The idea that donor dendritic cells (DCs) transplanted with the grafts and mobilized to draining lymphoid tissues (dLTs) present donor MHC molecules to allo-reactive naïve T cells has been challenged. New evidence indicates that lymph node (LN)-resident recipient DCs cross-dressed with donor extracellular vesicles (EVs) (i.e. exosomes) bearing donor MHC, prime directly allo-reactive T cells in dLTs. However, (i) if allo-sensitization occurs via release of donor EVs by donor DCs that reach dLTs, or non-migrating cells of the grafts; and (ii) the fate of the donor EVs in dLTs, remain unknown. Results: To answer the first question, we tested if allo-sensitization occurs in the absence of donor DC migration or alternatively, requires mobilization of donor DCs that are eliminated by recipient NK and CTLs, once they reach the dLTs. After transplantation of skin allografts [CD11c-YFP C57Bl/6 (B6) → wt BALB/c], no donor DCs (YFP⁺) were detected by FACS in dLTs on post-operative days (PODs) 2, 7,14 and 28. By contrast, after syngeneic skin transplantation (CD11c-YFP B6 → wt B6), donor DCs were found in dLTs on PODs 14 and 28. Thus, during allo-sensitization (first week after surgery), the lack of donor DCs in dLTs that drain skin allografts is not due to their elimination by host NK and CTLs, as graft-derived DCs were also absent in dLTs during the first week after transplantation of syngeneic grafts. This was confirmed by grafting CCR7^KO B6 skin in BALB/c mice, which exhibited a level of allo-sensitization against donor MHC Ag and graft survival similar to those in BALB/c mice grafted with wt B6 skin. Consistent with the idea that skin allografts promote allo-sensitization via EVs that traffic to dLTs, we found that (i) skin allografts release abundant exosome-like EVs ex vivo, and that (ii) macrophages lining the sub-capsular sinus (SCS) of graft dLTs capture donor MHC⁺ material. Analysis by electron microscopy revealed that such content corresponds to clusters of EVs (82.3 nm in size), carrying donor MHC Ag and the exosome-marker CD63, trapped in a system of vesicles / channels in the SCS macrophages. Conclusions: Migration of passenger leukocytes to the dLTs is not required to trigger allo-sensitization against skin allografts. Allo-sensitization is mediated via release by the graft of EVs that are efficiently captured by SCS macrophages in the dLTs.

CITATION INFORMATION: Zeng F., Shufesky W., Yang M., Liu Q., Larregina A., Morelli A. Origin and Traffic of Donor-Derived Extracellular Vesicles during Allo-Sensitization Am J Transplant. 2017;17 (suppl 3).

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