CD4+ T cell-released exosomes inhibit CD8+ cytotoxic T-lymphocyte responses and antitumor immunity

Abstract

T cells secrete bioactive exosomes (EXO), but the potential immunoregulatory effect of T-cell EXO is largely unknown. In this study, we generated activated ovalbumin (OVA)-specific CD4(+) T cells in vitro via coculture of OVA-pulsed dendritic cells (DC(OVA)) with naive CD4(+) T cells derived from OVA-specific T-cell receptor (TCR) transgenic OTII mice. CD4(+) T-cell EXO were then purified from the CD4(+) T-cell culture supernatants by differential ultracentrifugation. CD4(+) T-cell EXO exhibited the 'saucer' shape that is characteristic of EXO with a diameter between 50 and 100nm, as assessed by electron microscopy, and contained the EXO-associated proteins LAMP-1, TCR and lymphocyte function associated antigen-1 (LFA-1), as determined by western blot. Flow cytometric analysis showed that CD4(+) T-cell EXO expressed CD4(+) T-cell markers (CD4, TCR, LFA-1, CD25 and Fas ligand), but to a lesser extent than CD4(+) T cells. We demonstrated that DC(OVA) took up CD4(+) T-cell EXO via peptide/major histocompatibility complex (pMHC) II/TCR and CD54/LFA-1 interactions. OVA-specific CD4(+) T-cell EXO from OTII mice, but not ConA-stimulated polyclonal CD4(+) T-cell EXO from wild-type C57BL/6 mice inhibited DC(OVA)-stimulated in vitro CD4(+) T-cell proliferation and in vivo CD8(+) cytotoxic T lymphocyte (CTL) responses and antitumor immunity against OVA-expressing B16 melanoma BL6-10(OVA) cells. In addition, EXO derived from a T-cell hybridoma cell line, MF72.2D9, expressing an OVA-specific CD4(+) TCR, had a similar inhibitory effect as OTII CD4(+) T-cell EXO on CTL-mediated antitumor immunity. Taken together, our data indicate that antigen-specific T-cell EXO may serve as a new type of immunosuppressive reagent for use in transplant rejection and treatment of autoimmune diseases.