McElrath M J, Rabin M, Hoffman M, Klucking S, Garcia J V, Greenberg P D
Department of Medicine, University of Washington School of Medicine, Seattle.
J Virol. 1994 Aug;68(8):5074-83. doi: 10.1128/JVI.68.8.5074-5083.1994.
Analysis of major histocompatibility complex-restricted cytotoxic T lymphocytes (CTL) capable of killing human immunodeficiency virus type 1 (HIV-1)-infected targets is essential for elucidating the basis for HIV-1 disease progression and the potential efficacy of candidate vaccines. The use of primary CD4+ T cells with variable infectivity as targets for such studies has significant limitations, and immortal autologous cells with high levels of CD4 expression that can be consistently infected with HIV-1 would be of much greater utility. Therefore, we transduced Epstein-Barr-virus-transformed B-lymphoblastoid cell lines (LCL) with a retroviral vector, LT4SN, containing the human CD4 gene. Stable LCL in which more than 95% of cells expressed membrane CD4 were obtained. Aliquots were infected with HIV-1, and, after 4 to 7 days, nearly all of the cells contained cytoplasmic gag and produced high levels of p24 antigen. The ability of major histocompatibility complex-restricted CD8+ CTL to lyse such HIV-1-infected CD4-transduced LCL (LCL-CD4HIV-1) was evaluated. These autologous targets were lysed by CTL generated from an HIV-1-uninfected vaccinee over a broad range of effector-to-target ratios. Similarly, the LCL-CD4HIV-1 were efficiently lysed by fresh circulating CTL from HIV-1-infected individuals, as well as by CTL activated by in vitro stimulation. Both HIV-1 env- and gag-specific CTL effectors lysed LCL-CD4HIV-1, consistent with the cellular expression of both HIV-1 genes. The LCL-CD4HIV also functioned as stimulator cells, and thus are capable of amplifying CTL against multiple HIV-1 gene products in HIV-1-infected individuals. The ability to produce HIV-1-susceptible autologous immortalized cell lines that can be employed as target cells should enable a more detailed evaluation of vaccine-induced CTL against both homologous and disparate HIV-1 strains. Furthermore, the use of LCL-CD4HIV-1 should facilitate the analysis of the range of HIV-1 gene products recognized by CTL in seropositive persons.
分析能够杀伤人类免疫缺陷病毒1型（HIV-1）感染靶细胞的主要组织相容性复合体限制的细胞毒性T淋巴细胞（CTL），对于阐明HIV-1疾病进展的基础以及候选疫苗的潜在疗效至关重要。使用具有可变感染性的原代CD4 + T细胞作为此类研究的靶细胞有显著局限性，而能够持续感染HIV-1的、具有高水平CD4表达的永生化自体细胞将具有更大的用途。因此，我们用含有人类CD4基因的逆转录病毒载体LT4SN转导爱泼斯坦-巴尔病毒转化的B淋巴母细胞系（LCL）。获得了稳定的LCL，其中超过95%的细胞表达膜CD4。取等分试样用HIV-1感染，4至7天后，几乎所有细胞都含有细胞质gag并产生高水平的p24抗原。评估了主要组织相容性复合体限制的CD8 + CTL裂解此类HIV-1感染的CD4转导LCL（LCL-CD4HIV-1）的能力。这些自体靶细胞在广泛的效应细胞与靶细胞比例范围内被来自未感染HIV-1的疫苗接种者产生的CTL裂解。同样，LCL-CD4HIV-1被来自HIV-1感染个体的新鲜循环CTL以及通过体外刺激激活的CTL有效裂解。HIV-1 env和gag特异性CTL效应细胞均裂解LCL-CD4HIV-1，这与HIV-1两个基因的细胞表达一致。LCL-CD4HIV也作为刺激细胞发挥作用，因此能够在HIV-1感染个体中扩增针对多种HIV-1基因产物的CTL。产生可作为靶细胞使用的对HIV-1敏感的自体永生化细胞系的能力，应能更详细地评估疫苗诱导的针对同源和不同HIV-1毒株的CTL。此外，使用LCL-CD4HIV-1应有助于分析血清反应阳性者中CTL识别的HIV-1基因产物范围。
