Quantifying Cell-Cell Interactions in the Immune System by Trans-Synaptic Labeling

项目摘要 细胞-细胞相互作用对于免疫反应的特异性是必不可少的。然而，一种方法， 直接测量这些相互作用仍然缺乏，这给该领域造成了盲点。我的目标是发展一个 在体外和体内监测这种相互作用的广泛系统，具有分子特异性（即，包括 关于涉及哪些受体/配体的信息）和在单个细胞水平上。这个系统，我们称之为 细胞间接触分类标记免疫伙伴关系（LIPSTIC），使用酶标记 作为一种“细胞口红”，能够标记经历了与免疫突触相互作用的细胞。 另一个细胞群体具有可通过流式细胞术或显微镜检测的标记。我们最初的证据- 原理实验表明，该系统在体外和体内都能稳健地用于CD 40 L-CD 40相互作用， vivo.根据先锋奖，我建议将LIPSTIC技术扩展到多个受体配体， 其他模式的标记和实验方法，使它成为广泛有用的免疫学家， 将军这将在研究抗原诱导T细胞应答的科学背景下进行。 提呈细胞（APC）。我们将（i）扩展LIPSTIC调色板以标记多个受体之间的相互作用， 配体对参与T细胞-APC的串扰;（ii）开发双色系统，用于组合标记 涉及多个受体-配体对或细胞群体的相互作用;和（iii）使用LIPSTIC作为工具， 从全基因组文库中鉴定TCR配体。我们希望，开发LIPSTIC沿着这些 lines有可能使其成为免疫学家的关键工具，同时提供对T细胞生物学的深入了解。 在体内引发，不能通过任何其他手段获得。

Project Summary Cell-cell interactions are essential for the specificity of immunological reactions. However, an approach to measuring these interactions directly is still lacking, creating a blind spot for the field. My goal is to develop a broad-ranging system to monitor such interactions in vitro and in vivo, with molecular specificity (i.e., including information on which receptors/ligands are involved) and at the individual cell level. This system, which we call Labeling Immune Partnerships by SorTagging Intercellular Contacts (LIPSTIC), uses enzymatic labeling across immune synapses as a “cellular lipstick” capable of marking cells that have undergone interactions with another cell population with labels that are detectable by flow cytometry or microscopy. Our initial proof-of- principle experiments show that this system works robustly for the CD40L-CD40 interaction, both in vitro and in vivo. Under the Pioneer award, I propose to expand the LIPSTIC technology to multiple receptor ligands and to other modes of labeling and experimental approaches, so that it becomes widely useful to immunologists in general. This will be done within the scientific context of studying the priming of T cell responses by antigen presenting cells (APCs). We will (i) extend the LIPSTIC palette to label interactions between multiple receptor- ligand pairs involved in T cell-APC cross-talk; (ii) develop dual-color systems for combinatorial labeling of interactions involving multiple receptor-ligand pairs or cell populations; and (iii) use LIPSTIC as a tool to identify TCR ligands from among whole-genome libraries. We expect that developing LIPSTIC along these lines has the potential to make it a key tool for immunologists, while providing insight into the biology of T cell priming in vivo that could not be obtained by any other means.