Tetramer-assisted identification and characterization of epitopes recognized by HLAA*2402-restricted Epstein-Barr virus-specific CD8+ T cells
Tetramer-assisted identification and characterization of epitopes recognized by HLAA*2402-restricted Epstein-Barr virus-specific CD8+ T cells
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DOI：
10.1182/blood-2002-04-1240
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发表时间：
2003-02-15
期刊：
影响因子：
20.3
通讯作者：
Tsurumi, T
中科院分区：
文献类型：
--
作者：
Kuzushima, K;Hayashi, N;Tsurumi, T
我们通过计算机辅助算法和酶联免疫斑点（ELISPOT）试验筛选细胞毒性T淋巴细胞（CTL）表位，使用体外再活化的多克隆EB病毒（EBV）特异性CD 8（+）T细胞作为应答者。此外，为了证实表位是在内源性加工和呈递EBV蛋白后产生的，引入了一种新的T细胞受体（TCR）下调测定法，其中采用荧光四聚体主要组织相容性复合物（MHC）/肽复合物来检测抗原呈递细胞上呈递的表位刺激后TCR的下调。通过这样的筛选，鉴定了3个HLAA*2402限制性表位：IYVLVMLVL、TYPVLEEMF和DYNFVKQLF，分别来源于LMP 2、BRLF 1和BMLF 1蛋白。TCR下调分析揭示，与其他2个表位相反，IYVLVMLVL在用表达LMP 2的重组牛痘病毒感染的HLAA 24阳性成纤维细胞上不呈递。此外，ELISPOT测定与表位特异性CTL克隆表明，该介绍是部分恢复预处理的成纤维细胞与干扰素-γ。该表位被呈递在与抗原加工（TAP）阴性T2细胞相关的转运蛋白上，该T2细胞用编码HLA A*2402和最小表位的质粒转染，表明该呈递是TAP独立的。总之，由此定义的3个表位可用于研究HLAA*2402阳性人群中EBV特异性CD 8（+）T细胞应答。(C)2003年，美国血液学会。
We determined cytotoxic T lymphocyte (CTL) epitopes through screening with a computer-assisted algorithm and an enzyme-linked immunospot (ELISPOT) assay using in vitro-reactivated polyclonal Epstein-Barr virus (EBV)-specific CD8(+) T cells as responders. In addition, to confirm that the epitopes were generated after endogenous processing and presentation of the EBV proteins, a novel T-cell receptor (TCR) down-regulation assay was introduced, in which a fluorescent tetrameric major histocompatibility complex (MHC)/peptide complex was employed for detecting TCR down-regulation after stimulation with the epitope presented on antigen-presenting cells. Through such screening, 3 HLAA*2402-restricted epitopes were identified: IYVLVMLVL, TYPVLEEMF, and DYNFVKQLF, derived from LMP2, BRLF1, and BMLF1 proteins, respectively. TCR down-regulation assays disclosed that, in contrast to the other 2 epitopes, IYVLVMLVL was not presented on HLAA24-positive fibroblast cells infected with recombinant vaccinia viruses expressing LMP2. Furthermore, ELISPOT assays with an epitope-specific CTL clone demonstrated that the presentation was partially restored by pretreatment of the fibroblast cells with interferon-gamma. The epitope was presented on transporters associated with antigen processing (TAP)-negative T2 cells transfected with plasmids encoding HLA A*2402 and the minimal epitope, indicating that the presentation is TAP independent. In conclusion, the 3 epitopes thus defined could be useful for studying EBV-specific CD8(+) T-cell responses among populations positive for HLAA*2402. (C) 2003 by The American Society of Hematology.