Dynamics of Membrane Trafficking Downstream of B and T Cell Receptor Engagement: Impact on Immune Synapses

The onset of an adaptive immune response requires the activation of T and B lymphocytes by antigen-presenting cells, through a specialized form of intercellular communication, known as the immunological synapse (IS). In B lymphocytes the IS promotes efficient recognition and acquisition of membrane-bound Ags, while in T cells, it modulates the T cell response upon exposure to peptide-major histocompatibility complexes. In this review, we highlight the similarities that determine B and T cell activation, focusing on immune receptor downstream signaling events that lead to synapse formation. We stress the notion that polarization of T and B lymphocytes characterized by global changes in cytoskeleton and membrane trafficking modulates synapse structure and function, thus determining lymphocyte effector functions and fate.     

Modulation of immunological synapse by membrane-bound and soluble ligands

An efficient adaptive immune response should prevent pathogen infections and tumor growth without causing significant damage to host constituents. A crucial event determining the balance between tolerance and immunity is antigen recognition by T cells on the surface of antigen presenting cells (APC). Several molecular contacts at the interface between T cells and APCs contribute to define the nature of the adaptive immune response against a particular antigen. Upon TCR engagement by a peptide-MHC complex (pMHC) on the surface of an APC, a specialized supra-molecular structure known as immunological synapse (IS) assembles at the interface between these two cells. This structure involves massive re-distribution of membrane proteins, including TCR and pMHC complexes, as well as co-stimulatory and adhesion molecules. Furthermore, IS assembly leads to several important intracellular events necessary for T cell activation, such as recruitment of signaling molecules and cytoskeleton rearrangements. Because IS assembly leads to major consequences on the function of T cells, several studies have attempted to identify both soluble and membrane-bound molecules that could contribute to modulate the IS function. Here we describe recent literature on the regulation of IS assembly and modulation by TCR/pMHC binding kinetics, chemokines and cytokines focusing on their role at controlling the balance between adaptive immunity and tolerance.     

The role of B cells in regulating the magnitude of immune response

Recently, accumulating evidence has suggested that B cell depletion therapy with rituximab is effective not only in autoantibody‐associated, but also in T cell‐mediated, autoimmune diseases. It is likely that B cells play an important role in regulating the extent of immune response in both physiological and pathological conditions. When a severe infection occurs, pathogens spread throughout the bloodstream. B cells in the blood capture the pathogens, via their specific antigen receptors (surface immunoglobulins), then present the specific antigen to T cells in the spleen, thus increasing the degree of T‐cell immune responses to systemic infection. Similarly, in the exacerbation stage of autoimmunity, a large amount of autoantigens may be released into the blood and be captured by autoantigen specific B cells, and this may be followed by presentation of the antigen to CD4 positive autoreactive T cells resulting in extensive activation and proliferation of autoreactive T cells. Thus, it has been suggested that B‐cell depletion therapy for autoimmune diseases is most useful for the “vicious cycle” phase of autoreactive immune response. The recognition of this paradigm for the role of B cells in regulating the magnitude of immune response will help to facilitate both basic and clinical research on the regulation of immune responses.     

CD19 and BAFF‐R can signal to promote B‐cell survival in the absence of Syk

The development and function of B lymphocytes is regulated by numerous signaling pathways, some emanating from the B‐cell antigen receptor (BCR). The spleen tyrosine kinase (Syk) plays a central role in the activation of the BCR, but less is known about its contribution to the survival and maintenance of mature B cells. We generated mice with an inducible and B‐cell‐specific deletion of the Syk gene and found that a considerable fraction of mature Syk‐negative B cells can survive in the periphery for an extended time. Syk‐negative B cells are defective in BCR, RP105 and CD38 signaling but still respond to an IL‐4, anti‐CD40, CpG or LPS stimulus. Our in vivo experiments show that Syk‐deficient B cells require BAFF receptor and CD19/PI3K signaling for their long‐term survival. These studies also shed a new light on the signals regulating the maintenance of the normal mature murine B‐cell pool.     

The Ca(2+)-activated K(+) channel KCa3.1 compartmentalizes in the immunological synapse of human T lymphocytes

T cell receptor engagement results in the reorganization of intracellular and membrane proteins at the T cell-antigen presenting cell interface forming the immunological synapse (IS), an event required for Ca²⁺ influx. KCa3.1 channels modulate Ca²⁺ signaling in activated T cells by regulating the membrane potential. Nothing is known regarding KCa3.1 membrane distribution during T cell activation. Herein, we determined whether KCa3.1 translocates to the IS in human T cells using YFP-tagged KCa3.1 channels. These channels showed electrophysiological and pharmacological properties identical to wild-type channels. IS formation was induced by either anti-CD3/CD28 antibody-coated beads for fixed microscopy experiments or Epstein-Barr virus-infected B cells for fixed and live cell microscopy. In fixed microscopy experiments, T cells were also immunolabeled for F-actin or CD3, which served as IS formation markers. The distribution of KCa3.1 was determined with confocal and fluorescence microscopy. We found that, upon T cell activation, KCa3.1 channels localize with F-actin and CD3 to the IS but remain evenly distributed on the cell membrane when no stimulus is provided. Detailed imaging experiments indicated that KCa3.1 channels are recruited in the IS shortly after antigen presentation and are maintained there for at least 15–30 min. Interestingly, pretreatment of activated T cells with the specific KCa3.1 blocker TRAM-34 blocked Ca²⁺ influx, but channel redistribution to the IS was not prevented. These results indicate that KCa3.1 channels are a part of the signaling complex that forms at the IS upon antigen presentation. T cell activation; ion channels; membrane distribution     

Epac1‐induced cellular proliferation in prostate cancer cells is mediated by B‐Raf/ERK and mTOR signaling cascades

cAMP‐dependent, PKA‐independent effects on cell proliferation are mediated by cAMP binding to EPAC and activation of Rap signaling. In this report, we employed the analogue 8‐CPT‐2‐O‐Me‐cAMP to study binding to EPAC and subsequent activation of B‐Raf/ERK and mTOR signaling in human cancer cells. This compound significantly stimulated DNA synthesis, protein synthesis, and cellular proliferation of human 1‐LN prostate cancer cells. By study of phosphorylation‐dependent activation, we demonstrate that EPAC‐mediated cellular effects require activation of the B‐Raf/ERK and mTOR signaling cascades. RNAi directed against EPAC gene expression as well as inhibitors of ERK, PI 3‐kinase, and mTOR were employed to further demonstrate the role of these pathways in regulating prostate cancer cell proliferation. These studies were then extended to several other human prostate cancer cell lines and melanoma cells with comparable results. We conclude that B‐Raf/ERK and mTOR signaling play an essential role in cAMP‐dependent, but PKA‐independent, proliferation of cancer cells. J. Cell. Biochem. 108: 998–1011, 2009. © 2009 Wiley‐Liss, Inc.     

Arrestin makes T cells stop and become active

T‐cell activation requires signaling by T‐cell receptors (TCRs) that bind antigen on the antigen‐presenting cells (APCs) at the immunological synapse (IS). Sustained signaling requires continuous supply of new TCRs to the IS. In this issue of The EMBO Journal, Fernández‐Arenas et al ( 2014 ) describe a novel role of β‐arrestin‐1 at the IS periphery: endocytosis of TCRs and chemokine CXCR4 receptors. Internalized TCRs are then delivered to the IS, where they engage antigen and support prolonged signaling, whereas CXCR4 internalization stops T‐cell migration.     

Continuous signaling of CD79b and CD19 is required for the fitness of Burkitt lymphoma B cells

Expression of the B‐cell antigen receptor (BCR) is essential not only for the development but also for the maintenance of mature B cells. Similarly, many B‐cell lymphomas, including Burkitt lymphoma (BL), require continuous BCR signaling for their tumor growth. This growth is driven by immunoreceptor tyrosine‐based activation motif (ITAM) and PI3 kinase (PI3K) signaling. Here, we employ CRISPR/Cas9 to delete BCR and B‐cell co‐receptor genes in the human BL cell line Ramos. We find that Ramos B cells require the expression of the BCR signaling component Igβ (CD79b), and the co‐receptor CD19, for their fitness and competitive growth in culture. Furthermore, we show that in the absence of any other BCR component, Igβ can be expressed on the B‐cell surface, where it is found in close proximity to CD19 and signals in an ITAM‐dependent manner. These data suggest that Igβ and CD19 are part of an alternative B‐cell signaling module that use continuous ITAM/PI3K signaling to promote the survival of B lymphoma and normal B cells.     

Co‐ordination of Incoming and Outgoing Traffic in Antigen‐Presenting Cells by Pattern Recognition Receptors and T Cells

Dendritic cells are innate sentinels of the immune system and potent activators of naÏve T cells. Mechanisms must exist to enable these cells to achieve maximal activation of T cells specific for microbial antigens, while avoiding activation of T cells specific for self‐antigens. Here we discuss how a combination of signals from pattern recognition receptors and T cells co‐ordinates subcellular trafficking of antigen with both major histocompatibility complex class I and class II molecules and T‐cell costimulatory molecules, resulting in the preferential presentation of microbial peptides within a stimulatory context.      

Activation—induced cell death in B lymphocytes

Upon encountering the antigen(Ag),the immune system can either develop a specific immune response of enter a specific state of unresponsiveness,tolerance.The response of B cells to their specific Ag can be activation and proliferation,leading to the immune response,or anergy and activation-induced cell death(AICD),leading to tolerance.AICD in B lymphocytes is a highly regulated event initiated by crosslinking of the B cell receptor (BCR).BCR engagement initiates several signaling events such as activation of PLCγ,Ras,and PI3K,which generally speaking,lead to survival.However,in the absence of survival signals(CD40 or IL-4R engagement),BCR crosslinking can also promote apoptotic signal transduction pathways such as activation of effector caspases,expression of pro-apoptotic genes,and inhibition of pro-survival genes.The complex interplay between survival and death signals determines the B cell fate and, consequently,the immune response.     

Synaptic transfer by human gamma delta T cells stimulated with soluble or cellular antigens

B, alpha beta T, and NK lymphocytes establish immunological synapses (IS) with their targets to enable recognition. Transfer of target cell-derived Ags together with proximal molecules onto the effector cell appears also to occur through synapses. Little is known about the molecular basis of this transfer, but it is assumed to result from Ag receptor internalization. Because human gamma delta T cells recognize soluble nonpeptidic phosphoantigens as well as tumor cells such as Daudi, it is unknown whether they establish IS with, and extract molecules from, target cells. Using flow cytometry and confocal microscopy, we show in this work that Ag-stimulated human V gamma 9/V delta 2 T cells conjugate to, and perform molecular transfer from, various tumor cell targets. The molecular transfer appears to be linked to IS establishment, evolves in a dose-dependent manner in the presence of either soluble or cellular Ag, and requires gamma delta TCR ligation, Src family kinase signaling, and participation of the actin cytoskeleton. Although CD45 exclusion characterized the IS performed by gamma delta T cells, no obvious capping of the gamma delta TCR was detected. The synaptic transfer mediated by gamma delta T cells involved target molecules unrelated to the cognate Ag and occurred independently of MHC class I expression by target cells. From these observations, we conclude that despite the particular features of gamma delta T cell activation, both synapse formation and molecular transfer of determinants belonging to target cell characterize gamma delta T cell recognition of Ags.     

Retinoic Acid-induced Gene-1 (RIG-I) Associates with the Actin Cytoskeleton via Caspase Activation and Recruitment Domain-dependent Interactions

The actin cytoskeleton serves as a barrier that protects mammalian cells from environmental pathogens such as bacteria, fungi, and viruses. Several components of antimicrobial signaling pathways have been shown to associate directly with the actin cytoskeleton, indicating that the cytoskeleton may also serve as a platform for immune-associated molecules. Here we report that retinoic acid-induced gene-I (RIG-I), an important viral RNA recognition molecule, is associated with the actin cytoskeleton and localizes predominantly to actin-enriched membrane ruffles in non-polarized epithelial cells. Subcellular localization and fractionation experiments revealed that the association between RIG-I and the actin cytoskeleton was mediated by its N-terminal caspase activation and recruitment domains (CARDs), which were necessary and sufficient to induce cytoskeletal association. We also show that RIG-I plays a role in cellular migration, as ectopic expression of RIG-I enhanced cellular migration in a wound healing assay and depletion of endogenous RIG-I significantly reduced wound healing. We further show that in both cultured intestinal epithelial cells (IEC) and human colon and small intestine biopsies, RIG-I is enriched at apico-lateral cell junctions and colocalizes with markers of the tight junction. Depolymerization of the actin cytoskeleton in polarized IEC led to the rapid relocalization of RIG-I and to the induction of type I interferon signaling. These data provide evidence that RIG-I is associated with the actin cytoskeleton in non-polarized epithelial cells and with the junctional complex in polarized IECs and human intestine and colon biopsies and may point to a physiological role for RIG-I in the regulation of cellular migration.The actin cytoskeleton is not only a fundamental component of cellular homeostasis, but in many ways also serves as the first line of host defense against an invading pathogen. Cortical actin filaments directly below the cell membrane form a complex network that provides a barrier to the penetration of viral and bacterial pathogens. This network must therefore be modulated for a pathogen to gain entry into the cell cytoplasm, most commonly via endocytic vesicles. It is therefore not surprising that many pathogens have evolved highly varied strategies to dissolve or modulate the cortical actin meshwork to facilitate cell entry and/or trafficking (1, 2). Thus, the actin cytoskeleton would be ideally suited to act as a platform for immune-associated molecules to sense an invading pathogen and mount an immediate response to promote an antimicrobial state.Consistent with this, previous studies have shown that several components of the inflammatory pathway interact either directly or indirectly with the actin cytoskeleton. The Gram-positive and -negative bacterial recognition molecule nucleotide oligimerization domain 2 (NOD2)² associates with the actin cytoskeleton (3) and localizes to cell-cell junctions in intestinal epithelial cells (IECs) (4). Moreover, the proinflammatory caspase caspase-11 directly interacts with the actin interacting protein (Aip1) to promote actin depolymerization mediated by cofilin (5) and its activity is regulated by interaction with Flightless-1, a component of actin remodeling (6). The p65 subunit of nuclear factor (NF)-κB has also been shown to interact with actin filaments, suggesting its activity may also be regulated via this association (7). These findings suggest that components of the inflammatory response tightly associate with the actin cytoskeleton and that this association may regulate inflammatory signal activation. Disruption of the actin cytoskeleton by the use of actin depolymerizing agents (such as cytochalasin D (cytoD)) activates NFκB signaling in monocytes (8), human embryonic kidney (3), and polarized intestinal epithelial cells (9). Activation of NFκB often correlates with the induction of an inflammatory state, indicating that inflammatory signals are generated in response to dramatic alterations in actin cytoskeletal architecture. Taken together, these findings support a model of inflammatory signaling in response to perturbations of the actin cytoskeleton, possibly due to the activation of actin-associated inflammatory components.The innate immune system responds to essential functional components of microorganisms, which are thus broadly expressed within classes of pathogens (10). Two functionally related intracellular viral recognition molecules recognize cytoplasmic double-stranded RNA that is produced as a replication intermediate in the life cycle of many RNA viruses. Retinoic acid-induced gene-I (RIG-I) and melanoma differentiation associated gene 5 (MDA5) binding to double-stranded RNA initiates signaling events resulting in translocation of interferon (IFN) regulatory factors (IRF)-3 and -7 into the nucleus and subsequent induction of type I IFNs, a key component of antiviral host defense (11, 12). RIG-I and MDA5 each contain two N-terminal caspase activation and recruitment domains (CARDs) and a C-terminal DEXD/H-box RNA helicase domain. The C-terminal domain serves as a regulatory repressor domain that masks the exposure of the CARDs to prevent downstream signaling in the absence of stimulus. Upon RNA binding, structural changes release this domain leading to exposure of the CARD domains and in the induction of downstream antiviral signals.It remains unclear if either RIG-I or MDA5 serve functions beyond that of the antimicrobial response. Deletion of RIG-I in mice leads to the development of a colitis-like phenotype characterized by severe inflammation of the colon mucosa (13) (in another study, RIG-I-deficient mice die in utero (14)). RIG-I has also been implicated as a negative regulator of granulocytic differentiation in mice (15). These studies would suggest that RIG-I may serve an essential role beyond that of viral RNA recognition and may actively participate in some aspect of development and/or mucosal signaling. However, the precise nature of this role remains undefined.In this study, we show that RIG-I is concentrated at sites of actin-rich membrane ruffles in non-polarized and polarized epithelial cells and plays a role in cell migration. We further show that RIG-I is enriched at apico-lateral cell junctions of both cultured IECs and human colon biopsies where it colocalizes with markers of the apical tight junction (TJ) complex. The localization of RIG-I to lamellipodia and its association with the actin cytoskeleton is mediated by CARD-dependent interactions, as these domains are both necessary and sufficient to promote membrane ruffle association. Our results show that in addition to serving an important role in innate immune signaling, RIG-I is closely associated with the actin cytoskeleton and may participate in the regulation of cellular motility and migration.     

Sorting nexin 27 interactome in T‐lymphocytes identifies zona occludens‐2 dynamic redistribution at the immune synapse

T Lymphocyte recognition of antigens leads to the formation of a highly organized structure termed immune synapse (IS) by analogy with the neuronals synapse. Sorting nexin 27 (SNX27) controls the endosomal traffic of PSD95, Dlg1, ZO‐1 (PDZ) domain‐interacting proteins, and its alteration is associated with impaired synaptic function and neurological diseases. In T‐lymphocytes, SNX27‐positive vesicles polarize to the IS, the identity of SNX27 interactors in these conditions nonetheless remains unknown. Here we used proteomics to analyze the SNX27 interactome purified from IS‐forming T cells, and confirmed the conserved nature of the SNX27/WASH/retromer association in hematopoietic cells. Furthermore, our comparative interactome analysis of SNX27 wild‐type and a mutant‐deficient for PDZ cargo recognition identified the epithelial cell‐cell junction protein zona occludens‐2 (ZO‐2) as an IS component. Biochemistry and microscopy approaches in T cells confirmed SNX27/ZO‐2 PDZ‐dependent interaction, and demonstrated its role controlling the dynamic localization of ZO‐2 at the IS. This study broadens our knowledge of SNX27 function in T lymphocytes, and suggests that pathways that delimit polarized structures in nervous and epithelial systems also participate in IS regulation.      

Lymphocyte function-associated antigen-1-mediated T cell adhesion is impaired by low molecular weight phosphotyrosine phosphatase-dependent inhibition of FAK activity

Protein tyrosine phosphorylation is one of the earliest signaling events detected in response to lymphocyte function-associated antigen-1 (LFA-1) engagement during lymphocyte adhesion. In particular, the focal adhesion kinase p125FAK, involved in the modulation and rearrangement of the actin cytoskeleton, seems to be a crucial mediator of LFA-1 signaling. Herein, we investigate the role of a FAK tyrosine phosphatase, namely low molecular weight phosphotyrosine phosphatase (LMW-PTP), in the modulation of LFA-1-mediated T cell adhesion. Overexpression of LMW-PTP in Jurkat cells revealed an impairment of LFA-1-dependent cell-cell adhesion upon T cell receptor (TCR) stimulation. Moreover, in these conditions LMW-PTP causes FAK dephosphorylation, thus preventing the activation of FAK downstream pathways. Our results also demonstrated that, upon antigen stimulation, LMW-PTP-dependent FAK inhibition is associated to a strong reduction of LFA-1 and TCR co-clustering toward a single region of T cell surface, thus causing an impairment of receptor activity by preventing changes in their avidity state. Because co-localization of both LFA-1 and TCR is an essential event during encounters of T cells with antigen-presenting cells and immunological synapse (IS) formation, we suggest an intriguing role of LMW-PTP in IS establishment and stabilization through the negative control of FAK activity and, in turn, of cell surface receptor redistribution.     

B cell lipid rafts regulate both peptide-dependent and peptide-independent APC-T cell interaction

Formation of an immunological synapse (IS) between APCs and T CD4(+) lymphocytes is a key event in the initiation and the termination of the cognate immune response. We have analyzed the contribution of the APC to IS formation and report the implication of the actin cytoskeleton, the signaling proteins and the lipid rafts of B lymphocytes. Recruitment of MHC class II molecules to the IS is concomitant with actin cytoskeleton-dependent B cell raft recruitment. B cell actin cytoskeleton disruption abrogates both IS formation and T cell activation, whereas protein kinase C inhibition only impairs T cell activation. Pharmacological B cell lipid raft disruption inhibited peptide-dependent T lymphocyte activation and induced peptide-independent but HLA-DR-restricted APC-T cell conjugate formation. Such peptide-independent conjugates did not retain the ability to activate T cells. Thus, B cell lipid rafts are bifunctional by regulating T cell activation and imposing peptide stringency.     

2D DIGE analysis of the bursa of Fabricius reveals characteristic proteome profiles for different stages of chicken B‐cell development

Antibody producing B‐cells are an essential component of the immune system. In contrast to human and mice where B‐cells develop in the bone marrow, chicken B‐cells develop in defined stages in the bursa of Fabricius, a gut associated lymphoid tissue. In order to gain a better understanding of critical biological processes like immigration of B‐cell precursors into the bursa anlage, their differentiation and final emigration from the bursa we analyzed the proteome dynamics of this organ during embryonic and posthatch development. Samples were taken from four representative developmental stages (embryonic day (ED) 10, ED18, day 2, and day 28) and compared in an extensive 2D DIGE approach comprising six biological replicates per time point. Cluster analysis and PCA demonstrated high reliability and reproducibility of the obtained data set and revealed distinctive proteome profiles for the selected time points, which precisely reflect the differentiation processes. One hundred fifty three protein spots with significantly different intensities were identified by MS. We detected alterations in the abundance of several proteins assigned to retinoic acid metabolism (e.g. retinal‐binding protein 5) and the actin‐cytoskeleton (e.g. vinculin and gelsolin). By immunohistochemistry, desmin was identified as stromal cell protein associated with the maturation of B‐cell follicles. Strongest protein expression difference (10.8‐fold) was observed for chloride intracellular channel 2. This protein was thus far not associated with B‐cell biology but our data suggest an important function in bursa B‐cell development.     

综述与专论: 免疫突触形成的生物学特点及其光学成像研究

免疫突触(immunological synapse,IS)是抗原提呈细胞与T细胞免疫识别时,多种分子参与、分阶段不断变化的过程,涉及黏附分子、细胞因子、信号传导分子、细胞骨架蛋白等多分子的聚集或离散．其形成不仅促进T细胞和抗原提呈细胞的稳定接触,而且激活T细胞信号传导途径,促进T细胞的活化和增殖．对IS的研究可以从分子水平解释免疫激活、免疫耐受、病原微生物感染与免疫细胞相互作用的机制,为进一步揭示疾病发生的分子机制,寻求疾病防治的靶向分子提供新的思路．近年来,光学成像的发展为可视化研究IS形成与T细胞活化的关系提供了有力帮助,为研究生理病理状态下的免疫应答提供了有力工具．     

Cutting edge: dendritic cell actin cytoskeletal polarization during immunological synapse formation is highly antigen-dependent

Dendritic cells (DC) actively rearrange their actin cytoskeleton to participate in formation of the immunological synapse (IS). In this study, we evaluated the requirements for DC participation in the IS. DC rearrange their actin cytoskeleton toward naive CD4(+) T cells only in the presence of specific MHC-peptide complexes. In contrast, naive CD4(+) T cells polarized their cytoskeletal proteins in the absence of Ag. DC cytoskeletal rearrangement occurred at the same threshold of peptide-MHC complexes as that required for T cell activation. Furthermore, T cell activation was inhibited by specific blockade of DC cytoskeletal rearrangement. When TCR-MHC interaction was bypassed by using Con A-activated T cells, DC polarization was abrogated. In addition, directional ligation of MHC class II resulted in DC cytoskeletal polarization. Our findings suggest that a high Ag specificity is required for DC IS formation and that MHC class II signaling plays a central role in this process.