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.
Proteins released from cells form a hidden map that defines most of health and disease, but scientists have no straightforward tools to visualize this map in living, unmodified tissue. This project creates (i) an innovative method to detect the distribution of any protein, in any soft tissue, at a chosen time point, and (ii) a suite of small peptides that selectivity bind proteins of interest. If successful, these tools will make the protein cross-talk between cells visible for the first time, ultimately enabling new research into diseases such as autoimmunity, cancer, and neurodegeneration.
