PE-H-2K(b)/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 DC(OVA)-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 DC(OVA) (p<0.05) in immunized mice with co-injection of Tr cells and EXO(Tr), 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.