Abstract
Cell-to-cell communication engages signaling and spatiotemporal reorganization events driven by highly context-dependent and dynamic intercellular interactions, which are difficult to capture within heterogeneous primary cell cultures. Here, we present a straightforward correlative imaging approach utilizing commonly available instrumentation to sample large numbers of cell-cell interaction events, allowing qualitative and quantitative characterization of rare functioning cell-conjugates based on calcium signals. We applied this approach to examine a previously uncharacterized immunological synapse, investigating autologous human blood CD4+ T cells and monocyte-derived macrophages (MDMs) forming functional conjugates in vitro. Populations of signaling conjugates were visualized, tracked and analyzed by combining live imaging, calcium recording and multivariate statistical analysis. Correlative immunofluorescence was added to quantify endogenous molecular recruitments at the cell-cell junction. By analyzing a large number of rare conjugates, we were able to define calcium signatures associated with different states of CD4+ T cell-MDM interactions. Quantitative image analysis of immunostained conjugates detected the propensity of endogenous T cell surface markers and intracellular organelles to polarize towards cell-cell junctions with high and sustained calcium signaling profiles, hence defining immunological synapses. Overall, we developed a broadly applicable approach enabling detailed single cell- and population-based investigations of rare cell-cell communication events with primary cells.
Highlights
Contact-based cell communication is an essential mode of crosstalk in the functioning of mammalian organs, tissues, nervous and immune systems
To test the validity of such an approach, we investigated an uncharacterized form of T cell-antigen-presenting cell (APC) junction between human CD4+ T cells and macrophages in primary cell cultures
A time-staggered flow cytometry protocol was developed to investigate the extent to which addition of sAg affects the proportion of T cell–APC conjugates forming over time, and T cell signaling within these conjugates
Summary
Contact-based cell communication is an essential mode of crosstalk in the functioning of mammalian organs, tissues, nervous and immune systems. To gain a more in depth understanding of what defines IS formation, data from large numbers of conjugates need to be acquired, quantitatively and statistically analyzed Combining these methods in a correlative approach using calcium-sensitive reporter dyes and detection of endogenous cell markers would allow single cell- and population-based investigations of cell-cell junctions, even with primary cells. We describe an accessible approach that can deliver high throughput qualitative and quantitative assessments of rare dynamic events to obtain insights into the spectrum of antigen dependent cell-cell interactions between populations of immune cells and identify those that represent functional bona fide synapses. Since calcium signaling is a relatively ubiquitous readout of cell excitability, this approach can be extended to other cellular systems and more general studies of cell-cell communication
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