结核分枝杆菌耐热抗原激活的人γδT细胞Th2极性分化特征以及T-bet/GATA-3对分化的调控作用

Th2 differentiation features of Mycobacterium tuberculosis heat resistant antigen-activated human γδT cells and the regulation of transcription factor T-bet/GATA-3 on differentiation

目的研究结核杆菌耐热抗原(MTB-HAg)激活的人外周血γδT细胞Th2极性分化特点和表达于T细胞中的T盒(T-bet)和GATA结合蛋白3(GATA-3)转录因子的调控作用。方法用MTB-HAg刺激人外周血单个核细胞(PBMC)获得MTB-HAg激活的T细胞(MTBAT),分别在中性条件和Th2极化条件培养后,再经10 ng/m L佛波醇酯(PMA)、500 ng/m L离子霉素(ionomycin)和2.5μmol/L莫能菌素(monensin)刺激6 h,四色荧光抗体染色流式细胞术检测γδT细胞和αβT细胞内细胞因子γ干扰素(IFN-γ)和白细胞介素4(IL-4)的表达。流式细胞术分选28 d MTBAT中的γδT细胞和CD4+T细胞,反转录PCR(RT-PCR)检测T-bet和GATA-3 mRNA的表达。结果在中性条件和Th2极化条件下培养的MTBAT中,γδT细胞一直优势表达IFN-γ,而Th2极化条件下,随培养时间增加IFN-γ+αβT细胞百分率显著下降。与中性条件培养比较,Th2极化条件下MTBAT培养28 d,Th0型(IFN-γ+IL-4+)γδT细胞显著增加;而Th2型(IFN-γ-IL-4+)αβT细胞显著增加。RT-PCR结果显示,Th2极化的γδT细胞仍高表达T-bet mRNA,而在CD4+T细胞T-bet mRNA表达被显著下调;同时,GATA-3 mRNA在γδT细胞和CD4+T细胞的表达均显著上调。结论 Th2极化条件下,大部分γδT细胞仍为Th1型,仅部分极性分化为Th0型细胞;Th2极性分化的γδT细胞转录因子T-bet和GATA-3未表现出交互调节功能。

Objective To investigate Th2 differentiation features of Mycobacterium tuberculosis heat resistant antigens （MTB-HAg）-activated human γδT cells and the regulation of transcription factor T-box expression in T cells （T-bet） and GATA-binding protein 3 （GATA-3） on differentiation. Methods Peripheral blood mononuclear cells （PBMCs） were stimulated with MTB-HAg to generate MTB-HAg-activated T cells （MTBAT） and expanded in the neutral condition or Th2 polarizing condition. After restimulation for 6 hours with phorbol myristate acetate （PMA, 10 ng/mL）, ionomycin （500 ng/mL） and monensin （2.5 μmol/L）, intracellular cytokines （IFN-γ, IL-4） of γδT cells and γδT cells among MTBAT were detected by four-color fluorescence mAb staining combined wiht flow cytometry. The highly purified T cells and CD4＋ T cells were sorted by flow cytometer from MTBAT that were cultured in neutral and Th2 polarizing conditions for 28 days. The expressions of T-bet and GATA-3 mRNA in purified T cells and CD4＋ T cells were determined by reverse transcription PCR （RT-PCR） technique. Results γδT cells among MTBAT cultured in the neutral or Th2 polarizing condition predominantly produced IFN-γ, whereas the percentage of IFN-γ T cells significantly decreased in the Th2 polarizing condition as the culture time went by. Compared with the neutral condition, Th0 type （IFN-γ IL-4 ＋ ） T cells significantly increased, and Th2 type （IFN-γ IL-4＋） T cells also significantly increased in the Th2 polarizing condition. RT-PCR showed that mRNA expression of T-bet was still at a high level in T cells that were expanded in the Th2 polarizing condition, but at a low level in Th2 polarized CD4＋ T cells. Moreover, the mRNA expressions of GATA-3 in both Th2 polarized γδT cells and γδT cells were up-regulated. Conclusion In the Th2 polarizing condition, the majority of γδT cells in MTBAT still remained Th1 profile, whereas the portion of γδT cells differentiated into Th0 type cells. Both transcription factor T-bet and GATA-3 failed to display a fully cross-regulation function in Th2 polarized γδT cells.