The Role Of Different Source Of Regulatory T Cells In Corneal Allograft Rejection

Purpose:To describe the difference of innate Treg and adaptive Treg in cell phenotype, cytokines, antigen specificity and function mechanism and investigate the different effect of innate Treg and adaptive Treg in immunomodulation of murine corneal allograft rejection.Methods:1.iTregs were generated and expanded in vivo by antigen-loaded immature DCs which had been induced by transforming growth factor beta-2 (TGFβ-2) for 5 days. CD4+CD25+Tregs were isolated by 2-step magnetic cell sorting (MACS) and the Treg–specific phenotypes were identified by FACS. Three days after the injection, the difference of the number and phenotype of different source of Treg (nTreg & iTreg) in SPL were analyzed by FACS in mice of difference groups.2.Two kinds of mechanism were used to observe the effects of nTreg and iTreg function. PBS, TGF-β2 DCs, nTregs or iTregs were injected into the murine tail veins. After that, allogeneic corneal transplantation was performed. Observe the grafts'survival rate and the time when rejection occurred.3.Purified and adoptive transfer different resource of Treg. 3 days later, performed corneal transplantation which the receptor were C57BL/6 or C3H mouse, respectively. Antigen-Specific suppression of graft rejection were observed in MLR by Ex Vivo Stimulated CD4+CD25+ Treg Cells.Results:1.TGF-β2 DC can enhance the number of CD4+CD25+T cells in SPL of mice(P<0.05); Foxp3 and CTLA-4 was preferentially expressed by iTreg cells and CD28 expression decreased in adaptive Terg cells. MLR showed that the suppression to the proliferation of effector T cells of iTreg was significantly higher than other groups.2.TGF-β2 DC increased the number and function of CD4+CD25+Tregs in vivo.The corneal allograft rejection was suppressed notably in iTreg injection group. In iTreg injection group, all the allografts remained clear until 60 days after transplantation and there was statistical difference between the four groups analyzed by NPar test(P<0.05). After adoptive Transfer of TGFβ2-DC, nTregs or iTregs , there was an upregulation in the percentage of CD4+CD25+, CD4+CD25+Foxp3+, CD4+CD25+CTLA-4+T cells in SPL 3 days after corneal allograft transplantation, and there was statistical difference between the these groups (P<0.01). Adaptive Tregs were more potent in suppressing effector than innate Treg (P<0.01).3.CD4+CD25+Treg can acquire alloantigen-specific. iTreg acquired donor antigenic specificity with high inhibition ratio to donator antigen at 86.7%.iTreg can prolong grafts'survival time. Compared with the other 4 groups,the survival time of transplanted corneal graft in adaptive Treg groupwas prolonged significantly (P<0.05).Conclusions:In summary, we indicate that iTreg have more suppressive function than nTreg. Alloantibody was an important effector mechanism for corneal allograft rejection. The suppressor regulatory T cells were involved in the induction of ACAID contribution to suppressing corneal allograft rejection which was through their well-established role in delayed-type hypersensitivity (DTH). Antigen-loaded TGF-β2 DCs could increase the number and function of Foxp3+CD4+CD25+Tregs. CD4+CD25+Tregs with indirect allospecificity were more potent than antigen-loaded TGF-β2 DCs or innate CD4+CD25+Tregs in suppressing effector in allograft rejection specific for the same antigen. Our results add a new important perspective for Treg-based immunotherapeutic strategies.