Savoldo Barbara, Cubbage Michael L, Durett April G, Goss John, Huls M Helen, Liu Zhensheng, Teresita Lopez, Gee Adrian P, Ling Paul D, Brenner Malcolm K, Heslop Helen E, Rooney Cliona M
Center for Cell and Gene Therapy and Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA.
J Immunol. 2002 Jan 15;168(2):909-18. doi: 10.4049/jimmunol.168.2.909.
Adoptive immunotherapy with EBV-specific CTL (EBV-CTL) effectively prevents and treats EBV-driven lymphoproliferation in immunocompromised hosts. EBV-seronegative solid organ transplant recipients are at high risk of EBV-driven lymphoproliferation because they lack EBV-specific memory T cells. For the same reason, standard techniques for generating EBV-CTL in vitro from EBV-naive individuals are unsuccessful. To overcome this problem, we compared several methods of expanding EBV-CTL from seronegative adults and children. First, the standard protocol, using EBV-transformed lymphoblastoid B cell lines (LCL) as the source of APC, was compared with protocols using EBV-Ag-loaded dendritic cells as APC. Surprisingly, the standard protocol effectively generated CTL from all seronegative adults. The additional finding of EBV-DNA in the peripheral blood of three of these four adults suggested that some individuals may develop cellular, but not humoral, immune responses to EBV. By contrast, LCL failed to reactivate EBV-CTL from any of the six EBV-seronegative children. EBV-Ag-loaded dendritic cells could expand EBV-CTL, but only in a minority of children. However, the selective expansion of CD25-expressing T cells, 9-11 days after activation with LCL alone, proved to be a simple and reliable method for generating EBV-CTL from all seronegative children. The majority of these CTL were CD4(+) (71 +/- 26%) and demonstrated HLA class II-restricted, EBV-specific killing. Our results suggest that a negative EBV serology does not accurately identify EBV-negative individuals. In addition, our method for selecting EBV-specific CTL from naive individuals by precursor cell enrichment may be applicable to the immunotherapy of cancer patients with a low frequency of tumor- or virus-specific CTL.
采用EBV特异性CTL（EBV-CTL）进行过继性免疫治疗可有效预防和治疗免疫功能低下宿主中由EBV驱动的淋巴细胞增殖。EBV血清学阴性的实体器官移植受者由于缺乏EBV特异性记忆T细胞，因而有很高的由EBV驱动的淋巴细胞增殖风险。出于同样的原因，从EBV初免个体体外产生EBV-CTL的标准技术并不成功。为克服这一问题，我们比较了从血清学阴性的成人和儿童中扩增EBV-CTL的几种方法。首先，将使用EBV转化的淋巴母细胞B细胞系（LCL）作为抗原呈递细胞（APC）来源的标准方案与使用负载EBV抗原的树突状细胞作为APC的方案进行了比较。令人惊讶的是，标准方案能有效地从所有血清学阴性的成人中产生CTL。这四名成人中有三名外周血中出现EBV-DNA这一额外发现表明，一些个体可能对EBV产生细胞免疫反应，但无体液免疫反应。相比之下，LCL未能从六名EBV血清学阴性儿童中的任何一名中重新激活EBV-CTL。负载EBV抗原的树突状细胞能够扩增EBV-CTL，但仅在少数儿童中有效。然而，在用LCL单独激活9-11天后，选择性扩增表达CD25的T细胞被证明是从所有血清学阴性儿童中产生EBV-CTL的一种简单可靠的方法。这些CTL中的大多数是CD4(+)（71±26%），并表现出HLA II类限制性的、EBV特异性杀伤作用。我们的结果表明，EBV血清学阴性并不能准确识别EBV阴性个体。此外，我们通过前体细胞富集从初免个体中选择EBV特异性CTL的方法可能适用于肿瘤或病毒特异性CTL频率较低的癌症患者的免疫治疗。
