磷脂酰肌醇3激酶特异性抑制剂LY294002对结核杆菌低分子多肽抗原激活γδT细胞的影响

Effects of phosphatidylinositol 3-kinase specific inhibitor LY294002 on activation of γδTcells by low molecular weight peptide antigen of mycobacterium tuberculosis

目的观察磷脂酰肌醇3激酶（P13K）特异性抑制剂LY294002对CD3单克隆抗体（CD3mAb）活化的T细胞及结核杆菌低分子多肽抗原（Mtb-Ag）激活γδT细胞的影响，探讨P13K在CD3mAb活化的T细胞及Mtb-Ag启动的78T细胞TCR途径信号转导中作用。方法分离获取健康人外周血单个核细胞（PBMC），用CD3mAb、佛波醇酯＋离子霉素（PMA＋IM）、Mtb-Ag刺激PBMC，采用流式细胞术检测CD3’T细胞和78T细胞0、6、12、24、48h和72h的CD69分子的表达情况。PBMC经LY294002（0、0．4、2、10~tmol／L）处理后再刺激培养，用EIA试剂盒检测CD3mAb和PMA＋IM刺激组CD3’T细胞白细胞介素（IL）一2的含量；经CD3PE和CD69FITC、v6PE和CD69FITC双染后，检测LY294002对CD3mAb活化T细胞和Mtb．Ag活化的γδT细胞增殖的影响，并计数培养扩增10d的细胞数量。结果用CD3mAb刺激24hCD3’T细胞和γδT细胞CD69的表达均达高峰（均在56％左右），用PMA＋IM刺激6h，均达高峰（均在99％左右），用Mtb-Ag刺激24h，亦均达高峰，但CD3^＋T细胞和γδT细胞CD69的表达分别为（16．0±0．0）％和（75．2±0．7）％，3组同时间点CD3^＋T细胞CD69的表达之间差异均有统计学意义（均P〈0．05），而78T细胞CD69的表达在PMA＋IM刺激组高于CD3mAb刺激组和Mtb-Ag刺激组（均P〈0．05），后两组差异则没有统计学意义（均P〉0．05）。用LY294002终浓度分别为0、0．4、2、10μmol／L处理的样本，CD3mAb刺激组CD3^＋T细胞CD69的表达分别为（52．0±0．5）％、（46．3±0．4）％、（33．2±0．4）％和（19．1±0．0）％；Mtb-Ag刺激组78T细胞CD69的表达分别为（66．2±0．6）％、（58．4＋0．5）％、（38．1±0．3）％和（19．3±0．1）％。0、0．4、2、10μmol／LLY294002处理CD3＋T细胞后，CD3mAb刺激组IL-2的含量分别为（561．1±86．2）、（477．0±75.5）、（280．3±53．9）、（36．0±8．7）ng／L，PMA＋IM刺激组为（1922．2±371．3）、（1928．3±381．7）、（1938．1±262．1）、（1915．4±201．6）ng／L。CD3^＋T细胞的总数由培养前的1．5×10^6分别增殖到（35．6±8．4）×10^6、（31．1±7．3）×10^6、（18．7＋5．0）×10^6和（7．0±2．0）×10^6；γδT细胞的总数由培养前的1．5×10^6分别增殖到（7．0±1．1）×10^6、（4．7＋1．0）×10^6、（1．3＋0．8）×10^6和（0.2±0．1）×10^6。结论Mtb-Ag可特异性的激活78T细胞，其活化信号转导需通过TCR通路，LY294002对CD3mAb活化的T细胞及Mtb-Ag启动的γδT细胞具有明显的抑制作用。

Objective To observe the effects of phosphatidylinositol 3- kinase （PI3K） specific inhibitor LY294002 on CD3 monoclonal antibody （CD3 mAb） activated T cells and γδT ceils activated by low molecular peptide antigen of mycobacterium tuberculosis （Mtb-Ag）, and to investigate the role of PI3K in TCR signal transduction pathway in CD3 mAb activated T cells and Mtb- Ag activated γδT cells. Methods Human peripheral blood mononuclear cells （PBMC） were isolated from healthy subjects, and stimulated with CD3 mAb, phorbol myristate acetate＋ionomycin （PMA＋IM）, and Mtb-Ag, respectively. Flow cytometry was used to measure expression of CD69 molecules in the CD3^＋ T cells and γδT cells at 0, 6, 12, 24, 48 h and 72 h of stimulation. The stimulation culture was repeated after treatment of PBMC with various levels of LY294002 （0, 0.4, 2, and 10 μmol/L）. EIA kit was used to measure the interleukin （IL）-2 levels in CD3 T cells in the CD3 mAb and PMA ＋ IM stimulation groups. The effects of LY294002 on proliferation CD3 mAb activated T cells and Mtb-Ag activated γδT cells were determined after CD3PE/ CD69FITC and γδPE/CD69FITC double staining. The number of these ceils was calculated after 10 days of cultivation. Results The CD69 expression peaked in CD3^＋ T cells and γδT cells stimulated with CD3 mAb for 24 h （both around 56% ）, in those stimulated with PMA＋IM for 6 h （both around 99% ）, and in those stimulated with Mtb-Ag stimulation for 24 h [ （16.0 ± 0.0）% in CD3^＋ T cells vs （75.2 ± 0.7）% in γδT cells） ]. At any time point, the CD69 expression level in CD3^＋ T cells was statistically significant among groups with different stimulating agents （P〈0.05） ; in contrast, the expression level of CD69 in γδT cells was higher in PMA ＋ IM stimulation group than in the CD3 mAb stimulation group or Mtb-Ag stimulation group （P〈0.05） , and did not differ statistically between the latter two groups （P〉0.05）. After treatment with 0, 0.4, 2 and 10 p.mol/L （final concentration） LY294002, the CD69 expression in CD3 mAb- stimulated CD3^＋ T cells were （52.0±0.5）%, （46.3±0.4）%, （33.2±0.4）% and （19.1 ±0.0）% , respectively; and in Mtb-Ag stimulated γδT cells, （66.2±0.6）%, （58.4±0.5）%, （38.1±0.3）% and （19.3± 0.1 ）%, respectively. LY294002 did not inhibit the CD69 expression in CD3^＋ T ceils or γδT cells stimulated with PMA＋IM, with the expression level 〉 99% in both cells. After treatment with 0, 0.4, 2 and 10 p.mol/L （final concentration） LY294002, the IL-2 level in CD3 mAb-stimulated CD3^＋ T ceils were （561.1±86.2）, （477.0±75.5）, （280.3±53.9） and （36.0±8.7） ng/L, respectively; and in PMA＋IM stimulated CD3^＋ T cells, （1 922.2±371.3）, （1 928.3±381.7）, （1 938.1±262.1）, and （1 915.4±201.6）ng/L, respectively. Moreover, the total counts of CD3^＋ T cells increased from 1.5×10^6 before culture to （35.6±8.4）×10^6, （31.1±7.3）×10^6,（18.7±5.0） ×10^6 and （7.0±2.0） ×10^6, respectively ; those of yST cells from 1.5×10^6 before culture to （7.0±1.1 ） ×10^6, （4.7 ± 1.0 ） ×10^6, （ 1.3 ± O. 8 ） ×10^6 and （0.2 ± O. 1 ） ×10^6, respectively. Conclusion Mtb-Ag mayspecifically activate TST cells by using the TCR pathway for transduction of the activation signals. LY294002 exhibits obviously inhibitory effects on CD3 mAb activated T cells and Mtb-Ag activated γδT ceils.