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    Wiskott-Aldrich Syndrome at the nexus of autoimmune and primary immunodeficiency diseases. Cleland Sophia Y,Siegel Richard M FEBS letters Wiskott-Aldrich Syndrome (WAS) is a X-linked primary immunodeficiency disorder also marked by a very high (up to 70%) incidence of autoimmunity. Wiskott-Aldrich Syndrome arises from mutations in the Wiskott-Aldrich Syndrome protein (WASp), a cytoplasmic protein that links signaling by cell surface receptors such as the T-cell receptor and integrins to actin polymerization. WASp promotes the functions of multiple cell types that support immune responses, but also is important for the function of regulatory T cells and in TCR-induced apoptosis, two negative mechanisms of immune regulation that maintain peripheral immune tolerance. Here we review the nature of immune defects and autoimmunity in WAS and WASp deficient mice and discuss how this single gene defect can simultaneously impair immune responses to pathogens and promote autoimmunity. The myriad cellular immune defects found in WAS make this Mendelian syndrome an interesting model for the study of more complex immune diseases that arise from the interplay of environmental and multiple genetic risk factors. 10.1016/j.febslet.2011.10.031
    Autoimmunity in wiskott-Aldrich syndrome: an unsolved enigma. Catucci Marco,Castiello Maria Carmina,Pala Francesca,Bosticardo Marita,Villa Anna Frontiers in immunology Wiskott-Aldrich Syndrome (WAS) is a severe X-linked Primary Immunodeficiency that affects 1-10 out of 1 million male individuals. WAS is caused by mutations in the WAS Protein (WASP) expressing gene that leads to the absent or reduced expression of the protein. WASP is a cytoplasmic protein that regulates the formation of actin filaments in hematopoietic cells. WASP deficiency causes many immune cell defects both in humans and in the WAS murine model, the Was(-/-) mouse. Both cellular and humoral immune defects in WAS patients contribute to the onset of severe clinical manifestations, in particular microthrombocytopenia, eczema, recurrent infections, and a high susceptibility to develop autoimmunity and malignancies. Autoimmune diseases affect from 22 to 72% of WAS patients and the most common manifestation is autoimmune hemolytic anemia, followed by vasculitis, arthritis, neutropenia, inflammatory bowel disease, and IgA nephropathy. Many groups have widely explored immune cell functionality in WAS partially explaining how cellular defects may lead to pathology. However, the mechanisms underlying the occurrence of autoimmune manifestations have not been clearly described yet. In the present review, we report the most recent progresses in the study of immune cell function in WAS that have started to unveil the mechanisms contributing to autoimmune complications in WAS patients. 10.3389/fimmu.2012.00209
    Differential roles for Wiskott-Aldrich syndrome protein in immune synapse formation and IL-2 production. Cannon Judy L,Burkhardt Janis K Journal of immunology (Baltimore, Md. : 1950) Wiskott-Aldrich syndrome protein (WASP)-deficient T cells exhibit defects in IL-2 production that are widely believed to stem from primary defects in actin remodeling and immune synapse formation. Surprisingly, however, we find that WASP-deficient T cells responding to Ag-specific APCs polymerize actin and organize talin and PKC theta normally, forming an immune synapse that is stable for at least 3 h. At low doses of peptide, WASP-deficient T cells show less efficient talin and PKC theta polarization. Thus, although WASP may facilitate immune synapse formation at low peptide concentrations, WASP is not required for this process. Defects in IL-2 production are observed even under conditions in which immune synapse formation proceeds normally, suggesting that the role of WASP in regulating IL-2 production is independent of its role in immune synapse formation. 10.4049/jimmunol.173.3.1658
    Signal Integration during T Lymphocyte Activation and Function: Lessons from the Wiskott-Aldrich Syndrome. Cotta-de-Almeida Vinicius,Dupré Loïc,Guipouy Delphine,Vasconcelos Zilton Frontiers in immunology Over the last decades, research dedicated to the molecular and cellular mechanisms underlying primary immunodeficiencies (PID) has helped to understand the etiology of many of these diseases and to develop novel therapeutic approaches. Beyond these aspects, PID are also studied because they offer invaluable natural genetic tools to dissect the human immune system. In this review, we highlight the research that has focused over the last 20 years on T lymphocytes from Wiskott-Aldrich syndrome (WAS) patients. WAS T lymphocytes are defective for the WAS protein (WASP), a regulator of actin cytoskeleton remodeling. Therefore, study of WAS T lymphocytes has helped to grasp that many steps of T lymphocyte activation and function depend on the crosstalk between membrane receptors and the actin cytoskeleton. These steps include motility, immunological synapse assembly, and signaling, as well as the implementation of helper, regulatory, or cytotoxic effector functions. The recent concept that WASP also works as a regulator of transcription within the nucleus is an illustration of the complexity of signal integration in T lymphocytes. Finally, this review will discuss how further study of WAS may contribute to solve novel challenges of T lymphocyte biology. 10.3389/fimmu.2015.00047
    Immunological synapses: breaking up may be good to do. Krummel Matthew F Cell Activated T cells form stable immunological synapses with antigen-presenting cells whereas naïve T cells initially engage in more transient interactions. Sims et al. (2007) demonstrate that these transient interactions are due to the kinase PKCtheta, which serves to destabilize the synapse thereby permitting T cells to migrate elsewhere. They also show that re-establishment of a synapse involves the actin regulator WASp. 10.1016/j.cell.2007.05.008
    Involvement of the Wiskott-Aldrich syndrome protein and other actin regulatory adaptors in T cell activation. Badour Karen,Zhang Jinyi,Siminovitch Katherine A Seminars in immunology The actin cytoskeleton is a dynamic structure recognized for many years as integral to the coupling of external stimuli to cell activation and ensuing changes in morphology and movement. It is only recently, however, that a molecular understanding of actin involvement in these activities has emerged coincident with the identification of cytosolic signaling effectors that couple extracellular stimuli to induction of actin nucleation. Notable among these actin regulatory effectors are members of the Wiskott-Aldrich syndrome protein (WASp) family, a group of cytoskeletal adaptors imbued with the capacity to connect various signal transduction pathways to the Arp 2/3 complex and Arp 2/3-mediated actin polymerization. In T cells, the functional characterization of WASp and other actin-modulatory adaptors has proved instrumental in delineating the molecular interactions evoking actin cytoskeletal reorganization downstream of antigen receptor engagement and in clarifying the influence of actin-based processes on T cell activation. In this review, the structural and functional properties of the major actin regulatory cytoskeletal adaptors in T cells are described with an emphasis on the roles of these proteins in fostering the TCR actin cytoskeletal interplay required for induction of T cell activation and expression of dynamic effector responses. 10.1016/j.smim.2004.08.019
    Defective actin reorganization and polymerization of Wiskott-Aldrich T cells in response to CD3-mediated stimulation. Gallego M D,Santamaría M,Peña J,Molina I J Blood The Wiskott-Aldrich syndrome (WAS) is a severe immunodeficiency and platelet deficiency disease arising from mutation(s) in the WASP gene, which in normal cells encodes an intracellular protein able to interact with other proteins relevant to the control of cytoskeleton organization. Immunodeficiency is mainly due to T-cell progressive malfunction. Salient defects of WAS T cells are a CD3-restricted impairment in proliferative responses and cytoskeletal abnormalities, including the frequent appearance of T cells with atypical morphology. We have investigated the possibility that the CD3-restricted defect and some of the cytoskeletal defects of WAS T cells are linked. For this purpose, we immortalized by means of infection with Herpesvirus Saimiri a number of previously described allospecific WAS T-cell lines. The resulting cells preserve the surface, molecular, and functional phenotypes of their parental lines, including a negligible WASP mRNA expression as well as the CD3-restricted defect and cytoskeleton abnormalities. Results show that, in CD3-stimulated WAS T cells, the pattern of temporal changes in cell shape and F-actin distribution is substantially different from that of control cells. Furthermore, polymerization of actin, a critical step in the CD3-mediated cytoskeleton reorganization, does not occur in WAS T-cell lines in response to OKT3 stimulation. In conclusion, our data link both CD3 and cytoskeletal defects in WAS T cells, strongly suggesting that cytoskeleton abnormalities are an underlying cause for WAS immunodeficiency.
    The Wiskott-Aldrich syndrome protein acts downstream of CD2 and the CD2AP and PSTPIP1 adaptors to promote formation of the immunological synapse. Badour Karen,Zhang Jinyi,Shi Fabio,McGavin Mary K H,Rampersad Vik,Hardy Lynne A,Field Deborah,Siminovitch Katherine A Immunity The Wiskott-Aldrich syndrome protein (WASp) couples actin cytoskeletal rearrangement to T cell activation, but the mechanisms involved are unknown. Here, we show that antigen-induced formation of T cell:APC conjugates and synapses is abrogated in WASp-deficient T cells and that CD2 engagement evokes interactions between the proline-rich region required for WASp translocation to the synapse and the PSTPIP1 adaptor SH3 domain and between the PSTPIp1 coiled-coil domain and both CD2 and another CD2-binding adaptor, CD2AP. The induced colocalization of these proteins at the synapse is disrupted by expression of coiled-coil domain-deleted PSTPIP1. These data, together with the impairment in CD2-induced actin polymerization observed in WASp-deficient cells, suggest that PSTPIP1 acts downstream of CD2/CD2AP to link CD2 engagement to the WASp-evoked actin polymerization required for synapse formation and T cell activation.
    The Wiskott-Aldrich Syndrome Protein Contributes to the Assembly of the LFA-1 Nanocluster Belt at the Lytic Synapse. Houmadi Raïssa,Guipouy Delphine,Rey-Barroso Javier,Vasconcelos Zilton,Cornet Julie,Manghi Manoel,Destainville Nicolas,Valitutti Salvatore,Allart Sophie,Dupré Loïc Cell reports T lymphocyte cytotoxicity relies on a synaptic ring of lymphocyte function-associated antigen 1 (LFA-1), which permits polarized delivery of lytic granules. How LFA-1 organization is controlled by underlying actin cytoskeleton dynamics is poorly understood. Here, we explored the contribution of the actin cytoskeleton regulator WASP to the topography of LFA-1 using a combination of microscopy modalities. We uncover that the reduced cytotoxicity of Wiskott-Aldrich syndrome patient-derived CD8 T lymphocytes lacking WASP is associated with reduced LFA-1 activation, unstable synapse, and delayed lethal hit. At the nanometric scale, WASP constrains high-affinity LFA-1 into dense nanoclusters located in actin meshwork interstices. At the cellular scale, WASP is required for the assembly of a radial belt composed of hundreds of LFA-1 nanoclusters and for lytic granule docking within this belt. Our study unravels the nanoscale topography of LFA-1 at the lytic synapse and identifies WASP as a molecule controlling individual LFA-1 cluster density and LFA-1 nanocluster belt integrity. 10.1016/j.celrep.2017.12.088
    Critical requirement for the Wiskott-Aldrich syndrome protein in Th2 effector function. Morales-Tirado Vanessa,Sojka Dorothy K,Katzman Shoshana D,Lazarski Christopher A,Finkelman Fred D,Urban Joseph F,Fowell Deborah J Blood Patients with Wiskott-Aldrich syndrome (WAS) have numerous immune cell deficiencies, but it remains unclear how abnormalities in individual cell types contribute to the pathologies of WAS. In T cells, the WAS protein (WASp) regulates actin polymerization and transcription, and plays a role in the dynamics of the immunologic synapse. To examine how these events influence CD4 function, we isolated the WASp deficiency to CD4(+) T cells by adoptive transfer into wild-type mice to study T-cell priming and effector function. WAS(-/-) CD4(+) T cells mediated protective T-helper 1 (Th1) responses to Leishmania major in vivo, but were unable to support Th2 immunity to Nippostrongylus brasiliensis or L major. Mechanistically, WASp was not required for Th2 programming but was required for Th2 effector function. WAS(-/-) CD4(+) T cells up-regulated IL-4 and GATA3 mRNA and secreted IL-4 protein during Th2 differentiation. In contrast, cytokine transcription was uncoupled from protein production in WAS(-/-) Th2-primed effectors. WAS(-/-) Th2s failed to produce IL-4 protein on restimulation despite elevated IL-4/GATA3 mRNA. Moreover, dominant-negative WASp expression in WT effector T cells blocked IL-4 production, but had no effect on IFNgamma. Thus WASp plays a selective, posttranscriptional role in Th2 effector function. 10.1182/blood-2009-07-235754
    A role for Wiskott-Aldrich syndrome protein in T-cell receptor-mediated transcriptional activation independent of actin polymerization. Silvin C,Belisle B,Abo A The Journal of biological chemistry Wiskott-Aldrich syndrome protein (WASP) plays a key role in cytoskeletal rearrangement and transcriptional activation in T-cells. Recent evidence links WASP and related proteins to actin polymerization by the Arp2/3 complex. To study whether the role of WASP in actin polymerization is coupled to T-cell receptor (TCR)-mediated transcriptional activation, we made a series of WASP deletion mutants and tested them for actin co-localization, actin polymerization, and transcriptional activation of NFAT. A WASP mutant with a deletion in the C-terminal region (WASPDeltaC) that is defective in actin polymerization potentiated NFAT transcription following TCR activation by anti-CD3 and anti-CD3/CD28 antibodies, but not by phorbol 12-myristate 13-acetate/ionomycin. Furthermore, cotransfection of a dominant-active mutant (WASP-WH2-C) for Arp2/3 polymerization did not inhibit NFAT activation. Finally, by analyzing a series of WASP double-domain deletion mutants, we determined that the WASP homology-1 domain is responsible for NFAT transcriptional activation. Our results suggest that WASP activates transcription following TCR stimulation in a manner that is independent of its role in Arp2/3-directed actin polymerization. 10.1074/jbc.M010729200
    T cell receptor-triggered nuclear actin network formation drives CD4 T cell effector functions. Tsopoulidis N,Kaw S,Laketa V,Kutscheidt S,Baarlink C,Stolp B,Grosse R,Fackler O T Science immunology T cell antigen receptor (TCR) signaling triggers selective cytokine expression to drive T cell proliferation and differentiation required for immune defense and surveillance. The nuclear signaling events responsible for specificity in cytokine gene expression upon T cell activation are largely unknown. Here, we uncover formation of a dynamic actin filament network in the nucleus that regulates cytokine expression for effector functions of CD4 T lymphocytes. TCR engagement triggers the rapid and transient formation of a nuclear actin filament network via nuclear Arp2/3 complex, induced by elevated nuclear Ca levels and regulated via N-Wasp and NIK. Specific interference with TCR-induced formation of nuclear actin filaments impairs production of effector cytokines and prevents generation of antigen-specific antibodies but does not interfere with immune synapse formation and cell proliferation. Ca-regulated actin polymerization in the nucleus allows CD4 T cells the rapid conversion of TCR signals into effector functions required for T cell help. 10.1126/sciimmunol.aav1987
    R-loops cause genomic instability in T helper lymphocytes from patients with Wiskott-Aldrich syndrome. Sarkar Koustav,Han Seong-Su,Wen Kuo-Kuang,Ochs Hans D,Dupré Loïc,Seidman Michael M,Vyas Yatin M The Journal of allergy and clinical immunology BACKGROUND:Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), and X-linked neutropenia, which are caused by WAS mutations affecting Wiskott-Aldrich syndrome protein (WASp) expression or activity, manifest in immunodeficiency, autoimmunity, genomic instability, and lymphoid and other cancers. WASp supports filamentous actin formation in the cytoplasm and gene transcription in the nucleus. Although the genetic basis for XLT/WAS has been clarified, the relationships between mutant forms of WASp and the diverse features of these disorders remain ill-defined. OBJECTIVE:We sought to define how dysfunctional gene transcription is causally linked to the degree of T cell deficiency and genomic instability in the XLT/WAS clinical spectrum. METHODS:In human T1- or T2-skewing cell culture systems, cotranscriptional R-loops (RNA/DNA duplex and displaced single-stranded DNA) and DNA double-strand breaks (DSBs) were monitored in multiple samples from patients with XLT and WAS and in normal T cells depleted of WASp. RESULTS:WASp deficiency provokes increased R-loops and R-loop-mediated DSBs in T1 cells relative to T2 cells. Mechanistically, chromatin occupancy of serine 2-unphosphorylated RNA polymerase II is increased, and that of topoisomerase 1, an R-loop preventing factor, is decreased at R-loop-enriched regions of IFNG and TBX21 (T1 genes) in T1 cells. These aberrations accompany increased unspliced (intron-retained) and decreased spliced mRNA of IFNG and TBX21 but not IL13 (T2 gene). Significantly, increased cellular load of R-loops and DSBs, which are normalized on RNaseH1-mediated suppression of ectopic R-loops, inversely correlates with disease severity scores. CONCLUSION:Transcriptional R-loop imbalance is a novel molecular defect causative in T1 immunodeficiency and genomic instability in patients with WAS. The study proposes that cellular R-loop load could be used as a potential biomarker for monitoring symptom severity and prognostic outcome in the XLT-WAS clinical spectrum and could be targeted therapeutically. 10.1016/j.jaci.2017.11.023
    Cutting edge: selective requirement for the Wiskott-Aldrich syndrome protein in cytokine, but not chemokine, secretion by CD4+ T cells. Morales-Tirado Vanessa,Johannson Sara,Hanson Elaine,Howell Alan,Zhang Jinyi,Siminovitch Katherine A,Fowell Deborah J Journal of immunology (Baltimore, Md. : 1950) The mechanism of cytokine secretion is not well understood, but cytokines appear to be synthesized and released in a polarized fashion toward an Ag-specific target cell. In this study, we demonstrate that the Wiskott-Aldrich syndrome protein (WASp) is an essential component of the cytokine secretory pathway in CD4(+) T cells. Murine WASp-deficient CD4(+) T cells fail to polarize cytokines toward a target and show an unexpected and striking block in cytokine secretion. In contrast, chemokine secretion and trafficking of plasma membrane proteins, transported via the constitutive secretory pathway, are unaffected by the lack of WASp. These results suggest that CD4(+) T cell cytokines require a specialized, WASp-dependent pathway for cellular traffic and/or vesicle release that is distinct from that required for chemokine release. We propose that the use of different secretory pathways for cytokines and chemokines enables CD4(+) T cell activity to be further fine-tuned to serve specialized effector functions. 10.4049/jimmunol.173.2.726
    Wiskott-Aldrich syndrome protein-deficient mice reveal a role for WASP in T but not B cell activation. Snapper S B,Rosen F S,Mizoguchi E,Cohen P,Khan W,Liu C H,Hagemann T L,Kwan S P,Ferrini R,Davidson L,Bhan A K,Alt F W Immunity The Wiskott-Aldrich syndrome (WAS) is a human X-linked immunodeficiency resulting from mutations in a gene (WASP) encoding a cytoplasmic protein implicated in regulating the actin cytoskeleton. To elucidate WASP function, we disrupted the WASP gene in mice by gene-targeted mutation. WASP-deficient mice showed apparently normal lymphocyte development, normal serum immunoglobulin levels, and the capacity to respond to both T-dependent and T-independent type II antigens. However, these mice did have decreased peripheral blood lymphocyte and platelet numbers and developed chronic colitis. Moreover, purified WASP-deficient T cells showed markedly impaired proliferation and antigen receptor cap formation in response to anti-CD3epsilon stimulation. Yet, purified WASP-deficient B cells showed normal responses to anti-Ig stimulation. We discuss the implications of our findings regarding WASP function in receptor signaling and cytoskeletal reorganization in T and B cells and compare the effects of WASP deficiency in mice and humans.
    Wasp recruitment to the T cell:APC contact site occurs independently of Cdc42 activation. Cannon J L,Labno C M,Bosco G,Seth A,McGavin M H,Siminovitch K A,Rosen M K,Burkhardt J K Immunity Cdc42 and WASP are critical regulators of actin polymerization whose function during T cell signaling is poorly understood. Using a novel reagent that specifically detects Cdc42-GTP in fixed cells, we found that activated Cdc42 localizes to the T cell:APC contact site in an antigen-dependent manner. TCR signaling alone was sufficient to induce localization of Cdc42-GTP, and functional Lck and Zap-70 kinases were required. WASP also localized to the T cell:APC contact site in an antigen-dependent manner. Surprisingly, WASP localization was independent of the Cdc42 binding domain but required the proline-rich domain. Our results indicate that localized WASP activation requires the integration of multiple signals: WASP is recruited via interaction with SH3 domain-containing proteins and is activated by Cdc42-GTP concentrated at the same site.
    Lentiviral-mediated gene therapy restores B cell tolerance in Wiskott-Aldrich syndrome patients. Pala Francesca,Morbach Henner,Castiello Maria Carmina,Schickel Jean-Nicolas,Scaramuzza Samantha,Chamberlain Nicolas,Cassani Barbara,Glauzy Salome,Romberg Neil,Candotti Fabio,Aiuti Alessandro,Bosticardo Marita,Villa Anna,Meffre Eric The Journal of clinical investigation Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by microthrombocytopenia, eczema, and high susceptibility to developing tumors and autoimmunity. Recent evidence suggests that B cells may be key players in the pathogenesis of autoimmunity in WAS. Here, we assessed whether WAS protein deficiency (WASp deficiency) affects the establishment of B cell tolerance by testing the reactivity of recombinant antibodies isolated from single B cells from 4 WAS patients before and after gene therapy (GT). We found that pre-GT WASp-deficient B cells were hyperreactive to B cell receptor stimulation (BCR stimulation). This hyperreactivity correlated with decreased frequency of autoreactive new emigrant/transitional B cells exiting the BM, indicating that the BCR signaling threshold plays a major role in the regulation of central B cell tolerance. In contrast, mature naive B cells from WAS patients were enriched in self-reactive clones, revealing that peripheral B cell tolerance checkpoint dysfunction is associated with impaired suppressive function of WAS regulatory T cells. The introduction of functional WASp by GT corrected the alterations of both central and peripheral B cell tolerance checkpoints. We conclude that WASp plays an important role in the establishment and maintenance of B cell tolerance in humans and that restoration of WASp by GT is able to restore B cell tolerance in WAS patients. 10.1172/JCI82249
    Abnormalities of follicular helper T-cell number and function in Wiskott-Aldrich syndrome. Zhang Xuan,Dai Rongxin,Li Wenyan,Zhao Hongyi,Zhang Yongjie,Zhou Lina,Du Hongqiang,Luo Guangjin,Wu Junfeng,Niu Linlin,An Yunfei,Zhang Zhiyong,Ding Yuan,Song Wenxia,Liu Chaohong,Zhao Xiaodong Blood Wiskott-Aldrich syndrome protein (WASp) is a hematopoietic-specific regulator of actin nucleation. Wiskott-Aldrich syndrome (WAS) patients show immunodeficiencies, most of which have been attributed to defective T-cell functions. T follicular helper (Tfh) cells are the major CD4(+) T-cell subset with specialized B-cell helper capabilities. Aberrant Tfh cells activities are involved in immunopathologies such as autoimmunity, immunodeficiencies, and lymphomas. We found that in WAS patients, the number of circulating Tfh cells was significantly reduced due to reduced proliferation and increased apoptosis, and Tfh cells were Th2 and Th17 polarized. The expression of inducible costimulator (ICOS) in circulating Tfh cells was higher in WAS patients than in controls. BCL6 expression was decreased in total CD4(+) T and Tfh cells of WAS patients. Mirroring the results in patients, the frequency of Tfh cells in WAS knockout (KO) mice was decreased, as was the frequency of BCL6(+) Tfh cells, but the frequency of ICOS(+) Tfh cells was increased. Using WAS chimera mice, we found that the number of ICOS(+) Tfh cells was decreased in WAS chimera mice, indicating that the increase in ICOS(+) Tfh cells in WAS KO mice was cell extrinsic. The data from in vivo CD4(+) naive T-cell adoptive transfer mice as well as in vitro coculture of naive B and Tfh cells showed that the defective function of WASp-deficient Tfh cells was T-cell intrinsic. Consistent findings in both WAS patients and WAS KO mice suggested an essential role for WASp in the development and memory response of Tfh cells and that WASp deficiency causes a deficient differentiation defect in Tfh cells by downregulating the transcription level of BCL6. 10.1182/blood-2015-06-652636
    Coordinate control of cytoskeletal remodeling and calcium mobilization during T-cell activation. Babich Alexander,Burkhardt Janis K Immunological reviews Ca(2+) mobilization and cytoskeletal reorganization are key hallmarks of T-cell activation, and their interdependence has long been recognized. Recent advances in the field have elucidated the molecular pathways that underlie these events and have revealed several points of intersection. Ca(2+) signaling can be divided into two phases: initial events leading to release of Ca(2+) from endoplasmic reticulum stores, and a second phase involving STIM 1 (stromal interaction molecule 1) clustering and CRAC (calcium release activated calcium) channel activation. Cytoskeletal dynamics promote both phases. During the first phase, the actin cytoskeleton promotes mechanotransduction and serves as a dynamic scaffold for microcluster assembly. Proteins that drive actin polymerization such as WASp (Wiskott-Aldrich syndrome protein) and HS1 (hematopoietic lineage cell-specific protein 1) promote signaling through PLCγ1 (phospholipase Cγ1) and release of Ca(2+) from endoplasmic reticulum stores. During the second phase, the WAVE (WASP-family verprolin homologous protein) complex and the microtubule cytoskeleton promote STIM 1 clustering at sites of plasma membrane apposition, opening Orai channels. In addition, gross cell shape changes and organelle movements buffer local Ca(2+) levels, leading to sustained Ca(2+) mobilization. Conversely, elevated intracellular Ca(2+) activates cytoskeletal remodeling. This can occur indirectly, via calpain activity, and directly, via Ca(2+) -dependent cytoskeletal regulatory proteins such as myosin II and L-plastin. While it is true that the cytoskeleton regulates Ca(2+) responses and vice versa, interdependence between Ca(2+) and the cytoskeleton also encompasses signaling events that occur in parallel, downstream of shared intermediates. Inositol cleavage by PLCγ1 simultaneously triggers both endoplasmic reticulum store release and diacylglycerol-dependent microtubule organizing center reorientation, while depleting the pool of phosphatidylinositol-4,5-bisphosphate, an activator of multiple actin-regulatory proteins. The close interdependence of Ca(2+) signaling and cytoskeletal dynamics in T cells provides positive feedback mechanisms for T-cell activation and allows for finely tuned responses to extracellular cues. 10.1111/imr.12123
    Defective thymic output in WAS patients is associated with abnormal actin organization. Li Wenyan,Sun Xiaoyu,Wang Jinzhi,Zhao Qin,Dai Rongxin,Wang Yanping,Zhou Lina,Westerberg Lisa,Ding Yuan,Zhao Xiaodong,Liu Chaohong Scientific reports Wiskott-Aldrich syndrome protein (WASp) is a key regulator of the actin cytoskeleton. Defective T - cell function is a major cause for immune deficiency in Wiskott-Aldrich syndrome (WAS) patients. T cells originate in the bone marrow and develop in the thymus, and then migrate to peripheral tissues. TCR excision circles (TRECs) present in thymic output cells stably, which is used as a molecular marker for thymic output. We found that CD8 T naïve cells of classic WAS patients were significantly reduced, and TRECs in patients with classic WAS and X-linked thrombocytopenia (XLT) dramatically decreased compared with that of HCs. TRECs were also reduced in WAS (KO) mice. These suggest that defective thymic output partially accounts for T cell lymphopenia in WAS patients. However, the correlation between the defect of thymic output and actin organization still remains elusive. We found that the subcellular location and the levels of of F-actin were altered in T cells from both WAS and XLT patients compared to that of HCs with or without stimulation. Our study shows that WASp plays a critical role in thymic output, which highly correlates with the subcellular location and level of F-actin in T cells. 10.1038/s41598-017-12345-z
    The intersectin 2 adaptor links Wiskott Aldrich Syndrome protein (WASp)-mediated actin polymerization to T cell antigen receptor endocytosis. McGavin M K,Badour K,Hardy L A,Kubiseski T J,Zhang J,Siminovitch K A The Journal of experimental medicine Induction of T cell antigen receptor (TCR) endocytosis has a significant impact on TCR signaling and T cell behavior, but the molecular interactions coordinating internalization of the activated TCR are poorly understood. Previously we have shown that TCR endocytosis is regulated by the Wiskott Aldrich Syndrome protein (WASp), a cytosolic effector which, upon interaction with the cdc42 Rho GTPase, couples TCR engagement to Arp 2/3 complex-mediated actin polymerization. Here we report that WASp associates in T cells with intersectin 2, an endocytic adaptor containing multiple domains including a Dbl homology (DH) domain with the potential to activate Rho GTPases. Intersectin 2 association with WASp increases after TCR engagement, and its overexpression in Cos-7 cells induces WASp translocation to endocytic vesicles within which intersectin 2 colocalizes with both WASp and cdc42. Intersectin 2, but not a DH domain-deleted (DeltaDH) form of intersectin 2, and stimulation via the TCR also trigger the activation of cdc42. Induction of TCR internalization is also augmented by intersectin 2 and severely impaired by latrunculin B treatment. Thus, intersection 2 appears to function cooperatively with WASp and cdc42 to link the clathrin endocytic machinery to WASp-mediated actin polymerization and ultimately to occupancy-induced TCR endocytosis. 10.1084/jem.194.12.1777
    Defective nuclear translocation of nuclear factor of activated T cells and extracellular signal-regulated kinase underlies deficient IL-2 gene expression in Wiskott-Aldrich syndrome. Cianferoni Antonella,Massaad Michel,Feske Stefan,de la Fuente Miguel A,Gallego Lola,Ramesh Narayanaswamy,Geha Raif S The Journal of allergy and clinical immunology BACKGROUND:Proliferation and IL-2 production in response to T-cell receptor ligation are impaired in patients with Wiskott-Aldrich syndrome (WAS). The transcription factors nuclear factor-kappaB (NF-kappaB), nuclear factor of activated T cells (NF-AT), and activating protein-1 (AP-1) play a critical role in IL-2 gene expression. OBJECTIVE:To investigate the mechanisms of impaired IL-2 production after T-cell receptor ligation in T cells deficient in WAS protein (WASP). METHODS:T cells from WASP-/- mice were stimulated with anti-CD3 and anti-CD28. Nuclear NF-kappaB, NF-AT, and AP-1 DNA-binding activity was examined by electroshift mobility assay. NF-ATp dephosphorylation and nuclear localization were examined by Western blot and indirect immunofluorescence. Phosphorylation of the mitogen-activated protein kinases Erk and Jnk, and of their nuclear substrates Elk-1 and c-Jun, was examined by Western blot. Expression of mRNA for IL-2 and the NF-kappaB-dependent gene A20 and of the AP-1 components c-fos and c-Jun was examined by quantitative RT-PCR. RESULTS:Nuclear translocation and activity of NF-kappaB were normal in T cells from WASP-/- mice. In contrast, NF-ATp dephosphorylation and nuclear localization, nuclear AP-1 binding activity, and expression of c-fos, but not c-Jun, were all impaired. Phosphorylation of Jnk, c-Jun, and Erk were normal. However, nuclear translocation of phosphorylated Erk and phosphorylation of its nuclear substrate Elk1, which activates the c-fos promoter, were impaired. CONCLUSION:These results suggest that WASP is essential for NF-ATp activation, and for nuclear translocation of p-Erk, Elk1 phosphorylation, and c-fos gene expression in T cells. These defects underlie defective IL-2 expression and T-cell proliferation in WAS. 10.1016/j.jaci.2005.09.006
    WASp verprolin homology, cofilin homology, and acidic region domain-mediated actin polymerization is required for T cell development. Zhang Jinyi,Shi Fabio,Badour Karen,Deng Yupu,McGavin Mary K H,Siminovitch Katherine A Proceedings of the National Academy of Sciences of the United States of America All members of the Wiskott-Aldrich syndrome protein (WASp) family contain a carboxyl-terminal verprolin homology, cofilin homology, and acidic region (VCA) domain that binds and activates the Arp2/3 complex, thereby linking these proteins to the induction of actin polymerization. Although the VCA domain imbues WASp and other WASp family members with the capacity to modulate cytoskeletal organization, little is known about the impact of this domain activity on lymphoid cell function. Here we demonstrate that T cell-restricted expression of VCA domain-deleted WASp (WASpdeltaVCA) in WAS(-/-) mice engenders a severe early block in T lymphopoiesis associated with impaired T cell antigen receptor alphabeta expression and a consequent failure to generate single-positive CD4(+) and CD8(+) T cells. These latter defects, which are not observed in WAS(-/-) mice, are associated with impaired induction of cellular actin polymerization and a failure in the terminal differentiation of double-negative thymocytes. These findings indicate that WASp family proteins play an essential role in modulating the signaling events required for early thymocyte development and reveal their capacity to subserve this role to depend on VCA domain-mediated actin polymerization. 10.1073/pnas.042686099
    Systemic autoimmunity and defective Fas ligand secretion in the absence of the Wiskott-Aldrich syndrome protein. Nikolov Nikolay P,Shimizu Masaki,Cleland Sophia,Bailey Daniel,Aoki Joseph,Strom Ted,Schwartzberg Pamela L,Candotti Fabio,Siegel Richard M Blood Autoimmunity is a surprisingly common complication of primary immunodeficiencies, yet the molecular mechanisms underlying this clinical observation are not well understood. One widely known example is provided by Wiskott-Aldrich syndrome (WAS), an X-linked primary immunodeficiency disorder caused by mutations in the gene encoding the WAS protein (WASp) with a high incidence of autoimmunity in affected patients. WASp deficiency affects T-cell antigen receptor (TCR) signaling and T-cell cytokine production, but its role in TCR-induced apoptosis, one of the mechanisms of peripheral immunologic tolerance, has not been investigated. We find that WASp-deficient mice produce autoantibodies and develop proliferative glomerulonephritis with immune complex deposition as they age. We also find that CD4(+) T lymphocytes from WASp-deficient mice undergo reduced apoptosis after restimulation through the TCR. While Fas-induced cell death is normal, WASp deficiency affects TCR-induced secretion of Fas ligand (FasL) and other components of secretory granules by CD4(+) T cells. These results describe a novel role of WASp in regulating TCR-induced apoptosis and FasL secretion and suggest that WASp-deficient mice provide a good model for the study of autoimmune manifestations of WAS and the development of more specific therapies for these complications. 10.1182/blood-2009-08-237560
    Wiskott-Aldrich syndrome protein controls antigen-presenting cell-driven CD4+ T-cell motility by regulating adhesion to intercellular adhesion molecule-1. Lafouresse Fanny,Cotta-de-Almeida Vinicius,Malet-Engra Gema,Galy Anne,Valitutti Salvatore,Dupré Loïc Immunology T-cell scanning for antigen-presenting cells (APC) is a finely tuned process. Whereas non-cognate APC trigger T-cell motility via chemokines and intercellular adhesion molecule-1 (ICAM-1), cognate APC deliver a stop signal resulting from antigen recognition. We tested in vitro the contribution of the actin cytoskeleton regulator Wiskott-Aldrich syndrome protein (WASP) to the scanning activity of primary human CD4(+)  T cells. WASP knock-down resulted in increased T-cell motility upon encounter with non-cognate dendritic cells or B cells and reduced capacity to stop following antigen recognition. The high motility of WASP-deficient T cells was accompanied by a diminished ability to round up and to stabilize pauses. WASP-deficient T cells migrated in a normal proportion towards CXCL12, CCL19 and CCL21, but displayed an increased adhesion and elongation on ICAM-1. The elongated morphology of WASP-deficient T cells was related to a reduced confinement of high-affinity lymphocyte function-associated antigen 1 to the mid-cell zone. Our data therefore indicate that WASP controls CD4(+) T-cell motility upon APC encounter by regulating lymphocyte function-associated antigen 1 spatial distribution. 10.1111/j.1365-2567.2012.03620.x
    The expression of Wiskott-Aldrich syndrome protein (WASP) is dependent on WASP-interacting protein (WIP). Konno Akihiro,Kirby Martha,Anderson Stacie A,Schwartzberg Pamela L,Candotti Fabio International immunology The Wiskott-Aldrich syndrome protein (WASP) is a key molecule for transduction of extracellular signals that induce a variety of critical biological events involving actin cytoskeleton rearrangement. Among the cellular partners of WASP, the Wiskott-Aldrich syndrome protein-interacting protein (WIP) has been speculated to play a critical role in the pathophysiology of Wiskott-Aldrich syndrome since WASP mutation hot spots map to the WIP-binding region. The notion that WIP promotes WASP function, however, conflicts with evidence that WIP inhibits WASP-mediated actin polymerization and IL-2 production and suggests a complex regulation of WASP function by WIP. Here we show that WASP gene transfer results in high WASP expression only when WIP is concomitantly expressed in K562 cells. Furthermore, WIP-knockdown experiments demonstrated that T cells with reduced WIP expression show a concordant reduction of WASP levels. Mapping studies using WIP mutants showed that the minimal WIP region able to rescue WASP expression in WIP-knockdown cells was the WASP-binding domain. However, expression of such a minimal domain of WIP failed to rescue WASP-dependent, nuclear factor of activated T-cells-mediated IL-2 transcriptional activity. These results demonstrate that expression of WIP is necessary for functional WASP expression in human cells and provide a new paradigm for understanding the function of these two molecules. 10.1093/intimm/dxl135
    Cytoskeletal tension actively sustains the migratory T-cell synaptic contact. Kumari Sudha,Mak Michael,Poh Yeh-Chuin,Tohme Mira,Watson Nicki,Melo Mariane,Janssen Erin,Dustin Michael,Geha Raif,Irvine Darrell J The EMBO journal When migratory T cells encounter antigen-presenting cells (APCs), they arrest and form radially symmetric, stable intercellular junctions termed immunological synapses which facilitate exchange of crucial biochemical information and are critical for T-cell immunity. While the cellular processes underlying synapse formation have been well characterized, those that maintain the symmetry, and thereby the stability of the synapse, remain unknown. Here we identify an antigen-triggered mechanism that actively promotes T-cell synapse symmetry by generating cytoskeletal tension in the plane of the synapse through focal nucleation of actin via Wiskott-Aldrich syndrome protein (WASP), and contraction of the resultant actin filaments by myosin II. Following T-cell activation, WASP is degraded, leading to cytoskeletal unraveling and tension decay, which result in synapse breaking. Thus, our study identifies and characterizes a mechanical program within otherwise highly motile T cells that sustains the symmetry and stability of the T cell-APC synaptic contact. 10.15252/embj.2019102783
    Defective Th1 cytokine gene transcription in CD4+ and CD8+ T cells from Wiskott-Aldrich syndrome patients. Trifari Sara,Sitia Giovanni,Aiuti Alessandro,Scaramuzza Samantha,Marangoni Francesco,Guidotti Luca G,Martino Silvana,Saracco Paola,Notarangelo Luigi D,Roncarolo Maria-Grazia,Dupré Loïc Journal of immunology (Baltimore, Md. : 1950) Wiskott-Aldrich syndrome (WAS) protein (WASP) plays a key role in TCR-mediated activation and immunological synapse formation. However, the effects of WASP deficiency on effector functions of human CD4+ and CD8+ T cells remain to be determined. In this study, we report that TCR/CD28-driven proliferation and secretion of IL-2, IFN-gamma, and TNF-alpha are strongly reduced in CD8+ T cells from WAS patients, compared with healthy donor CD8+ T cells. Furthermore, WAS CD4+ T cells secrete low levels of IL-2 and fail to produce IFN-gamma and TNF-alpha, while the production of IL-4, IL-5, and IL-10 is only minimally affected. Defective IL-2 and IFN-gamma production persists after culture of naive WAS CD4+ T cells in Th1-polarizing conditions. The defect in Th1 cytokine production by WAS CD4+ and CD8+ T cells is also present at the transcriptional level, as shown by reduced IL-2 and IFN-gamma mRNA transcripts after TCR/CD28 triggering. The reduced transcription of Th1 cytokine genes in WAS CD4+ T cells is associated with a defective induction of T-bet mRNA and a reduction in the early nuclear recruitment of NFAT-1, while the defective activation of WAS CD8+ T cells correlates with reduced nuclear recruitment of both NFAT-1 and NFAT-2. Together, our data indicate that WASP regulates the transcriptional activation of T cells and is required specifically for Th1 cytokine production. 10.4049/jimmunol.177.10.7451
    WASp-deficient B cells play a critical, cell-intrinsic role in triggering autoimmunity. Becker-Herman Shirly,Meyer-Bahlburg Almut,Schwartz Marc A,Jackson Shaun W,Hudkins Kelly L,Liu Chaohong,Sather Blythe D,Khim Socheath,Liggitt Denny,Song Wenxia,Silverman Gregg J,Alpers Charles E,Rawlings David J The Journal of experimental medicine Patients with the immunodeficiency Wiskott-Aldrich syndrome (WAS) frequently develop systemic autoimmunity. Here, we demonstrate that mutation of the WAS gene results in B cells that are hyperresponsive to B cell receptor and Toll-like receptor (TLR) signals in vitro, thereby promoting a B cell-intrinsic break in tolerance. Whereas this defect leads to autoantibody production in WAS protein-deficient (WASp(-/-)) mice without overt disease, chimeric mice in which only the B cell lineage lacks WASp exhibit severe autoimmunity characterized by spontaneous germinal center formation, class-switched autoantibodies, renal histopathology, and early mortality. Both T cell help and B cell-intrinsic TLR engagement play important roles in promoting disease in this model, as depletion with anti-CD4 antibodies or generation of chimeric mice with B cells deficient in both WASp and MyD88 prevented development of autoimmune disease. These data highlight the potentially harmful role for cell-intrinsic loss of B cell tolerance in the setting of normal T cell function, and may explain why WAS patients with mixed chimerism after stem cell transplantation often develop severe humoral autoimmunity. 10.1084/jem.20110200
    Structure-function analysis of the WIP role in T cell receptor-stimulated NFAT activation: evidence that WIP-WASP dissociation is not required and that the WIP NH2 terminus is inhibitory. Dong Xiaoyun,Patino-Lopez Genaro,Candotti Fabio,Shaw Stephen The Journal of biological chemistry WASP and its binding partner WIP play important roles in T cells both in actin polymerization and in interleukin-2 transcription. Aberrations thereof contribute to the pathology of Wiskott-Aldrich syndrome (WAS). To directly evaluate the cooperativity of WIP and WASP in interleukin-2 transcription, we investigated how the WIP-WASP complex regulates NF-AT-mediated gene transcription. We developed an improved model system for analysis, using WIP and WASP cotransfection into Jurkat cells, in which strong induction of NFAT reporter activation is observed with anti-T cell receptor (TCR) antibody without the phorbol 12-myristate 13-acetate usually used previously. Using this system, our findings contradict a prevailing conceptual model of TCR-induced WIP-WASP dissociation by showing in three ways that the WIP-WASP complex mediates TCR-induced NFAT activation without dissociation. First, phosphorylation of WIP Ser(488) does not cause dissociation of the WIP-WASP complex. Second, WIP-WASP complexes do not dissociate demonstrably after TCR stimulation. Third, a fusion protein of WIP to WASP efficiently mediates NFAT activation. Next, our studies clarify that WIP stabilization of WASP explains otherwise unexpected results in TCR-induced NFAT activation. Finally, we find that the NH(2) terminus of WIP is a highly inhibitory region for TCR-mediated transcriptional activation in which at least two elements contribute: the NH(2)-terminal polyproline and the NH(2)-terminal actin-binding WH2 domain. This suggests that WIP, like WASP, is subject to autoinhibition. Our data indicate that the WIP-WASP complex plays an important role in WASP stabilization and NFAT activation. 10.1074/jbc.M704972200
    Fyn and PTP-PEST-mediated regulation of Wiskott-Aldrich syndrome protein (WASp) tyrosine phosphorylation is required for coupling T cell antigen receptor engagement to WASp effector function and T cell activation. Badour Karen,Zhang Jinyi,Shi Fabio,Leng Yan,Collins Michael,Siminovitch Katherine A The Journal of experimental medicine Involvement of the Wiskott-Aldrich syndrome protein (WASp) in promoting cell activation requires its release from autoinhibitory structural constraints and has been attributed to WASp association with activated cdc42. Here, however, we show that T cell development and T cell receptor (TCR)-induced proliferation and actin polymerization proceed normally in WASp-/- mice expressing a WASp transgene lacking the cdc42 binding domain. By contrast, mutation of tyrosine residue Y291, identified here as the major site of TCR-induced WASp tyrosine phosphorylation, abrogated induction of WASp tyrosine phosphorylation and its effector activities, including nuclear factor of activated T cell transcriptional activity, actin polymerization, and immunological synapse formation. TCR-induced WASp tyrosine phosphorylation was also disrupted in T cells lacking Fyn, a kinase shown here to bind, colocalize with, and phosphorylate WASp. By contrast, WASp was tyrosine dephosphorylated by protein tyrosine phosphatase (PTP)-PEST, a tyrosine phosphatase shown here to interact with WASp via proline, serine, threonine phosphatase interacting protein (PSTPIP)1 binding. Although Fyn enhanced WASp-mediated Arp2/3 activation and was required for synapse formation, PTP-PEST combined with PSTPIP1 inhibited WASp-driven actin polymerization and synapse formation. These observations identify key roles for Fyn and PTP-PEST in regulating WASp and imply that inducible WASp tyrosine phosphorylation can occur independently of cdc42 binding, but unlike the cdc42 interaction, is absolutely required for WASp contributions to T cell activation. 10.1084/jem.20030976
    Nuclear role of WASp in the pathogenesis of dysregulated TH1 immunity in human Wiskott-Aldrich syndrome. Taylor Matthew D,Sadhukhan Sanjoy,Kottangada Ponnappa,Ramgopal Archana,Sarkar Koustav,D'Silva Sheryl,Selvakumar Annamalai,Candotti Fabio,Vyas Yatin M Science translational medicine The clinical symptomatology in the X-linked Wiskott-Aldrich syndrome (WAS), a combined immunodeficiency and autoimmune disease resulting from WAS protein (WASp) deficiency, reflects the underlying coexistence of an impaired T helper 1 (TH1) immunity alongside intact TH2 immunity. This suggests a role for WASp in patterning T(H) subtype immunity, yet the molecular basis for the TH1-TH2 imbalance in human WAS is unknown. We have discovered a nuclear role for WASp in the transcriptional regulation of the TH1 regulator gene TBX21 at the chromatin level. In primary TH1-differentiating cells, a fraction of WASp is found in the nucleus, where it is recruited to the proximal promoter locus of the TBX21 gene, but not to the core promoter of GATA3 (a TH2 regulator gene) or RORc (a TH17 regulator gene). Genome-wide mapping demonstrates association of WASp in vivo with the gene-regulatory network that orchestrates TH1 cell fate choice in the human TH cell genome. Functionally, nuclear WASp associates with H3K4 trimethyltransferase [RBBP5 (retinoblastoma-binding protein 5)] and H3K9/H3K36 tridemethylase [JMJD2A (Jumonji domain-containing protein 2A)] proteins, and their enzymatic activity in vitro and in vivo is required for achieving transcription-permissive chromatin dynamics at the TBX21 proximal promoter in primary differentiating TH1 cells. During TH1 differentiation, the loss of WASp accompanies decreased enrichment of RBBP5 and, in a subset of WAS patients, also of filamentous actin at the TBX21 proximal promoter locus. Accordingly, human WASp-deficient TH cells, from natural mutation or RNA interference-mediated depletion, demonstrate repressed TBX21 promoter dynamics when driven under TH1-differentiating conditions. These chromatin derangements accompany deficient T-BET messenger RNA and protein expression and impaired TH1 function, defects that are ameliorated by reintroducing WASp. Our findings reveal a previously unappreciated role of WASp in the epigenetic control of T-BET transcription and provide a new mechanism for the pathogenesis of WAS by linking aberrant histone methylation at the TBX21 promoter to dysregulated adaptive immunity. 10.1126/scitranslmed.3000813
    Interaction of the Wiskott-Aldrich syndrome protein with sorting nexin 9 is required for CD28 endocytosis and cosignaling in T cells. Badour Karen,McGavin Mary K H,Zhang Jinyi,Freeman Spencer,Vieira Claudia,Filipp Dominik,Julius Michael,Mills Gordon B,Siminovitch Katherine A Proceedings of the National Academy of Sciences of the United States of America The Wiskott-Aldrich syndrome protein (WASp) plays a major role in coupling T cell antigen receptor (TCR) stimulation to induction of actin cytoskeletal changes required for T cell activation. Here, we report that WASp inducibly binds the sorting nexin 9 (SNX9) in T cells and that WASp, SNX9, p85, and CD28 colocalize within clathrin-containing endocytic vesicles after TCR/CD28 costimulation. SNX9, implicated in clathrin-mediated endocytosis, binds WASp via its SH3 domain and uses its PX domain to interact with the phosphoinositol 3-kinase regulatory subunit p85 and product, phosphoinositol (3,4,5)P3. The data reveal ligation-induced CD28 endocytosis to be clathrin- and phosphoinositol 3-kinase-dependent and TCR/CD28-evoked CD28 internalization and NFAT activation to be markedly enhanced by SNX9 overexpression, but severely impaired by expression of an SNX9 mutant (SNX9DeltaPX) lacking p85-binding capacity. CD28 endocytosis and CD28-evoked actin polymerization also are impaired in WASp-deficient T cells. These findings suggest that SNX9 couples WASp to p85 and CD28 so as to link CD28 engagement to its internalization and to WASp-mediated actin remodeling required for CD28 cosignaling. Thus, the WASp/SNX9/p85/CD28 complex enables a unique interface of endocytic, actin polymerizing, and signal transduction pathways required for CD28-mediated T cell costimulation. 10.1073/pnas.0610543104
    Interfacial actin protrusions mechanically enhance killing by cytotoxic T cells. Tamzalit Fella,Wang Mitchell S,Jin Weiyang,Tello-Lafoz Maria,Boyko Vitaly,Heddleston John M,Black Charles T,Kam Lance C,Huse Morgan Science immunology Cytotoxic T lymphocytes (CTLs) kill by forming immunological synapses with target cells and secreting toxic proteases and the pore-forming protein perforin into the intercellular space. Immunological synapses are highly dynamic structures that boost perforin activity by applying mechanical force against the target cell. Here, we used high-resolution imaging and microfabrication to investigate how CTLs exert synaptic forces and coordinate their mechanical output with perforin secretion. Using micropatterned stimulatory substrates that enable synapse growth in three dimensions, we found that perforin release occurs at the base of actin-rich protrusions that extend from central and intermediate locations within the synapse. These protrusions, which depended on the cytoskeletal regulator WASP and the Arp2/3 actin nucleation complex, were required for synaptic force exertion and efficient killing. They also mediated physical deformation of the target cell surface during CTL-target cell interactions. Our results reveal the mechanical basis of cellular cytotoxicity and highlight the functional importance of dynamic, three-dimensional architecture in immune cell-cell interfaces. 10.1126/sciimmunol.aav5445
    Wiskott-Aldrich syndrome protein may be critical for CD8 T cell function following MCMV infection. Li Sha,Huang Jing,Zhang Yu-Lin,Zhu Yan,An Yun-Fei,Du Juan,Zhang Zai-Li,Xia Yu,Liu Lin,Wang Li,Luo Xiao-Hua Cellular immunology Wiskott-Aldrich syndrome (WAS) patients are characterized by immunodeficiency and viral infections. T cells derived from WAS patients and WAS protein (WASP)-deficient mice have various defects. However, whether WASP plays a role in immune control of cytomegalovirus (CMV) infection remains unclear. We analyzed the distribution of CD8 T subsets and the pathological damage to various organs and tissues in MCMV infected Was knockout (KO) mice. A relatively high number of MCMV-specific cytotoxic T cells (CTLs) were observed in the spleen of Was KO mice. In MCMV infected Was KO mice, the late differentiated CD8 T subset (CD27CD28) decreased in lungs, compared with those in the spleen and peripheral blood. Additionally, we found that the most severe pathological lesions occurred in the lungs, the main target organ of MCMV infection. By stimulating the spleen-derived CD8 T lymphocytes of Was KO mice, we found that IL-2 and granzyme B production declined compared with that in wild- type mice. Moreover, the number of apoptotic CD8 T cells increased in Was KO mice compared with the number in wild-type mice. Therefore, our results demonstrate that WASP may be involved in regulating cytotoxic function and apoptosis in CD8 T cells following MCMV infection, which is supported by the distribution and memory compartment of MCMV-specific T cells in MCMV infected WAS mice. 10.1016/j.cellimm.2019.03.004
    Triple-color FRET analysis reveals conformational changes in the WIP-WASp actin-regulating complex. Fried Sophia,Reicher Barak,Pauker Maor H,Eliyahu Shani,Matalon Omri,Noy Elad,Chill Jordan,Barda-Saad Mira Science signaling Wiskott-Aldrich syndrome protein (WASp) is a key regulator of the actin cytoskeletal machinery. Binding of WASp-interacting protein (WIP) to WASp modulates WASp activity and protects it from degradation. Formation of the WIP-WASp complex is crucial for the adaptive immune response. We found that WIP and WASp interacted in cells through two distinct molecular interfaces. One interaction occurred between the WASp-homology-1 (WH1) domain of WASp and the carboxyl-terminal domain of WIP that depended on the phosphorylation status of WIP, which is phosphorylated by protein kinase C θ (PKCθ) in response to T cell receptor activation. The other interaction occurred between the verprolin homology, central hydrophobic region, and acidic region (VCA) domain of WASp and the amino-terminal domain of WIP. This latter interaction required actin, because it was inhibited by latrunculin A, which sequesters actin monomers. With triple-color fluorescence resonance energy transfer (3FRET) technology, we demonstrated that the WASp activation mechanism involved dissociation of the first interaction, while leaving the second interaction intact. This conformation exposed the ubiquitylation site on WASp, leading to degradation of WASp. Together, these data suggest that the activation and degradation of WASp are delicately balanced and depend on the phosphorylation state of WIP. Our molecular analysis of the WIP-WASp interaction provides insight into the regulation of actin-dependent processes. 10.1126/scisignal.2005198
    A DOCK8-WIP-WASp complex links T cell receptors to the actin cytoskeleton. Janssen Erin,Tohme Mira,Hedayat Mona,Leick Marion,Kumari Sudha,Ramesh Narayanaswamy,Massaad Michel J,Ullas Sumana,Azcutia Veronica,Goodnow Christopher C,Randall Katrina L,Qiao Qi,Wu Hao,Al-Herz Waleed,Cox Dianne,Hartwig John,Irvine Darrell J,Luscinskas Francis W,Geha Raif S The Journal of clinical investigation Wiskott-Aldrich syndrome (WAS) is associated with mutations in the WAS protein (WASp), which plays a critical role in the initiation of T cell receptor-driven (TCR-driven) actin polymerization. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 (DOCK8) deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. Here, we demonstrated that the WASp-interacting protein (WIP) bridges DOCK8 to WASp and actin in T cells. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp. These results indicate that DOCK8 and WASp are in the same signaling pathway that links TCRs to the actin cytoskeleton in TCR-driven actin assembly. Further, they provide an explanation for similarities in the clinical phenotypes of WAS and DOCK8 deficiency. 10.1172/JCI85774
    X-linked thrombocytopenia causing mutations in WASP (L46P and A47D) impair T cell chemotaxis. Jain Neeraj,Tan Jun Hou,Feng Shijin,George Bhawana,Thanabalu Thirumaran Journal of biomedical science BACKGROUND:Mutation in the Wiskott-Aldrich syndrome Protein (WASP) causes Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT) and X-linked congenital neutropenia (XLN). The majority of missense mutations causing WAS and XLT are found in the WH1 (WASP Homology) domain of WASP, known to mediate interaction with WIP (WASP Interacting Protein) and CIB1 (Calcium and Integrin Binding). RESULTS:We analyzed two WASP missense mutants (L46P and A47D) causing XLT for their effects on T cell chemotaxis. Both mutants, WASPRL46P and WASPRA47D (S1-WASP shRNA resistant) expressed well in JurkatWASP-KD T cells (WASP knockdown), however expression of these two mutants did not rescue the chemotaxis defect of JurkatWASP-KD T cells towards SDF-1α. In addition JurkatWASP-KD T cells expressing these two WASP mutants were found to be defective in T cell polarization when stimulated with SDF-1α. WASP exists in a closed conformation in the presence of WIP, however both the mutants (WASPRL46P and WASPRA47D) were found to be in an open conformation as determined in the bi-molecular complementation assay. WASP protein undergoes proteolysis upon phosphorylation and this turnover of WASP is critical for T cell migration. Both the WASP mutants were found to be stable and have reduced tyrosine phosphorylation after stimulation with SDF-1α. CONCLUSION:Thus our data suggest that missense mutations WASPRL46P or WASPRA47D affect the activity of WASP in T cell chemotaxis probably by affecting the turnover of the protein. 10.1186/s12929-014-0091-1
    WASp family verprolin-homologous protein-2 (WAVE2) and Wiskott-Aldrich syndrome protein (WASp) engage in distinct downstream signaling interactions at the T cell antigen receptor site. Pauker Maor H,Reicher Barak,Joseph Noah,Wortzel Inbal,Jakubowicz Shlomi,Noy Elad,Perl Orly,Barda-Saad Mira The Journal of biological chemistry T cell antigen receptor (TCR) engagement has been shown to activate pathways leading to actin cytoskeletal polymerization and reorganization, which are essential for lymphocyte activation and function. Several actin regulatory proteins were implicated in regulating the actin machinery, such as members of the Wiskott-Aldrich syndrome protein (WASp) family. These include WASp and the WASp family verprolin-homologous protein-2 (WAVE2). Although WASp and WAVE2 share several structural features, the precise regulatory mechanisms and potential redundancy between them have not been fully characterized. Specifically, unlike WASp, the dynamic molecular interactions that regulate WAVE2 recruitment to the cell membrane and specifically to the TCR signaling complex are largely unknown. Here, we identify the molecular mechanism that controls the recruitment of WAVE2 in comparison with WASp. Using fluorescence resonance energy transfer (FRET) and novel triple-color FRET (3FRET) technology, we demonstrate how WAVE2 signaling complexes are dynamically regulated during lymphocyte activation in vivo. We show that, similar to WASp, WAVE2 recruitment to the TCR site depends on protein-tyrosine kinase, ZAP-70, and the adaptors LAT, SLP-76, and Nck. However, in contrast to WASp, WAVE2 leaves this signaling complex and migrates peripherally together with vinculin to the membrane leading edge. Our experiments demonstrate that WASp and WAVE2 differ in their dynamics and their associated proteins. Thus, this study reveals the differential mechanisms regulating the function of these cytoskeletal proteins. 10.1074/jbc.M114.591685
    Wiskott-Aldrich syndrome protein (WASP) is a tumor suppressor in T cell lymphoma. Menotti Matteo,Ambrogio Chiara,Cheong Taek-Chin,Pighi Chiara,Mota Ines,Cassel Seth H,Compagno Mara,Wang Qi,Dall'Olio Riccardo,Minero Valerio G,Poggio Teresa,Sharma Geeta Geeta,Patrucco Enrico,Mastini Cristina,Choudhari Ramesh,Pich Achille,Zamo Alberto,Piva Roberto,Giliani Silvia,Mologni Luca,Collings Clayton K,Kadoch Cigall,Gambacorti-Passerini Carlo,Notarangelo Luigi D,Anton Ines M,Voena Claudia,Chiarle Roberto Nature medicine In T lymphocytes, the Wiskott-Aldrich Syndrome protein (WASP) and WASP-interacting-protein (WIP) regulate T cell antigen receptor (TCR) signaling, but their role in lymphoma is largely unknown. Here we show that the expression of WASP and WIP is frequently low or absent in anaplastic large cell lymphoma (ALCL) compared to other T cell lymphomas. In anaplastic lymphoma kinase-positive (ALK+) ALCL, WASP and WIP expression is regulated by ALK oncogenic activity via its downstream mediators STAT3 and C/EBP-β. ALK+ lymphomas were accelerated in WASP- and WIP-deficient mice. In the absence of WASP, active GTP-bound CDC42 was increased and the genetic deletion of one CDC42 allele was sufficient to impair lymphoma growth. WASP-deficient lymphoma showed increased mitogen-activated protein kinase (MAPK) pathway activation that could be exploited as a therapeutic vulnerability. Our findings demonstrate that WASP and WIP are tumor suppressors in T cell lymphoma and suggest that MAP-kinase kinase (MEK) inhibitors combined with ALK inhibitors could achieve a more potent therapeutic effect in ALK+ ALCL. 10.1038/s41591-018-0262-9
    Nuclear Wiskott-Aldrich syndrome protein co-regulates T cell factor 1-mediated transcription in T cells. Kuznetsov Nikolai V,Almuzzaini Bader,Kritikou Joanna S,Baptista Marisa A P,Oliveira Mariana M S,Keszei Marton,Snapper Scott B,Percipalle Piergiorgio,Westerberg Lisa S Genome medicine BACKGROUND:The Wiskott-Aldrich syndrome protein (WASp) family of actin-nucleating factors are present in the cytoplasm and in the nucleus. The role of nuclear WASp for T cell development remains incompletely defined. METHODS:We performed WASp chromatin immunoprecipitation and deep sequencing (ChIP-seq) in thymocytes and spleen CD4 T cells. RESULTS:WASp was enriched at genic and intergenic regions and associated with the transcription start sites of protein-coding genes. Thymocytes and spleen CD4 T cells showed 15 common WASp-interacting genes, including the gene encoding T cell factor (TCF)12. WASp KO thymocytes had reduced nuclear TCF12 whereas thymocytes expressing constitutively active WASp and WASp had increased nuclear TCF12, suggesting that regulated WASp activity controlled nuclear TCF12. We identify a putative DNA element enriched in WASp ChIP-seq samples identical to a TCF1-binding site and we show that WASp directly interacted with TCF1 in the nucleus. CONCLUSIONS:These data place nuclear WASp in proximity with TCF1 and TCF12, essential factors for T cell development. 10.1186/s13073-017-0481-6
    Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway. Kumari Sudha,Depoil David,Martinelli Roberta,Judokusumo Edward,Carmona Guillaume,Gertler Frank B,Kam Lance C,Carman Christopher V,Burkhardt Janis K,Irvine Darrell J,Dustin Michael L eLife Wiscott Aldrich Syndrome protein (WASP) deficiency results in defects in calcium ion signaling, cytoskeletal regulation, gene transcription and overall T cell activation. The activation of WASP constitutes a key pathway for actin filament nucleation. Yet, when WASP function is eliminated there is negligible effect on actin polymerization at the immunological synapse, leading to gaps in our understanding of the events connecting WASP and calcium ion signaling. Here, we identify a fraction of total synaptic F-actin selectively generated by WASP in the form of distinct F-actin 'foci'. These foci are polymerized de novo as a result of the T cell receptor (TCR) proximal tyrosine kinase cascade, and facilitate distal signaling events including PLCγ1 activation and subsequent cytoplasmic calcium ion elevation. We conclude that WASP generates a dynamic F-actin architecture in the context of the immunological synapse, which then amplifies the downstream signals required for an optimal immune response. 10.7554/eLife.04953
    WASp Is Essential for Effector-to-Memory conversion and for Maintenance of CD8T Cell Memory. Liu Qiao,Zhang Liang,Shu Zhou,Yu Tingting,Zhou Lina,Song Wenxia,Zhao Xiaodong Frontiers in immunology Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by recurrent infections, micro thrombocytopenia, eczema, and a high incidence of autoimmunity and malignancy. A defect in the T cell compartment is thought to be a major cause of immunodeficiency in patients with WAS; However, whether the antigen specific T memory cell is altered has not been extensively studied. Here, we examined the expansion/contraction kinetics of CD8 memory T cells and their maintenance in WASp mice. The results showed that WAS protein (WASp) is not required for differentiation of CD8 effector T cells; however, CD8 T cells from WASp mice were hyperactive, resulting in increased cytokine production. The number of CD8 T memory cells decreased as mice aged, and CD8 T cell recall responses and protective immunity were impaired. WASp-deficient CD8 T cells in bone marrow chimeric mice underwent clonal expansion, but the resulting effector cells failed to survive and differentiate into CD8 memory T cells. Taken together, these findings indicate that WASp plays an intrinsic role in differentiation of CD8 memory T cells. 10.3389/fimmu.2019.02262
    The role of WASp in T cells and B cells. Sun Xizi,Wei Yin,Lee Pamela P,Ren Boxu,Liu Chaohong Cellular immunology Wiskott-Aldrich syndrome (WAS) is a form of primary immunodeficiency (PIDs) resulting from mutations of the gene that encodes Wiskott-Aldrich syndrome protein (WASp). WASp is the first identified and most widely studied protein belonging to the actin nucleation-promoting factor family and plays significant role in integrating and transforming signals from critical receptors on the cell surface to actin remodeling. WASp functions in immune defense and homeostasis through the regulation of actin cytoskeleton-dependent cellular processes as well as processes uncoupled with actin polymerization like nuclear transcription programs. In this article, we review the mechanisms of WASp activation through an understanding of its structure. We further discuss the role of WASp in adaptive immunity, paying special attention to some recent findings on the crucial role of WASp in the formation of immunological synapse, the regulation of T follicular helper (Tfh) cells and in the prevention of autoimmunity. 10.1016/j.cellimm.2019.04.007
    Molecular difference between WASP and N-WASP critical for chemotaxis of T-cells towards SDF-1α. Jain Neeraj,Thanabalu Thirumaran Scientific reports Wiskott-Aldrich Syndrome protein (WASP) integrates cell signaling pathways to the actin cytoskeleton, which play a critical role in T-cell activation and migration. Hematopoietic cells express both WASP and neural-WASP (N-WASP) which share similar domain structure, yet WASP deficiency causes Wiskott-Aldrich syndrome, suggesting that N-WASP present in the cells is not able to carry out all the functions of WASP. We have identified a unique internal thirty amino acid region (I30) in WASP, which regulates its function in chemotaxis of Jurkat T-cells. Deletion of the I30 region altered the WASP's closed conformation and impaired its ability to rescue the chemotactic defect of WASP-deficient (Jurkat(WKD)) T-cells. Expression of N-WASP in Jurkat(WKD) T-cells using WASP promoter restored the migration velocity without correcting the chemotactic defect. However, insertion of I30 region in N-WASP (N-WASP-I30) enabled N-WASP to rescue the chemotactic defect of Jurkat(WKD) T-cells. N-WASP-I30-EGFP displayed a punctate localization in contrast to the predominant nuclear localization of N-WASP-EGFP. Thus, our study has demonstrated that the I30 region of WASP is critical for localization and chemotaxis. This suggests that N-WASP's failure to compensate for WASP in rescuing chemotaxis could be due to the absence of this I30 region. 10.1038/srep15031