The Mechanisms Of B Cells And Follicular Dendritic Cells Capturing Graft-derived Exosomes

Transplant rejection is an important factor affecting the survival of transplanted organs.Various kinds of immune cells and molecules are the main participants in the occurrence and development of transplantation rejection.Recent studies have demonstrated that exosomes can also act as vectors for the delivery of allogenic antigens,immunity activation or regulation of molecule expression among transplantation,whch is independent of leukocyte migration.As a subtype of dendritic cell(DC),follicular dendritic cell(FDC)is the only known cell that can preserve intact antigen and play an important role in the formation of germinal centers.However,we have not investigated whether FDCcan retain intact exosomes,generate germinal center responses and specific B cell activation.Aims:(1)To investigate whether FDC in the B cell follicle can retain graft-derived exosomes.(2)To clarify the mechanisms of B cell binding with exosomes,and whether B cells can present exosmes to FDC.(3)To investigate the roles of marginal zone(MZ)B cells and follicle(Fo)B cells in the transferring of exosomes in the spleen.(4)To investigate whether graft-derived exosomes can activate specific B cells.Methods:(1)We did mice skin transplantation and heart transplantation from B6 to BALB/c mouse,respectively.We did immunofluorescence staining to investigate whether FDC can capture MHC molecules from B6 mice in secondary lymphoid organs or tissues,and calculated the survival time of heart grafts.Next,we injected exosomes secreted by B6-derived DC into the footpad of BALB/c mice;we did immunofluorescence staining to detect whether FDC can capture B6-derived MHC molecules in popliteal lymph nodes,and the subsequent germinal center respones.(2)We used flow cytometry to compare which factors would affect the binding between B cells and exosomes,such as complement,complement receptor,serum Ig M and antigen-specificity,and to analyze the binding rate between FDC and exosomes after incubation for 16 hours in vitro.The morphology of FDC and exosomes was reconstructed by STED microscope.(3)We used flow cytometry to compare the binding rate of MZ B cells and Fo B cells with exosomes in vivo and in vitro experiments.Confocal microscopy was used to observe whether allogenic exosomes could be transported from cognate B cells to FDCs.(4)We compared the differences of mean velocity and arrest coefficient between BALB/c 3-83-Ig B cell and BALB/c B cell when they encountered allogenic exosomes on the surface of FDCs,and verifed whether graft-derived exosomes could elicit B cell activation by means of two-photon and Imaris software.Results:(1)We found that FDC could retain donor MHC molecules in the secondary organ on post-operation day(POD)1,3,7,21,42.After injection of allogenic exosomes into the mice footpad,the allogenic exosomes were captured by FDC in the draining lymph nodes at day 1,3,7,14,21 and 42;germinal center reponses were observed at day 7,14,21 and 42.(2)The binding rate of B cells with exosome was significantly increased in the presence of complement,complement receptor and specific antigen.FDCs cultured in vitro have high affinity with exosomes.(3)Both vitro and in vivo experiments demonstrated the binding rate of MZ B cells with exosomes was significantly higher than that of Fo B cells.Imaging in vivo through two-photon showed that non-cognate B cells delivered allogeneic exosomes to FDC.(4)Graft-derived exosomes can be uptaken by B cell antigen receptor(BCR)transgenic B cells after internalization on the surface by FDC.Conclusion: FDC can retain graft-derived exosomes and loaded donor-derived MHC molecules.B cells can capture graft-derived exosomes,and this process is complement-dependent.MZ B cells are transporters of exosomes in the secondary lymphoid organ,and can present captured exosomes to FDC.Exosomes retained by FDC can further activate donor-specific B cells.