体外制备和增殖烟曲霉特异性T细胞的研究

目的体外制备和增殖烟曲霉特异性T淋巴细胞。方法从健康志愿者外周抗凝血中分离并体外扩增DC,利用加热灭活的烟曲霉孢子作为抗原,体外共孵育制备烟曲霉孢子负载的DC,进一步将此成熟DC与源自同一个体的去除了DC细胞的外周血细胞共培养,体外诱导并扩增烟曲霉特异性T淋巴细胞。应用ELISPOT(酶联免疫斑点)技术检测活化T细胞IFN-γ的分泌情况,流式细胞仪检测细胞因子胞内合成情况,并分析功能细胞的类型和比例。结果 ELISPOT分析显示:PBMC+DC+Conidia实验组IFN-γ分泌(87.33±1.33/4.0×105)高于其他对照组,具有统计学意义(P0.05)。细胞因子流式细胞仪分析显示:PBMC+DC+Conidia组中,2.76%的细胞分泌IFN-γ,其中1.61%为CD4+T细胞,与各对照组相比具有统计学意义(P0.05)。获得的烟曲霉特异性T细胞可以在体外可进行大量增殖。结论本文结果显示烟曲霉孢子在体外可以作为变应原诱导产生烟曲霉特异性CD4+T细胞介导的Th1型免疫反应,为未来制备和扩增烟曲霉特异性T细胞及过继免疫治疗侵袭性曲霉病提供实验基础。

Generation and expansion of Aspergillus fumigatus specific T cells in vitro

Objective To generate and proliferate Aspergillus fumigatus （AF） specific T cells for immunotherapy of aspergillosis in vitro.Methods Monocyte-derived dendrictic cells （DCs） were seperated and proliferated from whole blood of healthy individuals.Aspergillus fumigatus loaded DCs were generated and cultured with peripheral blood mononuclear cells （PBMCs） descreted with DCs.Then Aspergillus fumigatus specific T lymphocytes were induced.IFN-γ secreation,cytokine synthesis,type and ratio of function cells were examined.Results ELISPOT analysis showed IFN-γ secretion in experimental group （87.33±1.33/4.0×105） was significant higher than that in control groups （P0.05）.Flow cytometric analysis showed 2.76% of total cells in PBMC＋DC＋Conidia group secreted IFN-γ,including 1.61% of CD4 positive,which is significantly higher than those in other groups （P0.05）.AF-specific T cells expanded to a large number in vitro.Conclusions Conidia of AF is a potent immunogen to induce CD4＋ T cell mediated specific Th1 type immune response in vitro and provide basic data on generation and expansion of AF-specific T cells for immunotherapy of IA in the future.