Study On The Mechanism Between Regulatory B Cells And T Cells In Transplantation Tolerance Induction

Purpose:Immune regulatory cells are a promising new approach for immunotherapy in inducing immune tolerance in allogeneic transplantation.According to research findings,the adoptive transfer of tolerant B cells can also induce transplantation tolerance.Therefore,inducing the generation and expansion of regulatory B cells(Bregs)with antigen replacement is an effective means to induce immune tolerance.In this paper,first tries to activate and expand B cells through the TLR signaling pathway in vitro(Bregs-TLR).To clarify the cell phenotype of the amplified Bregs-TLR and the expression of the cytokine IL-10 related to the regulatory function,and compare it with the verified Bregs.To explore and study the immune tolerance induction effect of this Bregs-TLR in skin and islet transplantation model.Give a further study of the relationship between Bregs-TLR and CD4+T,CD8+T,and Tregs in the process of transplantation tolerance induction and related mechanisms of action,respectively.To provide a theoretical basis for a new treatment plan for inducing transplantation tolerance.Methods:To establish a culture system to activate and amplify B6 B and OBI B cells in vitro.The phenotype and cytokines of the expanded Bregs-TLR cells were detected by flow cytometry.To establish the islet and skin transplantation models and adoptive transfer the Bregs-TLR to verify its regulatory function.The suppression effects of Bregs-TLR on proliferation and function of CD4+T and CD8+T were studied through in vitro MLR and establishing antigen-specific immune-transplantation models in vivo.The Transwell culture system was used to verify the mode of action between Bregs-TLR and T cells,SCID mice were used to establish a skin transplantation model,and IL-10 and TGF-? were neutralized by related antibodies to verify the inducing effect and the mechanism of Bregs-TLR on Tregs.Finally,B cells from IL-10-/-mice and CD19cre/WTTGF-?fl/fl mice were used to isolate and expand IL-10-/-Bregs-TLR and TGF-?-/-Bregs-TLR,and then adoptive transfer to islet transplant model to further confirm the mechanism of Bregs-TLR on tolerance induction.Results:1)Successfully established a culture system for B cell expansion in vitro.B cells from B6 or OBI mouse spleen were activated and expanded through the TLR signaling pathway,and the number of cells increased by about two times,and the cell volume increased significantly.2)The cell phenotype identification results showed that the phenotype of B6 Bregs-TLR was mainly IgMhiIgDint/lo and similar to IgM+IgD-CD21-CD23 TIB cell,and the phenotype of OBI Bregs-TLR was similar to CD19+CD21hiCD23loMZ B cell.The marker MHC-II,LAP,CD40,CD80,CD86,PD-L1,and IL-10 which related to B cell activation and regulatory functions,were all significantly up-regulated.The difference between OBI and B6 was that the expression of IgM and IgD of OBI was deficient,and the others were all similar.3)Adoptive transfer of Bregs-TLR to the transplanted animal model found that Bregs-TLR can prolong the survival of skin grafts and islet grafts.4)Establishing antigen-specific transplantation models in vivo and in vitro to explore the interaction between Bregs-TLR and CD4+T,the results showed that Bregs-TLR could inhibit the proliferation of CD4+T cells and its cytokines:TNF-? and IFN-? both in vivo and in vitro.They also could induce the apoptosis of CD4+T cells.Bregs-TLR inhibited the reaction of CD4+T cells through the direct cell to cell contact,and this inhibition was antigen-specific and dose-dependent.5)Exploring the interaction relationship between Bregs-TLR and CD8+T cells in a model similar to CD4+T showed that the relationship between Bregs-TLR and CD8+T cells is contradictory in vitro and in vivo.They inhibited the proliferation and the cytokine IFN-? secretion of CD8+T in vitro in cell to cell contact way.They also induced the apoptosis of CD8+T cells,but they failed to inhibit the proliferation of CD8+T cells in vivo.6)In the skin transplantation model,under the same type and same dose of antigen stimulation,CD8+T showed a faster and more potent immune response than CD4+T.CD8+T mediated systemic immune response,and CD4+T mediated local immune response.7)In the skin transplant model,Bregs-TLR could up-regulate CD4+Foxp3+Tregs and converted CD4+CD25 T into CD4+CD25+Foxp3+Tregs through TGF-? instead of IL-10.8)In the islet-transplant model,rejection occurred rapidly after the adoptive transfer of TGF-?-/-Bregs-TLR,but IL-10-/-Bregs-TLR could maintain at least 40%of pancreatic islet transplanted mice for long-term survival.Conclusion:The combined stimuli of CPG,LPS,PMA,and ionomycin significantly activated and amplified B cells through the TLR signaling pathway in vitro,and the cell phenotypes and cytokine IL-10 secretion,which related to regulatory function,were similar to verified Bregs.Except that,Bregs-TLR prolonged the survival of skin and islet grafts.In the process of transplantation tolerance induction,Breg-TLR controls the secretion of cytokine effectors and induces CD4+T cells apoptosis to control the immune response of CD4+T cells in cell to cell contact way.At last,Bregs-TLR through TGF-? rather than IL-10 to up-regulates Tregs to induce transplantation immune tolerance.This tolerance induction by Bregs-TLR depends on antigen specificity.