小鼠补体调节蛋白对CD4^＋T淋巴细胞的调控作用及其机制

Complement regulatory protein Crry regulates CD4^＋T lymphocytes and induces allograft immune hyporesponsiveness

目的研究小鼠补体调节蛋白Crry对CD4^＋T淋巴细胞的调控作用及诱导同种移植免疫低反应性的机制。方法分离C57BL／6小鼠脾淋巴细胞，用免疫磁珠法分选出CD4^＋T淋巴细胞后，将CD4^＋T淋巴细胞分为A、B、C、D、E和F组，分别用抗小鼠CD3、CD28、Crry、CD3／CD28、CD3／Crry和CD3／CD28／Crry抗体共刺激通路与CD4^＋T淋巴细胞进行反应，采用噻唑蓝（MTT）法检测各组CD4^＋T淋巴细胞的增殖情况，并采用酶联免疫吸附试验检测CD4^＋T淋巴细胞培养上清中白细胞介素2（IL-2）、γ干扰素（γ-IFN）、IL-4和IL-10的水平；另外，以BALB／c小鼠和C57BL／6小鼠的脾细胞分别作为刺激细胞和反应细胞，建立同种混合淋巴细胞反应（MLR）体系并加入抗小鼠Crry抗体，通过MTT法观察Crry对MLR的影响。结果D、E、F组的CD4^＋T淋巴细胞均出现明显增殖，增殖活性显著高于A、B、C组（P〈0．05），其中F组显著高于D组和E组（P〈0．05），D组和E组间增殖活性的差异无统计学意义。D组CD4^＋T淋巴细胞经抗CD3／CD28抗体共刺激后，培养上清中γ-IFN和IL-2的水平显著升高，与A、B、C和E组比较，差异均有统计学意义（P〈0．05），但与F组的差异无统计学意义；E组CD4^＋T淋巴细胞经抗CD3／Crry抗体共刺激后，IL－4的水平显著升高，与A、B、C、D组比较，差异均有统计学意义（P〈0．05），但显著低于F组（P〈0．05）；各组间IL-10水平的差异无统计学意义。Crry可以明显抑制MLR中的细胞增殖（P〈0．05）。结论补体调节蛋白Crry能刺激CD4^＋T淋巴细胞的增殖，并使其IL-4的表达升高及抑制IL-2和γ-IFN的表达，从而诱导同种移植免疫低反应性。

Objective To investigate the allograft immune hyporesponsiveness induced by complement regulatory protein Crry as well as CD4^＋T lymphocytes involved. Methods CD4^＋T cells were isolated from spleens of C57BL/6 mice by magnetic-activated cell sorting, and CD4^＋T cell proliferation was induced by anti-CD3, anti-CD28, anti-Crry, co-stimulations of anti-CD3/anti-CD28, anti-CD3/anti-Crry, or the monoclonal antibody panel against CD3/CD28/Crry. Production of interleukin-2 （IL-2）, y-interferon （y-IFN）, interleukin-10 （IL-10） and interleukin-4 （IL-4） was detected in the supernatants of different groups. Allo-MLR was examined by MTT colorimetry with spleen cells from BALB/c mice as the stimulators and spleen cells of C57BL/6 mice as responders. Results Compared with CD3 group, CD3/CD28, CD3/Crry and CD3/CD28/Crry co-stimualtion all could significantly induce more proliferation （P〈0. 05）. CD3/CD28/Crry co-stimulation significantly increased the proliferation compared with CD3/CD28 or CD3/Crry co-stimulation （P〈0. 05）. IL-2 and y-IFN levels were significantly increased in CD3/CD28 co-stimulation as compared with CD3 or CD28 or Crry or CD3/Crry group （P-0. 05）. IL-4 levels were significantly increased in CD3/Crry co- stimulation as compared with CD3 or CD28 or Crry or CD3/CD28 groups （P〈0. 05）. There was no significant difference in IL-10 level among the different groups （P〈0. 05）. CD3/Crry co-stimulation significantly inhibited proliferation in mixed lymphocyte culture （P〈0. 05）. Conclusion Complement regulatory protein Crry induces IL4 in CD4^＋T ceils, suppresses IL2 and γ-IFN production, and induces all ograft immune hyporesponsiveness