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.