新型冠状病毒肺炎康复个体HLA-A^(*)02限制性CD8^(+)T细胞免疫应答研究

Research on HLA-A^(*) 02 restricted CD8^(+) T cell immune responses in recovered individuals with COVID-19

目的 研究严重急性呼吸综合征冠状病毒2(SARS-CoV-2)感染个体康复4~5个月后外周血中HLA-A^(*)02限制性SARS-CoV-2表位特异性CD8^(+)T细胞应答水平及规律。方法 本研究在以往鉴定获得SARS-CoV-2刺突蛋白来源HLA-A^(*)0201限制性CD8^(+)T细胞9肽表位ALNTLVKQL和YLQPRTFLL的基础上,应用流式细胞术筛选入组HLA-A^(*)02阳性SARS-CoV-2灭活疫苗接种后感染且康复4~5个月后个体共27例,构建HLA-A^(*)02/9肽四聚体,分别应用酶联免疫斑点实验、四聚体(tetramer)染色技术及胞内细胞因子染色等方法,对康复个体外周血中SARS-CoV-2表位特异性的功能性CD8^(+)T细胞频率、表型以及分泌细胞因子和杀伤介质的能力进行检测。结果 HLA-A^(*)02^(+)康复个体外周血中可分别检测到分泌IFN-γ的SARS-CoV-2 9肽表位ALNTLVKQL和YLQPRTFLL特异性CD8^(+)T细胞应答,且tetramer^(+)CD8^(+)T细胞频率仍维持较高水平。表位特异性CD8^(+)T细胞主要表现为CCR7-CD45RA-效应记忆表型和CD27-CD28-高分化表型,可分泌IL-2、IFN-γ和TNF-α等多种细胞因子,以及颗粒酶B和穿孔素杀伤介质。结论 SARS-CoV-2感染康复后外周血中存在着HLA-A^(*)02限制性SARS-CoV-2表位特异性CD8^(+)T细胞应答,且其应答频率可至少持续至康复后4~5个月,仍具有抗病毒的功能和效应。

Objective To investigate the HLA-A^(*)02 restricted severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)epitope-specific CD8+T cell responses in peripheral blood of individuals infected with SARS-CoV-2 after 45 months of rehabilitation.Methods Based on the previously identified HLA-A^(*)0201 restricted SARS-CoV-2 spike protein CD8+T cell 9-mer peptide epitopes ALNTLVKQL and YLQPRTFLL,flow cytometry was used to screen a total of 27 HLA-A^(*)02 positive individuals who had received SARS-CoV-2 inactivated vaccines and then infected with SARS-CoV-2 and recovered for 45 months after infection.HLA-A^(*)02/SARS-CoV-29-mer peptide tetramers were constructed.Enzyme-linked immunospot assay,tetramer staining and intracellular cytokine staining were used to detect the frequency,phenotype,and ability for cytokines and cytotoxic mediator secretion of SARS-CoV-2 epitope-specific functional CD8+T cells in the peripheral blood of recovered individuals,respectively.Results SARS-CoV-29-mer peptide epitopes ALNTLVKQL and YLQPRTFLL specific CD8+T cell responses with IFN-γsecretion could be detected respectively in the peripheral blood of HLA-A^(*)02+recovered individuals,and the frequency of tetramer+CD8+T cells remained at a high level in the recovered individuals.SARS-CoV-2 epitope-specific CD8+T cells mainly exhibited the CCR7-CD45RA-effector memory phenotype and the CD27-CD28-highly differentiated phenotype,which could secrete a variety of cytokines such as IL-2,IFN-γand TNF-α,as well as cytotoxic mediators granzyme B and perforin.Conclusion HLA-A^(*)02 restricted SARS-CoV-2 epitope-specific CD8+T cell responses can be detected in peripheral blood after recovery from SARS-CoV-2 infection,and its response frequency can last at least 45 months after recovery,which still has antiviral functions and effects.