Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes.
Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes.
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DOI：
10.1084/jem.187.9.1383
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发表时间：
1998-05-04
期刊：
影响因子：
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通讯作者：
Zinkernagel R
中科院分区：
文献类型：
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作者：
Gallimore A;Glithero A;Godkin A;Tissot AC;Plückthun A;Elliott T;Hengartner H;Zinkernagel R
本研究描述了由小鼠I类分子H-2Db、化学生物素化的β2微球蛋白和来自淋巴细胞性脉络丛脑膜炎病毒（LCMV）糖蛋白（GP;氨基酸33-41）的肽表位组成的可溶性主要组织相容性复合物的构建。四聚体I类复合物，这是通过混合的I类复合物与藻蓝蛋白标记的中性抗生物素蛋白，允许直接分析的病毒特异性细胞毒性T淋巴细胞（CTL）的流式细胞术。通过（a）对转基因小鼠脾脏中的CD 8+细胞进行染色和（B）对颅内感染LCMV-DOCILE的C57 BL/6（B6）小鼠的脑脊液中的病毒特异性CTL进行染色来验证该技术，所述转基因小鼠表达与肽GP 33 -41相关的H-2Db特异性T细胞受体（TCR）。从B6小鼠分离的脾细胞的染色显示，在LCMV感染的初始阶段，高达40%的CD 8 + T细胞是GP 33四聚体+。相比之下，GP 33四聚体不染色从B6小鼠的脾脏分离的CD 8 + T细胞，所述B6小鼠在2个月前用高于在幼稚小鼠中发现的背景水平的LCMV感染。在用高或低剂量的LCMV-DOCILE颅内和静脉内攻击的小鼠中，在感染的急性期期间分析病毒特异性CTL的命运。研究结果表明，LCMV感染的结果仅由抗原载量决定。此外，数据表明在过量抗原存在下病毒特异性CTL的缺失在TCR下调之前，并且依赖于穿孔素。
This study describes the construction of soluble major histocompatibility complexes consisting of the mouse class I molecule, H-2Db, chemically biotinylated β2 microglobulin and a peptide epitope derived from the glycoprotein (GP; amino acids 33–41) of lymphocytic choriomeningitis virus (LCMV). Tetrameric class I complexes, which were produced by mixing the class I complexes with phycoerythrin-labeled neutravidin, permitted direct analysis of virus-specific cytotoxic T lymphocytes (CTLs) by flow cytometry. This technique was validated by (a) staining CD8+ cells in the spleens of transgenic mice that express a T cell receptor (TCR) specific for H-2Db in association with peptide GP33–41, and (b) by staining virus-specific CTLs in the cerebrospinal fluid of C57BL/6 (B6) mice that had been infected intracranially with LCMV-DOCILE. Staining of spleen cells isolated from B6 mice revealed that up to 40% of CD8+ T cells were GP33 tetramer+ during the initial phase of LCMV infection. In contrast, GP33 tetramers did not stain CD8+ T cells isolated from the spleens of B6 mice that had been infected 2 mo previously with LCMV above the background levels found in naive mice. The fate of virus-specific CTLs was analyzed during the acute phase of infection in mice challenged both intracranially and intravenously with a high or low dose of LCMV-DOCILE. The results of the study show that the outcome of infection by LCMV is determined by antigen load alone. Furthermore, the data indicate that deletion of virus-specific CTLs in the presence of excessive antigen is preceded by TCR downregulation and is dependent upon perforin.