The Emerging Role Of Tim3 And Tim4 In Immunoregulation In An Experimental Mouse Heart Transplant Model

Objective To introduce a simple and efficient method for isolation of intragraft infiltrating lymphocytes and examine the subsets of lymphocytes by immunofluorescent staining and flow cytometer analysis.Methods The murine cardiac transplant model was built. Experimental group (Balb/c to C57BL/6) and control group (C57BL/6 to C57BL/6) were designed in this study. Cardiac grafts harvested on 3,5 and 7 days after operation respectively were cut into small pieces. These pieces were then digested with modified enzyme digestion singly using collagenaseⅡ(250 U/ml,30-40 min). Mononuclear cells were isolated by Ficoll density gradient centrifugation (800g×20 min). Ca2+fluorescence dye Indo-1 was used to analyze cell viability. Histological slices were made to observe the rejection. Subsets of lymphocytes were identified by flow cytometry after surface antigen and intracellular cytokine immunofluorescent staining.Results The harvested mononuclear cells were more than 1×106, which contained (31.9±2.3)%lymphocytes with viability of (95.1±2.1)%. The number of the mononuclear cells from the graft reached a peak on 7 days after transplantation and was closely correlated with the severity of rejection. Flow cytometry can analyze surface and intracellular antigens of the intragraft infiltrating lymphocytes at the same time. Most of the intragraft infiltrating lymphocytes were effector T cells. The ratio of CD4+/CD8+decreased gradually as the progression of rejection.Conclusion The modified method of isolating intragraft infiltrating lymphocytes with high viability is simple, reliable and efficient. It can be used widely in transplant immunology research.Objective To detect the changes of Tim3 expression in cardiac graft and T lymphocytes from different sites in mice heart-transplant recipients and explore the relationship of Tim3 expression level with acute rejection.Methods The murine cardiac transplant model was built. Experimental group (Balb/c to C57BL/6) and control group (C57BL/6 to C57BL/6) were designed in this study. Cardiac grafts were harvested on 3,5,7 and 9 days after operation respectively. Real-time quantitative RT-PCR was used to detect the local expression of Tim3 mRNA in graft. Lymphocytes were isolated from peripheral blood, spleen, draining lymph nodes and allograft on 3 days and 6 days after transplantation. The expression of Tim3 on T lymphocyte subsets CD4+and CD8+T cells was detected by flow cytometry.Results The expression of Tim3 was very low in isograft group. In allograft group, as the acute rejection occurred, the Tim3 expression increased significantly on 3 days after transplantation, which was related to the severity of rejection (P<0.01). It reached its peak on 7 days and then decreased gradually after the completely rejection. No significant increase of Tim3+cells was observed in peripheral blood and spleen after transplantation (P > 0.05). The expression of Tim3 on CD4+and CD8+of graft infiltrating T cells was obviously increased (P<0.01). While the ratio of Tim3+/CD4+had a slight elevation in draining lymph node (P<0.05). There was no obvious difference in Tim3+/CD4+ratio in draining lymph node between 3 days and 6 days (P> 0.05). But the ratios of Tim3+/CD4+ and Tim3+/CD8+in graft infiltrating T cells on 6 days were significantly (P<0.01) higher than that on 3 days.Conclusion The expression of Tim3 incresed in cardiac graft and T lymphocytes from draining lymph node and allograft after transplantation, which was correlated with the progression of acute rejection in mice.Objective To investigate the effect of Tim3 ligand (Galectin-9) on the survival of allograft and explore its immunomodulating role in an allogeneic heart transplant model.Methods Murine cardiac transplant model from BALB/c to C57BL/6 was built. The study was divided into experimental group and control group. Experimental group were administered with Galectin-9 for 7 days since day 1 posttransplant while control group with PBS. The complete cessation of heart beating was defined as the observation endpoint. The survival of allograft was observed in two groups. Histology and immunohistochemistry were performed to estimate the severity of rejection on 7 days posttransplant. Real-time quantitative RT-PCR was utilized to analyze the Tim3, IFN-γand IL-17 mRNA expression in allograft. The phenotype and cytokine profile of lymphocytes from peripheral blood, draining lymph nodes and allograft was analyzed by flow cytometry.Results The survival of allograft treated with Galectin-9 (mean survival time, MST= 22.7 days) was significantly prolonged compared with the control group (MST= 7.2 days), which was associated with reduced infiltration of CD4 and CD8 lymphocytes in allograft. Galectin-9 decreased the expression of Tim3, IFN-γand IL-17 mRNA in allograft notably. The proportion of Tim3 positive cells in draining lymph nodes and allograft and Thl and Th17 positive cell in peripheral blood was obviously lower than the control group.Conclusion A short-course administration of Galectin-9 significantly prolonged the survival of fully allogeneic cardiac allografts, which was associated with the suppression of Th1 and Th17 immune responses.Objective To study the effect of Tim4 blockade on the survival of cardiac allograft and explore the role of Tim4 in Tim4-Tim1 pathway in inducing immune tolerance.Methods Murine cardiac transplant model from BALB/c to C57BL/6 was built. The survival was compared between the recipients treated with anti-Tim4 Ab and PBS. MLC was used to assess the effect of anti-Tim4 Ab on cell proliferation. The survival of Tim4-/-and wild-type graft in recipients was observed. Histology was performed to estimate the severity of rejection. Immunofluorescent staining was carried out for detecting lymphocytes in allograft. The expression of immune-related genes was determined by Real-time quantitative RT-PCR. The subsets of intragraft infiltrating lymphocytes were analyzed by flow cytometry. Effector T cells and Tregs from the recipients were sorted by FACS. These cells were then transfused into Balb/c SCID mice followed by implantation of skin allograft. The rejection and survival of the allograft were observed. The effect of low dose rapamycin on the survival of Tim4-/- allograft was studied. The role of Tregs in the model was evaluated by administering anti-CD25 mAb before transplantation.Results Recipients conditioned with anti-Tim4 mAb exhibited prolonged cardiac allograft survival with suppressing the T cell proliferation in MLC. The survival of Tim4-/- allograft was significantly prolonged compared with wild-type group. The prolonged survival time was associated with reduced lymphocytes and increased Treg in allografts. Strikingly, when combined with a low dose of rapamycin, Tim4-/- heart allografts survived indefinitely. The absence of Tim4 led to the decreased recruitment, but not decreased effect, in the effector T cells compartment. Similarly, it increased recruitment, but not increased function, in the Treg compartment. Moreover, depletion of Treg with anti-CD25 Ab completely abrogated the tolerogenic effects of Tim4 deficiency alone or when combined with rapamycin.Conclusion A blockade of the Tim4-Tim1 axis improves allograft survival as a monotherapy and synergizes with low-dose rapamycin to induce complete transplantation tolerance by enhancing immunoregulation.