A mouse to track dual TCR T cells

追踪双 TCR T 细胞的小鼠

• 批准号: 8563789
• 负责人: Bryce Binstadt
• 金额: $ 7.6万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-15 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8563789
• 关键词: A Mouse; Address; Alleles; Antigen Receptors; Antigens; Autoimmune Diseases; Autoimmune Process; Autoimmunity; B-Lymphocytes; Back; Biological; Biological Models; Cell Line; Cell surface; Cells; Clone Cells; Complement; Detection; Development; Engineering; Ensure; Epitopes; Exclusion; Flow Cytometry; Frequencies; Gene Targeting; Genes; Genetic Variation; Goals; Graft Rejection; Growth; Heavy-Chain Immunoglobulins; Host Defense; Human; Immunity; Immunoglobulin Constant Region; Immunoglobulin Gene Rearrangement; Immunologic Memory; Immunologics; Immunology; In Vitro; Infection; Investigation; Knock-in Mouse; Light; Light-Chain Immunoglobulins; Location; Lymphocyte; Monoclonal Antibodies; Mus; Organ Transplantation; Pathogenesis; Population; Reagent; Receptors, Antigen, B-Cell; Research; Research Personnel; Risk; Role; Site; Specificity; System; T-Cell Receptor; T-Lymphocyte; Thymus Gland; Transgenic Organisms; Transplantation; base; graft vs host disease; kappa-Chain Immunoglobulins; novel strategies; public health relevance; receptor; receptor expression; theories

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.

描述（由申请人提供）：等位基因排斥确保大多数T和B淋巴细胞仅表达一种抗原受体特异性-克隆选择理论的中心原则。在正常小鼠和人类中的一些T和B细胞表达两种不同的抗原受体特异性的发现表明等位基因排除是不完美的。这导致了双受体淋巴细胞对自身免疫的风险的担忧-通过一个受体识别外来抗原可能会激活另一个抗原受体是自身反应性的淋巴细胞。 事实上，双受体淋巴细胞在自身反应性淋巴细胞中富集，并且可以在实验环境中促进自身免疫性疾病的发病机制。双重T细胞受体（TCR）T细胞也在同种异体反应性细胞中富集，并导致移植物抗宿主病。另一方面，双受体淋巴细胞可以通过增加库中抗原受体特异性的数量来扩大宿主对感染的保护。 理解双重受体淋巴细胞在这些免疫学环境中的作用的主要障碍仅仅是这些细胞难以识别。对于B的情况 细胞，现在有可能鉴定双重免疫球蛋白重链表达细胞（通过利用天然等位基因变体）和双重κ轻链表达细胞（通过工程改造的 人κ恒定区的敲入）。目前还不存在类似的鉴定双重TCR T细胞的方法。 小鼠中的双重T细胞受体T细胞的鉴定是具有挑战性的，这是由于 缺乏TCR的等位基因标记？链，因为单克隆抗体只存在约16%的TCR V？片段因此，通过数学外推而不是真正的定量来估计淋巴细胞群体中双重TCR T细胞的数量。当前项目的目标是设计一种小鼠，允许准确识别、计数和表征双TCR？在正常多克隆库中表达T细胞。 其他研究人员已经成功地将表位标签引入TCR？链和TCR？在不破坏TCR的表达或功能的情况下，我们建议在这里首先使用逆转录病毒TCR表达系统，以确定一个最佳的网站插入表位标签在小鼠TCR？恒定区然后，我们将设计两个基因靶向敲入小鼠系：一个表达myc标记的TCR，另一个表达FLAG标记的TCR？恒定区将这些小鼠杂交将产生双TCR？表达T细胞可以容易地基于它们两个表位标签的表达来鉴定。这些小鼠将为在广泛的免疫环境中研究双重TCR T细胞提供一种强大的新方法。