Cardiac Allograft Tolerance Induced By Transfusion Of Donor B Cells With Impaired Inducible Costimulator/B7h Allorecognition

Background:For a long time, the induction of transplantation tolerance, has always been the biggest challenge in transplantation immunology research. Donor-specific transfusion (DST) is a strategy widely applied in allograft transplantation for the last 3 decades. The infusion of whole blood, splenocytes or other cells from donors into recipients can prolong allograft survival in humans and also in mice. The infusion of donor leukocytes with an impaired costimulation signal (e.g. CD28/B7, CD40/CD40L) has been proven to be an effective way to induce donor specific tolerance in rodent models. ICOS is the third member of the CD28 superfamily, and its ligand B7 homologous protein (B7h), is constitutively expressed on B cells and monocytes. ICOS/B7h signaling has been proven to play a critical role in the transplantation immunity. B cells have been employed as the donor antigen for DST in several studies, and B cells with the impaired costimulation have been proven to be tolerogenic. To test the role of ICOS signaling during DST, we employed ICOSIg-targeted B cells as a donor antigen to challenge the allogeneic response in vivo and in vitro.Methods:1. In Vitro, the binding of ICOSIg with B7h on splenic B cells were confirmed by flow cytometry analysis (FCM). To evaluate the capacity of ICOSIg-targeted B cells to elicit an allogeneic response in vitro, mixed lymphocyte reactions (MLR) were performed.2. A murine cardiac allograft model was employed using BALB/c donors and C57BL/6 recipients, while the patterns of ICOS expression in recipients'peripheral lymphatic organs and in allografts were examined by flow cytometry analysis and immunohistochemistry staining respectively.3. ICOSIg-targeted B cells transfusions and/or ICOSIg injections were performed on the day of transplantation. Recipient mice exhibiting long term survival of cardiac allografts (>100 days) were given a transplant again with donor (BALB/c) and third-party (C3H/He) skins.4. Concentrations of serum Th1/Th2 cytokines of tolerant mice were analyzed by sandwich ELISA.5. Expressions of Th1/Th2 cytokines mRNA were determined by semi-quantitative RT-PCR.6. Percentage of CD4⁺CD25⁺ T cells in tolerant recipients'peripheral blood were confirmed by FCM. Percentage of the T cells apoptosis in recipients'draining lymph nodes were also detected by FCM.7. Subsets of tolerated splenocytes (TSCs) were harvested by microbeads selections respectively. The indicated number of splenocytes from selective subsets was adoptive transferred into 300rad 60Co -irradiated naive C57BL/6 mice, and followed 24 hours later by a donor (BALB/c) cardiac transplant. Results:1. The binding of B7h on splenic B cells by ICOSIg was confirmed at a saturing concentration of 100μg/ml. Blockade of ICOS/B7h in direct allorecognition resulted in depressed proliferation of alloreactive T cells in vitro.2. Allografts survival was prolonged by infusions of ICOSIg-targeted B cells, however allograft tolerance could not be achieved, unless systemic injections of ICOSIg were given simultaneously. This tolerance exhibited in a donor-specific fashion.3. ICOS⁺ expressions were evident not only in tolerant recipients'peripheral lymphatic organs, but also in the long term surviving allografts. Compared with control, percentage of CD4⁺CD25⁺T cells in tolerant recipients'peripheral blood were upregulated significantly at 15.6±5.69 %, P<0.01.4. When ITBT and ICOSIg injection were simultaneously given, percentage of CD8⁺ T cells apoptosis in recipients'draining lymph nodes were upregulated significantly at 19.53±5.10%, P<0.05.5. In comparison, recipients in post-trans 7d group, showed the higher serum concentration of IFN-γthan that of naive B6 mice(600.2±400.0 pg/ml VS 172.6±70.7 pg/ml,P<0.05). However, tolerant recipients exhibited the decresed serum concentration of IL-10, compared with na?ve B6 mice(563.5±153.7 pg/ml VS 2063.3±367.9 pg/ml,P<0.005).6. Compared with rejected grafts, long term surviving grafts showed the decreased intragraft mRNA for IL-2(IL-2_Ratio:0.344±0.021,P<0.05) and increased mRNA for IL-10(IL-10_Ratio:0.910±0.038,P<0.05).7. Adoptive transfer of 5×10⁶ splenic CD4⁺ but not CD4⁺CD25^- subsets from tolerated mice to lightly-irradiated naive recipients resulted in subsequent BALB/c allograft acceptance without additional immunosuppression. However, decay of this transferrable tolerance occurred within 1 month following the removal of tolerated allografts.Conclusion:ICOS/B7h signaling during direct allorecognition plays an important role in eliciting an allogeneic response both in vitro and in vivo, and donor specific tolerance can be induced by DST with blockade of ICOS/B7h in allorecognition. Data showed that exact mechanisms of this tolerance were complicated, which involved the dynamic balance of apoptosis and regulation. However, this tolerance is transferrable and maintained at least partly by the immune regulation of CD4+CD25+T cells. We also found that the persistent presence of tolerated allografts was important for maintenance of stable DST-induced Tregs clones. These findings may help to design a potential therapeutic treatment to prevent allograft rejection by DST in combination with a costimulation blockade.