Tetramer Core

肽：MHC（pMHC）四聚体是可以直接基于其抗原受体识别T细胞的试剂 的特异性这些试剂的产生和使用的最新发展使它们成为一种强有力的试剂。 研究抗原特异性T细胞应答的新工具，特别是在实验性T细胞应答的人类中， T细胞的操作受到可行性问题的极大限制。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标 将是1）产生pMHCII四聚体，以确定在拟议项目中研究的过敏原，以及2） 提供四聚体富集技术方面的专门技术知识， 过敏原特异性T细胞最先进的四聚体将结合最先进的 技术改进至今。编码可溶形式的pMHCII分子的重组DNA构建体 与来自确定的过敏原的免疫原性肽表位融合的抗体将在昆虫细胞中克隆和表达 表达系统然后，纯化的生物素化pMHCII复合物将通过它们的相互作用四聚化 用荧光标记的链霉亲和素分子。对于在人类受试者中研究的每种过敏原， 将产生对应于几种常见HLA-DR等位基因的四聚体， 这些试剂对HLA多样性研究人群的适用性。因为抗原特异性T细胞 群体通常以非常低的频率存在，四聚体通常与 抗体包被的磁珠，以能够富集四聚体阳性细胞。这将允许高分辨率 从少量人类细胞样本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，以便他们能够快速掌握这些技术。总的来说， 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， 强大的技术，否则可能在许多年内无法以实际规模获得。

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.