The Therapeutic Effect On Allograft Rejection Of Suppressor Of Cytokine Signaling 1 Gene-modified Bone Marrow Dendritic Cells And Its Related Mechanisms

Organ transplantation has become the only effective treatment for end-stage organ disease at present and many patients have gained new life through the transplantation of allogeneic organs. However, the immune system will be inevitably stimulated by the allograft and a series of immune response and rejection will be generated against the transplanted organ. The incidence of organ transplant rejection is lowered after the application of a variety of new clinical immunosuppressants, but two major problems are still remained:first, new immunosuppressants can not completely suppress the occurrence of rejection; second, many adverse reactions will occur after long-term application of immunosuppressive agents, such as drug toxicity, infection and neonatal immunity induced tumors. Therefore, inducing specific immune tolerance for allo-antigen is the best way to solve the transplant rejection, with witch the recipients maintain normal immunity to resist pathogens and other microbial induced infections and tumors, and has become the hot spots of transplantation research.Dendritic cells (DC), which are potent professional antigen presenting cells (APC), have appeared to be central for immune systems because of their abilities to prime naive T cells and initiate a primary immune response. Recent studies have outlined that DC may function as immune-stimulating cells as well as tolerance-inducing cells. The immunophenotype and function of DC will change during maturation. Immature DC (imDC) display tolerance-inducing activities, though they turn to exhibit their immunogenicity after maturation. imDC lacking CD80 (B7-1) and CD86 (B7-2) could induce antigen-specific anergy in vitro. However, there is a natural mechanism of immature for DC in vivo, and at the same time, a variety of inflammatory mediators produced by recipients after organ transplant including inflammatory cytokines, LPS, etc promote DC differentiation and maturation, which make DC possessing strong immunogenicity. Gene modifications using immunosuppressive molecules, such as 1,25-dihydroxyvitamin D, IL-10, TGF-β, will confer DCs with greater tolerance-inducing effect, making DCs an attractive candidate for tolerance induction and rejection prevention.Suppressor of cytokine signaling 1 (SOCS1), which was discovered as a negative regulator of cytokine signaling proteins, plays an important role on the maturation of DC in resent research. By highly expressing costimulatory molecules, splenic DC of SOCS1-/- mice responded significantly higher on IL-4, IFN-γand intensely stimulated the proliferation of B cell. Antigen loaded SOCS1-/-DC and normal DC were injected into the different foot pad of one mice, the former could cause stronger Thl-type response in foot pad draining lymph nodes, proved by that the isolated lymph node T cells could produce higher levels of IFN-y. SOCS gene silenced DC were also more effective in triggering the proliferation and cytotoxic function of OVA-specific cytotoxic T cells than normal DC, both in vitro and in vivo. SOCS1 gene silencing DC, as well as SOCS1-/- DC have a stronger immune-stimulating functions to break self-tolerance.Therefore, that using genetic engineering techniques exogenously increase the expression of SOCS1 within DC to inhibit DC maturation and immunostimulatory function is likely to be an effective approach to prevent rejection and induce immune tolerance.In the present study, bone marrow-derived DC was cultivated, expanded and enriched in vitro. After SOCS1 gene transfer by adenovirus vector, the phenotype and function of SOCS1 gene modificated DC was investigated and the effect on inducing allogeneic T cells hyporesponsiveness in vitro was studied. By means of cervical heterotopic heart transaplantation model in mice, we also analyzed effects of SOCS 1 gene modificated immature DC in inducting alloantigen-specific immune tolerance in vivo and the related mechanism.PARTⅠCulturing mouse bone marrow dendritic cells in vitro and SOCS1 gene transferDC derived from bone marrow, are widely distributed in the body with the extremely low percentage less than 1% in peripheral blood mononuclear cells. In vitro, DC culture technology can provide a rich DC source for basic research and clinical treatment. We cultured freshly isolated mouse bone marrow-derived DC with rmGM-CSF and rmIL-4. Given that mouse bone marrow-derived DC relatively highly expressed specific surface molecules CD11c, we used immunomagnetic bead separating kits to enrich CD11+ DC. The results showed that newly prepared bone marrow cells were very rare in clusters or dendritic processes of cells, cultured for 3 days scattered colony formation can be clearly seen, and dendritic processes gradually increased, for 5 days the a larger cloning and partially connected pieces, cell processes became clear, performance of a typical immature DC. After immunomagnetic bead separation and enrichment, a sufficient number of DC with more than 93% purity could be obtained.Adenovirus vector possessing simple structure of the genome, high viral titers, high infection efficiency, wide range of host cells, non-insertion mutation and other advantages, so AdEasyTM Adenoviral vector system was selected to constructure of adenovirus vector for encoding SOCS1 genes (Ad-SOCSl).The resuts from PCR, specific enzyme digestion, sequencing, and Western Blotting have confirmed that Ad-SOCS1 vector was successfully constructed and could express SOCS1 protein. After amplification, a higher titer of Ad-SOCS1 vector was obtained. The enriched imDC was transfected with Ad-SOCS1 or Ad-GFP at 1:100 MOI, and imDC without virus transfection served as control.Accordingly, the resulting three groups of DC were known as DC-SOCS1, DC-GFP and imDC. Then, we detected the transfection efficiency of control viral vector in DC-GFP group by Flow cytometry, and examined the mRNA and protein expression of SOCS1 with real-time quantitative PCR and Western Blotting. The results showed the transfection efficiency of control Ad-GFP vector could reach more than 70%. The mRNA and protein expression of SOCS1 in DC-SOCS1 group was significantly increased as compared with that in imDC group and DC-GFP group (P<0.01). The above results demonstrated that our constructed Ad-SOCS1 vector could effectively transfect DC, and significantly up-regulated SOCS1 gene expression in DC.PART II The phenotype and immunologic function of SOCS1 gene modified DC in vitroStudies have shown that SOCS1 knock-out mice (SOCS1-/-) DC, expressing high levels of MHC molecules and costimulatory molecules, possessed significantly enhanced immunostimulation capability than normal DC. SOCS1-/- DC could stimulate immune response against tumors after loading tumor antigens, and was assumed as an effective adjuvant treatment of cancer. In view of the important role of SOCS1 gene in immune function, we assumed that increasing the expression of SOCS1 within DC in vitro by biotechnology could prevent the DC maturation process and contribute to induce an efficient and long-time donor-specific immunologic tolerance. In this section, we used Ad-SOCS1 to transfect the enriched imDC, observed the phenotypic features and functional characteristics of SOCS1 gene modified DC, and further investigated the effect of inducing immune tolerance and its mechanism.RmGM-CSF and rmIL-4 were applied to culture bone marrow-derived DC (C57BL/6) in vitro, and imDC were enriched with immunomagnetic beads separating kits.Then, SOCS1 gene was transferred into the imDC by Ad-SOCS1, and at the same time, Ad-GFP modified imDC and same period cultured imDC were set up as the control group. We analyzed the phenotype, cytokine secretion, antigen phagocytic capacity and antigen-presenting ability of DC-SOCS1, DC-GFP and imDC in the absence or the presence of LPS stimulation.The allostimulation of DC-SOCS1, DC-GFP and imDC was examined with MLR, and the cytokines in supernatant of MLR was detected with ELISA.The generation of IL-10 producing T cells and antigen-specific hyporesponseness was also studied.The results from analyzing DC phenotype showed that before LPS stimulation, DC-SOCS1 and imDC expressed low levels of CD40, CD80 and MHC molecules with no significant difference. Hower, SOCS1 gene modification could significantly inhibit the up-regulating effect of these molecules by LPS stimulation. For cytokines secretion, there had no significant difference of IL-12 before LPS stimulation in the groups (p>0.05), but DC-SOCS1 secreted lower levels of IL-12 in comparison with imDC and DC-GFP after LPS stimulation (p<0.01). High levels of IL-10 were found in the supernatant of DC-SOCSl group as compared with DC-GFP and imDC group in spite of LPS stimulation (p<0.01). These results suggested that increasing SOCS1 expression could block DC from maturation to exogenous LPS stimulation and maintained a stable immature condition.FITC-Dextran endocytosis examination showed that DC in each group all had strong phagocytic capacity before LPS stimulation. After that, imDC and DC-GFP significantly reduced their phagocytic capacity, hower, there was a slight decline in phagocytic capacity of DC-SOCS1. Testing antigen-presenting function of DC in each group, we found that antigen presenting ability of DC-SOCS1 was significantly lower than imDC and DC-GFP group (p<0.01). The results showed that SOCS1 gene modification could maintain the DC of a strong antigen phagocytosis and inhibit DC antigen-presenting function.In MLR, we found that allogeneic CD4+ T cell proliferation stimulated by SOCS1 modified DC were markedly reduced (p<0.01). After detecting cytokine levels in supernatants, we found that Thl subsets cytokines IFN-γlevels were significantly lower in DC-SOCS1 group (P＜0.01), while Th2 subsets cytokines IL-10 levels were significantly higher (P＜0.01). Results from intracellular cytokine staining showed that IL-10 producing CD4+ T in DC-SOCS1 group was significantly increased as compared with that in imDC and DC-GFP group. These results indicated that SOCS1 gene modified DC had lower levels of immune stimulating function, and could inhibit Thl differentiation, divert thl to Th2 bias, and promote IL-10 producing T cells generation in MLR.Further studied the alloantigenic T cells hyporesponsiveness, we found that CD4+ T stimulated with DC-SOCS1 in primary MLR showed low reactivity when re-stimulation with allogeneic antigen in secondary MLR, which was in contrast to CD4+ T stimulated with imDC and DC-GFP (P＜0.01). Hower, CD4+ T stimulated with DC-SOCS1, imDC or DC-GFP in primary MLR showed similar responsiveness to thirt part antigen (p>0.05). The results suggested that SOCS1 modified DC could induce alloantigenic specific hyporesponsiveness, and possessed the potential to induce allograft immune tolerance.PARTⅢThe therapeutic effect on allograft rejection of DC-SOCS1 and its related immunological mechanisms in vivoTo further study whether SOCS1 gene modified DC could induce immune tolerance in vivo, we immunized BALB/c mice with imDC, DC-GFP or DC-SOCS1 dirived from C57BL/6 mice, observed the allograft survival time utilizing cervical heterotopic heart transplantation mode and investigated the related mechanism.Same week-old BALB/c mice (20-22g) were divided into four groups randomly, and three groups were intravenously injected 2×106 imDC, DC-GFP or DC-SOCS1 seven days before heart transplant, respectively. Recipient received PBS injection served as blank group. Four groups recipient received the cervical heterotopic heart transplantation under same conditions, and were palpated everyday to record allograft survival. A sister group recipient were sacrificed at seven days and fifteen days to analyze pathology and immune parameters. The results showed that allograft survival in imDC and DC-GFP group was 10.9±1.4 days and 9.5±1.1 days, respectively, which was moderately extended as compared with PBS group with 7.1±0.9 days survival (P<0.01). However, allograft survival in DC-SOCS1 group with 25.5±6.9 days was significantly longer as compared with imDC and DC-GFP group (P <0.01). The results demonstrated that imDC and DC-GFP immunization could prolong allograft survival with limited effect. However, DC-SOCS1 immunization could induce allograft immune tolerance and significantly prolong allograft survival.Histological examination revealed heart graft of PBS control group were significantly congestive and swelling, and myocardial cells degeneration, necrosis, fiber fracture, interstitial edema and inflammatory cell infiltration in the muscle bundle and perivascular in light microscope. imDC and DC-GFP group showed the lesion was slightly reduced compared with PBS control group. In contrast, Allograft in DC-SOCS1 group showed no significant degeneration of myocardial cells, edema, and slight perivascular lymphocytic infiltration.Analysis of immunological mechanisms revealed that there was no difference in antigenic stimulus response of spleen lymphocytes in imDC and DC-GFP recipient (P>0.05), which was lower that in in PBS recipient (P＜0.01). However, spleen lymphocytes in DC-SOCS1 group showed significantly reduced alloresponsiveness as compared with that in in imDC and DC-GFP recipient (P＜0.01). At the same time, spleen lymphocytes in DC-SOCS1 group exhibited low cytotoxicity in CTL response (P＜0.01). The results showed that immunization with SOCS1 gene modified DC could reduce alloantigenic stimulation and alloantigenic killing activity of recipient lymphocytes.Detecting the generation of CD4+CD25+Treg in the spleen and cervical lymph node in each group, we found that the proportion of CD4+CD25+Treg in imDC and DC-GFP was moderately increased than that in PBS group (P＜0.05), however, the proportion of CD4+CD25+Treg in DC-SOCS1 group was significantly increased than that in imDC and DC-GFP group (P<0.01). Real-time quantitative PCR was used to detect the Foxp3 mRNA in CD4+ T in each group, and the results showed that Foxp3 mRNA levels in spleen and cervical lymph node CD4+ T in DC-SOCS1 group was significantly higher than that in other groups (P<0.01). The results demonstrated that immunization with SOCS1 gene modified DC could induce the generation of CD4+CD25+ Treg in vivo and promote the expression of Foxp3 within CD4+ T cells.ConclusionReplication defective adenovirus vector Ad-SOCS1 can effectively transfect DC, and increase the expression of SOCS1 mRNA and protein with harmlessness to DC. In spite of exogenous stimulus, SOCS1 gene modified immature DC can still stabilize in the immature state, expressing low levels of MHC molecules and costimulatory molecules, secreting high levels of immune suppressive cytokines IL-10, and has a stronger antigen phagocytosis, a weaker antigen stimulation activity, which can induce allogeneic T-cell antigenic specific hyporesponsiveness in vitro. Immunization with SOCS1 gene modified immature DC in vivo could prolong the allograft survival time, inhibit the allostimulatory and cytotoxicity activity of recipient lymphocyte, and induce CD4+CD25+Treg generation and promote the expression of Foxp3, exhibiting potential to induce antigen-specific immune tolerance in organ transplantation.