After organ transplantation, T cells are primed against donor MHC molecules by donor dendritic cells (DCs) mobilized from the graft. Nevertheless, evidence suggests that donor DCs home in low numbers in recipient lymphoid tissues, are short-lived, and are killed by recipient NK cells and CTLs. How then, relatively few donor DCs prime so efficiently recipient T cells against donor MHC molecules? Here, we demonstrate that after heart transplantation, donor DCs migrated to the spleen transfer functional MHC molecules (cross-dressing) and DC-activation signals to a high number of recipient conventional DCs (cDCs). This transfer is mediated via passage of extracellular vesicles (EVs) of endocytic origin, ranging 70-120nm in size, known as exosomes. Methods: Heterotopic (abdomen) heart allograft transplantation in mice, PCR, FACS, ELISPOT, and electron and confocal microscopy. Results: After transplantation of CD45.2 BALB/c hearts in CD45.1 C57Bl/6 (B6) mice, the few donor DCs that migrated to the recipient spleen (1127 ± 372 cells / spleen / on day 3) transferred donor MHC molecules to a higher number recipient splenic cDCs via clusters of exosome-like EVs (75±32 nm). The transferred MHC molecules were functional, since FACS-sorted recipient cDCs induced proliferation and effector cell differentiation of 2C CD8 T cells, specific for the BALB/c intact H2L^d molecule. Cultures of BALB/c DCs with B6 DCs with inhibitors of exosome release (Rab27a siRNA) confirmed that passage of BALB/c MHC occurred via exosomes. Importantly, transfer of exosomes released by donor mature DCs (unlike other types of EVs) promoted maturation of recipient DCs, in vitro and in vivo. By high resolution confocal microscopy, BALB/c DCs, engineered to release RFP-tagged exosomes and injected s.c. or i.v. in CD11c-YFP B6 mice, transferred RFP⁺ exosomes in clusters to recipient YFP⁺ DCs in lymph nodes and spleen. Accordingly, depletion of recipient DCs in diphtheria toxin (DT)-treated CD11c-DTR B6 recipients prevented presentation of donor intact MHC molecules to T cells and significantly delayed rejection of BALB/c cardiac grafts. Conclusion: These findings identify cross-dressing via exosomes as an explanation for the potency of alloimmunity, unveil a new role for EVs in the semi-direct pathway, and open new possibilities for development of therapies to treat transplant rejection.

CITATION INFORMATION: Liu Q, Rojas-Canales D, Divito S, Shufesky W, Larregina A, Morelli A. Transfer of Donor-Derived Exosomes to Recipient Ag-Presenting Cells Is Critical for Allograft Recognition. Am J Transplant. 2016;16 (suppl 3).

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