Immune-Privileged Sertoli Cells Survive Allotransplantation by Altering the Cellular Immune Response.

Abstract

Sertoli cells (SC) are important for immune privilege in the testis and survive long-term when transplanted across immunological barriers, as allografts or xenografts. On the other hand, MSC-1 cells (a mouse Sertoli cell line) lack some of the immunoprotective abilities associated with primary SC (pSC), as they are unable to survive in naive immune competent animals. The objective of this study was to compare the cell survival rate and immune response to these cells, to further understand the mechanism for SC immune-privilege. Aggregated pSC or aggregated MSC-1 cells were transplanted as allografts into the renal subcapsular space of naive BALB/c mice and cell survival was analyzed by immunohistochemistry (IHC). pSC grafts survived throughout the study and were not rejected, whereas, very few MSC-1 cells were detected by day 11 and MSC-1 cells were completely rejected within 20 days. Next, we focused on the possible mechanisms behind allograft rejection, antibody-mediated (complement) and/or cell-mediated (apoptosis) death. For the antibody-mediated rejection of allografts, pSC or MSC-1 cell grafts and serum from the transplanted animals were collected at days 1-20, analyzed for antibody production and deposition by ELISA and IHC (IgG & IgM), respectively. Further, to look for complement deposition, grafts were immunostained for complement factor 4 (C4), complement factor 3 (C3) and membrane attack complex (MAC). No IgG production was detected in serum whereas there was an IgM response against the grafted cells. Antibody deposition was not detected until day 14 post-transplantation and no complement deposition was observed in pSC or MSC-1 cell grafts throughout the study. This led us to the conclusion that antibody mediated cell death is not playing a major role in this allograft rejection model. We thus, hypothesized that SC were surviving as allografts by inhibiting cell-mediated death. Grafts were analyzed for cell-mediated death (apoptosis) by TUNEL assay. Significant apoptosis was observed in MSC-1 cell grafts as compared to pSC grafts. We further analyzed the grafts for immune cell infiltration by IHC for CD8 (cytotoxic T cell), CD4 (T helper cell), CD11b (macrophage) and Foxp3 (T regulatory cell) markers. CD4 T cells were observed in both sets of grafts, while little to no CD8 T cells were detected in MSC-1 cell grafts as compared to pSC grafts. A huge infiltration of macrophages was seen in MSC-1 cell grafts at day 2, whereas very few macrophages were detected until day 11 in pSC grafts. This coincided with Foxp3+ cells, which were detected in pSC grafts at day 11 and increased by day 20. The Foxp3+ cells were either absent or very few were detected in MSC-1 cell grafts. Overall, this led to the conclusion that cell-mediated death plays an important role in allograft rejection of MSC-1 cells. SC delayed the migration of immune destructive macrophages and, possibly by secreting certain cytokines, led to an increase in immunoregulatory cells (Foxp3+) within the graft site. Thus, by altering the immune cell response from immune destructive to immunoprotective, pSC enjoy long-term survival. Studies are ongoing to analyze MSC-1 and pSC grafts for cytokines and immune regulating molecules, which may further increase our understanding of the mechanism by which SC establish an immune-privilege environment. (platform)