Unutmaz D, KewalRamani V N, Marmon S, Littman D R
Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York, USA.
J Exp Med. 1999 Jun 7;189(11):1735-46. doi: 10.1084/jem.189.11.1735.
Lentiviral vectors have been advocated to be effective vehicles for the delivery and stable expression of genes in nondividing primary cells. However, certain cell types, such as resting T lymphocytes, are resistant to infection with HIV-1. Establishing parameters for stable gene delivery into primary human lymphocytes and approaches to overcome the resistance of resting T cells to HIV infection may permit potential gene therapy applications, genetic studies of primary cells in vitro, and a better understanding of the stages of the lentiviral life cycle. Here we demonstrate that an HIV-1-derived vector can be used for stable delivery of genes into activated human T cells as well as natural killer and dendritic cells. Remarkably, a sizeable fraction of resting T cells was stably transduced with the HIV-1 vector when cultured with the cytokine interleukin (IL)-2, IL-4, IL-7, or IL-15, or, at a lower level, with IL-6, in the absence of any other stimuli. Resting T cells stimulated with these cytokines could also be infected with replication-competent HIV-1. To test the utility of this system for performing structure-function analysis in primary T cells, we introduced wild-type as well as a mutant form of murine CD28 into human T cells and showed a requirement for the CD28 cytoplasmic domain in costimulatory signaling. The ability to stably express genes of interest in primary T cells will be a valuable tool for genetic and structure-function studies that previously have been limited to transformed cell lines. In addition, the finding that cytokine signals are sufficient to permit transduction of resting T cells with HIV may be relevant for understanding mechanism of HIV-1 transmission and pathogenesis.
慢病毒载体被认为是在非分裂原代细胞中传递基因并实现稳定表达的有效载体。然而，某些细胞类型，如静息T淋巴细胞，对HIV-1感染具有抗性。确定将基因稳定导入原代人淋巴细胞的参数以及克服静息T细胞对HIV感染抗性的方法，可能有助于潜在的基因治疗应用、体外原代细胞的遗传学研究，以及更好地理解慢病毒生命周期的各个阶段。在此，我们证明一种源自HIV-1的载体可用于将基因稳定导入活化的人T细胞以及自然杀伤细胞和树突状细胞。值得注意的是，在没有任何其他刺激的情况下，当与细胞因子白细胞介素（IL）-2、IL-4、IL-7或IL-15一起培养时，相当一部分静息T细胞能够被HIV-1载体稳定转导，与IL-6一起培养时转导水平较低。用这些细胞因子刺激的静息T细胞也能被具有复制能力的HIV-1感染。为了测试该系统在原代T细胞中进行结构-功能分析的实用性，我们将野生型以及小鼠CD28的突变形式导入人T细胞，并表明共刺激信号传导需要CD28胞质结构域。在原代T细胞中稳定表达感兴趣基因的能力将成为遗传和结构-功能研究的宝贵工具，这些研究以前仅限于转化细胞系。此外，细胞因子信号足以使HIV转导静息T细胞这一发现，可能与理解HIV-1传播和发病机制有关。
