Yolanda R. Carrasco

LFA-1/ICAM-1 Interaction Lowers the Threshold of B Cell Activation by Facilitating B Cell Adhesion and Synapse Formation

The integrin LFA-1 and its ligand ICAM-1 mediate B cell adhesion, but their role in membrane-bound antigen recognition is still unknown. Here, using planar lipid bilayers and cells expressing ICAM-1 fused to green fluorescence protein, we found that the engagement of B cell receptor (BCR) promotes B cell adhesion by an LFA-1-mediated mechanism. LFA-1 is recruited to form a mature B cell synapse segregating into a ring around the BCR. This distribution is maintained over a wide range of BCR/antigen affinities (10(6) M(-1) to 10(11) M(-1)). Furthermore, the LFA-1 binding to ICAM-1 reduces the level of antigen required to form the synapse and trigger a B cell. Thus, LFA-1/ICAM-1 interaction lowers the threshold for B cell activation by promoting B cell adhesion and synapse formation.

B‐cell activation by membrane‐bound antigens is facilitated by the interaction of VLA‐4 with VCAM‐1

VCAM‐1 is one of the main ligands of VLA‐4, an integrin that is highly expressed on the surface of mature B cells. Here we find that coexpression of VCAM‐1 on an antigen‐bearing membrane facilitates B‐cell activation. Firstly, this is achieved by mediating B‐cell tethering, which in turn increases the likelihood of a B cell to be activated. Secondly, VLA‐4 synergizes with the B‐cell receptor (BCR), providing B cells with tight adhesion and enhanced signalling. This dual role of VCAM‐1 in promoting B‐cell activation is predominantly effective when the affinity of the BCR for the antigen is low. In addition, we show that the VCAM‐1 ectodomain alone is sufficient to carry out this function. However, it requires the transmembrane domain to segregate properly into a docking structure characteristic of the B‐cell immunological synapse (IS). These results show that the VLA‐4/VCAM‐1 interaction during membrane antigen recognition enhances B‐cell activation and this function appears to be independent of its final peripheral localization at the IS.

The mitochondrial fission factor dynamin-related protein 1 modulates T-cell receptor signalling at the immune synapse

During antigen‐specific T‐cell activation, mitochondria mobilize towards the vicinity of the immune synapse. We show here that the mitochondrial fission factor dynamin‐related protein 1 (Drp1) docks at mitochondria, regulating their positioning and activity near the actin‐rich ring of the peripheral supramolecular activation cluster (pSMAC) of the immune synapse. Mitochondrial redistribution in response to T‐cell receptor engagement was abolished by Drp1 silencing, expression of the phosphomimetic mutant Drp1S637D and the Drp1‐specific inhibitor mdivi‐1. Moreover, Drp1 knockdown enhanced mitochondrial depolarization and T‐cell receptor signal strength, but decreased myosin phosphorylation, ATP production and T‐cell receptor assembly at the central supramolecular activation cluster (cSMAC). Our results indicate that Drp1‐dependent mitochondrial positioning and activity controls T‐cell activation by fuelling central supramolecular activation cluster assembly at the immune synapse.

Translocation dynamics of sorting nexin 27 in activated T cells.

Sorting nexin 27 (SNX27) belongs to the sorting nexin family of proteins, which participate in vesicular and protein trafficking. Similarly to all sorting nexin proteins, SNX27 has a functional PX domain that is important for endosome binding, but it is the only sorting nexin with a PDZ domain. We identified SNX27 as a partner of diacylglycerol kinase ζ (DGKζ), a negative regulator of T cell function that metabolises diacylglycerol to yield phosphatidic acid. SNX27 interacts with the DGKζ PDZ-binding motif in early/recycling endosomes in resting T cells; however, the dynamics and mechanisms underlying SNX27 subcellular localisation during T cell activation are unknown. We demonstrate that in T cells that encounter pulsed antigen-presenting cells, SNX27 in transit on early/recycling endosomes polarise to the immunological synapse. A fraction of SNX27 accumulates at the mature immunological synapse in a process that is dependent on vesicular trafficking, binding of the PX domain to phosphatidylinositol 3-phosphate and the presence of the PDZ region. Downmodulation of expression of either SNX27 or DGKζ results in enhanced basal and antigen-triggered ERK phosphorylation. These results identify SNX27 as a PDZ-containing component of the T cell immunological synapse, and demonstrate a role for this protein in the regulation of the Ras–ERK pathway, suggesting a functional relationship between SNX27 and DGKζ.

Early Events of B Cell Activation by Antigen

B cells undergo membrane spreading and contraction during activation in response to antigen-presenting cells. The activation of B cells confers long-lasting protection from a plethora of infectious diseases through the generation of plasma cells that produce high-affinity antibodies and memory cells. Engagement of the B cell receptor (BCR) with cognate antigen initiates intracellular signaling and subsequent internalization of antigen. Membrane-bound antigens are now considered the predominant forms that initiate B cell activation in vivo. We have shown that upon recognition of antigen on the surface of a presenting cell, the B cell undergoes a dramatic change in morphology characterized by rapid spreading followed by more prolonged contraction along the presenting surface. This two-phase response increases the amount of antigen that the B cell accumulates, internalizes, and subsequently presents to T cells. Thus, the spreading and contraction response shapes the outcome of B cell activation. We used a combination of planar lipid bilayers and total internal reflection fluorescence microscopy to investigate the early events that occur after engagement of the BCR and before B cell spreading. We observed the rapid formation of BCR-antigen microclusters, which we redefine as “microsignalosomes” because they mediate the coordinated recruitment of intracellular effectors, such as the kinases Lyn and Syk, the adaptor Vav, and phospholipase C–γ2 (PLC-γ2). We identified an essential role for the co-receptor CD19 in mediating spreading, and thus B cell activation, in response to membrane-bound antigen. Preliminary evidence suggests that the cellular morphology changes described in vitro are likely to occur upon recognition of antigen presented on the surface of macrophages in lymph nodes in vivo.

The role of integrins and coreceptors in refining thresholds for B-cell responses

Summary:  Despite compelling evidence that a large proportion of antigens encountered in vivo by B cells are membrane bound, the general view is that B cells are mainly activated by soluble antigens. This notion may have been biased somewhat over the years because the high affinity of the B‐cell receptor (BCR) for soluble intact ligands allows efficient B‐cell stimulation in vitro. In vivo, however, even soluble antigens are likely to be deposited on the surface of antigen‐presenting cells, either by complement or Fc receptors in the form of immune complexes, thus becoming more potent stimulators of B‐cell activation. In this framework, the BCR works in a complex environment of integrins and coreceptors, as well as the B‐cell cytoskeleton. Over the last few years, we have focused on B‐cell membrane‐bound antigen recognition. Here, we discuss some of our findings in the context of what is currently known in this exciting new field.

Regulation of surface expression of the human pre-T cell receptor complex

Considerable progress has recently been made in defining the role that pre-antigen receptor complexes, namely the pre-T and pre-B cell receptors, play in lymphocyte development. It is now established that these receptors direct, in a similar way, the survival, expansion, clonality and further differentiation of pre-T and pre-B lymphocytes, respectively. However, less is known about the mechanisms which ensure that only minute amounts of pre-TCR and pre-BCR reach the plasma membrane of developing lymphocytes. In this review, we discuss the implications of recent experimental approaches which address the developmental regulation of human pre-TCR expression and the molecular mechanisms that control surface pre-TCR expression levels.

Differential Developmental Regulation and Functional Effects on Pre-TCR Surface Expression of Human pTαa and pTαb Spliced Isoforms

Functional rearrangement at the TCRβ locus leads to surface expression on developing pre-T cells of a pre-TCR complex composed of the TCRβ-chain paired with the invariant pre-TCRα (pTα) chain and associated with CD3 components. Pre-TCR signaling triggers the expansion and further differentiation of pre-T cells into TCRαβ mature T cells, a process known as β selection. Besides the conventional pTα transcript (termed pTαa), a second, alternative spliced, isoform of the pTα gene (pTαb) has been described, whose developmental relevance remains unknown. In this study, phenotypic, biochemical, and functional evidence is provided that only pTαa is capable of inducing surface expression of a CD3-associated pre-TCR complex, which seems spontaneously recruited into lipid rafts, while pTαb pairs with and retains TCRβ intracellularly. In addition, by using real-time quantitative RT-PCR approaches, we show that expression of pTαa and pTαb mRNA spliced products is differentially regulated along human intrathymic development, so that pTαb transcriptional onset is developmentally delayed, but β selection results in simultaneous shutdown of both isoforms, with a relative increase of pTαb transcripts in β-selected vs nonselected pre-T cells in vivo. Relative increase of pTαb is also shown to occur upon pre-T cell activation in vitro. Taken together, our data illustrate that transcriptional regulation of pTα limits developmental expression of human pre-TCR to intrathymic stages surrounding β selection, and are compatible with a role for pTαb in forming an intracellular TCRβ-pTαb complex that may be responsible for limiting surface expression of a pTαa-containing pre-TCR and/or may be competent to signal from a subcellular compartment.

TLR4 Signaling Shapes B Cell Dynamics via MyD88-Dependent Pathways and Rac GTPases

B cells use a plethora of TLR to recognize pathogen-derived ligands. These innate signals have an important function in the B cell adaptive immune response and modify their trafficking and tissue location. The direct role of TLR signaling on B cell dynamics nonetheless remains almost entirely unknown. In this study, we used a state-of-the-art two-dimensional model combined with real-time microscopy to study the effect of TLR4 stimulation on mouse B cell motility in response to chemokines. We show that a minimum stimulation period is necessary for TLR4 modification of B cell behavior. TLR4 stimulation increased B cell polarization, migration, and directionality; these increases were dependent on the MyD88 signaling pathway and did not require ERK or p38 MAPK activity downstream of TLR4. In addition, TLR4 stimulation enhanced Rac GTPase activity and promoted sustained Rac activation in response to chemokines. These results increase our understanding of the regulation of B cell dynamics by innate signals and the underlying molecular mechanisms.

WIP regulates persistence of cell migration and ruffle formation in both mesenchymal and amoeboid modes of motility.

The spatial distribution of signals downstream from receptor tyrosine kinases (RTKs) or G-protein coupled receptors (GPCR) regulates fundamental cellular processes that control cell migration and growth. Both pathways rely significantly on actin cytoskeleton reorganization mediated by nucleation-promoting factors such as the WASP-(Wiskott-Aldrich Syndrome Protein) family. WIP (WASP Interacting Protein) is essential for the formation of a class of polarised actin microdomain, namely dorsal ruffles, downstream of the RTK for PDGF (platelet-derived growth factor) but the underlying mechanism is poorly understood. Using lentivirally-reconstituted WIP-deficient murine fibroblasts we define the requirement for WIP interaction with N-WASP (neural WASP) and Nck for efficient dorsal ruffle formation and of WIP-Nck binding for fibroblast chemotaxis towards PDGF-AA. The formation of both circular dorsal ruffles in PDGF-AA-stimulated primary fibroblasts and lamellipodia in CXCL13-treated B lymphocytes are also compromised by WIP-deficiency. We provide data to show that a WIP-Nck signalling complex interacts with RTK to promote polarised actin remodelling in fibroblasts and provide the first evidence for WIP involvement in the control of migratory persistence in both mesenchymal (fibroblast) and amoeboid (B lymphocytes) motility.