健康人外周血烟曲霉硫氧还蛋白还原酶抗原特异性T淋巴细胞检测与结果评价

Detection of Aspergillus fumigatus thioredoxin reductase Gli T antigen-specific T cells by ELISPOT assay in healthy individuals and result evaluation

目的为评估烟曲霉硫氧还蛋白还原酶(thioredoxin reductase,TR)抗原是否具有免疫保护作用,文中建立并优化检测健康人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中TR抗原特异性T淋巴细胞(TR/AST)分泌干扰素γ(interferonγ,IFN-γ)与白细胞介素-4(interleukin-4,IL-4)的酶联免疫斑点法(enzyme linked immunospot assay,ELISPOT)检测技术,以探讨TR抗原特异性T淋巴细胞在侵袭性曲霉病(invasive aspergillosis,IA)中的作用。方法采用方阵滴定法优化ELISPOT反应条件。以烟曲霉TR为特异性刺激物,阳性细胞刺激剂为对照,用ELISPOT技术检测20名健康人外周血PBMC分泌IFN-γ、IL-4的阳性斑点形成细胞(spot forming cell,SFC)频数。结果方阵滴定法结果显示,10μg TR抗原、健康人PBMC终浓度为3×105/孔时ELISPOT检测结果最佳。20名健康人特异性分泌IFN-γ和IL-4的SFC频数分别为15(3.5,59.5)个和0(0,0)个,IFN-γ的SFC频数显著高于IL-4,差异有统计学意义(P<0.001)。TR可刺激所有20名健康人产生IFN-γ应答,其中9例产生强IFN-γ应答(SFC>20个/3×105PBMC),占45%;而19名健康人在TR刺激后不产生IL-4应答,仅1例产生弱IL-4应答(SFC=1)。结论烟曲霉TR抗原诱导的T细胞免疫以Th1型免疫应答占绝对优势,因此,该抗原具有成为保护性抗原的潜能。

Objective The purpose of this study was to evaluate the protective potential of the Aspergillus fumigatus thioredoxin reduetase GliT（TR） antigen by establishing and optimizing ELISPOT assay for TR antigen-specific T cells （TR/AST） secreting IFN-γ and IL-4 in peripheral blood mononuclear ceils （PBMCs） and explore the role of TR/AST in invasive aspergillosis （IA）. Methods We optimized the reaction conditions of ELISPOT by preliminary checkerboard titration and determined the frequencies of positive spot-forming cells （SFCs） specifically secreting IFN-γ and IL-4 in the PBMCs of 20 healthy individuals with TR as specific stimulant and with PHA and PMA as positive controls,. Results Checkerboard titration demonstrated the best result of ELISPOT with the TR antigen at the final concentration of 10 μg/well and PBMCs at 3 × 105/well. The median frequency of IFN- γSFCs was significantly higher （ 15 [3.5, 59.5 ] ） than that of IL-4 SFCs （0 [0, 0] ） （ P 〈 0. 001 ）. TR induced IFN-γ responses in all the 20 healthy donors, including 9 cases of strong IFN-γ response （SFCs 〉 20/3 × 10^5 PBMCs）, accounting for 45% , but failed to induce IL-4 response in 19 of the healthy individuals. Conclusion The Aspergillus fumigatus TR antigen could induce an immunodominant Thl response, and therefore might be a potential protective antigen.