Multimolecular Analysis of Stable Immunological Synapses Reveals Sustained Recruitment and Sequential Assembly of Signaling Clusters

Abstract

The formation of the immunological synapse between T cells and antigen-presenting cells (APC) begins within minutes of contact and can take hours for full T-cell activation. Although early phases of the synapse have been extensively studied for a select number of proteins, later phases have not yet been examined in detail. We studied the signaling network in stable synapses by measuring the simultaneous localization of 25 signaling and structural molecules over 2 h at the level of individual synapses using multi-epitope ligand cartography (MELC). Signaling proteins including phospho(p)ZAP70, pSLP76, pCD3, and pLAT, along with proteins that influence synapse structure such as F-actin, tubulin, CD45, and ICAM-1, were localized in images of synapses and revealed the multidimensional construction of a mature synapse. The construction of the stable synapse included intense early TCR signaling, a phase of recruitment of structural proteins, and a sustained increase in signaling molecules and colocalization of TCR and pLAT signaling clusters in the center of the synapse. Consolidation of TCR and associated proteins resulted in formation of a small number of discrete synaptic microclusters. Development of synapses and cSMAC composition was greatly affected by the absence of Vav1, with an associated loss in PLC␥1 recruitment, pSLP76, and increased CXCR4. Adaptive immune responses are initiated by the meeting of a T cell and an antigen-presenting cell (APC) 1 bearing peptide-MHC (pMHC) complexes that are a specific fit for the T-cell receptor (TCR) on the T-cell surface. Within seconds, TCR signaling starts with a sequence of phosphorylation and de-phosphorylation events of membrane-proximal and -distal TCR-signaling molecules and their spatial reorganization into protein multiclusters (1). Together with the rearrangement of structural molecules at the cell-cell interface, these signals lead to the formation of a supramolecular structure termed the immunological synapse (1-3). The synapse can differ substantially in size and composition, but comprises several common structural motifs (4 -6). In the classical synapse, these structural motifs are organized in domains that form a target

TCR signalling network organization at the immunological synapses of murine regulatory T cells

“…Since we (i) revealed Treg cell‐specific and diverging signalling of cytoskeleton regulators and (ii) characterized a variant MTOC positioning and impaired PKCθ recruitment in Treg cells, we next aimed to study the spatial organization of selected TCR signalling components in stimulated Treg cells and Tconv cells. For this, we employed the multi‐epitope ligand cartography (MELC) technology , and evaluated a total of about 300 OVA‐specific BT‐pairs using a panel of 25 antibodies (see Material and Methods, Supporting Information Fig. 1C and Supporting Information Video 1).…”

“…At this moment, however, the knowledge on how TCR signalling, protein recruitment and IS formation are differentially organized in Treg cells is far from complete. In the present study, we have now systematically studied the activity and spatial organization of TCR signalling components in ex vivo isolated Treg cells and Tconv cells by employing accurate mass spectrometry and microscopy supported by multi‐epitope ligand cartography (MELC) . Together, our data reveal that TCR engagement harmonizes the activity status within the TCR signalling network, but in parallel induces a distinct diverging signalling pattern at regulators of cytoskeletal dynamics.…”

“…Since we (i) revealed Treg cell-specific and diverging signalling of cytoskeleton regulators and (ii) characterized a variant MTOC positioning and impaired PKCθ recruitment in Treg cells, we next aimed to study the spatial organization of selected TCR signalling components in stimulated Treg cells and Tconv cells. For this, we employed the multi-epitope ligand cartography (MELC) technology [21,22] of key TCR signalling components as well as their specific phosphovariants. Following 30 and 120 min after BT-pair formation, robust fluorescence intensities could be detected and quantified to calculate the abundances of components in seven distinct spatial regions.…”

Multiplexed fluorescence microscopy reveals heterogeneity among stromal cells in mouse bone marrow sections

“…2, Supporting Information Fig. With the combination of MELC and object based segmentation, we were able to quantify cells of interest with the gain of spatial information, which is lost in flow cytometry experiments (60). MELC is based on sequential immunofluorescent staining and bleaching of multiple markers.…”

T cell adhesion triggers an early signaling pole distal to the immune synapse

“…However, this pole has not been analyzed as a discrete structure. More recently, a multimolecular analysis of the distribution of 25 signaling molecules in T-cell-APC contacts has been reported, revealing the existence of sequential signaling clusters; unfortunately, the analysis focused on the synapse and only 5 min after contact was formed (Philipsen et al, 2013).…”