Multiplex IP-FCM (immunoprecipitation-flow cytometry): Principles and guidelines for assessing physiologic protein-protein interactions in multiprotein complexes.

There is significant interest in the development of methods with the potential to increase access to 'the interactome' for both experimental and clinical applications. Immunoprecipitation detected by flow cytometry (IP-FCM) is a robust, biochemical method that can be used for measuring physiologic protein-protein interactions (PPI) in multiprotein complexes (MPC) with high sensitivity. Because it is based on antibody-mediated capture of protein complexes onto microspheres, IP-FCM is potentially compatible with a multiplex platform that could allow simultaneous assessment of many physiologic PPI. Here, we consider the principles of ambient analyte conditions (AAC) and inter-bead independence, and provide a template set of experiments showing how to convert singleplex IP-FCM to multiplex IP-FCM, including assays to confirm the validity of the experimental conditions for data acquisition. We conclude that singleplex IP-FCM can be successfully upgraded to multiplex format, and propose that the unique strengths of multiplex IP-FCM make it a method that is likely to facilitate the acquisition of new PPI data from primary cell sources.

IP-FCM measures physiologic protein-protein interactions modulated by signal transduction and small-molecule drug inhibition.

Protein-protein interactions (PPI) mediate the formation of intermolecular networks that control biological signaling. For this reason, PPIs are of outstanding interest in pharmacology, as they display high specificity and may represent a vast pool of potentially druggable targets. However, the study of physiologic PPIs can be limited by conventional assays that often have large sample requirements and relatively low sensitivity. Here, we build on a novel method, immunoprecipitation detected by flow cytometry (IP-FCM), to assess PPI modulation during either signal transduction or pharmacologic inhibition by two different classes of small-molecule compounds. First, we showed that IP-FCM can detect statistically significant differences in samples possessing a defined PPI change as low as 10%. This sensitivity allowed IP-FCM to detect a PPI that increases transiently during T cell signaling, the antigen-inducible interaction between ZAP70 and the T cell antigen receptor (TCR)/CD3 complex. In contrast, IP-FCM detected no ZAP70 recruitment when T cells were stimulated with antigen in the presence of the src-family kinase inhibitor, PP2. Further, we tested whether IP-FCM possessed sufficient sensitivity to detect the effect of a second, rare class of compounds called SMIPPI (small-molecule inhibitor of PPI). We found that the first-generation non-optimized SMIPPI, Ro-26-4550, inhibited the IL-2:CD25 interaction detected by IP-FCM. This inhibition was detectable using either a recombinant CD25-Fc chimera or physiologic full-length CD25 captured from T cell lysates. Thus, we demonstrate that IP-FCM is a sensitive tool for measuring physiologic PPIs that are modulated by signal transduction and pharmacologic inhibition.

2B4 utilizes ITAM-containing receptor complexes to initiate intracellular signaling and cytolysis.

2B4 is a member of the SLAM receptor family capable of activating NK cell cytotoxicity in the context of EBV infection. SAP (SLAM Associated Protein) deficiency causes defective signaling downstream of SLAM family receptors and high susceptibility to EBV. 2B4 costimulates natural cytotoxicity receptor (NCR) and TCR initiated signals to induce cellular cytotoxicity and cytokine release. The 2B4-SAP signal transduction pathway is not predicted to overlap with the TCR-ITAM pathway, although SAP is required for some TCR-induced signals. We therefore examined the functional relationship between SLAM family receptor 2B4 and ITAM-containing adaptor complexes. Removal of FcɛRIγ or CD3ζ-containing complexes, using genetically manipulated cell lines or siRNA specific suppression, significantly reduces 2B4-initiated functions in NK and T cells, respectively. Consistent with this relationship, Syk and ZAP-70 are capable of transducing 2B4 signals for calcium mobilization and cytolysis. Furthermore, ITAM-containing molecules constitutively associate with SAP. These results suggest a potential physical association between 2B4 and the ITAM receptor complexes that is required for 2B4-initiated signaling and cell-mediated killing.