T细胞IFN-g表达水平检测方法的建立及其在马传染性 贫血疫苗免疫机理研究中的应用

A flow cytometric assay for the expression of interferon gamma in T lymphcytes and its application in the study of EIAV-induced immune response

摘要:

【目的】马传染性贫血病毒（EIAV）弱毒疫苗致弱机制和免疫保护机理的研究可以为慢病毒疫苗的研究提供重要的模型。为探讨IFN-g 表达水平与疫苗保护性免疫的关系，本研究旨在建立一种准确、有效地检测EIAV感染马不同T细胞亚型表达IFN-g 水平的方法。【方法】我们将分离的马传贫弱毒疫苗免疫马（FDDV）、强毒感染马（LV）和健康马的外周血单核细胞(PBMC)，体外分别经病毒（FDDV）和PMA/Inomycin激活、 BFA 阻断蛋白分泌、荧光标记马的特异性表面抗体和IFN-g 抗体等过程后，进行流式检测。【结果】疫苗免疫马产生的特异性IFN-g 水平为CD4+1.7±0.9%/CD8+ 6.1±1.2%，而强毒组则为CD4+ 0.6±0.1%/CD8+ 2.4±0.9%。【结论】本研究建立的多荧光参数流式细胞术同时检测细胞内IFN-g 染色和淋巴细胞亚型的方法，具有良好的特异性，稳定性和重复性。为研究EIAV弱毒疫苗免疫保护机制奠定了基础。

Abstract:

[Objective] The attenuated vaccine of equine infectious anemia virus (EIAV) is the first lentiviral vaccine that provides solid protection against the infection of EIAV virulent strains. Study of the immune response induced by EIAV vaccine is an important approach to understand the immunity to other lentiviruses. IFN-g expressed by specifically stimulated lymphocytes is an important indicator for the evaluation of T cell-mediated immunity. A flow cytometry based assay was established in this study to accurately and effectively detect IFN-g expression in different subtypes of T lymphocytes in EIAV-infected horses. [Methods] Peripheral blood mononuclear cells (PBMC) were prepared from horses inoculated with EIAV vaccine stain FDDV (fetal donkey dermal-attenuated virus vaccine), virulent strain LV or saline (health control), were stimulated in vitro with FDDV or PMA + Inomycin. Brefeldin A was added into the cell culture to block protein secretions. Stimulated cells were then labeled with monoclonal antibodies specific for equine CD4 and CD8 surface markers, as well as an IFN-g-specific monoclonal antibody. Flow cytometry was applied to detect CD4+ and CD8+ lymphocytes expressing IFN-g. [Results] IFN-g positive cell population isolated from FDDV-immunized horses was 1.7±0.9% in CD4+ T cells and 6.1±1.2% in CD8+ T cells (n=4). In contrast, only 0.6±0.1% of IFN-g positive cells in CD4+ subset and 2.4±0.9% of IFN-g positive cells in CD8+ subset of T cells were detected for PBMC isolated from LV-infected horses (n=4). [Conclusion] The multi-fluorescence cell flowmetry detecting both the IFN-g expressing cells and subsets of T lymphocytes simultaneously, is specific, stable and reproducible in evaluating antigen-specific response of IFN-g- producing lymphocytes. This is an essential approach to study the protective immunity induced by EIAV vaccine.