酶联免疫斑点直接法与预孵育法检测胰岛抗原特异性T细胞反应的比较

Comparison of islet autoantigen-specific T cell response detected by direct enzyme-linked immunospot(ELISPOT)assay and accelerated co-cultured dendritic cells(acDCs)assay

目的探讨酶联免疫斑点(ELISPOT)加速树突状细胞成熟的预孵育法(acDCs)和直接检测法检测1型糖尿病(T1DM)患者外周血中胰岛全长抗原特异性T细胞反应的优劣。方法选取2012年3月至2014年8月中南大学湘雅二医院代谢内分泌科就诊的T1DM患者16例(男9例,女7例),年龄(28.5±9.4)岁,病程6.0(2.8~8.3)个月;另纳入年龄、性别匹配的健康对照12名;ELISPOT-acDCs法和直接检测法检测3种胰岛抗原谷氨酸脱羧酶65(GAD65)、C肽、胰岛素(INS)特异性干扰素-γ(IFN-γ)分泌性CD4+T细胞反应的斑点数,比较不同检测方法下各种胰岛抗原及联合3种抗原特异性T细胞反应的阳性比例。结果ELISPOT-acDCs法检测T1DM患者GAD65特异性IFN-γ分泌性CD4+T细胞反应的阳性比例为1/16,INS反应阳性比例为6/16,C肽反应阳性比例为4/16,其中INS反应阳性比例高于健康对照组的0/12(P=0.024);联合3种抗原的T细胞阳性比例为9/16,明显高于健康对照组的1/12(P=0.016)。ELISPOT直接法检测GAD65特异性T细胞反应阳性比例为2/16,INS反应阳性比例为1/16,C肽反应阳性比例7/16,其中C肽反应阳性比例高于健康对照组的1/12,但差异无统计学意义(P=0.088)。联合3种抗原检测T细胞反应阳性比例8/16,高于健康对照组的1/12(P=0.039);与直接法比较,仅acDCs法检测下的INS特异性T细胞反应阳性比例较高(P=0.041);两种方法在余各抗原间比较差异均无统计学意义(均P>0.05)。结论多种全长胰岛抗原ELISPOT-acDCs法及直接法检测胰岛抗原特异性T细胞反应均可为T1DM患者提供细胞免疫学分型诊断价值;ELISPOT-acDCs法检测INS特异性T细胞反应的灵敏度优于ELISPOT直接检测法。

Objective To investigate the effect of enzyme-linked immunospot(ELISPOT)on accelerated co-cultured dendritic cells(acDCs)and direct detection of islet full-length antigen-specific T cell response in peripheral blood of patients with type 1 diabetes mellitus(T1DM).Methods Sixteen patients with T1DM[9 males,7 females,mean age(28.5±9.4)years]and 12 age-and sex-matched healthy controls were selected in the Department of Metabolism and Endocrinology,the Second Xiangya Hospital between March 2012 and August 2014.The numbers of IFN-γsecreting CD4+T cells responding to glutamic acid decarboxylase 65(GAD65),C-peptide(CP)and insulin(INS)were detected by ELISPOT-acDCs and ELISPOT-direct assays,respectively.The positive rate of islet autoantigen and associated antigen reactive T cells under different detection assays were compared.Results The positive rate for GAD65,INS,and CP antigen reactive T cells detected by ELISPOT-acDCs was 1/16,6/16 and 4/16,respectively,and T cells positive for INS in T1DM patients were higher than that in the controls(0/12)(P=0.024).Combining GAD65,CP and INS-ELISPOT-acDCs detection,the positive rate for CD4+T cells in T1DM patients was higher than that in the controls(9/16 vs 1/12,P=0.016).The positive rate for GAD65,INS,and CP antigen reactive T cells detected by ELISPOT-direct detection was 2/16,1/16 and 7/16,respectively,and T cells positive for CP was higher than that in the controls(1/12),but the difference was not statistically significant(P=0.088).Likewise,the positive rate for CD4+T cells was higher in T1DM patients than that in the controls by combined GAD65,CP and INS-ELISPOT-direct detection(8/16 vs 1/12,P=0.039).Compared with the ELISPOT-direct assay,the positive rate of INS antigen specific T cell response detected by ELISPOT-acDCs was higher(P=0.041).No statistical differences of other antigens were found between the two groups(all P>0.05).Conclusions Both multiple islet antigens-combined CD4+-ELISPOT-acDCs and direct assays could provide diagnostic value of cellular immunology for T1DM patients.The ELISPOT-acDCs assay is superior to the ELISPOT-direct assay in the detection of INS antigen-specific T cell response.