A correlative and quantitative imaging approach enabling characterization of primary cell-cell communication: Case of human CD4+ T cell-macrophage immunological synapses

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.

Next‐generation pathology detection of T cell–antigen‐presenting cell immune synapses in human liver allografts

“…These data indicate that ai-iSYN lobular lymphocytes have a physiological phenotype of SMAC formation with APCs. The large percentage of lobular lymphocytes with a "transient" phenotype is consistent with the probing and monitoring nature of T cells traversing the APC-lined sinusoids, [36,38,42] the reported effects of T cell activation state, and the APC type in immune synapse dynamics. [43,44] A higher density of CD8 + T cells, drivers of effector mechanisms and TCMR, was associated with unsuccessful ISW.…”

“…and A.G.) for evidence of polarization from HiRes images. Lymphocyte polarization was defined by published morphological descriptions of immune synapse formation [8,19,[35][36][37][38] as "stable," "transient," or "unbound" (Figure 2B). All pairings were classified as CD8 + or CD4 + (CD3 + /CD8 − ).…”

“…(B) Lobular lymphocytes identified (or not) by the ai-iSYN algorithm were evaluated for polarization of CD3 and CD45 from high-resolution images, including detection of supramolecular activation complex (SMAC) formation. The phenotype of the lymphocytes engaged in ai-iSYNs were defined based on binding stability depicted in published literature [8,19,[35][36][37][38] : "stable," "transient," or "unbound". "Stable" lymphocytes have complete polarization of CD3 to one cytoplasmic location with CD45 exclusion from that region, indicative of SMAC formation.…”

Considerations of Antibody Geometric Constraints on NK Cell Antibody Dependent Cellular Cytotoxicity

“…Fluorescent light microscopy provides the unique advantage of being able to observe live cells, illuminating IS dynamics such as the spatial arrangement of receptors and ligands over time. Work in this area has been pioneered by the study of the T cell IS, which has been extensively reviewed elsewhere (7,9,76,(114)(115)(116)(117)(118)(119). Study of the T cell IS has been in part driven by a more reductionist approach to parse the very complex IS into more digestible pieces.…”

Single-molecule and super-resolved imaging deciphers membrane behaviour of onco-immunogenic CCR5

“…Cell samples were fixed with a solution of 3% formaldehyde in Phosphate Buffer Saline (PBS) for 20 mins at room temperature, before being extensively washed in PBS. Coverslips were mounted in Mowiol, as previously described in preceding studies (Kasprowicz et al, 2018).…”