HLA-A2+供者外周血hCMV特异性CTL的定量及其表型分析

目的:利用加载人巨细胞病毒(hCMV)结构蛋白pp65衍生抗原肽NLV(pp65495-503)的HLA-A0201(A2-NLV)四聚体,探讨四聚体染色条件的优化及其在特异性CTL表型分析中的应用。方法:取HLA-A2 供者外周血,以A2-NLV四聚体/PE在不同条件下染色,然后以anti-CD3-FITC和anti-CD8-APC标记,流式细胞术分析染色结果,寻找四聚体最佳染色条件,并分析特异性CTL的表型和活化抗原表达。结果:以全血样进行四聚体染色结果优于分离的PBMC。A2-NLV四聚体用量为0.3μg时,与100μL全血于4℃温育1h,特异性染色效果最佳,而与CD8-T细胞非特异性结合很低。以此优化条件进行特异性CTL表型分析,结果显示CD28阳性的A2-NLV四聚体特异性CTL的百分率较非特异性CTL低,表达CD57的百分率较非特异性CTL高,CD25分子只表达于活化的特异性CTL上。结论:通过对染色条件进行优化可明显提高四聚体染色的特异性,降低非特异性结合,优化的四聚体染色法能用于抗原特异性CTL的表型和功能分析。

Quantification and phenotypic analysis of hCMV specific CTL in peripheral blood from HLA-A2+ donors

AIM: To optimize tetramer staining condition using HLA-A*0201 tetramer (A2-NLV tetramer) loaded with NLV peptide (pp65(495-503)) derived from structural protein pp65 of human cytomegalovirus and to investigate its application in phenotyping of specific cytotoxic T lymphocytes (CTL). METHODS: Peripheral blood from HLA-A2(+) donors was first stained with A2-NLV tetramer/PE under different conditions and then labeled with anti-CD3-FITC and anti-CD8-APC. The stained samples were analyzed with flow cytometry to find out the optimized staining condition. Meanwhile, the phenotype and activation antigen expression were determined. RESULTS: Tetramer staining with whole blood was superior to peripheral blood mononuclear cells. The optimized condition for tetramer staining was incubating 100 muL of whole blood with 0.3 mug of A2-NLV tetramer for 1 h at 4 degrees Celsius. Under this condition the specific staining was strong while unspecific staining of CD8(-) T cells was quite weak. Phenotypic analysis under this condition showed that the ratio of CD28 positive A2-NLV tetramer specific CTL was lower than that of nonspecific CTL, whereas the ratio of CD57 positive specific CTL was higher than that of nonspecific CTL. CD25 molecules were only expressed on the activated specific CTL. CONCLUSION: The optimized tetramer staining condition can increase the specificity of tetramer staining and decrease unspecific binding, therefore it is applicable for phenotyping and functional analysis of antigen-specific CTL.