*Purpose: The recognition of donor-MHC molecules by recipient T cells triggers the immune response leading to rejection of allografts. Our recent studies have documented the presence of high numbers of recipient APCs displaying donor-MHC molecules (cross-dressed) on their surface in the lymphoid organs of mice after skin, heart or pancreatic islet transplantation. In addition, we have reported that acquisition of allogeneic MHC molecules by host APCs (MHC cross-dressing) is mediated by donor-derived extracellular vesicles (EVs) trafficking through blood and lymphatic vessels (Marino et al. Science Immunology, 2016). In the present study, we investigated the ability of allogeneic EVs and allo-MHC-cross-dressed cells to initiate a T cell alloresponse in vitro and in vivo.

*Methods: EVs were isolated (using differential centrifugation) from BALB/c Bone Marrow Derived Dendritic Cells (BMDCs). These EVs were used to cross-dress B6 splenocytes in vitro. The transfer of donor MHC class I and II on B6 cells was analyzed by imaging flow cytometry. Next, T cells from B6 mice were cultured in vitro with either allogeneic BMDC-derived BALB/c EVs or B6 spleen cells cross-dressed with allogeneic BALB/c MHC. Alternatively, 2x 10⁹ BALB/c or B6 BM derived EVs or 20x 10⁶ BALB/c BM cells were injected IV to B6 mice. In both cases, the T cell response was assessed by activation markers detection, INFg production and cell proliferation.

*Results: APCs cross-dressed with allogeneic MHC molecules can trigger a pro-inflammatory direct alloresponse by T cells in vitro and in vivo. On the other hand, allogeneic EVs alone were only able to induce early T cell activation but not proliferation in vitro. Furthermore, injection of mice with allogeneic EVs alone could induce some but suboptimal alloresponse in vivo and only when administered with complete Freund’s adjuvant.

*Conclusions: Blocking donor EVs release and subsequent recipient APC cross-dressing may represent a promising target to selectively inhibit anti-donor T cell inflammatory responses thus achieving long-term allograft survival.

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