Travers H, Anderson G, Gentle D, Jenkinson E, Girdlestone J
Anatomy Department, Division of Immunity and Infection, MRC Centre for Immune Regulation, The Medical School, University of Birmingham, B15 2TT, Birmingham, UK.
J Immunol Methods. 2001 Jul 1;253(1-2):209-22. doi: 10.1016/s0022-1759(01)00391-x.
Viral vectors have the potential to provide a fast and economic alternative to transgenic methods for manipulating gene expression in studies of immune system development and function. Although protocols exist for the infection of hematopoietic precursors and peripheral T cells in vitro, critical stages of T cell differentiation are strictly dependent upon a three-dimensional thymic architecture and their analysis poses unique technical challenges. Whole fetal thymic lobes have been used as targets for retroviral and adenoviral infection, both in situ and in vitro, but this approach does not allow for discrimination between lymphoid and stromal components. Isolated thymocytes have been infected by co-culture with viral producer cells, but under these conditions they rapidly lose their developmental potential. To overcome these problems we have combined a number of efficient techniques for retroviral production, concentration, and infection that allow us to rapidly achieve significant transduction rates of purified populations of double-negative (DN) and double-positive (DP) thymocytes, single-positive (SP) T lymphocytes, as well as fetal thymic MHC II(+) epithelial cells without the need for co-culture with viral producer cells. Reaggregate thymic organ culture (RTOC) techniques were used to assess the development and function of transduced cells in defined cellular environments. As a demonstration of the utility of these methods, CD80 (B7.1) was transduced into thymic epithelial cells and shown to allow them to mediate negative selection of DP thymocytes, and to act as antigen-presenting cells (APC) to mature T cells. The ability to genetically manipulate primary cells of a specified type and differentiation stage provides a powerful complement to RTOC techniques for the study of T cell development.
在免疫系统发育和功能的研究中，病毒载体有可能为操纵基因表达的转基因方法提供一种快速且经济的替代方案。尽管存在体外感染造血前体和外周T细胞的方案，但T细胞分化的关键阶段严格依赖于三维胸腺结构，并且对其进行分析面临独特的技术挑战。完整的胎儿胸腺叶已被用作逆转录病毒和腺病毒感染的靶标，包括原位和体外感染，但这种方法无法区分淋巴细胞和基质成分。分离的胸腺细胞已通过与病毒生产细胞共培养进行感染，但在这些条件下它们会迅速丧失其发育潜能。为了克服这些问题，我们结合了多种用于逆转录病毒生产、浓缩和感染的高效技术，这些技术使我们能够快速实现对纯化的双阴性（DN）和双阳性（DP）胸腺细胞、单阳性（SP）T淋巴细胞以及胎儿胸腺MHC II(+)上皮细胞群体的显著转导率，而无需与病毒生产细胞共培养。重新聚集胸腺器官培养（RTOC）技术用于评估转导细胞在特定细胞环境中的发育和功能。作为这些方法实用性的一个例证，将CD80（B7.1）转导到胸腺上皮细胞中，并显示其能够介导DP胸腺细胞的阴性选择，并作为抗原呈递细胞（APC）作用于成熟T细胞。对特定类型和分化阶段的原代细胞进行基因操作的能力为研究T细胞发育的RTOC技术提供了强大的补充。
