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）：等位基因排除确保大多数 T 和 B 淋巴细胞仅表达一种抗原受体特异性 - 这是克隆选择理论的核心原则。正常小鼠和人类中的一些 T 细胞和 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。这导致人们担心双受体淋巴细胞会带来自身免疫风险——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丰富，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 T 细胞受体 (TCR) T 细胞也在同种异体反应细胞中富集，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。 了解双受体淋巴细胞在任何这些免疫学环境中的贡献的主要障碍在于这些细胞难以识别。对于B的情况 细胞，现在可以识别双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 kappa 轻链表达细胞（通过工程改造） 人类 kappa 恒定区的敲入）。目前尚不存在类似的识别双 TCR T 细胞的方法。 小鼠双 T 细胞受体 T 细胞的鉴定具有挑战性，因为 缺乏 TCR 等位基因标记？链并且因为单克隆抗体仅存在于 TCR V 的约 16%？段。因此，淋巴细胞群中双 TCR T 细胞的数量的估计是通过数学外推而不是真正的定量来完成的。当前项目的目标是设计一种能够准确识别、计数和表征正常多克隆库中双 TCR 表达 T 细胞的小鼠。 其他研究人员已成功将表位标签引入 TCR 中？链条和TCR？细胞系中的链，而不破坏 TCR 的表达或功能。我们在这里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点？恒定区。然后，我们将设计两种基因靶向敲入小鼠品系：一种表达 myc 标签，另一种表达 FLAG 标签 TCR？恒定区。将这些小鼠杂交将产生这样的小鼠，其中可以根据两个表位标签的表达来容易地识别双 TCR 表达 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。