负载异体抗原对低剂量粒细胞巨噬细胞集落刺激因子诱导的树突状细胞免疫学性状的影响

The influence of antigen loading on the immunological characteristics of dendritic cells induced by low concentrations of granulocyte macrophage colony stimulating factor

目的观察低剂量粒细胞巨噬细胞集落刺激因子(GM-CSF)诱生的未成熟树突状细胞(GM^(low)DC)对异体抗原的摄取能力,以及负载异体抗原后细胞表型和功能的改变。方法对处于增殖期的C_(57)BL/6小鼠单个核细胞(MNC)作氚标记亮氨酸(~3H-Leu)掺入处理,制备~3H-Leu标记的抗原上清液。将此抗原上清液分别与昆明小鼠GM^(low)DC和成熟树突状细胞(DC)孵育30、60、90 min,检测细胞每分钟放射性荧光闪烁计数(cpm)值；用流式细胞仪检测负载异体抗原前后GM^(low)DC表面I^A/I^E、CD80分子的表达情况。分离C_(57)BL/6小鼠的T淋巴细胞,根据加入的刺激因素分组并进行同种混合淋巴细胞反应(MLR)：对照组(不加刺激因素)、GM^(low)DC组、GM^(low)DC+异体抗原组、GM^(low)DC+异体抗原+细胞毒性T淋巴细胞相关抗原4Ig(CTLA-4Ig)组。检测各组细胞cpm值,计算刺激指数(SI)。结果加入异体抗原上清液后30、60、90 min,GM^(low)DC的cpm值均明显高于同时相点的成熟DC(P＜0．05或0．01)。负载异体抗原前,GM^(low)DC细胞表面I^A/I^E的表达率为(32±8)%,CD80的表达率为(25±10)%；负载后两者分别为(54±10)%、(71±18)%,均明显升高(P＜0．05或0．01)。MLR：GM^(low)DC+异体抗原组细胞cpm值明显高于对照组(P＜0．05),SI＞2．0；GM^(low)DC组以及GM^(low)DC+异体抗原+CTLA-4Ig组细胞cpm值与对照组比较,差异无统计学意义(P＞0．05),AI均＜2．0。结论GM^(low)DC具有较强的抗原摄取能力,负载抗原后,细胞在表型及功能上渐趋成熟。应用CTLA-4Ig可阻断其免疫应答效应,建立免疫耐受。

Objective To investigate the influence of low doses of granulocyte macrophage colony stimulating factor on the allogeneic antigen （Ag） ingestion capacity of immature dendritic cells （GM^lowDC ）, and subsequently the changes in the cellular phenotype and function. Methods Mononuclear cells from C57BL/6 mice was labelled with 3H-Leu to make Ag supernatant. The Ag supernatant was cocuhured with GM^low DC or mature DC for 30,60 and 90 mins, then cpm value were determined. The changes in I^A/I^B and CD80 on cell surface after antigen ingestion were determined with flow cytometry （FCM）. By using mixed lymphocyte reaction（MLR） , the cells were divided into control（ non-sensitized T lymphocyte） , GM^low DC , GM^low DC and allogeneic antigen, GM^low DC and allogeneic antigen and CTLA-4 Ig groups. The cpm value in each group was recorded and the stimulation index （SI） was calculated. Results Upon 30,60 and 90 mins of allogeneic Ag stimulation, the cpm value of GM^lowDC was obviously higher than that of mature DC （ P 〈0.05 or 0.01 ）. In addition, the expression of I^A/I^B and CD80 before allogeneic Ag ingestion were significantly higher than those after Ag ingestion（ I^A/I^B ：32 ±8% vs 54 ± 10, P 〈 0.05; CD80：25 ± 10% vs 71 ± 18% , P 〈0.01 ）. MLR： Compared with control group, the cpm value in GM^low DC with allogeneic Ag group was increased markedly（ P 〈0.05） , with SI higher than 2.0, while no difference was found among control, GM^low DC group, GM^low DC and allogeneic Ag and CTLA-4Ig groups（ P 〉0.05）, with SI lower than 2.0 Conclusion Though GM^low DC exhibits powerful antigen ingestion capacity, the cell pheonotype and function will get mature gradually. Immune tolerance can be established by incubating GM^lowDC with CTLA-4Ig.