Targeted cloning of cytotoxic T cells specific for minor histocompatibility antigens restricted by HLA class I molecules of interest
Targeted cloning of cytotoxic T cells specific for minor histocompatibility antigens restricted by HLA class I molecules of interest
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DOI：
10.1097/01.tp.0000039166.86548.93
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发表时间：
2002-12-27
期刊：
影响因子：
6.2
通讯作者：
Takahashi, T
中科院分区：
文献类型：
--
作者：
Akatsuka, Y;Kondo, E;Takahashi, T
背景对次要组织相容性抗原（mHAgs）特异的T细胞在HLA相合骨髓移植（BMT）后的移植物抗宿主病和移植物抗肿瘤效应中起主要作用。然而，表征单个T细胞对mHAg的应答是困难的，并且通常涉及从BMT受体产生的大量T细胞培养物中分离的T细胞克隆的广泛筛选。在这份报告中，我们描述了一种新的方法，既允许直接可视化的CD 8（+）T细胞的mHAgs和克隆的T细胞与mHAgs提出的个别HLA等位基因的利益。Epstein-Barr病毒。将表达逆转录病毒转导的HLA cDNA的转化B细胞系（B-LCL）用作酶联免疫斑点（ELISPOT）测定中的刺激细胞，以鉴定从BMT后受体外周血产生的培养物中与mHAg反应的CD 8（+）T细胞。然后可以使用干扰素-γ分泌测定和磁珠选择捕获和克隆对由所选HLA等位基因呈递的mHAg特异的T细胞。由此分离的大多数T细胞克隆对用于ELISPOT测定的相同HLA转染的B细胞系表现出细胞溶解活性。ELISPOT试验可用于鉴定呈递由单个T细胞系识别的mHAg的HLA等位基因。这种用于分离mHAg特异性CD 8（+）T细胞克隆的方法应有助于表征受感兴趣的HLA等位基因限制的应答，这在某个种族群体中是常见的。
Background. T cells specific for minor histocompatibility antigens (mHAgs) play a major role in both graft-versus-host disease and the graft-versus-tumor effect after HLA-identical bone marrow transplantation (BMT). However, characterization of individual T-cell responses to mHAgs is difficult and has generally involved extensive screening of T-cell clones isolated from bulk T-cell cultures generated from BMT recipients. In this report, we describe a new approach that permits both direct visualization of CD8(+) T-cell responses to mHAgs and cloning of T cells reacting with mHAgs presented by individual HLA alleles of interest.Methods and Results. Panels of Epstein-Barr virus. transformed B-cell lines (B-LCL) expressing retrovirally transduced HLA cDNA were used as stimulator cells in an enzyme-linked immunospot (ELISPOT) assay to identify CD8(+) T cells reacting with mHAgs in cultures generated from postBMT recipient peripheral blood. T cells specific for mHAgs presented by selected HLA alleles could then be captured and cloned using an interferon-gamma secretion assay and magnetic bead selection. A majority of T-cell clones thus isolated exhibited cytolytic activity against the same HLA-transfected B-cell lines used for the ELISPOT assay.Conclusion. The ELISPOT assay was useful for identification of the HLA alleles presenting mHAgs recognized by individual T-cell lines. This approach for isolating mHAgs-specific CD8(+) T-cell clones should assist in characterizing responses restricted by HLA alleles of interest, which are common in a certain ethnic group.