D'Orsogna L J, Amir A L, Zoet Y M, van der Meer-Prins P M W, van der Slik A R, Kester M G D, Heemskerk M H M, Doxiadis I I N, Roelen D L, Claas F H J
Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, The Netherlands.
Tissue Antigens. 2009 Oct;74(4):290-7. doi: 10.1111/j.1399-0039.2009.01311.x. Epub 2009 Jul 15.
Accumulating evidence suggests that alloreactive memory T-cells may be generated as a result of viral infection. So far, a suitable tool to define the individual human leukocyte antigen (HLA) cross-reactivity of virus-specific memory T-cells is not available. We therefore aimed to develop a novel system for the detection of cross-reactive alloresponses using single HLA antigen expressing cell lines (SALs) as stimulator. Herein, we generated Epstein-Barr Virus (EBV) EBNA3A specific CD8 memory T-cell clones (HLA-B0801/FLRGRAYGL peptide restricted) and assayed for alloreactivity against a panel of SALs using interferon-gamma Elispot as readout. Generation of the T-cell clones was performed by single cell sorting based on staining with viral peptide/major histocompatibility complex-specific tetramer. Monoclonality of the T-cell clones was confirmed by T-cell receptor (TCR) polymerase chain reaction analysis. First, we confirmed the previously described alloreactivity of the EBV EBNA3A-specific T-cell clones against SAL-expressing HLA-B4402. Further screening against the entire panel of SALs also showed additional cross-reactivity against SAL-expressing HLA-B*5501. Functionality of the cross-reactive T-cell clones was confirmed by chromium release assay using phytohemagglutinin blasts as targets. SALs are an effective tool to detect cross-reactivity of viral-specific CD8 memory T-cell clones against individual class I HLA molecules. This technique may have important implications for donor selection and monitoring of transplant recipients.
越来越多的证据表明，同种异体反应性记忆T细胞可能是病毒感染的结果。到目前为止，尚无合适的工具来定义病毒特异性记忆T细胞的个体人类白细胞抗原（HLA）交叉反应性。因此，我们旨在开发一种新系统，以表达单个HLA抗原的细胞系（SAL）作为刺激物来检测交叉反应性同种异体反应。在此，我们生成了爱泼斯坦-巴尔病毒（EBV）EBNA3A特异性CD8记忆T细胞克隆（HLA-B0801/FLRGRAYGL肽受限），并使用干扰素-γ酶联免疫斑点法作为检测指标，检测其对一组SAL的同种异体反应性。T细胞克隆的生成是通过基于病毒肽/主要组织相容性复合体特异性四聚体染色的单细胞分选进行的。通过T细胞受体（TCR）聚合酶链反应分析证实了T细胞克隆的单克隆性。首先，我们证实了先前描述的EBV EBNA3A特异性T细胞克隆对表达SAL的HLA-B4402的同种异体反应性。对整个SAL组的进一步筛选还显示，对表达SAL的HLA-B*5501存在额外的交叉反应性。使用植物血凝素刺激的细胞作为靶细胞，通过铬释放试验证实了交叉反应性T细胞克隆的功能。SAL是检测病毒特异性CD8记忆T细胞克隆对单个I类HLA分子交叉反应性的有效工具。该技术可能对供体选择和移植受者的监测具有重要意义。
