Illuminating T Cell-APC interactions using imaging flow cytometry and Amnis AIDr: Haley Pugsley, Manager, Senior Scientist, Amnis Flow Cytometry, Luminex

• Interaction between antigen-specific T cells and antigen presenting cells (APCs) cognate ligand involves 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 via fluorescence microscopy. However, immunological synapses are rare, and are therefore difficult to analyze objectively and statistically using traditional microscopy methods. 

• To overcome these problems, we employed imaging flow cytometry to objectively collect imagery of a large number 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 for CD3, CD19, Actin, and DAPI. Using the FlowSight® Imaging Flow Cytometer and Amnis® AI, we demonstrate how image-based parameters were used to assess the frequency of conjugates with an organized immunological synapse.