Zhu Anyou, Lv Hezuo, Zhang Lunjun, Hu Jianguo, Wang Fengchao, Li Baiqing
Department of Clinical Laboratory, First Affiliated Hospital, Bengbu Medical College, Bengbu 233004; Department of Immunology, Bengbu Medical College, Bengbu 233030, China.
Department of Clinical Laboratory, First Affiliated Hospital, Bengbu Medical College, Bengbu 233004, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2015 Jan;31(1):72-6.
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.
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 αβT cells among MTBAT were detected by four-color fluorescence mAb staining combined with flow cytometry. The highly purified γδT 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 γδT cells and CD4⁺ T cells were determined by reverse transcription PCR (RT-PCR) technique.
γδT cells among MTBAT cultured in the neutral or Th2 polarizing condition predominantly produced IFN-γ, whereas the percentage of IFN-γ⁺ αβT 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⁺) γδT cells significantly increased, and Th2 type (IFN-γ⁻ IL-4⁺) αβT 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 γδT 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 CD4⁺T cells were up-regulated.
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.
研究结核分枝杆菌耐热抗原（MTB-HAg）激活的人γδT细胞的Th2分化特征以及转录因子T细胞T-box表达（T-bet）和GATA结合蛋白3（GATA-3）对分化的调节作用。
用MTB-HAg刺激外周血单个核细胞（PBMC），产生MTB-HAg激活的T细胞（MTBAT），并在中性条件或Th2极化条件下进行扩增。用佛波酯（PMA，10 ng/mL）、离子霉素（500 ng/mL）和莫能菌素（2.5 μmol/L）再刺激6小时后，采用四色荧光单克隆抗体染色结合流式细胞术检测MTBAT中γδT细胞和αβT细胞的细胞内细胞因子（IFN-γ、IL-4）。通过流式细胞仪从在中性和Th2极化条件下培养28天的MTBAT中分选高度纯化的γδT细胞和CD4⁺ T细胞。采用逆转录PCR（RT-PCR）技术检测纯化的γδT细胞和CD4⁺ T细胞中T-bet和GATA-3 mRNA的表达。
在中性或Th2极化条件下培养的MTBAT中的γδT细胞主要产生IFN-γ，而随着培养时间的延长，在Th2极化条件下IFN-γ⁺ αβT细胞的百分比显著降低。与中性条件相比，在Th2极化条件下Th0型（IFN-γ⁺ IL-4⁺）γδT细胞显著增加，Th2型（IFN-γ⁻ IL-4⁺）αβT细胞也显著增加。RT-PCR显示，在Th2极化条件下扩增的γδT细胞中T-bet的mRNA表达仍处于高水平，但在Th2极化的CD4⁺ T细胞中处于低水平。此外，Th2极化的γδT细胞和CD4⁺ T细胞中GATA-3的mRNA表达均上调。
在Th2极化条件下，MTBAT中的大多数γδT细胞仍保持Th1型，而部分γδT细胞分化为Th0型细胞。转录因子T-bet和GATA-3在Th2极化的γδT细胞中均未显示出完全的交叉调节功能。
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