| Antigen-specific lymphocytes can be stimulated by antigens presentated by dendritic cells. The T cells proliferate and then undergo activation-induced cell death (AICD). AICD is an important phenomenon in immune response and related with the downregulation of immune system. But the mechanisms involved in AICD induced downregulation are still in dispute. Some researches showed that macrophages and dendritic cells (DC) would phagocytose immediately apoptotic na?ve self-cells and then induced immune tolerance. The defects of this process were associated with etiopathogenisis of many diseases. But how the AICD cells impact dendritic cells or macrophages is not very clear.In recent years, researchers reported that mature DC can be transformed functionally by microenvironment, for example, many kinds of stromal cells and some cytokines could induce DC redifferentiate into regulatory dendritic cells (DCreg), which are immunesuppressive. All these results hinted that DC engulfing AICD cells (DCapo) may be have some changes in function. There were many reports that had proved regulatory DC (DCreg) could inhibit immune responses. And the mechanisms involved in the inhibitory function of DCreg are described below. First, secreting some suppressive cytokines and chemicals, such as IL-10, TGF-β, IDO, NO, etc. Second, inducing generation of regulatory T cells (Treg) and downregulating immune responses through Treg. The function changes of DC after phagocyting apoptotic cell is still confused for the diversity of apoptotic cells. AICD of T cells can induce immune tolerance. Some researchers reported that DC engulfing self na?ve cells can induce Treg to inhibit immune response. Ren et al. have recently demonstrated that DC could secret NO after phagocytosing apoptotic na?ve cells, which indicated that NO might also the main molecule of DCapo.In our study, we use antigen-loaded-DC to activate antigen specific CD4+ T cell and induce apoptotic T cells. Bone marrow derived DC will be used to coculture with the activation induced apoptotic T cells. The function of DCapo will be evaluated using an antigen presenting system in vitro. AICD on T cells was induced after antigen-specific CD4+ T cells (DO11.10×C57BL/6 F1) were stimulated for about 4 days by OVA peptide (323-339) loaded mature DC in vitro. The apoptotic T cells were valuated by FCM and transmission electron microscope. We cultured the bone marrow-derived DC with GM-CSF and IL-4 for 7-9 days. Then the mature DC were cocultured with apoptotic T cells for 48-72h, the cocultured DC were harvested as DCapo. The DCapo were identified by confocal fluorescent microscope. Apoptotic bodies of T cell can be observed in the cytoplasm of DC. The phenotype and phagocytotic function of DCapo were also evaluated by FCM and showed that DCapo express upregulated CD11b, downregulated CD11c, Ia, CD80, CD86, CD40 and have enhanced ability of phagocytosis as compared to mDC. DC could secret high level of NO after phagocytosis to apoptotic T cells. These data demonstrated that apoptotic T cells or apoptotic bodies can be phagocyte by DC, some characteristics of DC are changed.The antigen presenting ability of DCapo were evaluated using antigen presenting system in vitro. In the system, specific CD4+ T or CD8+ T cells were stimulated by mDC with or without DCapo in vitro, the quantities and CFSE dilution of T cells were assayed by FCM. The results showed that DCapo could not stimulate the proliferation and division of T cells compared with mDC. Nevertheless, DCapo could inhibit the proliferation and division of T cells simulated by mDC. To further confirm the inhibitory function of DCapo, specific CD4+ T or CD8+ T cells, peptide-loaded mDC and/or DCapo together were transferred into the recipient mice, after 3-4 days, the quantities and CFSE dilution of specific T cells in spleen were assayed by FCM. The similar results with in vitro were observerd in vivo. DCapo significantly inhibit the proliferation of antigen-specific CD4+ T cells stimulated by mDC, which leaded to a decrease in the number of specific CD4+ T cells in the blood. The data suggested that DCapo belonged to the immunosuppression DCreg.Then, the immunosuppresion mechanism of DCapo was investigated. The NO donor (NOC-18) and selective NO synthase inhibitor (PBIT) were added respectively into the experimental system. The results showed that the selective NO synthase inhibitor effectively reverse the DCapo inhibition to CD4+ T cell proliferation stimulated by mDC. On the other hand, the addition of NO donor NOC-18 to the antigen-specific CD4+ T cell-mDC coculture system significantly suppressed the proliferation of T cells. Thus, NO play an important role in the immunosuppression of DCapo. Then generation of Treg were tested using the transgenic mice with Foxp3EGFP×Do11.10 F1 mice CD4 T cells in the experiment in vitro. The proportions of CD4+CD25+Foxp3+ cells were not significantly increased in the coculture system with DCapo. In further test in vivo, statistical result among AICD, DCapo and PBS groups was not achieved when the proportions of CD4+CD25+Foxp3+ cells in the peripheral blood of mice were tested.At last, the effect of AICD cells on intracellular signal pathway in DC and relationship between the signal pathway and DCapo inhibition to immuno responses were investigated. In recent years many studies on basic immunology have focus on immunosuppressive DC, but the mechanism of differentiation of these DC remains to be elucidated. During the early stages of apoptosis, phosphatidylserine (PS) becomes exposed on the outside of the cell membrane and is an important signal for DC to identify. And the PS on the cell membrane of apoptotic cell binding the receptors on DC might activate the STAT3 signal pathway. On the other hand the STAT3 signal pathway were associated with expression of many cytokines, such as IL-6, IL-10, TGF-β. These cytokines were always involved in the immunoregulation of DC. Thus, we further analyze whether activation of the STAT3 signal pathway in DC will be induced by AICD cells and then mediate the immunosuppression of the DCapo. For answering these questions, we not only define the phenotype and function of DC phagocytosing AICD cells, but also investigate their immunosuppression mechanism and related intracellular signal transduction.We tested the p-STAT3 level in DC cocultured with AICD cells with western blot. The result suggested that the level of p-STAT3 protein in DC increased gradually along the coculture time. At the same time, the expression of iNOS protein was also enhanced and coincidence with p-STAT3. The protein expression and mRNA transcription of iNOS could be inhibited by the selective p-STAT3 inhibitor JSI-124. The NO secretion of DCapo could also be inhibited by JSI-124. These data showed that the generation of DCapo induced by AICD cells was associated with the STAT3 signal pathway in DC. The data demonstrated that the STAT3 signal pathway was associated with the expression of iNOS and the inhibitory function of DCapo.We demonstrated that DC could be induced to DCreg by coculturing with AICD cells, and have a phenotype with downregulated CD11c, Ia, co-stimulated molecules, upregulated CD11b and enhanced phagocytotic ability. They could significantly inhibit the proliferation and division of antigen specific T cells stimulated by mDC mainly through NO. We didn't found the generation of Treg involved in the immunosuppression of DCapo. The molecular mechanisms involved in DCapo inhibitory function induced by AICD cells was associated with activation of STAT3 signal pathway which enhanced the transcription of iNOS and secretion of NO. In conclusion, DC could be changed to a kind of inhibitory cells with immunosuppressive phenotype and inhibit immune responses mainly through NO after phagocytosing AICD cells. The STAT3 activation in DCapo induced by AICD cells was associated with iNOS expression and NO secreation. Our results elucidated how the AICD cells affected DC and the mechanisms under the immunosuppression induced by AICD.
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