OX40信号调节抑制小鼠CD4＋CD25＋调节性T淋巴细胞Foxp3的表达

OX40 stimulation down-regulates the expression of Foxp3 in CD4＋ CD25＋ regulatory T cells

目的探讨共刺激信号OX40对体外诱导的小鼠CD4＋CD25＋适应性调节性T淋巴细胞（iTreg）的Foxp3表达的影响。方法制备C57BL／6小鼠淋巴细胞悬液，经免疫磁珠法分选，获得CD4＋CD25一静息T淋巴细胞，与抗CD3单克隆抗体、抗CD28单克隆抗体、转化生长因子B1、白细胞介素2共孵育，诱导产生Foxp3＋iTreg。在此基础上，于培养体系中加入OX40激动型抗体及其对照抗体，利用流式细胞仪分析研究OX40信号刺激对iTregFoxp3表达的影响。结果C57BL／6小鼠淋巴结中CD4＋CD25＋天然调节性T淋巴细胞（Treg）比例为（5．0±0．4）％，体外诱导培养的CD4＋CD25＋Treg比例为（71．8±13．4）％，其中Foxp3阳性表达占（74．9±1．9）％。OX40激动型抗体组CD4＋CD25＋Treg细胞比例为（80．0±1．6）%，其中Foxp3表达水平为（59．2±0．7）％；OX40激动型抗体对照抗体组CD4＋CD25＋Treg细胞比例为（86．0±1．4）％，其中Foxp3表达水平为（70．0±0．8）％，两组问差异有统计学意义（P〈0．05）。结论静息T淋巴细胞可以在体外诱导培养获得高纯度iTreg；OX40信号刺激可以显著抑制CD25＋iTreg细胞Foxp3的表达。

Objective To evaluate the regulatory effect of OX40 co- stimulatory signal on the expression of Foxp3 in inductive regulatory T cells （iTreg） in vitro. Method CD4＋ CD25＋ naive T cells were isolated from C57BL/6 mouse lymphocyte suspension by MASC CD4＋CD25＋ regulatory T cell isolation kit. Inductive Tregs were generated by stimulation of naive T cells in the presence of transforming growth factor beta （TGFβ1）, anti-CD3, anti-CD28 and IL-2. The regulatory effect on iTregs was shown by use of OX40 stimulation monoclonal antibody （OX86） or control antibody. Using flow cytometric analysis （FACS）, we examined the antibody-based identification of Tregs surface markers CD4 and CD25, along with the intracellular activation marker FoxP3. Results The ratio of CD4＋ CD25＋ nTregs isolated from mouse lymphatic node was （5.0±0. 4）% vs. （71.8±13.4）% of TGFβ1-driven iTregs. The ratio of CD4＋ CD25＋ Tregs was （80. 0±1.6） % in OX40 stimulation McAb group vs. （86. 0±1. 4）% in control antibody group. Furthermore, the expression of Foxp3 was （59. 2±0. 7）% in OX40 stimulation McAb group vs. （70. 0± 0. 8）% in control antibody group （P〈0. 05）. Conclusion TGFβ1-dependent protocol may induce the conversion of naive CD4＋ T cells into CD25＋ Foxp3＋ iTregs. OX40 stimulation can down-regulate the expression of Foxp3 in CD4＋ CD25＋ iTreg significantly. Thus OX40 molecular may become an attractive target in Tregs-induced transplant tolerance. Further study should be performed to increase the suppressive activity of iTregs through blockade of OX40 signal.