Institute for Transfusion Medicine, Hannover Medical School, D-30625 Hannover, Germany.
J Immunother. 2010 Jan;33(1):60-72. doi: 10.1097/CJI.0b013e3181b56dcc.
The reactivation of the human cytomegalovirus (CMV) can be prevented or controlled by the adoptive transfer of ex vivo expanded donor-derived CMV-specific T lymphocytes. Several methods for expansion and adoptive transfer of CMV-specific T cells have been developed using either defined CMV peptides or peptide pools for antigen-specific T-cell stimulation. The majority of studies have focused on the lower matrix protein (pp65) and the immediate-early protein-1 (IE-1) of CMV as immunodominant targets. We investigated the behavior of secretory CMVpp65 (sCMVpp65) with respect to its capacity to stimulate pp65-specific T cells independently of human lymphocyte antigen (HLA) type and even in donors unresponsive to the immunodominant HLA-A0201-restricted CMVpp65495-503 peptide. To facilitate the eukaryotic expression and isolation procedures, we constructed an HLA-A0201/CMVpp65 fusion protein that is secreted into the supernatant of human embryonic kidney 293 (HEK293) cells. CMV-specific CD4 and CD8 T cells generated by culturing unfractionated peripheral blood mononuclear cells in the presence of recombinant sCMVpp65 did not differ in function with regard to cytotoxicity and interferon-gamma (IFN-gamma) production compared with cytotoxic T cells induced using the well-studied HLA-A*0201-restricted CMVpp65495-503 peptide. We demonstrated that polyclonal CMV-specific T cells could be generated from CMV-seropositive individuals expressing HLA alleles for which no immunogenic epitopes have been identified so far. The production of recombinant sCMVpp65 can easily be adapted to good manufacturing practice conditions and can be used to generate large numbers of immunogenic pathogen-derived proteins for therapeutic applications.
人巨细胞病毒（CMV）的再激活可以通过过继转移体外扩增的供体来源的 CMV 特异性 T 淋巴细胞来预防或控制。已经开发了几种用于 CMV 特异性 T 细胞扩增和过继转移的方法，这些方法使用定义的 CMV 肽或肽池进行抗原特异性 T 细胞刺激。大多数研究都集中在 CMV 的较低基质蛋白（pp65）和即刻早期蛋白-1（IE-1）作为免疫优势靶标。我们研究了分泌型 CMVpp65（sCMVpp65）的行为，其能够独立于人类淋巴细胞抗原（HLA）类型刺激 pp65 特异性 T 细胞，甚至在对免疫显性 HLA-A0201 限制性 CMVpp65495-503 肽无反应的供体中也是如此。为了便于真核表达和分离程序，我们构建了一种 HLA-A0201/CMVpp65 融合蛋白，该蛋白分泌到人胚肾 293（HEK293）细胞的上清液中。通过在存在重组 sCMVpp65 的情况下培养未分选的外周血单核细胞来产生的 CMV 特异性 CD4 和 CD8 T 细胞在细胞毒性和干扰素-γ（IFN-γ）产生方面与使用经过充分研究的 HLA-A*0201 限制性 CMVpp65495-503 肽诱导的细胞毒性 T 细胞没有差异。我们证明，可以从表达迄今尚未鉴定出免疫原性表位的 HLA 等位基因的 CMV 血清阳性个体中产生多克隆 CMV 特异性 T 细胞。重组 sCMVpp65 的生产可以很容易地适应良好的生产规范条件，并可用于产生大量用于治疗应用的免疫原性病原体衍生蛋白。
