| Objective:This study aims to investigate the effects of X-box-binding protein 1(XBP1)deletion on bone marrow-derived dendritic cells(BMDCs)and the underlying mechanisms involved,and to explore whether transfusion of XBP1-deficient BMDCs can protect heart allografts from allorejection.Methods:We generated mice with specific deletion of the XBP1 allele in bone marrow cells(XBPl-/-mice)and cultured XBP1-deficientg BMDCs(XBP1-/-BMDCs).We then tested the phenotype of XBP1-/-BMDCs,evaluated their capability to activate allogeneic T cells and investigated their mechanistic actions.We developed a mouse model of allogeneic heart transplantation in which recipients received PBS,XBP1-/-BMDCs,a suboptimal dose of cyclosporine A(CsA),or XBP1-/-BMDCs combined with a suboptimal dose of CsA to evaluate the effects of XBP1-/-BMDCs transfusion on alloimmunity and on the survival of heart allografts.Results:The deletion of XBP1 in BMDCs exploited the IRE 1-dependent decay(RIDD)of TAPBP mRNA to reduce the expression of MHC-I on the cell surface,altered the capability of BMDCs to activate CD8+T cells,and ultimately suppressed CD8+T-cell-mediated allogeneic rejection.The adoptive transfer of XBP1-/-BMDCs inhibited CD8+T-cellmediated rejection.In addition,XBP1-deficient BMDCs were weak stimulators of allogeneic CD4+T cells despite expressing high levels of MHC-Ⅱ and costimulatory molecules on their cell surface.Moreover,the adoptive transfer of XBP1-/-BMDCs inhibited the production of donor-specific IgG.The combination of XBP1-/-BMDCs and CsA treatment significantly prolonged the survival of allografts compared to CsA alone.Conclusions:The deletion of XBP1 induces immunosuppressive BMDCs,and treatment with these immunosuppressive BMDCs prevents alloimmune rejection and improves the outcomes of heart transplantation.These findings provide a promising therapeutic target for combating transplant rejection and expand knowledge of inducing therapeutic DCs.Part Ⅰ:XBP1-Deficiency Induces Immunosuppression in Bone MarrowDerived Dendritic CellsObjective:This study aims to first culture XBP1-/-BMDCs using XBP1-/-mice,and then investigate the effects of XBP1 deletion on BMDCs.Methods:The XBP1-/-mouse model was established using the Cre-loxP system.Mice harboring loxP site flanked Xbpl alleles(XBP1fl/fl mice)were served as controls.BMDCs were cultured from bone marrow cells isolated from the tibia and femur of these animals.Surface markers,including MHC-1,MHC-Ⅱ,CD40,CD80,CD86,and PD-L1,were detected to evaluate the effect of XBP1 deletion on the phenotype of BMDCs using flow cytometry.Naive T cells were extracted from the spleens of BALB/c mice and co-cultured with XBP1-/-or XBP1fl/fl BMDCs at a DC:T ratio of 1:5 cells for 4 days to evaluate the capability of XBP1-/-BMDCs to activate allogeneic T cells.Results:XBP1-/-BMDCs exhibited approximately 2 to 3-fold lower levels of MHC-Ⅰ on the cell surface than XBPlfl/fl controls.However,XBP1 deletion in BMDCs had no effect on the surface levels of MHC-Ⅱ,CD40,CD80,CD86,or PD-L1.Both CD8+T and CD4+T cells exhibited reduced levels of proliferation but increased levels of apoptosis in response to XBP1-/-BMDCs compared with those observed with XBPlfl/fl BMDCs.The production of IFN-γ,IL-4,and IL-17A was significantly inhibited by XBP1-/-BMDCs in the 4-day mixed lymphocyte reaction assay.XBP1-/-BMDCs induced significantly lower levels of both CD8+IFN-γ+effector T cells and CD4+IFN-γ+effector T cells than XBPlfl/fl BMDCs.The proliferation of both CD4+and CD8+ memory T cells was significantly reduced in response to XBP1-/-BMDCs compared with XBPlfl/fl control BMDCs.Conclusion:XBP1-deficient BMDCs expressed low levels of MHC-I on the cell surface and exhibited an immunosuppressive phenotype.Therefore,the targeting of XBP1 could be a promising approach to induce immunosuppressive DCs.Part Ⅱ:Transfusion of XBP1-Deficient Bone Marrow Derived Dendritic Cells Protects Heart AllograftsObjective:This study aims to investigate whether the transfusion of XBP1-/-BMDCs can protect heart allografts from allorejection.Methods:A fully MHC-mismatched heart transplantation model was established in BALB/c mice receiving hearts from C57BL/6 mice.Recipients received PBS(served as an untreated control),XBP1-/-BMDCs,a suboptimal dose of cyclosporine A(CsA),or a combination of XBP1-/-BMDCs and a suboptimal dose of CsA to evaluate the effects of XBP1-/-BMDC transfusion on the survival of heart allografts.Another group of recipients received PBS or XBP1-/-BMDCs and were sacrificed on postoperative day(POD)5 to evaluate the effects of XBP1-/-BMDCs transfusion on alloimmunity.Results:XBP1-/-BMDCs administration prolonged heart allograft survival from a median survival time(MST)of 7 to 15 days(P<0.0001,compared with untreated controls),which was similar to the suboptimal dose of CsA(MST=17 days,P=0.154).Treatment combining XBP1-deficient BMDCs with suboptimal dose of nonspecific immunosuppressive agent prolonged the survival of heart allografts from an MST of 17 to 41 days(P=0.0008,compared with suboptimal dose of CsA administration alone).Histological analyses of the allografts on POD 5 showed that XBP1-/-BMDCs administration significantly ameliorated acute cellular rejection compared to untreated controls.Immunofluorescence staining of the allografts showed that the levels of infiltrating CD8+T cells were significantly reduced in allografts treated with XBP1-/-BMDCs compared to untreated controls;however,the levels of infiltrating CD4+T cells appeared similar in both groups.Subsequent lymphocyte counts showed that the absolute numbers of CD8+T cells in allografts,spleens and peripheral blood were significantly reduced in recipients treated with XBP1-/-BMDCs compared with untreated controls,whereas the absolute numbers of CD4+T cells were unaltered.The absolute numbers of both CD4+and CD8+memory T populations were decreased in recipients treated with XBPl-/-BMDCs compared with untreated controls.Serum cytokine analyses showed that recipients treated with XBP1-/-BMDCs had significantly higher levels of IL-10 and lower levels of IFN-y,IL-2 and IL-4 than untreated controls.The donor-specific IgG level was decreased in recipients treated with XBP1-/-BMDCs compared to untreated controls.Conclusion:XBP1-deficient BMDCs transfusion can suppress allorejection and protect heart allografts.Part Ⅲ:Investigation on the Mechanism by Which XBPI-deficiency Induces Immunosuppressive BMDCsObjective:This study aims to explore the mechanism by which XBP1-deficiency induces immunosuppressive BMDCs.Methods:The subcellular distribution of MHC-I molecules in XBP1-/-BMDCs was first quantitatively detected using western blot,and then observed using immunoelectron microscopy.The expression of the components of the MHC-I antigen presentation machinery were evaluated using western blot.Next,endoplasmic reticulum homeostasis in XBP1-/-BMDCs was identified by first observing the morphology of endoplasmic reticulum using immunofluorescence staining and transmission electron microscopy,and then by quantitatively detecting endoplasmic reticulum stress markers(BiP and CHOP).The activity of IRE 1-dependent decay(RIDD)in XBP1-/-BMDCs was determined by measuring phosphorylated IRE1α(p-IRE1α),BLOC1S1,and GALNT2.After inhibiting RIDD activity in XBP1-/-BMDCs using the IRE1-specific inhibitor B-109,the expression of tapasin protein,the level of MHC-I on the cell surface,and the capability to activate CD8+T cells were evaluated.Results:XBP1-/-BMDCs express a similar level of total MHC-I as XBPlfl/fl BMDCs;however,in XBP1-/-BMDCs,the expression of MHC-I was significantly increased in the endoplasmic reticulum and significantly decreased in the cytoplasm.Among the components of the APM,only the expression of tapasin is significantly reduced in XBP1-/BMDCs compared with XBPlfl/fl BMDCs.The expansion and aberrant structure of the ER and the increased abundance of ER stress factors(BiP and CHOP)in XBP1-/-BMDCs demonstrated ERS.The increased levels of p-IRE1α and decreased levels of RIDD markers(BLOC1S1 and GALNT2)in XBP1-/-BMDCs demonstrated the activation of RIDD.Inhibition of RIDD activity in XBP1-/-BMDCs could restore the expression of tapasin,the surface level of MHC-I,and subsequent CD8+T-cell-mediated allogeneic immunity.Conclusion:Deletion of XBP1 exploited the RIDD of TAPBP mRNA to reduce the surface expression of MHC-I on BMDCs,alter their ability to activate allogeneic T cells,and ultimately suppress CD8+T-cell-mediated allogeneic rejection.This study presents a novel mechanism by which XBP1 modulates the phenotype and antigen presentation function of BMDCs. |
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