| Schistosomiasis, a parasitic disease, is one of the most important challenges to population living in the epidemic areas. At present, the mechanisms of schistosome antigen-induced immune response and immune protection have been well studied. Animal experiments have indicated that antigen-induced immunity was related to the antigen properties and immunization approach. In recent years, the inhibitory immune response of CD4~+CD25~+ regulatory T cells was recongnized. CD4~+CD25~+ regulatory T cells can not only be derived from the thymus, but also be transformed from CD4~+CD25~- precursor cells when stimulated by antigen and induced by IL-10 and TGF-β. Our previous experiments have established the relationship between the immunosuppression of Schistosoma japonicum chronic infection and the CD4~+CD25~+ T cell subsets, which indicated that CD4~+CD25~+ T cells may play an important role in regulation of natural infection of S. japonicum. But so far, little is known about the effects of worm or egg itself on immune responses in schistosoma infection. In this study, we prepared Sepharose4B coupling rSj22.6/26GST antigen just to mimic the form of early schistosomula and evaluated the effects of this antigen form on DCs and CD4~+CD25~+ T cells.We established BALB/c mice model immunized with Sepharose4B coupling rSj22.6/26GST antigen. Control groups were set up at the same time. At first, we measured the percentage of splenic CD4~+CD25~+ regulatory T cells by flow cytometry. The inhibitory function of CD4~+CD25~+ regulatory T cells was assessed by the [~3H] thymidine incorporation method on CD4~+CD25~- T cell proliferation and the ELISA on cytokine production with the stimulation of anti-CD3. To further understand the effect of Sepharose4B coupling antigen on CD4~+CD25~+ regulatory T cells, we measured the effect of Sepharose4B coupling antigen on phenotype of dendritic cells (DCs) by flow cytometry and function of DCs by ~3H-TdR. Finally, the effect of DCs pulsed with Sepharose 4B coupling antigen on the induction of CD4~+CD25~+ regulatory T cells was analyzed by flow cytometry.The main results we got are as follows:1. The effect of Sepharose 4B coupling rSj22.6/26GST antigen on CD4~+CD25~+ regulatory T cells. The results show that the proportion of CD4~+CD25~+Foxp3~+ T cells in spleen cells from group immunized with Sepharose 4B coupling rSj22.6/26GST antigen [(5.0±0.8)%] was significantly higher compared with that from group immunized with Freund's adjuvant emulsified rSj22.6/26GST antigen [(3.9±1.4)%]. The results suggest that Sepharose4B coupling antigen is easier to induce the increase of CD4~+CD25~+ regulatory T cells.2. Suppression of CD4~+CD25~+ regulatory T cells isolated from Sepharose 4B coupling rSj22.6/26GST antigen immunied mice on CD4~+CD25~- T cell proliferation. ~3H-TdR incorporation assay showed that CD4~+CD25~+ regulatory T cells isolated from the experimental groups could inhibit the proliferation of CD4~+CD25~- T cells isolated from normal mice when stimulated by anti-CD3. The suppression rate of CD4~+CD25~+ T cells separated from group immunized with Sepharose4B coupling antigen was higher than that of other groups. The results suggest that, compared to Freund's adjuvant emulsified antigen group, CD4~+CD25~+ T cells separated from Sepharose4B coupling antigen have stronger inhibition potential on CD4~+CD25~- T cell proliferation. 3. Suppression of CD4~+CD25~+ regulatory T cells isolated from Sepharose 4B coupling rSj22.6/26GST antigen immunied mice on cytokine production of CD4~+CD25~- T cells. ELISA detection of cytokines in culture supernatants showed that CD4~+CD25~+ T cells isolated from all experimental groups could inhibit the production of IFN-γof CD4~+CD25~- T cells when stimulated by anti-CD3. But this inhibition was less on the production of IL-4. Moreover, the inhibition of CD4~+CD25~+ T cells isolated from mice immunized with Sepharose 4B coupled antigen on IFN-γproduction was higher than that from mice immunized with Freund's adjuvant emulsified antigen (p<0.05).4. The effect of Sepharose 4B coupling rSj22.6/26GST antigen on DCs maturation. In in vitro experiments, the phenotypes of DCs detected by flow cytometry showed that, the expression of costimulatory molecules of CD40, CD80 and CD86 were lower in group when DCs pulsed with Sepharose4B coupling antigen, compared with that in other group (p<0.05). But MHCⅡexpression had no significant difference in each group (p> 0.05). Also, the number of DCs in inguinal lymph node from group immunized with Sepharose 4B coupled antigen was lower than that from group immunized with Freund's adjuvant emulsified antigen (p<0.05). The results suggest that Sepharose 4B coupling rSj22.6/26GST antigen can not effectively stimulate the maturation of DCs.5. The effect of Sepharose 4B coupling rSj22.6/26GST antigen on DCs function. Mixed lymphocyte reaction showed that DCs can stimulate proliferation of allogeneic CD4~+ T cells. But this effect was less on DCs pulsed with Sepharose4B coupling antigen, compared with that pulsed with soluble antigen (p<0.05). 6. The effect of DCs pulsed with Sepharose 4B coupling antigen on induction of CD4~+CD25~+ regulatory T cells. DCs were pulsed with Sepharose4B coupling rSj22.6/26GST antigen and Sepharose4B coupling OVA antigen, and co-culture with CD4~+ T cells, respectively. Flow cytometry analysis showed that the percentage of CD4~+CD25~+Foxp3~+ T cells in CD4~+ T cells was higher in group when DCs were pulsed with Sepharose4B coupling antigen, compared with that when DCs were pulsed with soluble antigen(p<0.05). The results suggest that DCs pulsed with Sepharose4B coupling antigen may induce CD4~+CD25~+Foxp3~+ T cells.
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