R848对胸腺基质淋巴细胞生成素调控Naive T细胞分化的影响

Investigation of R848 Implication on Thymic Stromal Lymphopoeitin Modulating T cells Differentiation

【目的】探讨咪喹莫特类似物R848对胸腺基质淋巴细胞生成素(TSLP)介导Naive T细胞分化的影响。【方法】从健康志愿者外周血中提取单个核细胞(PBMC),免疫磁珠分选出CD14+PBMC,随后用不同因素诱导分化为成熟树突状细胞(DC);另用免疫磁珠阴性分选出Na觙ve T细胞后,将其与诱导分化后的DC共培养,并分组:A组为Naive T+PBS/DC;B组为Naive T+TSLP/DC;C组为Naive T+(TSLP+R848)/DC;D组为Na觙veT+(TSLP+anti-OX40L)/DC。ELISA检测上清液细胞因子IFN-γ、IL-4和IL-13水平,荧光定量RT-PCR测定转录因子GATA-3及T-bet m RNA的表达。【结果】(1)TSLP诱导后DC表达OX40L增加,而R848可抑制OX40L表达(P<0.05)。(2)B、C、D三组T细胞表达细胞因子IL-4和IL-13水平均高于A组;B组IL-4和IL-13均较C组和D组高,而D组高于C组(均P<0.05);C组表达IFN-γ水平明显高于其余三组(均P<0.05);A、B、D三组之间IFN-γ无明显差异(P>0.05)。(3)C组和D组GATA-3 m RNA水平均低于B组,但高于空白对照A组,其中D组GATA-3 m RNA水平高于C组;C组表达T-bet m RNA水平高于其余三组(均P<0.05);A、B、D三组之间T-bet水平无明显差异(P>0.05)。【结论】TSLP通过上调人DC表达OX40L,在转录因子水平介导Na觙ve T细胞向Th2细胞分化;这种TSLP介导的Th2细胞优势反应可被R848所抑制。

[ Objective ] To investigate the role of R848 in the pathogenesis of thymic stromal lymphopoeitin （TSLP） modulating Naive T cell differentiation. [ Methods ] Peripheral blood mononuclear ceils （PBMC） were isolated by density gradient centrifugation from peripheral venous blood of healthy volunteers. Purified CD14＋ PBMC were obtained with microbead separation method and induced by TSLP to get matured DC. OX40L expressions on DC were measured by Flow cytometry assay. Purified CD4＋ CD45RA＋ T Naive T cell were also obtained from PBMC with microbead negative separation method, and used to cocultured with stimulated DC. The ceils were divided into 4 groups, Group A： Naive T＋PBS/DC; Group B： Naive T＋TSLP/DC; Group C： Naive T＋（TSLP＋R848） /DC; Group D：Naive T＋（TSLP＋ anti-OX40L）/DC. Then, the concentrations of cytokine IL-4, IL-13 and IFN-γ in culture supernatant were measured by ELISA method, and expressions of GATA-3 and T-bet mRNA were determined by real time PCR. [Results] （1） TSLP-induced DC produced increased OX40L but R848 inhibited the production of OX40L （P 〈 0.05）. （2） T cells of group B secreted more IL-4 and IL-13 than the other three groups, while the levels of these cytokines were increased in group D when compared to group C （P 〈 0.05）. T cells of Group C produced more IFN-γ than the other three groups （P 〈 0.05） while no significant difference among group A, B and D （P〉 0.05）. （3） T ceils of group D expressed more GATA-3 mRNA than group C, but the mRNA expressions of this transcript factor were lower in both group C and D when compared to group B （P 〈 0.05）. The expression of T-bet mRNA was increased in group C than the other three groups （P 〈 0.05）, while no significant difference among group A, B and D （P 〉 0.05）. [Conclusion] TSLP promoted Naive T cell differentiation into Th2 cells by modulating the OX40L expression on DC; R848 suppressed the induction of TSLP on Th2 response.