The Mechanisms Underlying Transplant Immune Tolerance Induced By Donor-derived Apoptotic Thymocytes

It has been shown that apoptotic cells possess the immune regulatory capacity, and play a role in the induction of the alloimmune tolerance. However, the mechanisms by which apoptotic cells downregulate the alloimmune response remains unclear. It is known that the apoptotic cells was first phagocyted by phagocytes (mainly by macrophages and dendritic cell), and then mediated the suppression of alloreactive T cells response either through the release of some immunosuppressive cytokines (such as TGF-β, IL-10, MIF, etc) or directly inactivated the alloreactive T cells by cell -cell contact mechanism, as a result, to prolong allograft survival. In this study, we are planning to compare the differential roles played by macrophages and DCs in terms of the capacity of phygocytosis of apoptotic cells and the immunomodulatory potency in the context of skin allograft transplantation.Objective: To investigate the mechanisms by which donor-derived apoptotic thymocytes infusion induced the transplant immune tolerance, and provide a foundation for clinical prevention and control allograft rejection and autoimmune diseases.Methods: The apoptosis of thymocytes originated from BALB/c mice was induced with 20μM dexamethasone, while necrosis of thymocytes was induced by incubation at 560C water bath for 1 hr. The living, necrotic or apoptotic thymocytes from BALB / c mice were injected to C57BL/6 mice via the tail vein at 2×10~7 cells per mouse. 7 days after infusion of the different thymocytes, the skin allograft transplantation was performed from BALB / c donor mice to the C57BL/6 recipient mice, and the mean survival time (MST) of skin allograft was monitored. The live, necrotic or apoptotic thymocytes from BALB/c mice were subjected to pre-staining with PKH67 or CFSE and injected into C57BL/6 mice via the tail vein. Thereafter the recipients were sacrificed at various time points, the phagotrophic efficiency of the infused donor thymocytes by different types of splenic macrophages or DCs was evaluated by flow cytometry using the following antibodies against cell surface markers: PE-F4/80+ for macrophages, APC-CD11c+ and PE-B220+ for pDC, CD11c low and B220 + for imDC, CD11c high and B220 + for mDC or CD11c + for DC. An MLR was established to determine the regulatory effects of macrophages or DCs which engualfed the allogeneic or syngeneic apoptotic thymocytes to alloreactive T cells proliferation (labelled with CFSE and analysis by flow cytometry ).Results:Ⅰ. The early apoptosis proportation of thymocytes induced by dexamethasone treatment were up to 61.9%. while the rate of thymocytes necrosis by water bath heating were almost 100% PI-positive.II. The mean survival time (MST) of skin allografts in recipients treated with various donor live, necrotic, or apoptotic thymocytes was as follows: apoptotic cells treatment group (15.889±2.088 days), live cells group ( 9.000±1.323 days), necrotic cells group (7.333±1.323 days) and PBS control group (10.111±1.616 days). The MST of skin allografts in apoptotic cells group was longer than other groups (N = 9, p <0.01), while the MST of skin allografts in PBS control group was longer than necrotic group (N = 9, p <0.01).Ⅲ. We found that the phagotrophic efficiency of the splenic DCs and macrophages in C57BL/6 recipients was peaked at 60 min after apoptotic thymocytes infusion, and the apoptotic cells phagotrophic efficiency by CD11clow B220- DC (imDC) was significantly higher than that of CD11chigh B220-DC (mDC) or the CD11c + B220 + DC (pDC) at any time points. Meanwhile F4/80low M? displayed a higher phagotrophic efficiency of apoptotic thymocytes than F4/80high M?. The apoptotic thymocytes in the spleens of C57BL/6 recipients mice were not detectabe 24 hours after infusion. An analysis of the phagocytosis of live, necrotic or apoptotic thymocytes originated from BALB/ c mice thymocytes in C57BL/6 recipient mice revealed that the phagotrophic efficiency of live was lower that that of apoptotic cells, it could be due to living cells in the body will not be swallowed, thus only a small number of natural apoptoic thymocytes in infused live thymocytes were engulfed by phagocytes. As compared with necrotic thymocytes, the apoptotic thymocytes was much faster to be engulfed by DCs. The phagotrophic rate of apoptotic thymocytes by F4/80low M? was 21.3% while the phagotrophic rate of necrotic thymocytes rate was 1.1%. The phagotrophic rate of apoptotic thymocytes by F4/80high M? was 9.5% while the phagotrophic rate of necrotic thymocytes was 12.1%. These two types of macrophages exhibited the opposite results in term of phagotrophic rate of apoptotic versus necrotic thymocytes .Ⅳ. It was showed that DCs or macrophages that engulfed apoptotic cells displayed a differences in immune regulatory effects on alloreative T cells proliferation in an MLR: Mixed lymphocyte culture show that phagocytosis of apoptosis by significantly inhibited T cells proliferation. The inhibitory efficiency of phagocytes that engulfed allogeneic apoptotic thymocytes on alloreactive T cells proliferation was stronger than the phagocytes that engulfed syngeneic apoptotic thymocytes. It indicated that the immune suppression induced by phagocytosis of apoptotic thymocytes is antigen specific.Conclusions:Ⅰ. Intravenous infusion of donor derived apoptotic thymocytes can significantly prolong the mean survival time of skin allograft. In contrast, live thymocytes or necrotic thymocytes infusion show no such effect.Ⅱ. Live cells in the body can not be swallowed, while necrotic cells can induce phagocytic inflammatory response and accelerate skin graft rejection. Analysis of phagocytes in the spleen with specific phagocytic capacity revealed that CD11clow B220 + (imDC) are the most efficient phagocyte in all of DC subsets. F4/80low macrophages were more efficient than F4/80high macrophages in phagocytosis of apoptotic thymocytes. Whereas these two types of macrophages exhibit opposite in the efficiency of phagocytosis of necrotic thymocytes.Ⅲ. Mixed lymphocyte culture show that phagocytosis of apoptosis by significantly inhibited T cells proliferation. The immune suppression induced by phagocytosis of apoptotic thymocytes has antigenic specificity.