Protective Mechanisms Against Pandemic Respiratory Virus (Resource D)

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PROJECT D: CELL ACTIVATION STATE ANALYSIS BY MULTI-PARAMETER FACS Mellins, E., P.I., Nolan, G., co-Pl. Overview: Previous immune cell phenotyping has relied heavily on subset identification on the basis of surface markers. The last two decades of immunological research using Flow Cytometry has advanced solely on this straightforward and accepted technology. However, cell activation typically results first in intracellular changes, through the sequential protein modification associated with signal transduction, and later in changes at the cell surface. Thus, the ability to detect and accurately measure these intracellular changes in cellular phenotype represents an important technological frontier. Measuring levels of phosphorylated molecules by intracellular FACS analysis provides a quantitative approach to assessing cell activation at the single cell level. The Nolan laboratory has recently developed this approach using monoclonal and polyclonal antibodies to measure levels of phosphorylated forms of key signaling molecules in immune cells by Flow Cytometry (1,2). This technology has been applied to the analysis of activation states of mature T lymphocyte subsets, B cells NK cells and macrophages, revealing previously unappreciated heterogeneity among cells (1) and shedding light on the contributions of signals from particular surface receptors as well as on the integration of signaling cascades initiated by their concurrent ligation (2). Here we propose to further develop this technology for application to the specific research projects of the U19 application. New tools for single cell activation state analysis by FACS will be developed. These tools will be utilized for comparative analysis of the activation phenotypes of influenza specific T cells (project 1 and 2), and B cells (project 3) generated in response to natural influenza A infection and in response to two forms of trivalent influenza immunization. The reagents and assays developed in this component will also be applicable to the analysis of NK cell activation in response to influenza-infected targets (project 4). In this way, we will work directly with the specific research projects to generate a comprehensive view of the immune response to influenza and to vaccine approaches to this pathogen. In addition, we will extend this technology development to new assays to probe activation states of antigen presenting cell (APC) types present in human peripheral blood. Signal transduction pathways in APCs are somewhat less well characterized than in T cells and B cells, particularly for those receptors that are unique to APCs, but this is an area of active current investigation. We postulate that APC signaling profiles will reflect the immune status of the host (with implications for appropriate vaccination strategies) or the presence of a pathogen, raising the possibility that rapid assay formats developed here, or modified for clinical utility, may yield information of significant value to biodefense efforts. Our long term goal is to develop tools and approaches for rapid phenotyping of complex populations of immune cells in multiple dimensions and to link data from FACS-based assays to clinically relevant outcomes.