ConA刺激对CD^4＋CD25^＋调节性T细胞趋化特性及其表面趋化因子受体CCR4／CCR6表达的影响

Exploration on the Chemotaxis and CCR4/CCR6 expression of CD4^+CD25^+ regulatory T cells activated by ConA in vitro

目的:体外动态观察ConA激活的调节性T细胞表面趋化因子受体的表达变化及其趋化特性,为利用调节性T细胞诱导免疫耐受提供线索。方法:①流式细胞仪分选出CD4hiCD127loCD25hi-int细胞。②纯化的调节性T细胞与CD4+CD25-T细胞分别用ConA(10μg/ml)刺激0、24和48小时后,用趋化因子CCL1、CCL5、CCL20、CCL22做趋化实验,观察各趋化因子作用下调节性T细胞与CD4+CD25-T细胞的趋化特性。同时,流式细胞仪检测CCR4与CCR6的表达。结果:①分离得到的调节性T细胞纯度为97.4%,活细胞率为95%,得率:4.1%。②CCL1、CCL20、CCL22均可趋化调节性T细胞,且在ConA激活后趋化效率随时间而改变。CCL1与CCL22对调节性T细胞的趋化指数显著高于CD4+CD25-T细胞;CCL20对调节性T细胞和CD4+CD25-T细胞趋化指数都很高;CCL5对调节性T细胞趋化性则显著弱于CD4+CD25-T细胞。③ConA刺激后,调节性T细胞趋化因子受体CCR4、CCR6的表达均明显高于对照组细胞(CD4+CD25-细胞)。随着ConA刺激时间延长,两组细胞CCR4的表达均持续增强;两组细胞CCR6的表达均在刺激24小时后表达明显增强,48小时后CCR6的表达略有减弱,呈下降趋势。结论:①磁珠阴性分选结合流式细胞仪技术可分选出较高纯度及活率的调节性T细胞。②调节性T细胞与CD4+CD25-T细胞相比,二者具有不同的趋化特性。CCL1对调节性T细胞的趋化作用较特异,CCL22趋化作用较强,CCL5趋化作用较弱。而CCL20对CD4+CD25-T细胞和调节性T细胞趋化作用都强。③ConA刺激后48小时内趋化因子CCL1、CCL20、CCL22对调节性T细胞的趋化作用随刺激时间而增强。④ConA刺激可以增强受体CCR4、CCR6表达。⑤提示趋化因子受体的表达与细胞活化状态有关,且不同受体表达变化趋势不同。

Objective：To provide a few hints for inducing immune tolerance with regulatory T cells,the chemotaxis and the changes of some chemokine receptor expression on activated CD4^＋ CD25 - regulatory T cells were observed dynamically in vitro. Methods： ①CD4hiCD127 loCD25hi-int T cell were sorted by flow cytometer. ②The chemotaxis of regulatory T cells was observed. Purified regulatory T cells and CD4^＋ CD25- T cells were induced by ConA（ 10μg/ml） for 0 h, 24 h and 48 h respectively. Chemokines CCL1, CCL5, CCL20, CCL22 were applied to do chemotaxis assay, and the chemotactic effects of those chemokines on regulatory T cells and CD4^＋ CD25 - T cells were observed. QObserving expression changes of chemokine receptor on regulatory T cells： After stimulated by ConA（ 10 pg/ml）for different duration as above, T cells were labelled with PE-Cy7-Anti CCR4, PE-Anti CCR6 and the changes in expression CCR4, CCR6 were detected by Flow cytometry. Results： ①The purity of sorted regulatory T cells was 97.4%, the survival rate was 95 %, with yield was 4.1%. ② CCL1, CCL20 and CCL22 all had the chemotactic effect on regulatory T cells. Furthermore, after cells were activated, chemotactic efficacy changed with time. The chemotactic index of CCL1 for regulatory T cells was high, whereas that of CCL1 was very low; Chemotactic index of CCL22 for regulatory T cells and CD4^＋ CD25 - T cells were high, with a stronger chemotactic effect on regulatory T cells. Chemotactic index of CCL20 for regulatory T cells and CD4^＋CIY25-T cells were high, whereas CCL5 had weaker chemotactic effect for regulatory T cells. QAfter stimulted by ConA, the expression of CCR4 and CCR6 on regulatory T cells were higher than that of CD4 ＋ CD2.5 - cells. Along with the constitutively stimulation by ConA, the expression of CCR4 on both CD4^＋ CD25- T cells group and the control group was continuously increased, The expression of CCR6 on CD4^＋ CD25- T cells increased evidently at 24 h and decreased at 48 h after ConA stimuation, qlae expression of CCR6 of control group was in the same trend. Conclusion： ①Differential charactors of chemotaxis are found in regulatory T cells and in CD4＋ CIY25- T cells,The action of chemokine CCL1 is more specific for regulatory T cells, with CCL22 stronger and CCL5 weaker. CCL20 had a higher chemotactic effect on both regulatory T cells and CD4 ^＋ CIY25 - T cells. ②Accompanying with activation of regulatory T cells by ConA in vitro, chemotactic effect of CCL1, CCL20 and CCL22 for the regulatory T cells is gradually enhanced.③The expression level of CCR4 and CCR6 can be incluced by ConA stimulation. ④The expression of chemokine receptor has a relation to the state of regulatory T cell activation. ⑤The expression of chemokines receptor is correlated to the duceration of stimulation,and different receptor has its unigue trends.