Objective:To examine the effect of cyclosporine A(CsA) on the differentiation of Th17 cells from splenocytes after stimulation with anti-CD3 mAb+IL-23.Methods:Total splenocytes were cultured with anti-CD3 mAb alone or anti-CD3 mAb plus IL-23 in the presence or absence of different concentrations of CsA.After stimulation,production of IL-17 and IFN-? was assayed by ELISA.The subsets of IL-17-producting T cells were examined at single-cell level by flow cytometry.Results:The splenocytes stimulated with anti-CD3 mAb alone could induce low level of IL-17 production and addition of IL-23 could promote high level of IL-17 in a dose-dependent manner.CsA inhibited the differentiantion of Th17 cells from splenocytes stimulated with anti-CD3 mAb+IL-23 in a dose-dependent manner.Kinetic studies indicated that CsA also inhibited the differentiantion of Th17 cells from activated T cells stimulated by anti-CD3 mAb+IL-23 for 48 hours.In addition,CsA inhibited production of IFN-? and IL-2.The analysis of subsets of T cells demonstrated that CD4+T cells but not CD8+T cells produced IL-17.Conclusion:CsA can inhibit Th17 cell differentiation.This observation shed a new light on the potential mechanisms for the immunosuppressive and anti-inflammatory functions of CsA and provideds a theoretical and experimental foundation for clinical application.

During the past few years,mounting evidence has proved that the protection induced by vaccines or the prognosis of diseases is correlated with the quantity and quality of T cell responses in both mouse models and human infectious diseases.Examination of one or a few effector functions might not fully reflect the antigen-specific immunity.Recent studies demonstrated that after antigen stimulation,T cells could be induced to produce two or more effector functions simultaneously at single cell level,including cytokines,chemokines production and cytotoxic related molecules,which are named as polyfunctional T cells.Further studies indicated that these polyfunctional T cells are linked to protection from infectious diseases.Therefore,measuring multiple functions of T cells simultaneously is essential for the proper evaluation of specific immunity and may have substantial implications for the design of vaccines.

Objective:To evaluate the role of IL-12 and CpG as adjuvants to promote the differentiation of antigen-specific CD4~+T cells in vivo.Methods:Naive OVA-TCR-Tg CD4~+T cells were isolated from spleens and lymph nodes of DO11.10 mice.The cells were labeled with CFSE in vitro and then were adoptively transferred into normal mice.One day after transfer,mice were immunized i.v.either with OVA,OVA+IL-12 or OVA+CpG.3 days post transfer,single cell suspensions were prepared from spleens,lymph nodes and lungs.The proliferation and IFN-? expression of transferred CD4~+T cells in different tissues were assessed by FACS.Results:No proliferation of antigen-specific CD4~+T cells was observed in the absence of immunization.After immunization with OVA for 3 days,cells were proliferated.The frequency of IFN-? producing cells in spleens,lymph nodes and lungs was at a range of 0.93%～2.17%.IL-12 promoted IFN-?~+ cells(4.53%～26.79%);Moreover,CpG enhanced the frequency of IFN-?-producing cells(3.84%) by the cells only from lymph nodes.In consistent with the results reported previously,cells divided 3-5 times revealed higher frequency of IFN-?-producing cells than the rest of them.Conclusion:IL-12 and CpG as adjuvants can promote the differentiation and expression of IFN-? by antigen-specific CD4~+T cells,and thus mediate cellular immune responses.

Objective:To evaluate the role of IL-23 in the production of IFN-?, cell subsets and regulation by human peripheral blood mononuclear cells(PBMCs).Methods:PBMCs were isolated from normal human peripheral blood and cultured with IL-23 in different culture conditions. The level of IFN-? in the culture supernatants was examined by ELISA. The subsets and frequency of IFN-?-producing cells were examined at a single cell level by flow cytometry.Results:IL-23 could directly induce IFN-? production by PBMCs and have synergistic effect with IL-2 on the induction of IFN-? production in a dose dependent manner. The data from Flow cytometric analysis indicated that IL-23 could induce IFN-? expression by CD3~-CD56~+NK cells but not CD3~+CD4~+ and CD8~+T cells. It is noted that IL-23 predominantly induced IFN-? expression by NK cells with high expression of CD56 molecules. Addition of Th2 cytokines or anti-IL-12R?1 mAbs resulted in the inhibition of IL-23-inducing IFN-? production.Conclusion:IL-23 can directly induce IFN-? production by PBMCs. The induction of IFN-? induced by IL-23 can be suppressed by Th2 cytokines and anti-IL-12R?1 mAbs. The data indicated that Th2 cytokines and anti-IL-12R?1 mAbs might have the potential application for the treatment of IL-23-mediated autoimmune diseases.

Objective To investigate whether R-848 could inhibit IgE production of mice immunized with OVA plus ALUM in vivo.Methods BALB/c mice were immunized s.c on the back with PBS,OVA,OVA plus R-848,OVA plus CpG,OVA plus ALUM,and OVA plus ALUM in R-848 or CpG every two weeks for three times.The blood from the mice was harvested after the last immunization,and the concentration of OVA specific or total IgE,IgG1 and IgG2a in the sera was determined;the splenocytes from the immunized mice were harvested and cultured with or without OVA for 3 days,and the concentration of IL-4 and IFNγ in the supernatants was determined.Results Firstly,we investigated that R-848 as Th1 adjuvant could enhance the production of OVA specific IgG2a in mice immunized with OVA.Secondly,further study showed that R-848 could inhibit the production of OVA specific IgE and total IgE,and promote the production of OVA specific IgG2a in the sera of mice immunized with OVA plus ALUM.Moreover,the results of ELISA showed that R-848 inhibiting IgE production was related to its reducing IL-4 production and enhancing IFNγ production.Conclusion R-848 could inhibit IgE production of mice immunized with OVA plus ALUM in vivo.

Objective:Understanding of subset、memory cell generation、effector function and organ distribution of Th1 cells in vitro and in vivo.Methods:Different stages of polarized Th1 cells are generated from antigen-specific CD4 +T cells.The expression of cell surface markers and intracellular cytokines(IL-2、IFN-? and IL-4) is determined at a single cell level by FACS. Equal numbers of CFSF-labeled naive CD4 +T cells and different stages of Th1 cells are adoptively transferred into mouse.The production of IFN-? and distribution of Th1 memory cells in lymphoid and non-lymphoid organs are measured. Results:Th1 cell populations can be subdivided into several subsets based upon the production of IL-2 and IFN-?. The percentage of IFN-?-producing cells is increased and IL-2-producing cells is decreased following repeatedly stimulation under Th1 culture condition. Moreover,the expression of CD62L on Th1 cells is reduced after activation and differentiation of CD4 +T cells. 2 weeks post-transfer,the frequency of naive CD4 +T cells is comparable from lymph nodes、spleens and lungs,whereas memory Th1 cells are preferentially detected in spleens and lungs and rapidly produce IFN-? following re-exposure to the same antigen.Conclusion:Th1 cell population is composed of distinct subsets of cells. In comparson with naive CD4 +T cells,memory Th1 cells reveal the changes of surface molecule expression、biologic function and distribution in tissues.

Objective:To investigate the correlation between CD4+T cell division and surface molecule expression or cytokine production after stimulation with antigen.Methods:Isolation of CD4+T cells from spleens and lymph nodes of OVA-T cell-receptor transgenic mouse (TCR-Tg ). After stimulation with OVA peptide antigen in the presence of antigen-presenting cells, CD4+T cell division, surface molecule expression and intracellular cytokine production are determined in flow cytometry.Results:CD4+T cells are divided 1-5 times after stimulation for 3 days with antigen. The cell division is associated with both up- and down-regulation of surface molecules such as CD25, CD44, CD62L and CD69 and of cytokines such as IFN-?, IL-4 and IL-10.IL-12 promotes cell division and enhances IFN-? but inhibits IL-4 and IL-10 production.Conclusion:After stimulation with antigen, CD4+T cells are divided in association with the quantitative and qualitative changes of surface molecules and cytokine production.

AIM: To study the effect and mechanism of BCG on human nature killer cells. METHODS: PBMC or purified NK cells were isolated from normal human peripheral blood with negative anti-Mycobacterium tuberculosis antibody and cultured with BCG, IL-12, BCG plus IL-12 and BCG plus anti-IL-12Rβ1 mAb (2B10), respectively. The levels of IFN-γ and IL-12p40 in the culture supernatants were measured by ELISA. The frequency of IFN-γ and granzyme B producing cells were analyzed by ELISpot. The cytolytic activity was detected by MTT reduction assay. The surface expression of IL-12Rβ1 on NK cells was detected by flow cytometry. RESULTS: BCG significantly induced IFN-γ production by PBMC in a dose-dependent manner. When PBMC was stimulated with BCG, the frequency of granzyme B producing cells was higher than that in unstimulated PBMC (P<0.05). BCG enhanced the cytotoxic activity of PBMC. BCG alone didn' t induce IFN-γ production by purified NK cells, but it can augment IL-12-induced IFN-γ production by purified NK cells. The cytotoxic activities of BCG-stimulated and unstimulated purified NK cells were not significantly different (P>0.05). BCG induced IL-12 production by PBMC in a dose-dependent manner and enhanced IL-12Rβ1 expression on different subsets of NK cells. Blocking the effect of IL-12 by anti-IL-12Rβ1 mAb (2B10) inhibited BCG-induced IFN-γ production and granzyme B releasing by PBMC. CONCLUSION: BCG can indirectly promote biologic activity of NK cells and the production of endogenous IL-12 combined with up-regulation IL-12Rβ1 expression on the surface of NK cells is a part of the mechanisms of IL-12 on human NK cells.

Objective: IFN-y is produced by both activated T and NK cells in response to mitogen or antigen and has a broad range of immunoregulatory activity. IL-12 has been described as a strong inducer of IFN-?production and promotes the differentiation of naive CD4+T cells toward the Th1 phenotype, priming them for IFN-?production, and consequent induction of cell-mediated immunity. Aim is to know endogenous production of IL,12 from PBMC inducing production of IFN-?in vitro, which is involving in mechanism of T cells to be activated. Methods: Induced IFT-? secretion from human PBMC by stimulated with anti-CD3 , PHA, anti-CD3 plus anti-CD28 and antigen(MLC) Also inhibited IFN-?production by neutralizing antibodies to IL-12 and IL-12R?1 significantly. Results: IFN-?secretion from human activated PBMC is endogenous IL-12dependent, and activated T cells induce the production of IL-12 from APC by a mechanism involving the interaction between CD40L on T cells and CD40 on APC. Conclusion: These results suggest that endogenous IL-12 plays an important role in the normal host defense against infection by a variety of intracellular pathogens and also plays a central role in the genesis of some forms of immunopathology including autoimmune diseases and transplantation rejections.

Objective:To investigate the effects of sinomenine(SIN)on the activation and function of T-cells from SLE patients and to elucidate the mechanisms for the immunosuppressive effect of SIN.Methods:PBMCs were isolated from SLE patients and stimulated with anti-CD3 in the presence or absence of different concentrations of SIN.The cytokines were measured by ELISIA,and the activation and function of T-cells were analyzed by flow cytometry.Results:SIN could inhibit IFN-?,IL-2 and IL-4 production of T cells in SLE patients' peripheral blood.In addition,SIN could significantly inhibit the expression of activation molecules CD69 and CD25 by CD4+ and CD8+ T cells.Conclusion:SIN could inhibit immune responses of SLE patients by suppressing T cells activation and cytokine secretion.