CD4^+CD25^+调节性T细胞的体外扩增

Amplification of CD4^+CD25^+Tregs in Vitro

【目的】探讨有效的体外培养扩增CD4+CD25+Treg的方法。【方法】收集健康志愿者外周血5例,免疫磁珠法分选出CD4+CD25+Treg。将实验分为3组,第1组在CD4+CD25+Treg中加入100nmol/Lrapamycin(1μg/mL)和包被了CD3/CD28单抗的磁珠培养3周,第8天开始加入1000U/mL的IL-2,第2组培养条件除不加rapamycin外其余与第1组相同,第3组培养细胞为CD4+CD25-T细胞,培养条件同第1组。培养第7、14、21天收集细胞计数,第21天收集细胞行流式细胞术三标法检测CD4、CD25、FOXP3的表达。MTT法检测体外扩增的CD4+CD25+Treg对自体和异体的CD4+T细胞增殖的抑制作用。【结果】三组细胞均有明显的扩增。加rapamycin培养的CD4+CD25+Treg细胞的扩增率比不加rapamycin扩增倍数低,但细胞的纯度明显增加。CD4+CD25-T细胞组在培养第1周扩增迅速,从第2周开始增殖减慢,其中CD4+CD25+Treg细胞的比例较培养前无明显的差别。三组至第3周时扩增倍数分别为89±21、338±78、216±55(F=484.655,P<0.0001),细胞的纯度分别为84.32%±4.62%、34.04%±5.78%、0.68%±0.39%(F=24.660,P<0.0001)。体外抑制实验显示体外扩增的CD4+CD25+T细胞对自体和异体的CD4+T细胞增殖均有明显的抑制作用,并且随着效靶比浓度的降低而降低。在CD4+T/Treg比例为8:1、4:1、2:1、1:1时对自体CD4+T细胞的抑制率分别为9.65%、16.43%、28.75%、55.34%,对异体CD4+T细胞的抑制率分别为6.43%、14.72%、26.47%、50.25%,而且在各浓度梯度其抑制作用在自体和异体之间均无明显的差异。【结论】我们采用rapamycin+CD3/CD28单抗标记的磁珠+IL-2在体外成功扩增了CD4+CD25+Treg,其扩增的倍数为89.4±20.53,具有免疫抑制功能,有望进一步应用于临床。

[Objective] The amplification of CD4^＋CD25^＋Tregs in vitro is a difficult problem in immunology, and it restricts the application of CD4^＋C25^＋Tregs in clinic. This research was designed to investigate an effective method to amplify CD4^＋CD25^＋Tregs in vitro. [Method] Peripheral blood was obtained from five healthy donors. CD4^＋CD25^＋Tregs were purified by MACS （Miltenyi Biotec）. We set up three groups in this experiment： in group one： CD4^＋CD25^＋Tregs were cultured with 100 nmol/L rapamycin （1 μg/mL） and CD3/CD28 antibody coated beads for 3 weeks, and 200 U/mL IL-2 was added in the second week. In group two, CD4^＋CD25^＋Tregs were cultured with CD3/CD28 antibody coated beads for 3 weeks and 1000 U/mL IL-2 was added in the second week. In group three, all culture condition was the same as group one except the cells were changed to CD4^＋CD25^＋T cells. The cell numbers were counted in day 7, 14, and 21. In day 21, cells were collected to test the expression of CD4, CD25, FOXP3 by FACS. The inhibitory effect of amplified CD4^＋CD25^＋T cells on auto and allo-CD4^＋T cells was tested by MTT method. [ Results ] All cells in the three groups amplified significantly. After three weeks, the proliferation rates in the three groups were 89 ± 21, 338 ± 78, and 216 ± 55, respeetively（F = 484.655, P 〈 0.0001）. The purity of CD4^＋CD25^＋Treg was significantly higher in group one （84.32% ± 4.62%, 34.04% ± 5.78%,and 0.68% ± 0.39%, respectively. F = 24.660,P 〈 0.0001）. The proliferation rate was lower in group 1 than that of group 2. In group 3, the cells amplified quickly, but slowed down from the second week. The inhibitory test showed these amplified CD4^＋CD25^＋T cells inhibited the proliferation of auto and allo CD4^＋T cells in vitro, and the inhibition ratio decreased with the decreasing of CD4^＋T/CD4^＋CD25^＋Tregs, when the CD4^＋T/CD4^＋CD25^＋Tregs was 8：1, 4： 1, 2： 1, 1：1. The inhibitory rates were 9.65%, 16.43%, 28.75%, and 55.34% for auto CD4^＋T cells and 6.43%, 14.72%, 26.47%, and 50.25% for allo CD4^＋T ceils, respectively. There was no significantly difference between the auto and allo groups. [Conclusion] We successfully amplified CD4^＋CD25^＋Tregs with rapamycin and CD3/CD28 antibody coated beads and IL-2 in vitro, and the cells had inhibitory effects in vitro, the amplified ratio was 89.4 ± 20.53. This method may be used in clinic.