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淋巴细胞仅表达一个抗原受体特异性 - 克隆选择理论的中心宗旨。正常小鼠和人类表达两种不同的抗原受体特异性的某些T和B细胞的发现表明，等位基因排除是不完善的。这导致人们担心双重受体淋巴细胞构成自身免疫性的风险 - 通过一种受体对外抗原的识别可能会激活其他抗原受体具有自动反应性的淋巴细胞。 实际上，双受体淋巴细胞在自动反应性淋巴细胞中富集，并且可以在实验环境中有助于自身免疫性疾病发病机理。双重T细胞受体（TCR）T细胞在同种反应性细胞中也富集，并导致移植物抗宿主疾病。另一方面，双重受体淋巴细胞可以通过增加曲目中抗原受体特异性的数量来扩大宿主保护的感染。 在任何这些免疫学环境中，了解双重受体淋巴细胞的贡献的主要障碍是很难鉴定出这种细胞。对于b 细胞，现在可以鉴定双重免疫球蛋白表达链式链式的细胞（通过利用天然等位基因变体）和双喀巴轻型链链的细胞（通过工程设计 人类卡帕常数区域的敲击）。识别双重TCR T细胞的类似方法尚不存在。 由于鉴定小鼠双重T细胞受体T细胞的鉴定，因此具有挑战性 TCR缺乏等位基因标记？链，并且因为单克隆抗体仅存在TCR V的约16％？细分市场。因此，通过数学外推而不是真实的定量来完成估计淋巴细胞中双重TCR T细胞的数量。当前项目的目的是设计一种允许在正常多克隆曲目中表达双重TCR？ - 表达TCR的TCR细胞的小鼠。 其他研究人员已成功将表位标签引入了TCR？链和TCR？细胞系中的链，而不会破坏TCR的表达或功能。我们在这里首先建议使用逆转录病毒TCR表达系统来识别最佳位点，以在小鼠TCR中插入表位标签？恒定区域。然后，我们将设计两个基因靶向敲蛋白小鼠系的基因：一种表达myc标记的 - 标记为标记的tcr？恒定区域。越过这些小鼠将产生小鼠，其中双重TCR？ - 表达T细胞可以根据它们的两个表位标签的表达轻松识别。这些小鼠将提供一种强大的新方法来研究在各种免疫学环境中的双重TCR T细胞。