CTLA4Ig可诱导T淋巴细胞对氧化修饰低密度脂蛋白的免疫无反应性

CTLA4Ig can induce T lymphocytes anergy to oxidized low density lipoprotein

目的研究融合蛋白CTLA4Ig对氧化修饰低密度脂蛋白(ox-LDL)诱导的T淋巴细胞特异性免疫反应的作用,探索预防和延缓动脉粥样硬化发生和发展的新途径。方法从健康成人外周血分离单核细胞,加入500IU/ml的重组人粒单细胞集落刺激因子(rhGM-CSF)和500IU/ml的重组人白细胞介素4(rhIL-4),培养6d。取1×106个细胞以流式细胞仪检测CD14的表达,其余细胞分为4组,分别加入细菌脂多糖(LPS)30ng/m(lLPS组)、LDL10μg/ml(LDL组)、ox-LDL10μg/ml(ox-LDL组)和相同体积的PBS(PBS组),作用48h,以流式细胞仪检测CD86和HLA-DR的阳性表达率和平均荧光强度(MFI)。将各组DC与同种异体T淋巴细胞以1:5和1:10的比例混合进行同种异体混合淋巴细胞增殖反应(MLR),其中ox-LDL组在1:5的试验中分为4个亚组,分别给予加入1.25、0.62、0.31μg/ml的CTLA4Ig和不加CTLA4Ig的处理,各组细胞继续培养4d,以噻唑蓝(MTT)比色法检测T淋巴细胞增殖活性,结果以刺激指数(SI)表示。结果细胞培养6d后CD14的阳性表达率为2.9%,证实已由单核细胞转化为DC。LPS组、ox-LDL组CD86阳性表达率(96.0%±8.8%,97.7%±11.4%)和HLA-DR阳性表达率(90.3%±8.8%,90.9%±7.0%)均明显高于LDL组(90.0%±10.2%,84.4%±9.6%)和PBS组(87.3%±8.4%,83.6%±7.0%)(均P<0.05),ox-LDL组CD86MF(I73.4±6.6)和HLA-DRMFI(87.0±7.1)均明显高于LDL组(40.0±7.4,55.0±7.7,均P<0.01)和PBS组(54.6±8.2,P<0.01;70.2±6.7,P<0.05)。在MLR中,DC:T细胞=1:5和1:10两种条件下,LPS组、ox-LDL组SI均明显高于LDL组和PBS组(均P<0.05)。在ox-LDL组的MLR中,以1.25、0.62、0.31和0μg/ml的CTLA4Ig处理的各亚组SI分别为0.96±0.30,1.12±0.33,1.29±0.28和1.64±0.37,CTLA4Ig浓度为1.25μg/ml时,ox-LDL激发的同种异体MLR已被完全抑制。结论ox-LDL可作为抗原被DC递呈,激发同种异体MLR;CTLA4Ig能明显抑制该作用,诱导T细胞对ox-LDL的免疫无反应。这一结果为动脉粥样硬化防治的研究提示了新的思路。

Objective To investigate the effect of CTLA4Ig on oxidized low density lipoprotein (ox-LDL) induced T lymphocyte-specific immune reaction. Methods Monocytes were separated from the peripheral blood of healthy adults and cultured with rhGM-CSF 500 IU/ml and rhIL-4 500 IU/ml for 6 days to form dentritic cells (DC). Then 1×106 of the cells were harvested to analyze the expression of CD14 by flow cytometry (FCM). The other cells were treated with LPS (30 ng/ml, LPS group), LDL (10 μg/ml, LDL group), ox-LDL (10 μg/ml, ox-LDL group), or PBS in the same volume (control group) respectively for 48 hours. The rates of positive expression of CD86 and HLA-DR and their mean fluorescence intensity (MFI) were detected by FCM. The treated DC were mixed with allogeneic T lymphocytes at ratios of 1:5 and 1:10, and cultured for another 4 days. In the ox-LDL group, the DC that were mixed with allogeneic T lymphocytes at a ratio of 1:5 were subdivided into 4 groups, treated with CTLA4Ig of different concentrations (0, 1.25, 0.62, and 0.31 μg/ml, respectively). The proliferation of T lymphocytes was analyzed by MTT method, and the result was expressed as stimulation index (SI). Results The rate of positive expression of CD14 was 2.9%. In the LPS and ox-LDL groups, the rates of positive expression of CD86 and HLA-DR (96.0%±8.8% and 97.7%±11.4% for CD86, and 90.3%±8.8% and 90.9%±7.0% for HLA-DR) were significantly higher than those in the LDL group (90.0%±10.0% for CD86, and 84.4%±9.6% for H2A-DR, P<0.05) and control group (87.3%±8.4% for CD86, and 83.6%±7.0% for H2A-DR, P<0.05). The MFI of CD86 and HLA-DR in the ox-LDL group (73.4±6.6 and 87.0±7.1) was significantly higher than those in the LDL (40.0±7.4, P<0.01; 55.0±7.7, P<0.05) and control groups (54.6±8.2, P<0.01; 70.2±6.7, P<0.05). The SI (DC:T=1:5) elicited by DC treated with PBS, LDL, ox-LDL, and LPS were 1.32±0.23, 1.42±0.29, 1.67±0.32, and 1.75±0.32 respectively. And the SI of DC treated with ox-LDL and LPS were significantly higher than that of DC treated with PBS and LDL (P<0.05). In the 3 subgroups of the ox-LDL group treated with different concentrations of CTLA4Ig, the SI were 0.96±0.30, 1.12±0.33, and 1.29±0.28, respectively, which were significantly lower than that in the subgroup treated without CTLA4Ig (1.64±0.37; P<0.01, P<0.01, and P<0.05, respectively). Conclusion CTLA4Ig can inhibit the ox-LDL induced allogeneic mixed lymphocyte reaction, and induce T lymphocytes anergy to ox-LDL in a dose-dependent manner.