HLA-A~*0201/WT1五聚体联合胞内IFNγ染色在检测白血病患者外周血WT1特异性T细胞中的应用

Application of HLA-A~*0201/WT1 Pentamer Combined with Intracellular IFNγ^+ Staining in Detecting Circulating WT1 Specific T Cells in Leukemia

本研究探讨五聚体及胞内因子染色在定性定量检测抗原特异性T细胞中的应用价值。用RT-PCR方法检测受试者WT1表达水平;用HLA-A*0201/WT1五聚体及胞内IFNγ染色检测14例表达HLA-A*0201的白血病患者全相合移植前后及其供者外周血中的WT1特异性T细胞。结果表明:14例供者中2例有低水平WT1表达,白血病患者均有不同水平的WT1表达。14例供者均未检测到WT1+CD8+CTL和WT1+IFNγ+细胞;移植前14例患者中2例可检出WT1+CD8+CTL,3例检出WT1+IFNγ+细胞,二者间无明显差异(p=0.357),而移植后均可检出WT1+CD8+CTL和WT1+IFNγ+细胞,明显高于移植前(p值均<0.01)。移植后大部分患者均在30天内检出WT1+CD8+CTL和WT1+IFNγ+细胞,但后者检出率高于前者(p=0.014)。14例患者中WT1+CD8+CTL峰值中位数为0.18%,而WT1+IFNγ+细胞峰值中位数为0.83%,明显高于前者(p=0.005);WT1+CD8+CTL峰值中位时间为移植后75天,WT1+IFNγ+细胞峰值中位时间为移植后105天,但二者间无明显差异(p=0.455)。结论:五聚体及胞内IFNγ染色能有效检测白血病患者外周血中白血病抗原特异性T细胞;两种方法联合检测有助于抗原特异性T细胞的准确定性定量检测。

This study was purposed to investigate the value of combination of pentamer and intracellular IFNγ staining in the qualitative and quantitative detection of circulating antigen-specific T cells. WT1 expressions in 14 HLA- A＊0201 ＋ patients and their matched donors were detected by RT-PCR, and circulating WT1 specific T cells were assayed by HLA-A＊ 0201/WT1 pentamer combined with intracellular IFNγ ＋ staining. The results showed that the low level of WTI expression was found only in 2 cases out of 14 donors, but different levels of WT1 expression could be observed in all leukemic patients. The WT1 ＋ CD8 ＋ CTL and WT1 ＋ IFNγ＋ cells did not detected in all 14 donors, but WT1 ＋ CD8 ＋ CTL cells in 2 patients and WT1＋ IFNγ＋ cells in 3 patients could be detected before transplantation respectivelly, there was no significant difference between them, while the WT1＋ CD8 ＋ CTL cells and WT1＋ IFNγ ＋ cells both could be detected in all 14 patients after transplantation, the positive detection rate after transplantation was obviously higher than that before transplantation. The WT1＋ CD8 ＋ and WT1＋ IFNγ＋ cells could be detected within 30 days after transplantation, but the positive detection rate of WT1 ＋ IFNγ cells was higher than that of WT1＋ CD8 ＋ CTL cells （p =0.014）. The median peak value of WT1 ＋ CD8 ＋ CTL cells was 0.18% in 14 patients, and the median peak value of WT1 ＋ IFNγ cells was 0.83% in 14 patients, the later was significantly higher than former. The median peak time of WT1＋ CD8＋ CTL cells was 75 days after transplatation, while the WT1＋ IFNγ＋ cells was 105 days after transplatation, there was no significant difference beween them. It is concluded that pentamer and intracellular IFNγ staining may effectively detect circulating WT1 specific T cells in leukemic patients, and the combination of these two methods profit to the exact qualitation and quantitation of circulating antigen-specific T cells.