Towards GLIMPSE: Enabling technology to map cytokines in inflamed tissue

免疫和炎症是多种疾病的基础，如癌症、自身免疫和免疫性疾病。 神经变性在这些系统中的每一个中，分泌的细胞因子协调了 淋巴细胞和组织驻留细胞，以正确地平衡增殖、分化、迁移和细胞死亡。 尽管细胞因子的关键机制作用及其作为药物靶点的前景， 以检测它们在没有遗传修饰的组织中的空间分布。我们推测， 当细胞因子分泌时， 揭示了细胞活动的区域然而，对细胞因子分布成像的尝试因细胞因子的快速降解而受挫。 小蛋白质的质量传递、大抗体基试剂的传递受阻以及 试剂不会改变活细胞和组织的状态。 该项目的目标是创建第一种方法来量化活组织中局部细胞因子浓度 样品离体，同时保留组织的状态和结构。我们的方法是轻轻地捕捉一个 将分泌的细胞因子的一部分直接分泌到其分泌位置附近的细胞上，用荧光标记的 抗体片段，并通过活组织荧光成像对蛋白质的分布进行成像。避免 如果在测定过程中无意中激活了组织，我们将使用抗体片段和小肽 结合剂，而不是可以结合Fc受体的完整抗体。淋巴结中的蛋白质信号将被 作为一个案例研究，因为参与炎症的关键蛋白质（细胞因子）已经得到了很好的研究， 可量化的效果，并且作为炎性疾病的药物靶标是令人感兴趣的。在这个试点项目中，我们将 开发一种概念验证试验，使用抗体片段检测T细胞特异性细胞因子分泌（Aim 1）， 并同时开发新的肽基试剂来检测淋巴细胞表面标志物和细胞因子 (Aim 2）。将根据固定组织的免疫染色、流式细胞术和ELISA对试验进行基准化。 将在两种不同的物种（小鼠和人）中测试检测试剂盒和新试剂，以证明 炎症性疾病期间细胞因子检测的原理。 如果成功，这个高风险、高回报的项目将展示第一个化学成像的可行性 方法，以产生在未固定的组织中的细胞因子分泌的“地图”。所提出的技术是故意 模块化以允许准备好的转座以检测其他组织中的其他蛋白质，并且易于多路复用用于多个 细胞因子同时原则上，该技术可以用于可视化任何蛋白质， 抗体对或肽结合剂是可用的，并用于任何软组织，以彻底改变对 免疫系统中基于蛋白质的串扰。这些数据将使未来的R01应用程序能够扩展 方法，以额外的细胞和细胞因子，使重复分析在同一组织随着时间的推移，并应用它 在炎症性疾病，特别是神经炎症中成像动态细胞因子信号传导。

Immunity and inflammation are fundamental to diseases as diverse as cancer, autoimmunity, and neurodegeneration. In each of these systems, secreted cytokines coordinate an intricate dance between lymphocytes and tissue-resident cells, to correctly balance proliferation, differentiation, migration, and cell death. Despite the critical mechanistic role of cytokines and their promise as a drug target, there are currently no tools to detect their spatial distribution in tissue without genetic modification. We speculate that knowledge of where and when cytokines are secreted would dramatically accelerate the design of effective immunotherapies by revealing pockets of cellular activity. However, attempts to image cytokine distribution are frustrated by the rapid mass transport of small proteins, the hindered delivery of large antibody-based reagents, and the requirement that the reagents not alter the state of live cells and tissue. The goal of this project is to create the first method to quantify local cytokine concentrations in live tissue samples ex vivo, while preserving the state and structure of the tissue. Our approach is to gently capture a fraction of the secreted cytokine directly onto cells near where it was secreted, label it with a fluorescently-tagged antibody fragment, and image the distribution of the protein by live-tissue fluorescence imaging. To avoid inadvertently activating the tissue during the assay, we will work with antibody fragments and small peptide binders, rather than intact antibodies that could bind Fc receptors. Protein signaling in the lymph node will be used as a case study because the key proteins (cytokines) involved in inflammation are well-studied, have quantifiable effects, and are of interest as drug targets for inflammatory diseases. In this pilot proposal, we will develop a proof-of-concept assay to detect T cell-specific cytokine secretion, using antibody fragments (Aim 1), and simultaneously develop novel peptide-based reagents to detect lymphocyte surface markers and cytokines (Aim 2). The assay will be benchmarked against immunostaining of fixed tissues, flow cytometry, and ELISA. The assay and new reagents will be tested in two different species, mouse and human, to demonstrate proof-of- principle of cytokine detection during inflammatory disease. If successful, this high-risk, high-reward project will show the feasibility of the first chemical imaging method to generate “maps” of cytokine secretion in unfixed tissue. The proposed technology is deliberately modular to allow ready transposition to detect other proteins in other tissues, and is easily multiplexed for multiple cytokines simultaneously. The technology can, in principle, be adopted to visualize any protein for which an antibody pair or peptide binder is available, and used in any soft tissue, to revolutionize the understanding of protein-based cross-talk in the immune system. The data will enable a future R01 application to expand the method to additional cells and cytokines, enable repeated analysis in the same tissue over time, and to apply it to image dynamic cytokine signaling in inflammatory disease, particularly in neuroinflammation.