The Role And Mechanism Of Graft-derived Extracellular Vesicles In Allograft Rejection

Objective The immune response against transplanted allografts is one of the major causes for graft dysfunction in solid organ transplantation.It is widely accepted that the donor dendritic cells（DCs）is the initial response and trigger allo-immune response subsequently.Donor-derived extracellular vesicles（EVs）transfer MHC molecules to recipient DCs.Despite the DCs,the latest studies have raised concerns that allo-reactive B cells and donor-specific antibodies（DSAs）play important roles in allo-immune response,remains unclear.Here,we aime to investigate the detail mechanism of donor-derived EVs for initiating the alloreactive T and B cell in graft-draining secondary lymphoid tissues（graft-d SLTs）.Methods All the experiment depend on the skin and heart transplantation model.（1）Flow cytometry was used to detected the donor DCs（YFP⁺）in the draining lymph nodes.Graft survival,T cell and B cell sensitization were measured after CCR7^ko B6heart grafts transplanted to BALB/c mice.Donor intact MHC molecules in the draining lymph nodes（skin）or the spleen（heart）were checked by immunofluorescence microscopy.Immuno-electron microscopy was used to determine the format in which the cell-free donor Ag is delivered to d SLTs.GW4869and Rab27a^ko were choosed to inhibit the release of extracellular vesicles,then measure the survival of heart graft.（2）Image Stream（？）and immuno-electron microscopy were used to detect cross-dressing between the recipient APCs and donor-derived EVs in graft-d SLTs.Photoconvertible mice and Image Stream（？）were used to determine the position on donor MHC cross-dressing of recipient leukocytes.GW4869 were choosed to inhibit the release of extracellular vesicles,then Image Stream（？）was used to detect the donor MHC cross-dressing of recipient leukocytes and to check if it has an effect of the titer of DSAs in serum.Flow cytometry was used to detect the activation of CD4⁺T cells by receptor DCs.Intravital 2-photon microscopy was used to visualize how allogeneic EVs disseminate in d SLTs and whether recognition by B cells of allogeneic MHC on EVs presented by SCS macrophages triggers B-cell activation.Immunofluorescence microscopy was used to check the mobilized to the B cell/T cell border area.STED microscopy was used to analyze if the allogeneic EVs concentrated and mobilized across SCS macrophage.Low dose clodronate liposomes were injected to test the relevance of SCS M（？）s in B-cell allo-sensitization in response to donor EVs.（3）Immunofluorescence microscopy,immuno-electron microscopy and Image Stream（？）were used to evaluate if our results are relevant to human transplants,we investigated in humice if human skin grafts shed cell-free donor Ag via EVs and if graft-derived EVs are captured in vivo by human SCS macrophages in graft-d SLTs.Results In the graft-d SLTs and spleens of the recipients,donor DCs were undetectable after skin transplant.Inhibition of donor DCs migration was unable to prolong the graft survival and reduce the sensitization of recipient T and B cells.Donor MHC molecules were carried by donor EVs and were detected in the d SLTs by immunofluorescence microscopy without donor DCs migration.Allograft survival was significantly prolonged by EVs inhibitor GW4869 but not Rab27a.EVs-related donor MHC were cross-dressed and present donor MHC to recipient DCs in recipient DLNs.Cross-dressing between EVs and recipient DCs as well as activation of alloreactive CD4⁺T cells were significantly inhibited by GW4869.EVs-related donor MHC were rapidly captured by SCS M（？）s and concentrated in d SLTs,which were transported across the SCS M（？）s in one-way.The donor MHC molecules on the EVs were presented to allo-reactive na（？）ve B cells,which contribute to B-cell activation and DSA production.The allo-reactive B cells were mobilized to the B cell/T cell border area.Finally,we demonstrated that grafts release cell-free donor Ag via EVs are captured by SCS M（？）s and cross-dressed recipient DCs relevant for expansion of CD4⁺T cell in humice.Conclusion Donor DCs migrate to d SLTs is unessassry for activation of immunological rejection.Graft-EVs transfer donor MHC molecules to recipient CDs by cross-dress in recipient d SLTs,and activat alloreactive T cells subsequently.Graft derived EVs captured and presented by recipient SCS M（？）s to activate allo-reactive B cells.These results reveal that inhibition of Graft derived EVs may provide a potential therapeutic tool for dampening the immune response to allografts.