摘要
目的通过研究中国恒河猴外周血淋巴细胞免疫表型及其功能,为以恒河猴为模型的研究提供数据参考。方法通过优选抗体克隆和荧光配色,确定了主要淋巴细胞亚群检测和T细胞功能检测Panel,并用此方法分析15只健康中国恒河猴外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)中T、B、NK等多种细胞亚群的比例,以及在非特异性刺激条件下T细胞分泌细胞因子的能力。结果成功建立了两套多色流式Panel,Panel 1可同时检测细胞毒性T淋巴细胞(cytotoxic T-lymphocyte,CTL)、滤泡辅助性T细胞(follicular helper T cell,Tfh)、调节性T细胞(regulatory T cells,Treg)、B细胞和NK细胞等多种淋巴细胞亚群,Panel 2可检测多种T细胞亚群功能和免疫检查点分子的表达。中国恒河猴PBMCs中主要淋巴细胞亚群比例的平均值分别为T细胞(75.32±7.73)%、B细胞(13.22±7.50)%、NK细胞(0.88±0.48)%、Tfh细胞(0.73±0.27)%、Treg细胞(0.75±0.43)%、CD16+NK细胞(47.87±22.35)%、CD56+NK细胞(10.69±12.41)%。非特异性刺激后,分泌IL-2和TNF-α的CD4+T细胞比例高于CD8+T细胞,分泌CD107a和IFN-γ的CD8+T细胞比例高于CD4+T细胞,而这两种细胞分泌IL-17A的比例均较低。结论本研究建立了可在单细胞水平同时检测多个细胞表面分子和分泌因子的多色流式检测方案,能够准确、全面地分析恒河猴PBMCs中免疫细胞亚群、免疫功能以及免疫检查点分子的表达,为利用恒河猴模型进行传染病疫苗及药物研发提供新的实验方法和基础数据。
Objective To provide data reference for using Chinese rhesus macaques as research model by studying the immunophenotype and function of peripheral blood lymphocytes in Chinese rhesus macaques.Methods By optimizing antibody clones and fluorescent colors,the lymphocyte subset assay and T cell function assay panels were determined.Then the panels were used to analyze the proportion of T,B,NK and other cell subsets in peripheral blood mononuclear cells(PBMCs)in 15 healthy Chinese rhesus monkeys,and the ability of T cells to secrete cytokines after non-specific stimulation.Results Two multi-color flow cytometry analytic panels were established.Panel 1 could simultaneously detect a variety of lymphocyte subsets,including cytotoxic T lymphocytes,follicular helper T cells,regulatory T cells,B cells and NK cells.Panel 2 could detect the functions of multiple T cell subsets and the expression of immune checkpoint moleculars.The mean percentages of T,B,NK,Tfh,Treg,CD16+NK and CD56+NK cells in PBMCs of the Chinese rhesus macaques were(75.32±7.73)%,(13.22±7.50)%,(0.88±0.48)%,(0.73±0.27)%,(0.75±0.43)%,(47.87±22.35)%and(10.69±12.41)%.After non-specific stimulation,the proportion of CD4+T cells secreting IL-2 and TNF-αwas higher than that of CD8+T cells,and the proportion of CD8+T cells secreting CD107a and IFN-γwas higher than that of CD4+T cells,while the proportion of CD4+and CD8+T cells secreting IL-17A was low.Conclusions This study established a multi-color flow detection scheme that could simultaneously detect multiple cellular surface molecules and cytokines at the single cell level and could accurately and comprehensively analyze the immune cell subsets,functions and the immune checkpoint molecules in peripheral blood of Chinese rhesus macaques,providing a new experimental method and basic data for the development of vaccines and drugs against infectious diseases.
作者
李静
王书晖
王硕
李丹
刘颖
Li Jing;Wang Shuhui;Wang Shuo;Li Dan;Liu Ying(Department of Virus and Immunity Research,Center for AIDS/STD Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2023年第1期1-9,共9页
Chinese Journal of Microbiology and Immunology
基金
国家重点研发计划(2016YFE0107600)。
关键词
多色流式细胞术
中国恒河猴
淋巴细胞亚群
T细胞功能
Multi-color flow cytometry
Chinese rhesus macaques
Lymphocyte subsets
T cell function
