Zhong Shi, Malecek Karolina, Perez-Garcia Arianne, Krogsgaard Michelle
NYU Cancer institute, New York University School of Medicine, USA.
J Vis Exp. 2010 Oct 22(44):2307. doi: 10.3791/2307.
T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs). This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity). It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment. Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.
T细胞受体（TCRs）在免疫系统中发挥着核心作用。T细胞表面的TCRs能够特异性识别抗原呈递细胞（APCs）呈递的肽抗原。这种识别会导致T细胞的激活以及一系列功能结果（如细胞因子产生、靶细胞杀伤）。了解TCRs的功能作用对于利用免疫系统的力量治疗各种免疫相关疾病（如癌症或自身免疫性疾病）至关重要。在小鼠模型中研究TCRs很方便，可通过多种方式实现。制作TCR转基因小鼠模型成本高且耗时，目前可用的数量有限。另外，可通过骨髓嵌合体产生具有抗原特异性T细胞的小鼠。这种方法也需要几周时间且需要专业知识。将TCRs逆转录病毒转导到体外激活的小鼠T细胞中是一种快速且相对容易的方法，可获得具有所需肽 - MHC特异性的T细胞。抗原特异性T细胞可在一周内产生并用于任何下游应用。研究转导的T细胞也直接应用于人类免疫治疗，因为用抗原特异性TCRs转导的人类T细胞的过继性转移是一种新兴的癌症治疗策略。在此，我们展示了一种将TCRs逆转录病毒转导到体外激活的小鼠T细胞中的方案。人类和小鼠的TCR基因均可使用。携带特定TCR基因的逆转录病毒被产生并用于感染用抗CD3和抗CD28抗体激活的小鼠T细胞。在体外扩增后，通过流式细胞术分析转导的T细胞。
