Khanna R, Jacob C A, Burrows S R, Moss D J
Queensland Institute of Medical Research, Bancroft Centre, Herston, Brisbane, Australia.
J Immunol Methods. 1993 Aug 26;164(1):41-9. doi: 10.1016/0022-1759(93)90274-b.
Cytotoxic T lymphocytes (CTLs) recognising viral antigens are an important host defence mechanism in restricting the proliferation of virus-infected cells. Previously, lymphoblastoid cell lines (LCLs) infected with vaccinia recombinants encoding viral proteins have been used to identify specific CTL epitopes. However, to localise the EBV CTL epitopes encoded by Epstein-Barr virus (EBV) transformants, LCLs are an inappropriate host for vaccinia recombinants. In the present study, an alternative host cell for vaccinia infection is described. Initial studies demonstrated that anti-CD40 stimulated human B cells replicated vaccinia virus and expressed EBV nuclear antigen(s) (EBNA) following infection with recombinant vaccinia encoding the appropriate region of the EBV genome. Recombinant vaccinia-infected anti-CD40 stimulated B cell lines were then used to localise target epitopes for a panel of EBV-specific CTL clones. Most importantly, in vitro stimulation of unfractionated mononuclear cells with recombinant vaccinia-infected anti-CD40 B cells activated a memory CTL response. Based on the vaccinia results, screening of peptides from EBNA6 defined the epitope for the EBNA6-specific CTL clones to the sequence KEHVIQNAFRK. This work clearly demonstrates that anti-CD40 stimulated B cell lines not only provide an efficient tool for localising CTL epitopes but also presents an alternative mechanism of reactivating a memory T cell response to any gene product expressed by recombinant vaccinia.
识别病毒抗原的细胞毒性T淋巴细胞（CTL）是限制病毒感染细胞增殖的重要宿主防御机制。此前，已使用感染了编码病毒蛋白的痘苗重组体的淋巴母细胞系（LCL）来鉴定特定的CTL表位。然而，对于定位由爱泼斯坦-巴尔病毒（EBV）转化体编码的EBV CTL表位而言，LCL并非痘苗重组体的合适宿主。在本研究中，描述了一种用于痘苗感染的替代宿主细胞。初步研究表明，抗CD40刺激的人B细胞在感染编码EBV基因组适当区域的重组痘苗后可复制痘苗病毒并表达EBV核抗原（EBNA）。然后，将重组痘苗感染的抗CD40刺激的B细胞系用于定位一组EBV特异性CTL克隆的靶表位。最重要的是，用重组痘苗感染的抗CD40 B细胞对未分离的单核细胞进行体外刺激可激活记忆CTL反应。基于痘苗实验结果，对来自EBNA6的肽段进行筛选，确定了EBNA6特异性CTL克隆的表位为序列KEHVIQNAFRK。这项工作清楚地表明，抗CD40刺激的B细胞系不仅为定位CTL表位提供了一种有效的工具，而且还提出了一种重新激活对重组痘苗表达的任何基因产物的记忆T细胞反应的替代机制。
