Abstract
A central process in immunity is the activation of T cells through interaction of T cell receptors (TCRs) with agonistic peptide-major histocompatibility complexes (pMHC) on the surface of antigen presenting cells (APCs). TCR-pMHC binding triggers the formation of an extensive contact between the two cells termed the immunological synapse, which acts as a platform for integration of multiple signals determining cellular outcomes, including those from multiple co-stimulatory/inhibitory receptors. Contributors to this include a number of chemokine receptors, notably CXC-chemokine receptor 4 (CXCR4), and other members of the G protein-coupled receptor (GPCR) family. Although best characterized as mediators of ligand-dependent chemotaxis, some chemokine receptors are also recruited to the synapse and contribute to signaling in the absence of ligation. How these and other GPCRs integrate within the dynamic structure of the synapse is unknown, as is how their normally migratory Gαi-coupled signaling is terminated upon recruitment. Here, we report the spatiotemporal organization of several GPCRs, focusing on CXCR4, and the G protein Gαi2 within the synapse of primary human CD4+ T cells on supported lipid bilayers, using standard- and super-resolution fluorescence microscopy. We find that CXCR4 undergoes orchestrated phases of reorganization, culminating in recruitment to the TCR-enriched center. This appears to be dependent on CXCR4 ubiquitination, and does not involve stable interactions with TCR microclusters, as viewed at the nanoscale. Disruption of this process by mutation impairs CXCR4 contributions to cellular activation. Gαi2 undergoes active exclusion from the synapse, partitioning from centrally-accumulated CXCR4. Using a CRISPR-Cas9 knockout screen, we identify several diverse GPCRs with contributions to T cell activation, most significantly the sphingosine-1-phosphate receptor S1PR1, and the oxysterol receptor GPR183. These, and other GPCRs, undergo organization similar to CXCR4; including initial exclusion, centripetal transport, and lack of receptor-TCR interactions. These constitute the first observations of GPCR dynamics within the synapse, and give insights into how these receptors may contribute to T cell activation. The observation of broad GPCR contributions to T cell activation also opens the possibility that modulating GPCR expression in response to cell status or environment may directly regulate responsiveness to pMHC.
Highlights
The adaptive immune system depends on the activation of antigen-specific lymphocytes to deliver an appropriate and coordinated response to infection or cellular dysfunction
CXCR4 distribution exhibited no obvious organization in contact with the non-activating supported lipid bilayers (SLBs), CXCR4 exhibited a clear exclusion from the center of the contact within minutes on activating SLB, and from both the cSMAC and pSMAC in the mature synapse (Figures 1A–D; Supplementary Figure 1A)
Three-dimensional confocal microscopy revealed large amounts of CXCR4 away from the planar bilayer interface that could be consistent with receptor endocytosis, and with presence in extracellular vesicles that accumulate between the cell and the SLB (Supplementary Figure 1B)
Summary
The adaptive immune system depends on the activation of antigen-specific lymphocytes to deliver an appropriate and coordinated response to infection or cellular dysfunction. The recognition of cognate peptideMHC (pMHC) by TCR leads to activation of the T cell and formation of a large interface with the APC; in either the form of a stable immunological synapse (synapse) or a motile kinapse (Dustin, 2007; Mayya et al, 2018) This involves spatial organization into distinct zones that correspond to transitions in an underlying filamentous actin (F-actin) network, described as supramolecular activation clusters (SMACs): the central (c)SMAC corresponds to sparse F-actin bundles that enable access for bidirectional vesicular budding and fusion; the actinomyosin- and talin-rich peripheral (p)SMAC stabilizes adhesion; and the dendritic F-actin-rich distal (d)SMAC is an important site for signal initiation (Freiberg et al, 2002; Sims et al, 2007; Fritzsche et al, 2017). Several GPCRs have important regulatory function during T cell-APC communication, including receptors for lysophosphatidic acid (Oda et al, 2013), adenosine (Linnemann et al, 2009), adrenaline (Fan and Wang, 2009), and dopamine (Papa et al, 2017); the most ubiquitous are members of the chemokine receptor family
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