Controlled WASp activity regulates the proliferative response for Treg cell differentiation in the thymus.

The Wiskott-Aldrich syndrome protein (WASp) regulates actin cytoskeletal dynamics and function of hematopoietic cells. Mutations in the WAS gene lead to two different syndromes; Wiskott-Aldrich syndrome (WAS) caused by loss-of-function mutations, and X-linked neutropenia (XLN) caused by gain-of-function mutations. We previously showed that WASp-deficient mice have a decreased number of regulatory T (Treg) cells in the thymus and the periphery. We here evaluated the impact of WASp mutations on Treg cells in the thymus of WAS and XLN mouse models. Using in vitro Treg differentiation assays, WAS CD4 single-positive thymocytes have decreased differentiation to Treg cells, despite normal early signaling upon IL-2 and TGF-ß stimulation. They failed to proliferate and express CD25 at high levels, leading to poor survival and a lower number of Foxp3+ Treg cells. Conversely, XLN CD4 single-positive thymocytes efficiently differentiate into Foxp3+ Treg cells following a high proliferative response to IL-2 and TGF-ß, associated with high CD25 expression when compared with WT cells. Altogether, these results show that specific mutations of WASp affect Treg cell development differently, demonstrating a critical role of WASp activity in supporting Treg cell development and expansion.

Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome.

Patients with Wiskott-Aldrich syndrome (WAS) lacking a human leukocyte antigen-matched donor may benefit from gene therapy through the provision of gene-corrected, autologous hematopoietic stem/progenitor cells. Here, we present comprehensive, long-term follow-up results (median follow-up, 7.6 years) (phase I/II trial no. NCT02333760 ) for eight patients with WAS having undergone phase I/II lentiviral vector-based gene therapy trials (nos. NCT01347346 and NCT01347242 ), with a focus on thrombocytopenia and autoimmunity. Primary outcomes of the long-term study were to establish clinical and biological safety, efficacy and tolerability by evaluating the incidence and type of serious adverse events and clinical status and biological parameters including lentiviral genomic integration sites in different cell subpopulations from 3 years to 15 years after gene therapy. Secondary outcomes included monitoring the need for additional treatment and T cell repertoire diversity. An interim analysis shows that the study meets the primary outcome criteria tested given that the gene-corrected cells engrafted stably, and no serious treatment-associated adverse events occurred. Overall, severe infections and eczema resolved. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. The results suggest that lentiviral gene therapy provides sustained clinical benefits for patients with WAS.

Clinical and molecular characteristics of Wiskott-Aldrich Syndrome in five unrelated Chinese families.

Wiskott-Aldrich syndrome (WAS) also called the eczema-thrombocytopenia-immunodeficiency syndrome is a primary immunodeficiency disease with X-linked recessive inheritance caused by mutations in the WAS protein (WASp) gene and characterized by thrombocytopenia with reduced platelet volume, eczema, immunodeficiency, and increased risk of malignant tumours. The mutations will lead to separate WAS severity which can be typical severe 'classical' WAS or less severe 'non-classical' WAS. This article will review and analyse clinical and immune characteristics of five unrelated Chinese families harbouring classical and non-classical WAS. The expression of WASp was detected in the peripheral blood monocytes (PBMC) by flow cytometry, and five mutations were found by WAS gene sequencing, one of which had not been reported in the literature, namely frameshift mutation c.1240_1247delCCACTCCC (p. P414Sfs*41).

Wiskott Aldrich Syndrome: A Multi-Institutional Experience From India.

Background: Wiskott Aldrich syndrome (WAS) is characterized by bleeding manifestations, recurrent infections, eczema, autoimmunity, and malignancy. Over the last decade, improved awareness and better in-house diagnostic facilities at several centers in India has resulted in increased recognition of WAS. This study reports collated data across major primary immunodeficiency diseases (PID) centers in India that are involved in care of children with WAS and highlights the varied clinical presentations, genetic profile, and outcomes of patients in India. Methods: Request to share data was sent to multiple centers in India that are involved in care and management of patients with PID. Six centers provided requisite data that were compiled and analyzed. Results: In this multi-institutional cohort, clinical details of 108 patients who had a provisional diagnosis of WAS were received. Of these, 95 patients with 'definite WAS' were included Fourteen patients were classified as XLT and 81 patients as WAS. Median age at onset of symptoms of patients was 3 months (IQR 1.6, 6.0 months) and median age at diagnosis was 12 months (IQR 6,48 months). Clinical profile included bleeding episodes (92.6%), infections (84.2%), eczema (78.9%), various autoimmune manifestations (40%), and malignancy (2.1%). DNA analysis revealed 47 variants in 67 cases. Nonsense and missense variants were the most common (28.4% each), followed by small deletions (19.4%), and splice site defects (16.4%). We also report 24 novel variants, most of these being frameshift and nonsense mutations resulting in premature termination of protein synthesis. Prophylactic intravenous immunoglobulin (IVIg) was initiated in 52 patients (54.7%). Hematopoietic stem cell transplantation (HSCT) was carried out in 25 patients (26.3%). Of those transplanted, disease-free survival was seen in 15 patients (60%). Transplant related mortality was 36%. Outcome details were available for 89 patients. Of these, 37% had died till the time of this analysis. Median duration of follow-up was 36 months (range 2 weeks- 12 years; IQR 16.2 months- 70 months). Conclusions: We report the first nationwide cohort of patients with WAS from India. Bleeding episodes and infections are common manifestations. Mortality continues to be high as curative therapy is not accessible to most of our patients.

Constitutive activation of WASp leads to abnormal cytotoxic cells with increased granzyme B and degranulation response to target cells.

X-linked neutropenia (XLN) is caused by gain-of-function mutations in the actin regulator Wiskott-Aldrich Syndrome protein (WASp). XLN patients have reduced numbers of cytotoxic cells in peripheral blood; however, their capacity to kill tumor cells remains to be determined. Here, we examined NK and T cells from 2 patients with XLN harboring the activating WASpL270P mutation. XLN patient NK and T cells had increased granzyme B content and elevated degranulation and IFN-γ production when compared with healthy control cells. Murine WASpL272P NK and T cells formed stable synapses with YAC-1 tumor cells and anti-CD3/CD28-coated beads, respectively. WASpL272P mouse T cells had normal degranulation and cytokine response whereas WASpL272P NK cells showed an enhanced response. Imaging experiments revealed that while WASpL272P CD8+ T cells had increased accumulation of actin upon TCR activation, WASpL272P NK cells had normal actin accumulation at lytic synapses triggered through NKp46 signaling but had impaired response to lymphocyte function associated antigen-1 engagement. When compared with WT mice, WASpL272P mice showed reduced growth of B16 melanoma and increased capacity to reject MHC class I-deficient cells. Together, our data suggest that cytotoxic cells with constitutively active WASp have an increased capacity to respond to and kill tumor cells.

Deficiency of Wiskott-Aldrich syndrome protein has opposing effect on the pro-oncogenic pathway activation in nonmalignant versus malignant lymphocytes.

Immunodeficiency is associated with cancer risk. Accordingly, hematolymphoid cancers develop in Wiskott-Aldrich syndrome (WAS), an X-linked primary immunodeficiency disorder (PID) resulting from the deficiency of WAS-protein (WASp) expressed predominantly in the hematolymphoid cell lineages. Despite the correlation between WASp deficiency and hematolymphoid cancers, the molecular mechanism underlying the oncogenic role of WASp is incompletely understood. Employing the WASp-sufficient and WASp-deficient cell-pair model of human T and B lymphocytes, we show that WASp deficiency differentially influences hyperactivation versus inhibition of both CDC42:ERK1/2 and NF-κB:AP-1 pro-oncogenic signaling pathways in nonmalignant versus malignant T and B lymphocytes. Furthermore, WASp deficiency induces a cell-type specific up/down-modulation of the DNA-binding activities of NF-κB, AP-1, and multiple other transcription factors with known roles in oncogenesis. We propose that WASp functions as a putative "tumor-suppressor" protein in normal T and B cells, and "oncoprotein" in a subset of established T and B cell malignancies that are not associated with the NPM-ALK fusion.

Wiskott-Aldrich Syndrome in four male siblings from a consanguineous family from Lebanon.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency disorder (PID) characterized by microthrombocytopenia, bloody diarrhea, eczema, recurrent infections, and a high incidence of autoimmunity and malignancy. OBJECTIVE: To investigate the mechanism of thrombocytopenia and infections in four boys of consanguineous parents from Lebanon. METHODS: Patient gDNA was studied using Next Generation Sequencing and Sanger Sequencing. Protein expression was determined by immunoblotting, and mRNA expression by semi-quantitative RT-PCR. F-actin polymerization and cellular proliferation were assayed by flow cytometry. RESULTS: We identified a threonine to a methionine change at position 45 (T45M) of the WAS protein (WASp) that abolished protein expression and disturbed F-actin polymerization and T cell proliferation, but not B cell proliferation. In addition, the levels of the WAS-interacting protein (WIP) were significantly decreased in the patients. CONCLUSION: The mutation identified severely destabilizes WASp and affects the downstream signaling events important for T cell function, but not B cell function. It was previously known that the stability of WASp depends on WIP. In this manuscript, we report that the stability of WIP also depends on WASp. Finally, it is important to suspect X-linked PIDs even in consanguineous families. CLINICAL IMPLICATIONS: The patients are above the optimal age for transplant in WAS, and it is difficult to identify one or more donors for four patients, therefore, they represent ideal candidates for gene therapy or interleukin-2 therapy.

Quality of Life of Patients with Wiskott Aldrich Syndrome and X-Linked Thrombocytopenia: a Study of the Primary Immune Deficiency Consortium (PIDTC), Immune Deficiency Foundation, and the Wiskott-Aldrich Foundation.

BACKGROUND: We undertook a study to determine the impact of Wiskott Aldrich Syndrome (WAS) and X-linked thrombocytopenia (XLT) and their therapies upon the health-related quality of life (HRQOL) of patients and their families. MATERIALS AND METHODS: We undertook a survey of patients and their families, who self-identified as having either WAS or XLT. We assessed the PedsQL™ 4.0, the parent proxy form, and the family impact module. These results were compared with normative data from previously published reports. RESULTS: Sixty-eight patients (29 patients completed both the PedsQL™ 4.0 and the parent proxy form; 21 completed only the PedsQL™ 4.0; and 18 completed only the parent proxy form) were included. In contrast to patient-reported outcomes, parents of patients who had a bone marrow transplant (BMT) reported that their children had better QOL scores compared with those who did not (82.6 vs. 73.3, p = 0.023). The QOL of patients vs. previously published normative data showed decreases in patient scores for psychosocial health (72.62 vs. 86.58, p = < 0.001), emotional functioning (69.91 vs. 82.64, p = < 0.001), social functioning (77.55 vs. 91.56, p = < 0.001), and school functioning (70.46 vs. 85.67, p = < 0.001). The family impact study revealed deficits in emotional, social, and cognitive functioning, communication, and worry. CONCLUSION: These results show that patients with WAS/XLT are significantly impacted with respect to QOL. BMT offered a better QOL for patients according to parents, but not as reported by the patients. Future studies should incorporate QOL to provide more data and a better understanding of outcomes for long-term survivors and decision-making regarding BMT.

When WAS Gene Diagnosis Is Needed: Seeking Clues Through Comparison Between Patients With Wiskott-Aldrich Syndrome and Idiopathic Thrombocytopenic Purpura.

Background: Wiskott-Aldrich syndrome (WAS) is a rare and severe X-linked disorder with variable clinical phenotypes correlating with the type of mutations in the WAS gene. The syndrome is difficult to differentiate from idiopathic thrombocytopenic purpura (ITP) before genetic diagnosis. We retrospectively reviewed patients suspected to have WAS who were referred to our hospital from 2004 to 2016 and compared the clinical features and laboratory examination of genetically confirmed WAS patients and of patients diagnosed with ITP in order to seek some clues to distinguish WAS and ITP before genetic diagnosis. Methods: Seventy-eight children suspected to have WAS from 78 unrelated families were enrolled in this study. The clinical data and laboratory examination of children were reviewed in the present study. The distribution of lymphocyte subsets from peripheral blood was examined by how cytometry. WASP mutations were identified by direct sequencing of PCR-amplified genomic DNA. Results: Forty-two patients were finally diagnosed with WAS genetically. The median onset age of these patients was 1 month (range: 1 day-10 months). The median diagnosis lag was 4.6 months (range: 0 months-9.42 years). Fifteen patients (35.71%) had positive family histories. More than half of the patients (n = 23, 54.76%) had diarrhea. Twenty-three (54.76%) had pneumonia, 7 with severe symptoms. Major bleeding events included skin spots or petechiae (n = 27, 64.29%), per-rectal bleeding (n = 21, 50.00%), epistaxis (n = 7, 16.67%) and intracranial bleeding (n = 2, 4.76%). Twenty-nine patients (69.05%) had eczema, and one patient had a drug allergy. Three patients had autoimmune diseases, among whom 2 had autoimmune hemolytic anemia and one had autoimmune hemolytic anemia and IgA nephropathy. A total of 42 mutations in WASP were identified, including 19 novel mutations. Eight patients received hematopoietic stem cell transplantation (HSCT) and all survived. Compared with the 30 patients diagnosed with ITP, the WAS patients had higher EOS counts and elevated IgE level, increased NK cell numbers but fewer CD8+T lymphocytes. Conclusion: The WAS gene diagnosis should be considered in all males with ITP-like features, especially for patients with a very early onset age, decreased MPV (<6.5 fl), higher EOS counts and elevated IgE level, increased NK cell number, diminished CD8+T lymphocyte count.

Wiskott-Aldrich syndrome protein may be critical for CD8+ T cell function following MCMV infection.

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 (CD27-CD28-) 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.

Advances in site-specific gene editing for primary immune deficiencies.

PURPOSE OF REVIEW: Conventional gene therapy has been a successful, curative treatment modality for many primary immune deficiencies with significant improvements in the last decade. However, the risk of leukemic transformation with viral-mediated gene addition still remains, and unregulated gene addition is not an option for certain diseases in which the target gene is closely controlled. The recent bloom in genome modification platforms has created the opportunity to site-specifically correct mutated DNA base pairs or insert a corrective cDNA minigene while maintaining gene expression under control of endogenous regulatory elements. RECENT FINDINGS: There is an abundance of ongoing research utilizing programmable nucleases to facilitate site-specific gene correction of many primary immune deficiencies including X-linked severe combined immune deficiency, X-linked chronic granulomatous disease, Wiskott-Aldrich syndrome, X-linked hyper-IgM syndrome, X-linked agammaglobulinemia, and immune dysregulation, polyendocrinopathy, enteropathy, X-linked. In all, these studies have demonstrated the ability to integrate corrective DNA sequences at a precise location in the genome at rates likely to either cure or ameliorate disease. SUMMARY: Gene editing for primary immune deficiency (PID) has advanced to the point to that translation to clinical trials is likely to occur in the next several years. At the current pace of research in DNA repair mechanisms, stem cell biology, and genome-editing technology, targeted genome modification represents the next chapter of gene therapy for PID.

WASP-mediated regulation of anti-inflammatory macrophages is IL-10 dependent and is critical for intestinal homeostasis.

Mutations in Wiskott-Aldrich syndrome protein (WASP) cause autoimmune sequelae including colitis. Yet, how WASP mediates mucosal homeostasis is not fully understood. Here we show that WASP-mediated regulation of anti-inflammatory macrophages is critical for mucosal homeostasis and immune tolerance. The generation and function of anti-inflammatory macrophages are defective in both human and mice in the absence of WASP. Expression of WASP specifically in macrophages, but not in dendritic cells, is critical for regulation of colitis development. Importantly, transfer of WT anti-inflammatory macrophages prevents the development of colitis. DOCK8-deficient macrophages phenocopy the altered macrophage properties associated with WASP deficiency. Mechanistically, we show that both WASP and DOCK8 regulates macrophage function by modulating IL-10-dependent STAT3 phosphorylation. Overall, our study indicates that anti-inflammatory macrophage function and mucosal immune tolerance require both WASP and DOCK8, and that IL-10 signalling modulates a WASP-DOCK8 complex.

Neutrophils drive type I interferon production and autoantibodies in patients with Wiskott-Aldrich syndrome.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is a rare primary immunodeficiency caused by mutations in Wiskott-Aldrich syndrome protein (WASp), a key regulator of cytoskeletal dynamics in hematopoietic cells. A high proportion of patients experience autoimmunity caused by a breakdown in T- and B-cell tolerance. Moreover, excessive production of type I interferon (IFN-I) by plasmacytoid dendritic cells (pDCs) contributes to autoimmune signs; however, the factors that trigger excessive innate activation have not been defined. OBJECTIVE: Neutrophil extracellular traps (NETs) emerged as major initiating factors in patients with diseases such as systemic lupus erythematosus and rheumatoid arthritis. In this study we explored the possible involvement of aberrant neutrophil functions in patients with WAS. METHODS: We evaluated the expression of a set of granulocyte genes associated with NETs in a cohort of patients with WAS and the presence of NET inducers in sera. Using a mouse model of WAS, we analyzed NET release by WASp-null neutrophils and evaluated the composition and homeostasis of neutrophils in vivo. By using depletion experiments, we assessed the effect of neutrophils in promoting inflammation and reactivity against autoantigens. RESULTS: Transcripts of genes encoding neutrophil enzymes and antimicrobial peptides were increased in granulocytes of patients with WAS, and serum-soluble factors triggered NET release. WASp-null neutrophils showed increased spontaneous NETosis, induced IFN-I production by pDCs, and activated B cells through B-cell activating factor. Consistently, their depletion abolished constitutive pDC activation, normalized circulating IFN-I levels, and, importantly, abolished production of autoantibodies directed against double-stranded DNA, nucleosomes, and myeloperoxidase. CONCLUSIONS: These findings reveal that neutrophils are involved in the pathogenic loop that causes excessive activation of innate cells and autoreactive B cells, thus identifying novel mechanisms that contribute to the autoimmunity of WAS.

Autonomous role of Wiskott-Aldrich syndrome platelet deficiency in inducing autoimmunity and inflammation.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by eczema, infections, and susceptibility to autoimmunity and malignancies. Thrombocytopenia is a constant finding, but its pathogenesis remains elusive. OBJECTIVE: To dissect the basis of the WAS platelet defect, we used a novel conditional mouse model (CoWas) lacking Wiskott-Aldrich syndrome protein (WASp) only in the megakaryocytic lineage in the presence of a normal immunologic environment, and in parallel we analyzed samples obtained from patients with WAS. METHODS: Phenotypic and functional characterization of megakaryocytes and platelets in mutant CoWas mice and patients with WAS with and without autoantibodies was performed. Platelet antigen expression was examined through a protein expression profile and cluster proteomic interaction network. Platelet immunogenicity was tested by using ELISAs and B-cell and platelet cocultures. RESULTS: CoWas mice showed increased megakaryocyte numbers and normal thrombopoiesis in vitro, but WASp-deficient platelets had short lifespan and high expression of activation markers. Proteomic analysis identified signatures compatible with defects in cytoskeletal reorganization and metabolism yet surprisingly increased antigen-processing capabilities. In addition, WASp-deficient platelets expressed high levels of surface and soluble CD40 ligand and were capable of inducing B-cell activation in vitro. WASp-deficient platelets were highly immunostimulatory in mice and triggered the generation of antibodies specific for WASp-deficient platelets, even in the context of a normal immune system. Patients with WAS also showed platelet hyperactivation and increased plasma soluble CD40 ligand levels correlating with the presence of autoantibodies. CONCLUSION: Overall, these findings suggest that intrinsic defects in WASp-deficient platelets decrease their lifespan and dysregulate immune responses, corroborating the role of platelets as modulators of inflammation and immunity.

Platelets in Wiskott-Aldrich syndrome: Victims or executioners?

Microthrombocytopenia is the clinical hallmark of WAS, a rare X-linked immunodeficiency that is characterized by eczema, autoimmunity, and cancer susceptibility. This disease is caused by mutations in the WAS gene, which is expressed in hematopoietic cells and regulates actin cytoskeleton remodeling thereby modulating various cellular functions, including motility, immunologic synapse assembly, and signaling. Despite extensive studies that have provided great insight into the relevance of this molecule to innate and cellular immunity, the exact mechanisms of microthrombocytopenia in WAS are still unknown. This review focuses on the recent progress made in dissecting the pathogenesis of platelet defects in patients with WAS and their murine counterparts. In parallel, we will provide an overview of the state-of-the art platelets as immune modulators at the interface between hemostasis and the immune system, which suggests that these cells may have a direct role in the pathogenesis of immune dysregulation in WAS.

Wiskott-Aldrich syndrome protein regulates autophagy and inflammasome activity in innate immune cells.

Dysregulation of autophagy and inflammasome activity contributes to the development of auto-inflammatory diseases. Emerging evidence highlights the importance of the actin cytoskeleton in modulating inflammatory responses. Here we show that deficiency of Wiskott-Aldrich syndrome protein (WASp), which signals to the actin cytoskeleton, modulates autophagy and inflammasome function. In a model of sterile inflammation utilizing TLR4 ligation followed by ATP or nigericin treatment, inflammasome activation is enhanced in monocytes from WAS patients and in WAS-knockout mouse dendritic cells. In ex vivo models of enteropathogenic Escherichia coli and Shigella flexneri infection, WASp deficiency causes defective bacterial clearance, excessive inflammasome activation and host cell death that are associated with dysregulated septin cage-like formation, impaired autophagic p62/LC3 recruitment and defective formation of canonical autophagosomes. Taken together, we propose that dysregulation of autophagy and inflammasome activities contribute to the autoinflammatory manifestations of WAS, thereby identifying potential targets for therapeutic intervention.