Tetramer Core

肽：MHC(PMHC)四聚体是一种可以直接根据T细胞的抗原受体识别T细胞的试剂 专一性。这些试剂的产生和使用的最新发展使它们成为一种强大的 研究抗原特异性T细胞反应的新工具，特别是在实验中的人类 T细胞的操控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 在老鼠和人类身上研究过敏原特异性T细胞的技术。四聚体核心的具体目标 将是1)产生pMHCII对拟议项目中研究的已定义过敏原的四聚体，以及2) 提供基于四聚体的富集技术方面的技术专长，以便能够识别低频 过敏原特异性T细胞。将产生最先进的四聚体，其中包括最先进的 到目前为止的技术改进。编码可溶性形式pMHCII分子的重组DNA构建 确定的过敏原与免疫原肽表位的融合将被克隆并在昆虫细胞中表达 表达系统。纯化的生物素化的pMHCII复合体将通过它们的相互作用进行四聚 带有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一种过敏原， 将产生与几个常见的HLA-DR等位基因相对应的四聚体，以确保足够的 这些试剂对人类白细胞抗原多样性研究人群的适用性。因为抗原特异性T细胞 种群的出现频率通常很低，四聚体经常与 抗体包被的磁珠，使四聚体阳性细胞能够浓缩。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。总而言之， 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不容易在实际规模上获得。

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.