RATG对CD4+细胞和CD8+细胞共刺激分子基因表达和细胞因子分泌的影响

目的 探讨兔抗人胸腺细胞多克隆抗体(RATG)在体外对CD4+细胞和CD8+细胞共刺激分子基因表达和细胞因子分泌的影响.方法 从正常成人外周血单个核细胞中分离和纯化CD4+细胞和CD8+细胞,加入RATG,37℃下培养.分别于24、48和72 h收集培养上清液和细胞,以不处理的细胞为正常对照,正常兔Ig处理的细胞作为阴性对照.采用实时聚合酶链反应技术检测培养细胞的细胞毒性T淋巴细胞相关抗原4(CTLA4)、CD154、Foxp3、OX40、γ干扰素(IFN-γ)、白细胞介素2(IL-2)、IL-10和IL-2受体(CD25)的基因表达水平,应用Multiplex检测技术测定培养上清液中IFIN-γ、IL-2、IL_4和IL-10的水平.结果 与正常CD4+细胞比较,加入RATG的CD4+细胞培养24 h,其CTLA-4、CD154、Foxp3、OX40、IFN-γ、IL-2、IL-10和CD25基因转录表达均上调,阴性对照CD4+细胞则无这些基因转录表达的上调.处理48 h后,CD4+细胞的CD154和IL-2的基因转录表达呈现下调现象,而CTLA4、Foxp3、0X40、IFN-γ、IL-10和CD25的基因转录水平均较24 h时明显降低.处理72 h后,CD4+细胞的CTLA4、Foxp3、OX40和CD25的基因转录表达再次呈现高水平,CD154和IFN-γ的基因转录表达上调表达不明显,而IL-2和IL-10的基因转录表达则呈现明显的下调.RATG处理的CD4+细胞培养上清液中的IFN-γ、IL-2、IL-4和IL-10浓度显著增加,以处理24 h的水平最高,而阴性对照者未测出.与正常CD8+细胞相比较,加入RATG处理的CD8+细胞培养24 h,其CTLA4、Foxp3、OX40、IFN-γ,、IL-2、IL-10和CD25的基因转录表达呈现明显上调,而CD154基因转录表达稍有下调.RATG处理48 h,CD8+细胞的CTLA4、Foxp3、OX40、IFN-γ和CD25基因转录表达仍维持在高水平,CD154基因转录表达仅仅呈现低水平的上调,IL-10基因转录表达水平显著下降,而IL-2的基因转录表达则明显下调.处理72 h,CD8+细胞的CTLA4、Foxp3、OX40、IFN-γ、IL-10和CD25的基因转录表达仍维持在高水平,CD154基因转录表达则呈现下调,而IL-2基因转录表达的下调更为显著.阴性对照CD8+细胞则未呈现这些基因转录的上调现象.RATG处理的CD8+细胞培养上清液中IFN-γ、IL-2和IL-10显著增多,以处理24 h的浓度最高,IL-4浓度升高的幅度较小,而正常CD8+细胞和阴性对照CD8+细胞几乎检测不到IFN-γ、IL-2、IL-4和IL-10.结论 在体外,RATG可以刺激CD4+细胞和CD8+细胞上调多种促进免疫抑制的共刺激分子基因表达,促进其分泌与免疫调节相关的IFN-γ、IL-2、IL-4和IL-10.

Effects of RATG on CD4+and CD8+ T cell eostimulatory molecule gene expression and productiun of immune-regulatory cytokines

Objective To investigate the immunological effects of thymoglobulin (RATG) on human CD4+and CD8+cells for costimulatory molecule gene expression and the production ofimmune-regulatory cytokines. Methods CD4+and CI8+T cells were isolated and purified fromnormal human peripheral blood mononuclear cells (PBMC) followed by incubation with RATG at37℃. Cells and culture supematants were collected at 24, 48, and 72 h after incubation, and analyzedby real-time quantitative polymerase chain reaction (RT-PCR) for CTLA-4, CD154, forkhead box P3(Foxp3), OX40, IFN-γ, IL-2, IL-10 and CD25 gene expression, and multiplex cytokine detectionassay for IFN-y, IL-2, IL-10, and IL-4 production. Untreated and rabbit isotype Ig-treated cells wereused as negative controls. Results RT-PCR demonstrated that RATG pre-treated CI+and CD8+cells upregulated the expression of CTLA-4, OX40, Foxp3, CD25, IFN-γ, IL-10 and IL-2 genes, anda dramatic increase of supernatant IFN-γ, IL-10, IL-2 and IL-4 was revealed 24 h after treatment asdetermined by multiplex cytokine detection assay when compared with negative controls. Theupre gulation of CTLA-4, Foxp3, OX40, IL-10 and CD25 was reduced, and a down-regulation ofCD154 and IL-2 gene expression was revealed 48 h after treatment. Cells, treated with RATG for 72h, demonstrated up-regulation of CTLA-4, Foxp3, OX40, IFN-y and CD25 gene expression, and theexpression of IL-2 and IL-10 genes was down-regulated. Additionally, supernatant IFN-γ, IL-2,IL-10 and IL-4 levels were decreased. Conclusion RATG stimulates CI4/CD8 T cells to up-regulatecostimulatory molecules and release immune regulation associated cytokines IF'N-γ, IL-2, IL-10in vitro. These results suggest that the unique effect of RATG on CD4+CD8+T cells may be animportant mechanism for its action in inducing immunoregulation, immunosuppression and transplanttolerance.