Blocking Tim-3 Or TLR Signaling Pathway In Innate Immune Promotes Transplant Tolerance Induction In Mice

[Aim] To explore the immunosuppressive effect of galectin-9 on fully allogeneic carcdiac allografts in mice.[Methods] CHO cells were engineerly overexpressed galectin-9 using adenovirus which contains the galecitn-9 gene. Immunocellular chemistry and flow cytometry staining were performed to ascertain the location of galectin-9. Preactivated or normal splenic CD4+or CD8+lymphocytes were subjected to flow cytometry analysis for mean fluoresence intensity. Galectin-9 protein was expressed and purified by pET E. Coli. FACS-sorted CD4+ CD25- T cells were cultured to differentiate to Th17 cells in the presence or absence of galectin-9. The percentage of Thl7 cells were detected three days later. BALB/c→C57BL/6 cervical cardiac transplant models were built. The recipients were treated with a short course of galectin-9 treantment. Compare the survival time of different recipients. The percentage of CD4+IFN-y+, CD4+IL-17+, CD8+IFN-γ+, CD8+IL-17+, CD4+Tim-3+and CD4+Tim-1+lymphocytes in the periphereal or draining lymphonode were analyzed by FACS. The percentage of CD4+CD25+Foxp3+T cells in spleen lymphocytes among different groups was compared.[Results] Galectin-9 was in the cytosol of CHO cells and actived CD4/CD8 cells. In vitro, Thl7 cell differnentitation was inhibited by the addition of galectin-9. The graft infiltrating cells were mainly CD8+T cells. The percentage of CD8+Tim-3+T lymphocytes in CD8+T lymphocytes was 40.5%±5.2%. A short course administration of galectin-9 promotes the survival of fully allogeneic cardiac allografs than the control group (MST= 23.0±1.0 days vs 7.2±0.4 days, respectively.). The prolonged survival was related with the reduction of CD8+IFN-γ+and CD8+IL-17+lymphocytes in the draining lymphonodes and the deviation from Th1/Th17(CD4+Tim-3+) to Th2(CD4+Tim-1+) immune responses.[Conclusion] Galectin-9 is in the cytosol of activated CD4+and CD8+lymphocytes. It may negatively regulate the overactivation of CD4+and CD8+lymphocyte immune responses. In vitro, galectin-9 inhibits the production of Thl7 cells. The immunosuppressive effect of galectin-9 on allografts is related with the reduced CD8+T cell responses and deviation from Th1/Th17 immune responses to Th2 immune responses. [Aim] To explore whether the the maturation promoting effect of galectin-9 on DCs could be reversed by Rapamycin. To explore the cardiac allograft tolerance induction mediated by galectin-9 and Rapamycin.[Methods] Infiltrating cells in the allografts were islated to analyze the Tim-3 expression level on monocyte/DC subgroups. The level of Tim-3 ligand galectin-9 in the allografts was detected by Western Blot. Costimulatory molecules CD80/CD86 on galectin-9/ Rapamycin-treated BMDCs were evaluated by flow cytometry. BALB/c→C56BL/6 vascularized cardiac allograft transplant model was built. Five groups were included in this study:1) syngeneic transplant control (C56BL/6→C56BL/6); 2) galectin-9-treated allogeneic transplantation; 3) Rapamycin-treated allogeneic transplantation; 4) combined treatment of galectin-9 and Rapamycin in allogeneic transplantation; 5) PBS-treated allogeneic transplantation. The complete cessation of palpation of the grafts was considered as observation endpoint. The recipients which long-term survived grafts were subjected to donor-specific assays on IFN-y and IL-4 secretion detection using ELISPOT.[Results] The percentage of Tim-3 positive cells in monocyte/DC of allograft infiltrating cells was around 47.3%±5.6%. Immature BMDCs constitutively express Tim-3 (around 6.7%). Galectin-9 could promote the upregulation of CD80/CD86 on BMDC, while the addition of Rapamycin reversed this process. Combined treatment of galectin-9 with Rapamycin promotes the permernant acceptance of fully allogeneic grafts (>180 days, n= 6). However, the protocol that single administration of Rapamycin is not potent enough to induce tolerance to all of the allografts. Galectin-9 treatment only prolonged the survival of allografs (MST= 23.5±2.2 days). The tolerant state induced by galectin-9 and Rapamycin was related with the donor-specific low secretion of IL-4 and IFN-γ.[Conclusion] High proportion of Tim-3 positive innate immune cells in the allografts may be involved in the initiation of transplant rejection. Rapamycin could be used to inhibit the proinflammatory effect of galectin-9 on DCs. Combined treatment of galectin-9 with Rapamycin promote the transplant tolerance induction which was associated with the reduced Th1 and Th2 responses. [Aim] To explore the immunosuppressive effect of MyD88 inhibitor which is the essential TLR signaling adaptor on cardiac allograft rejection.[Methods]①BMDC from BALB/c mice were cultured in the medium containing GM-CSF and IL-4. Various TLR agonists and cardiac homogenate were used to stimulated the maturation of BMDC in the presence or absence of ST2825. CD80/CD86 expression level on BMDC was evaluated.②Lymphonode lymphocytes from C57BL/6 were isolated to stimulate by BMDCs from BALB/c mice. CpG were added to the mixed culture system with or without the addition of ST2825. T cell proliferation was detected by flow cytometry.③BALB/c→C57BL/6 cardiac transplantation model was built. The recipient mice were administered with ST2825 at the dose of 250mg/kg/day two hours prior to transplantation. The treatment started from day 0 (the day of transplantation) to day 6. The complete cessation of the grafts was considered as rejection. On day 7, cardiac grafts were harvested for qRT-PCR analysis for transcription level of IL-1β,IL-6 and TNF-αand histological analysis. The proportion of CD4+IFN-y+, CD4+IL-17+and Treg in the spleen lymphocytes were detected by flow cytometry.[Results] Except for TLR3 ligands, ST2825 can inhibit the upregulation of CD80/CD86 on the BMDCs stimulated by varous TLR ligands and cardiac homogenate. ST2825 dose-dependently inhibit the proliferation of T lymphocytes in the one-way mixed culture system. A short term treatment with ST2825 significantly prolonged the survival of fully allogeneic cardiac grafts (MST= 19.4±0.98 days vs control 7.2±0.4 days). ST2825 mediated protection on allografts was related with the reduction of inflammatory cytokine IL-1βand IL-6. Moreover, ST2825 inhibits the upregulation of CD4+IL-17+and CD4+ IFN-γ+lymphocytes while promotes the propagation of Tregs in the spleens.[Conclusion] ST2825 can significantly prolong the survival of fully allogeneic cardiac grafts in mice. This effect was related with the immunosppressive effect of ST2825 on DC activation. Thus, ST2825 can inhibit the activation of T lymphocytes while promote the survival of Tregs. [Aim] To explore the possibility that robust skin transplant tolerance could be achieved by combined TLR signaling inhibition using MyD88 inhibitor with anti-CD 154 blockade.[Methods] BALB/c to C57BL/6 skin transplant model was established. The recipients were treated with anti-CD 154+ST2825 for a short term. Graft survival was monitored daily. Splenic lymphocytes from the recipients which habor the long-survived skin grafts were stimulated with splenic cells from the donor (BALB/c) or the third-party (C3H). ELISPOT technique was employed to evaluate the cytokine secretion (IL-4 and IFN-y) responses of the tolerant state.[Results]Single administration with anti-CD 154, ST2825 or CMC control cannot promote the prolongation of skin allografts. However, combined therapy using anti-CD 154 and ST2825 promote the permanent acceptance of fully allogeneic skin grafts (>150 days). The tolerant state was associated with reduced donor-specific IFN-y secretion while intact IL-4 secretion.[Conclusion] Tolerance induction protocol based on anti-CD154 combined with ST2825 may provide new manipulation to induce robust skin transplant tolerance. This tolerant state may be related to the reduced Th1 immune responses.