Efficient identification of HLA-A*2402-restricted cytomegalovirus-specific CD8+ T-cell epitopes by a computer algorithm and an enzyme-linked immunospot assay
Efficient identification of HLA-A*2402-restricted cytomegalovirus-specific CD8+ T-cell epitopes by a computer algorithm and an enzyme-linked immunospot assay
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DOI：
10.1182/blood.v98.6.1872
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发表时间：
2001-09-15
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
由病毒特异性细胞毒性T淋巴细胞（CTL）识别的抗原肽是研究CTL应答的有用工具，所述CTL应答仅在那些拥有呈递所述肽的主要组织相容性复合体（MHC）I类分子的人中进行。为了拓宽应用，非常需要在给定MHC的背景下确定此类表位的有效策略。提出了一种快速有效的策略，通过计算机辅助算法和MHC稳定化和酶联免疫斑点试验组成的多重筛选，在给定的MHC分子的背景下确定CTL表位。从83个候选肽中鉴定出HLA-A*2402分子递呈的巨细胞病毒（CMV）低基质65 kd磷蛋白（pp 65）的主要CTL表位QYDPVAALF。结果表明，CMV特异性CTL应答在HLA-A*2402的背景下高度集中于pp 65。用肽特异性CD 8（+）T细胞克隆作为效应细胞，用表达pp 65基因的重组痘苗病毒或CMV感染的自体成纤维细胞作为抗原呈递细胞，证实了内源性加工和呈递。细胞内干扰素-γ产生的流式细胞术分析显示，HLA-A24（+）和CMV血清阳性供体外周血中0.04%至0.27%的CD 8（+）T细胞对该肽具有特异性。四聚体MHC-肽复合物特异性结合来自血清阳性供体的外周血中的反应性T细胞克隆和0.79%的CD 8（+）T细胞。该肽可能是一个有用的试剂，研究CTL反应的CMV人群中的HLA-A*2402阳性。
Antigenic peptides recognized by virus-specific cytotoxic T lymphocytes (CTLs) are useful tools for studying the CTL responses exclusively among those who own the major histocompatibility complex (MHC) class I molecules that present the peptides. For widening the application, an efficient strategy to determine such epitopes in the context of a given MHC is highly desirable. A rapid and efficient strategy is presented for the determination of CTL epitopes in the context of given MHC molecules of interest through multiple screenings consisting of a computer-assisted algorithm and MHC stabilization and enzyme-linked immunospot assays. A major cytomegalovirus (CMV)-specific CTL epitope, QYDPVAALF, in the amino acid sequence of its lower matrix 65 kd phosphoprotein (pp65) presented by HLA-A*2402 molecules was identified from 83 candidate peptides. The results indicate that the CMV-specific CTL response is highly focused to pp65 in the context of HLA-A*2402. Endogenous processing and presentation was confirmed using a peptide-specific CD8(+) T-cell clone as the effectors and autologous fibroblast cells infected with recombinant vaccinia virus expressing pp65 gene or CMV as anti gen-presenting cells. Flow cytometric analysis of intracellular interferon-gamma production revealed 0.04% to 0.27% of CD8(+) T cells in peripheral blood of HLA-A24(+) and CMV-seropositive donors to be specific for the peptide. The tetrameric MHC-peptide complexes specifically bound to the reactive T-cell clone and 0.79% of CD8(+) T cells in peripheral blood from a seropositive donor. The peptide could be a useful reagent to study CTL responses to CMV among populations positive for HLA-A*2402.