Imaging the immunological synapse between dendritic cells and T cells

• Immunological synapses between ex vivo DC and responder T cells can be analysed using high-throughput imaging flow cytometry.
• This method allows for quantification and functional analysis of large numbers of DC/T cell pairs from within a single co-culture.
• Large sample sizes facilitate robust statistical analysis, which is not possible when microscopy-based techniques are used.

Immunological synapse formation between antigen-specific T cells and antigen presenting cells (APC) involves reorganization of the cellular cytoskeleton (polymerization of filamentous actin) and recruitment of adhesion molecules (e.g. LFA-1, ICAM-1). This engagement is critical for the generation of specific immune responses. Until recently, quantitative, high-throughput measurements of these interactions have not been possible. Instead, previous assessment was reliant on qualitative microscopy of live cells, where typically the APC is adhered to a surface and the suspended T cell is required to migrate to facilitate synapse formation. While this methodology can demonstrate the capacity for synapse formation, it cannot accommodate quantification of large numbers of interacting cell pairs, nor does it allow for statistically robust comparison between test conditions.We have developed a method for assessing immunological synapse formation between purified ex vivo dendritic cells (DCs) and responder antigen-specific CD4+ T cells using imaging flow cytometry, allowing us to quantify LFA-1 and f-actin rearrangement at the interface between DC/T cell pairs. This novel application of imaging flow cytometry represents a major advance in dendritic cell function and immunological synapse research as it facilitates quantitative, high throughput analysis of the interaction between live, ex vivo DC and T cells.