Illuminating T cell – APC interactions using multispectral imaging flow cytometry.

Abstract

Abstract Interaction between antigen-specific T cells and antigen presenting cells (APC) cognate ligand involve reorganization of the cytoskeleton and recruitment of adhesive and signaling molecules to the site of intercellular contact. Sustained adhesion of T cells to APCs and formation of the immunological synapse after T cell receptor stimulation are required for the antigen-specific response. One way to measure an immunological synapse is by fluorescently labeling the molecules that have been recruited to the synapse and imaging by fluorescence microscopy. However, immunological synapses are rare and therefore difficult to analyze objectively and statistically by traditional microscopy methods. To overcome these problems, we employed the Amnis brand ImageStream to objectively collect imagery of large numbers of cells. In this study, Raji B cells loaded with Staphylococcal enterotoxin B (SEB) were incubated with human T cells to create T cell-APC conjugates. Cells were stained in various combinations for CD3, CD19, Actin, LFA-1, Lck and NFkB. Using multispectral imaging flow cytometry we demonstrate image-based parameters that were used to assess the frequency of conjugates with an organized immunological synapse, evaluate the specific location of the adhesion and signaling molecules LFA-1 and Lck within the immunological synapse complex and measure the nuclear localization of NFkB in the T cell-APC conjugates.