Tetramer Core

四聚体核心

• 批准号: 8381647
• 负责人: James J Moon
• 金额: $ 9.14万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8381647
• 关键词: Alleles; Allergens; Allergic; Antibodies; Antigen Receptors; Antigens; Area; Asthma; Blood; Blood specimen; CD4 Positive T Lymphocytes; Cells; Cellular Structures; Complex; Detection; Development; Disease; Elements; Ensure; Epitope Mapping; Epitopes; Etiology; Feedback; Food Hypersensitivity; Frequencies; Generations; Goals; HLA-DR Antigens; Human; Hypersensitivity; Immune System Diseases; Immune response; Immunology; Individual; Inflammatory; Insecta; Investments; Label; Ligands; Lymphoid Tissue; Mus; Peptide/MHC Complex; Peptides; Performance; Population; Population Study; Positioning Attribute; Production; Reagent; Recombinant DNA; Research Personnel; Resolution; Resources; Role; Sampling; Screening procedure; Services; Specificity; Streptavidin; Study Subject; System; T cell response; T-Cell Receptor; T-Lymphocyte; T-Lymphocyte Epitopes; Technical Expertise; Techniques; Technology; Time; Tissues; United States National Institutes of Health; Work; adaptive immunity; base; design; flexibility; genetic manipulation; human subject; immunogenic; improved; magnetic beads; member; tool

Peptide:MHC (pMHC) tetramers are reagents that can identify T cells based directly on their antigen receptor specificity. Recent developments in the generation and use of these reagents have made them a powerful new tool with which to study antigen-specific T cell responses, particularly in humans where experimental manipulation of T cells is extremely limited by feasibility issues. The overall objective of the Tetramer Core is to provide researchers from each of the proposed projects with the resources necessary to use tetramer technology to study allergen-specific T cells in mice and humans. The specific aims of the Tetramer Core will be 1) to generate pMHCII tetramers to defined allergens studied in the proposed projects, and 2) to provide technical expertise in tetramer-based enrichment techniques to enable identification of low frequency allergen-specific T cells. State-of-the-art tetramers will be generated incorporating the most advanced technical refinements to date. Recombinant DNA constructs encoding soluble forms of pMHCII molecules fused to immunogenic peptide epitopes from defined allergens will be cloned and expressed in an insect cell expression system. Purified biotinylated pMHCII complexes will then be tetramerized via their interaction with fluorescently labeled streptavidin molecules. For each allergen studied in human subjects, a panel of tetramers corresponding to several common HLA-DR alleles will be generated to ensure adequate applicability of these reagents to the HLA-diverse study population. Because antigen-specific T cell populations are often present in very low frequencies, tetramers will often be used in conjunction with antibody-coated magnetic beads to enable enrichment of tetramer-positive cells. This will allow for high-resolution detection of allergen-specific T cells from small amounts of human cell samples. The Tetramer Core will provide technical support to researchers so they can quickly master these techniques. Collectively, the services provided by the Tetramer Core will enable researchers to immediately take advantage of this powerful technology that otherwise may not be readily available at a practical scale for many years.

肽：MHC（PMHC）四聚体是可以直接基于其抗原受体的T细胞的试剂 特异性。这些试剂的一代和使用的最新发展使它们成为了强大的 研究抗原特异性T细胞反应的新工具，尤其是在实验性的人类中 T细胞的操纵受到可行性问题的极限限制。四聚体核心的总体目标是 为每个拟议项目的研究人员提供使用四聚体所需的资源 研究小鼠和人类过敏原特异性T细胞的技术。四聚体核心的具体目的 将为1）生成PMHCII四聚体以在拟议项目中研究的定义过敏原，而2） 在基于四聚体的富集技术方面提供技术专长，以识别低频 过敏原特异性T细胞。最先进的四聚体将生成最先进的 迄今为止的技术改进。重组DNA构造编码PMHCII分子的可溶性形式 定义过敏原的免疫原性肽表位将被克隆并在昆虫细胞中表达 表达系统。然后将通过其相互作用将纯化的生物素化PMHCII复合物四分之一 带有荧光标记的链霉亲和素分子。对于每个在人类受试者中研究的过敏原， 将生成与几个常见HLA-DR等位基因相对应的四聚体，以确保足够 这些试剂适用于HLA多样性研究人群。因为抗原特异性T细胞 种群通常以非常低的频率存在，四聚体通常会与 抗体涂层的磁珠可以富集四聚体阳性细胞。这将允许高分辨率 从少量的人类细胞样品中检测过敏原特异性T细胞。四聚体 Core将为研究人员提供技术支持，以便他们可以快速掌握这些技术。共同 四聚体核心提供的服务将使研究人员能够立即利用这一点 多年来，否则可能无法在实践规模上不易获得的强大技术。