A mouse to track dual TCR T cells

描述(由申请人提供)：等位基因排斥确保大多数T和B淋巴细胞只表达一种抗原受体特异性-克隆选择理论的核心原则。在正常小鼠和人类中，一些T和B细胞表达两种不同的抗原受体特异性，这一发现表明等位基因排斥是不完美的。这引起了人们的担忧，即双重受体淋巴细胞对自身免疫构成了风险--通过一个受体识别外来抗原可能会激活另一个抗原受体具有自身反应的淋巴细胞。事实上，双受体淋巴细胞在自身反应性淋巴细胞中丰富，并在实验环境中有助于自身免疫性疾病的发病。双T细胞受体(TCR)T细胞也在同种异体反应细胞中丰富，并有助于移植物抗宿主病。另一方面，双受体淋巴细胞可以通过增加抗原受体特异性的数量来扩大宿主对感染的保护。理解双受体淋巴细胞在这些免疫学环境中的作用的主要障碍很简单，就是这种细胞很难识别。对于B的情况 细胞，现在可以鉴定双重免疫球蛋白重链表达细胞(通过利用天然的等位基因变体)和双重kappa轻链表达细胞(通过工程设计的 人类kappa恒定区的Knockin)。目前尚不存在识别双TCR T细胞的类似方法。在小鼠中鉴定双T细胞受体T细胞具有挑战性，因为 缺乏TCR的等位基因标记？因为只有16%的TCR V？细分市场。因此，估计淋巴细胞群体中双TCRT细胞的数量是通过数学外推而不是真正的量化来完成的。当前项目的目标是设计一种能够在正常的多克隆谱系中准确识别、计数和鉴定双TCR？表达的T细胞的小鼠。其他研究人员已经成功地将表位标签引入TCR？链条和TCR？在不破坏TCR的表达或功能的情况下，在细胞系中形成链。我们建议首先使用逆转录病毒TCR表达系统来确定在小鼠TCR中插入表位标签的最佳位置。恒定区域。然后我们将设计两个基因靶向敲击小鼠品系：一个表达带有myc标记的基因，另一个表达带有标志标记的TCR？恒定区域。将这些小鼠杂交将产生小鼠，在这些小鼠中，可以根据表达两个表位标签的T细胞很容易地识别出双重TCR？表达的T细胞。这些小鼠将为在广泛的免疫学环境中研究双TCR T细胞提供一种强大的新方法。

DESCRIPTION (provided by applicant): Allelic exclusion ensures that most T and B lymphocytes express only one antigen receptor specificity - a central tenet of clonal selection theory. The discovery that some T and B cells in normal mice and humans express two different antigen receptor specificities demonstrated that allelic exclusion was imperfect. This led to concern that dual receptor lymphocytes pose a risk for autoimmunity - recognition of a foreign antigen through one receptor might activate a lymphocyte whose other antigen receptor is autoreactive. Indeed, dual receptor lymphocytes are enriched among autoreactive lymphocytes and can contribute to autoimmune disease pathogenesis in experimental settings. Dual T cell receptor (TCR) T cells are also enriched among alloreactive cells and contribute to graft-versus-host disease. On the other hand, dual receptor lymphocytes may broaden host protection against infection by increasing the number of antigen receptor specificities in the repertoire. The primary barrier to understanding the contribution of dual receptor lymphocytes in any of these immunological settings is simply that such cells are difficult to identify. For the case of B cells, it is now possible to identify dual immunoglobulin heavy chain-expressing cells (by exploiting natural allelic variants) and dual kappa light chain-expressing cells (via an engineered knockin of the human kappa constant region). Similar approaches to identify dual TCR T cells do not yet exist. Identification of dual T cell receptor T cells in mice is challenging due to the lack of allelic markers of the TCR? chain and because monoclonal antibodies exist for only ~16% of TCR V? segments. Estimating the number of dual TCR T cells in a population of lymphocytes has thus been accomplished by mathematical extrapolation rather than true quantification. The goal of the current project is to engineer a mouse that permits accurate identification, enumeration, and characterization of dual TCR?-expressing T cells in a normal polyclonal repertoire. Other investigators have successfully introduced epitope tags into the TCR? chain and TCR? chain in cell lines without disrupting the expression or function of the TCR. We propose here first to use a retroviral TCR expression system to identify an optimal site to insert an epitope tag in the mouse TCR? constant region. We will then engineer two gene targeted knockin mouse lines: one expressing a myc-tagged- and the other a FLAG-tagged-TCR? constant region. Crossing these mice will produce mice in which dual TCR?-expressing T cells can readily be identified based on their expression of both epitope tags. These mice will provide a powerful new approach to study dual TCR T cells in a wide range of immunological settings.