Qualitative and quantitative analysis of PMN/T-cell interactions by InFlow and super-resolution microscopy

Neutrophils or polymorphonuclear cells (PMN) eliminate bacteria via phagocytosis and/or NETosis. Apartfrom these conventional roles, PMN also have immune-regulatory functions. They can transdifferentiateand upregulate MHCII as well as ligands for costimulatory receptors which enables them to behave asantigen presenting cells (APC). The initial step for activating T-cells is the formation of an immunesynapse between T-cells and antigen-presenting cells. However, the immune synapse that develops atthe PMN/T-cell contact zone is as yet hardly investigated due to the non-availability of methods foranalysis of large number of PMN interactions. In order to overcome these obstacles, we introduce herea workflow to analyse the immune synapse of primary human PMN and T-cells using multispectral imag-ing flow cytometry (InFlow microscopy) and super-resolution microscopy. For that purpose, we used CD3and CD66b as the lineage markers for T-cells and PMN, respectively. Thereafter, we applied and criticallydiscussed various ‘‘masks” for identification of T-cell PMN interactions. Using this approach, we foundthat a small fraction of transdifferentiated PMN (CD66b+CD86high) formed stable PMN/T-cell conjugates.Interestingly, while both CD3 and CD66b accumulation in the immune synapse was dependent on thematuration state of the PMN, only CD3 accumulation was greatly enhanced by the presence of superanti-gen. The actin cytoskeleton was weakly rearranged at the PMN side on the immune synapse upon contactwith a T-cell in the presence of superantigen. A more detailed analysis using super-resolution microscopy(structured-illumination microscopy, SIM) confirmed this finding. Together, we present an InFlow micro-scopy based approach for the large scale analysis of PMN/T-cell interactions and – combined with SIM – apossibility for an in-depth analysis of protein translocation at the site of interactions.