[不同细胞因子体外诱导大鼠CD4+ CD25+调节性T细胞增殖的研究]

[Proliferation of CD4+ CD25+ regulatory T cells of rat by different cytokines in vitro].

作者信息

Wang Zi-Han, Zhu Ji-Ye, Li Tao, Leng Xi-Sheng

机构信息

Peking University People's Hospital, Department of Hepatobiliary Surgery, Beijing 100044, China.

出版信息

Zhonghua Yi Xue Za Zhi. 2008 Mar 25;88(12):844-7.

PMID:18756991

Abstract

OBJECTIVE

To evaluate the effects of cytokines on the proliferation and function of CD4+ CD25+ regulatory T cell (Treg).

METHODS

Tregs were isolated from naive C57BL/6 mice spleen and lymph nodes. Mature dendritic cells (mDC) were isolated from DBA/2 mice, co-cultured with Tregs, and divided into 4 groups with or without interleukin-2 (IL-2), interleukin-4 (IL-4), and interleukin-15 (IL-15) added into the culture fluid. Fluorescence-activated cell sorting (FACS) was used to detect the Treg proliferation and apoptosis with CFSE and annexin-V staining. The co-culture increased Tregs were divided into 5 groups: CFSE labeled naïve CD4+ CD25- T cells, self-proliferated Treg, Treg mixedly cultured with IL-2 mDC, and Teff, Treg mixedly cultured with IL-4, mDC, and Teff, and Treg mixedly cultured with IL-15, mDC, and Teff, a control group included Teff co-cultured with mDC. FACS was used 5 d later to evaluate the suppressive function of the Treg on the Teff. The expression of Foxp3, indicating the phenotype of Treg was detected.

RESULTS

FASC showed that the values of precursor frequency (PF) of the Tregs stimulated by IL-2, IL-4, and IL-15 were 31.3%, 28.9%, and 34.5% respectively, all significantly higher than that of the control group (14.5% all P < 0.05), and the values of proliferation index (PI) of the Tregs stimulated by IL-2, IL-4, and IL-15 were 1.9, 1.7, and 1.8 respectively, all significantly higher than that of the control group (1.5, all P < 0.05). The apoptotic rates of the Tregs stimulated by IL-2, IL-4, and IL-15 were 12. 8% , 11.4%, and 12.7% respectively, all significantly lower than that of the control group (28.9%, P < 0.05). The Foxp3 expression rate of the Tregs stimulated by IL-2, IL-4, and IL-15 was 91.75%.

CONCLUSION

IL-2, IL-4, and IL-15 in the in vitro culture of Treg stimulate the Treg proliferation, reduce their apoptosis, and maintain their suppressive function. The proliferated Tregs still maintain their phenotype, highly expressing Foxp3.

摘要

目的

评估细胞因子对CD4+CD25+调节性T细胞（Treg）增殖及功能的影响。

方法

从未经免疫的C57BL/6小鼠脾脏和淋巴结中分离Tregs。从DBA/2小鼠中分离成熟树突状细胞（mDC），与Tregs共培养，并分为4组，培养液中分别添加或不添加白细胞介素-2（IL-2）、白细胞介素-4（IL-4）和白细胞介素-15（IL-15）。采用荧光激活细胞分选术（FACS），通过CFSE和膜联蛋白-V染色检测Treg的增殖和凋亡情况。将共培养后增多的Tregs分为5组：CFSE标记的未经免疫的CD4+CD25-T细胞、自身增殖的Treg、与IL-2 mDC混合培养的Treg以及效应性T细胞（Teff）、与IL-4 mDC和Teff混合培养的Treg、与IL-15 mDC和Teff混合培养的Treg，对照组为Teff与mDC共培养。5 d后采用FACS评估Treg对Teff的抑制功能。检测指示Treg表型的Foxp3的表达情况。

结果

FACS显示，IL-2、IL-4和IL-15刺激的Tregs的前体频率（PF）值分别为31.3%、28.9%和34.5%，均显著高于对照组（均为14.5%，P<0.05），IL-2、IL-4和IL-15刺激的Tregs的增殖指数（PI）值分别为1.9、1.7和1.8，均显著高于对照组（1.5，P<0.05）。IL-2、IL-4和IL-15刺激的Tregs的凋亡率分别为12.8%、11.4%和12.7%，均显著低于对照组（28.9%，P<0.05）。IL-2、IL-4和IL-15刺激的Tregs的Foxp3表达率为91.75%。