Critical role of WASp in germinal center tolerance through regulation of B cell apoptosis and diversification.

A main feature of Wiskott-Aldrich syndrome (WAS) is increased susceptibility to autoimmunity. A key contribution of B cells to development of these complications has been demonstrated through studies of samples from affected individuals and mouse models of the disease, but the role of the WAS protein (WASp) in controlling peripheral tolerance has not been specifically explored. Here we show that B cell responses remain T cell dependent in constitutive WASp-deficient mice, whereas selective WASp deletion in germinal center B cells (GCBs) is sufficient to induce broad development of self-reactive antibodies and kidney pathology, pointing to loss of germinal center tolerance as a primary cause leading to autoimmunity. Mechanistically, we show that WASp is upregulated in GCBs and regulates apoptosis and plasma cell differentiation in the germinal center and that the somatic hypermutation-derived diversification is the basis of autoantibody development.

An Autopsy Case of Wiskott-Aldrich Syndrome Revealing "FDC-Only Lymphoid Follicles" in Lymphoid Tissue: A Morphologic Correlate of Defective Immune Synapse.

Wiskott-Aldrich Syndrome (WAS) is an inherited disorder characterized by the classical triad of eczema, micro-thrombocytopenia, and immune deficiency. This disease affects the hematopoietic cells to a variable extent. The spectrum of clinical and laboratory data for WAS has been well described in the literature though there is a paucity of its histopathologic and immunohistochemical correlates. The current case describes the autopsy findings of this rare entity in an 8-year old male child with specific recognition of altered histology noticed in the lymphoreticular tissues. The predominant morphological finding in lymphoid tissue was atretic hyalinized germinal centers labeled as "the follicular dendritic cell (FDC)-only lymphoid follicles." Immunohistochemistry revealed a reduction in germinal-center B-cells, T-follicular helper cells, attenuated mantle zone, FDC proliferation, and paracortical plasmacytosis. This case highlights the crippled immune cell population in WAS, ultimately leading to the morphology of atretic follicles rich in FDCs.

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.

A Novel Mutation in WAS Gene Causing a Phenotypic Presentation of Wiskott-Aldrich Syndrome: A Case Report.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by immunodeficiency, thrombocytopenia, and atopic dermatitis. OBSERVATIONS: This infant presented at birth with petechiae and bruising, with severe neonatal thrombocytopenia. Genetic testing for WAS revealed a variant of unknown significance hemizygous missense mutation in the WAS gene. This variant has not previously been reported. On the basis of the patient's clinical course including bleeding, infection, abnormal immune evaluation, and dermatologic sequelae, he was diagnosed with WAS and underwent allogeneic hematopoietic stem cell transplantation. CONCLUSIONS: We report a novel mutation in the WAS gene that causes a phenotypic presentation of Wiskott-Aldrich Syndrome.

Successful Haploidentical Stem Cell Transplant With Posttransplant Cyclophosphamide in Wiskott-Aldrich Syndrome With Myeloablative Conditioning.

Hematopoietic stem cell transplant (HSCT) is the only curative treatment modality for Wiskott-Aldrich syndrome. Haploidentical HSCT with posttransplant cyclophosphamide (PTCy) is an upcoming option in children with nonmalignant conditions. However, only few cases have been reported for Wiskott-Aldrich syndrome HSCT with PTCy approach. Here we report a 4-year-old boy, treated successfully by haploidentical HSCT with myeloablative conditioning (busulfan, fludarabine, and thiotepa) and PTCy. Posttransplant chimerism was fully donor. Of 13 cases (current case and other 12 published cases) 10 are alive and disease free after haploidentical HSCT with PTCy. Haploidentical HSCT with PTCy using myeloablative conditioning is feasible and safe.

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.

Development of a rabbit model of Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is a severe recessive X-linked immunodeficiency resulting from loss-of-function mutations in the WAS gene. Mouse is the only mammalian model used for investigation of WAS pathogenesis. However, the mouse model does not accurately recapitulate WAS clinical phenotypes, thus, limiting its application in WAS clinical research. Herein, we report the generation of WAS knockout (KO) rabbits via embryo co-injection of Cas9 mRNA and a pair of sgRNAs targeting exons 2 and 7. WAS KO rabbits exhibited many symptoms similar to those of WAS patients, including thrombocytopenia, bleeding tendency, infections, and reduced numbers of T cell in the spleen and peripheral blood. The WAS KO rabbit model provides a new valuable tool for preclinical trials of WAS treatment.

Targeted gene correction of human hematopoietic stem cells for the treatment of Wiskott - Aldrich Syndrome.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with severe platelet abnormalities and complex immunodeficiency. Although clinical gene therapy approaches using lentiviral vectors have produced encouraging results, full immune and platelet reconstitution is not always achieved. Here we show that a CRISPR/Cas9-based genome editing strategy allows the precise correction of WAS mutations in up to 60% of human hematopoietic stem and progenitor cells (HSPCs), without impairing cell viability and differentiation potential. Delivery of the editing reagents to WAS HSPCs led to full rescue of WASp expression and correction of functional defects in myeloid and lymphoid cells. Primary and secondary transplantation of corrected WAS HSPCs into immunodeficient mice showed persistence of edited cells for up to 26 weeks and efficient targeting of long-term repopulating stem cells. Finally, no major genotoxicity was associated with the gene editing process, paving the way for an alternative, yet highly efficient and safe therapy.

MicroRNA-1253 Regulation of WASF2 (WAVE2) and its Relevance to Racial Health Disparities.

The prevalence of hypertension among African Americans (AAs) in the US is among the highest of any demographic and affects over two-thirds of AA women. Previous data from our laboratory suggest substantial differential gene expression (DGE) of mRNAs and microRNAs (miRNAs) exists within peripheral blood mononuclear cells (PBMCs) isolated from AA and white women with or without hypertension. We hypothesized that DGE by race may contribute to racial differences in hypertension. In a reanalysis of our previous dataset, we found that the Wiskott-Aldrich syndrome protein Verprolin-homologous protein 2 (WASF2 (also known as WAVE2)) is differentially expressed in AA women with hypertension, along with several other members of the actin cytoskeleton signaling pathway that plays a role in cell shape and branching of actin filaments. We performed an in silico miRNA target prediction analysis that suggested miRNA miR-1253 regulates WASF2. Transfection of miR-1253 mimics into human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAECs) significantly repressed WASF2 mRNA and protein levels (p < 0.05), and a luciferase reporter assay confirmed that miR-1253 regulates the WASF2 3' UTR (p < 0.01). miR-1253 overexpression in HUVECs significantly increased HUVEC lamellipodia formation (p < 0.01), suggesting the miR-1253-WASF2 interaction may play a role in cell shape and actin cytoskeleton function. Together, we have identified novel roles for miR-1253 and WASF2 in a hypertension-related disparities context. This may ultimately lead to the discovery of additional actin-related genes which are important in the vascular-related complications of hypertension and influence the disproportionate susceptibility to hypertension among AAs in general and AA women in particular.

Excellent outcomes following hematopoietic cell transplantation for Wiskott-Aldrich syndrome: a PIDTC report.

Wiskott-Aldrich syndrome (WAS) is an X-linked disease caused by mutations in the WAS gene, leading to thrombocytopenia, eczema, recurrent infections, autoimmune disease, and malignancy. Hematopoietic cell transplantation (HCT) is the primary curative approach, with the goal of correcting the underlying immunodeficiency and thrombocytopenia. HCT outcomes have improved over time, particularly for patients with HLA-matched sibling and unrelated donors. We report the outcomes of 129 patients with WAS who underwent HCT at 29 Primary Immune Deficiency Treatment Consortium centers from 2005 through 2015. Median age at HCT was 1.2 years. Most patients (65%) received myeloablative busulfan-based conditioning. With a median follow-up of 4.5 years, the 5-year overall survival (OS) was 91%. Superior 5-year OS was observed in patients <5 vs ≥5 years of age at the time of HCT (94% vs 66%; overall P = .0008). OS was excellent regardless of donor type, even in cord blood recipients (90%). Conditioning intensity did not affect OS, but was associated with donor T-cell and myeloid engraftment after HCT. Specifically, patients who received fludarabine/melphalan-based reduced-intensity regimens were more likely to have donor myeloid chimerism <50% early after HCT. In addition, higher platelet counts were observed among recipients who achieved full (>95%) vs low-level (5%-49%) donor myeloid engraftment. In summary, HCT outcomes for WAS have improved since 2005, compared with prior reports. HCT at a younger age continues to be associated with superior outcomes supporting the recommendation for early HCT. High-level donor myeloid engraftment is important for platelet reconstitution after either myeloablative or busulfan-containing reduced intensity conditioning. (This trial was registered at www.clinicaltrials.gov as #NCT02064933.).

[Ileitis as presentation of lymphoma in Wiskott-Aldrich syndrome]. / Ileítis como presentación de linfoma en síndrome de Wiskott-Aldrich.

Wiskott-Aldrich syndrome is a rare X chromosome-linked primary immunodeficiency syndrome associated with an increased incidence of infections, autoimmune disorders and neoplasms. We present the case of a 41-year-old man with a diagnosis of Wiskott-Aldrich syndrome with ileitis as a form of presentation of a lymphoproliferative syndrome. The ileitis, in the context of the patient, represents a clinical challenge given the large number of differential diagnoses (inflammatory bowel disease, infections, neoplasms and lymphoproliferative diseases), so it usually requires anatomopathological diagnosis and particular considerations regarding the subsequent specific treatment.

El síndrome de Wiskott-Aldrich (SWA) es un raro síndrome de inmunodeficiencia primaria ligado al cromosoma X que se asocia con aumento de incidencia de infecciones, trastornos autoinmunes y neoplasias. Se presenta el caso de un varón de 41 años con diagnóstico de síndrome de Wiskott-Aldrich y cuadro de ileítis como forma de presentación de un síndrome linfoproliferativo. La ileítis, en el contexto del paciente, representa un problema clínico dado el gran número de diagnósticos diferenciales (enfermedad inflamatoria intestinal, infecciones, neoplasias y enfermedades linfoproliferativas) por lo que suele requerir diagnóstico anatomopatológico y consideraciones particulares respecto al posterior tratamiento específico.

Ileítis como presentación de linfoma en síndrome de Wiskott-Aldrich / Ileitis as presentation of lymphoma in Wiskott-Aldrich syndrome

El síndrome de Wiskott-Aldrich (SWA) es un raro síndrome de inmunodeficiencia primaria ligado al cromosoma X que se asocia con aumento de incidencia de infecciones, trastornos autoinmunes y neoplasias. Se presenta el caso de un varón de 41 años con diagnóstico de síndrome de Wiskott-Aldrich y cuadro de ileítis como forma de presentación de un síndrome linfoproliferativo. La ileítis, en el contexto del paciente, representa un problema clínico dado el gran número de diagnósticos diferenciales (enfermedad inflamatoria intestinal, infecciones, neoplasias y enfermedades linfoproliferativas) por lo que suele requerir diagnóstico anatomopatológico y consideraciones particulares respecto al posterior tratamiento específico.

Wiskott-Aldrich syndrome is a rare X chromosome-linked primary immunodeficiency syndrome associated with an increased incidence of infections, autoimmune disorders and neoplasms. We present the case of a 41-year-old man with a diagnosis of Wiskott-Aldrich syndrome with ileitis as a form of presentation of a lymphoproliferative syndrome. The ileitis, in the context of the patient, represents a clinical challenge given the large number of differential diagnoses (inflammatory bowel disease, infections, neoplasms and lymphoproliferative diseases), so it usually requires anatomopathological diagnosis and particular considerations regarding the subsequent specific treatment.

Eltrombopag Therapy in Children With Rare Disorders Associated With Thrombocytopenia.

Eltrombopag (ELT) is a thrombopoietin receptor activator that has shown efficacy in chronic immune thrombocytopenia. We report the outcome of ELT therapy in 4 children who were treated for rare hematologic disorders, including Pearson syndrome, DiGeorge syndrome, posttransplant allogeneic poor graft function (PGF), and Wiskott-Aldrich syndrome. The ELT tolerance in the analyzed group was good, with the exception of the child with Pearson syndrome, who experienced an exacerbation of cataracts and had to discontinue treatment. Thromboembolic events were observed in one child, who continued ELT therapy despite achieving normalized platelet counts. Independence from PLT transfusions was observed at the 4-week timepoint of therapy in patients with DiGeorge syndrome and PGF who responded to ELT. Discontinuation of therapy was successful in one child, who sustained the normal CBC values afterward. In 2 patients, an increase in neutrophil counts was observed during ELT therapy without additional intervention, and a positive correlation between neutrophil and platelet values during ELT therapy was observed in the child with PGF. ELT is effective in rare pediatric disorders, but response patterns are determined by the underlying disease. ELT shows promising results in patients, but constitutional hematopoiesis defects reduce the chances of a response.

The role of WASp in T cells and B cells.

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.

Bond swapping from a charge cloud allows flexible coordination of upstream signals through WASP: Multiple regulatory roles for the WASP basic region.

Wiskott-Aldrich syndrome protein (WASP) activates the actin-related protein 2/3 homolog (Arp2/3) complex and regulates actin polymerization in a physiological setting. Cell division cycle 42 (Cdc42) is a key activator of WASP, which binds Cdc42 through a Cdc42/Rac-interactive binding (CRIB)-containing region that defines a subset of Cdc42 effectors. Here, using site-directed mutagenesis and binding affinity determination and kinetic assays, we report the results of an investigation into the energetic contributions of individual WASP residues to both the Cdc42-WASP binding interface and the kinetics of complex formation. Our results support the previously proposed dock-and-coalesce binding mechanism, initiated by electrostatic steering driven by WASP's basic region and followed by a coalescence phase likely driven by the conserved CRIB motif. The WASP basic region, however, appears also to play a role in the final complex, as its mutation affected both on- and off-rates, suggesting a more comprehensive physiological role for this region centered on the C-terminal triad of positive residues. These results highlight the expanding roles of the basic region in WASP and other CRIB-containing effector proteins in regulating complex cellular processes and coordinating multiple input signals. The data presented improve our understanding of the Cdc42-WASP interface and also add to the body of information available for Cdc42-effector complex formation, therapeutic targeting of which has promise for Ras-driven cancers. Our findings suggest that combining high-affinity peptide-binding sequences with short electrostatic steering sequences could increase the efficacy of peptidomimetic candidates designed to interfere with Cdc42 signaling in cancer.

Mapping Active Gene-Regulatory Regions in Human Repopulating Long-Term HSCs.

Genes that regulate hematopoietic stem cell (HSC) self-renewal, proliferation, and differentiation are tightly controlled by regulatory regions. However, mapping such regions relies on surface markers and immunophenotypic definition of HSCs. Here, we use γ-retroviral integration sites (γRV ISs) from a gene therapy trial for 10 patients with Wiskott-Aldrich syndrome to mark active enhancers and promoters in functionally defined long-term repopulating HSCs. Integration site clusters showed the highest ATAC-seq signals at HSC-specific peaks and strongly correlated with hematopoietic risk variants. Tagged genes were significantly enriched for HSC gene sets. We were able to map over 3,000 HSC regulatory regions in late-contributing HSCs, and we used these data to identify miR-10a and miR-335 as two miRNAs regulating early hematopoiesis. In this study, we show that viral insertion sites can be used as molecular tags to assess chromatin conformation on functionally defined cell populations, thereby providing a genome-wide resource for regulatory regions in human repopulating long-term HSCs.

Neuropsychological Profile of a Girl with Wiskott-Aldrich Syndrome.

We report the neuropsychological profile of a 6-year-old girl with Wiskott-Aldrich syndrome, a rare X-linked immunodeficiency disorder associated with thrombocytopenia, eczema, recurrent infections, and malignancy. Wiskott-Aldrich syndrome occurs almost exclusively in males and is extremely rare in females, with no known research focused on cognitive and academic functioning in this population. Our patient was referred due to concerns about her memory and academic functioning. She had a history of progressive thrombocytopenia and hematopoietic stem cell transplantation at age 15 months. Standardized measures of intellectual ability, language, visual-spatial and visual-motor skills, attention, memory, and academic achievement were administered. The results showed average to above-average performance in multiple areas of cognitive and academic functioning, with weaknesses in phonological awareness and rapid naming. The advent of hematopoietic stem cell transplantation has led to considerable improvement in the long-term prognosis of children with Wiskott-Aldrich syndrome. Although the impact of this syndrome and related conditions on neurocognitive development is presently unknown, this case highlights both the importance of considering base rates for commonly occurring conditions and the significant role neuropsychology can play in identifying cognitive strengths and weaknesses in the context of the developing brain.

A Conditioning Regimen with Plerixafor Is Safe and Improves the Outcome of TCRαß+ and CD19+ Cell-Depleted Stem Cell Transplantation in Patients with Wiskott-Aldrich Syndrome.

Our initial experience with hematopoietic stem cell transplantation (HSCT) from a matched unrelated donor (MUD; n = 12) or a haploidentical related donor (n = 6) with T cell receptor (TCR)αß+/CD19+ graft depletion in patients with Wiskott-Aldrich syndrome (WAS) (n = 18) showed a dramatic decrease in the incidence of graft-versus-host disease (GVHD) and transplantation-related mortality, with an increased overall survival (OS) of 88.9%. Unfortunately, the treatment was associated with mixed myeloid donor chimerism and secondary graft dysfunction (severe thrombocytopenia, n = 2; graft rejection, n = 5). To improve the outcome, we hypothesized that the addition of G-CSF and plerixafor to the conditioning chemotherapy would result in more complete donor stem cell engraftment. This trial was registered at www.clinicaltrials.gov (NCT03019809). A study group of patients with WAS (n = 16) underwent TCRαß+/CD19+-depleted HSCT (MUD, n = 6; haploidentical, n = 10). The conditioning regimen was treosulfan-fludarabine-rabbit antithymocyte globulin-melphalan (or thiophosphamide in 1 patient) with G-CSF (10 µg/kg/day for 5 days starting on day -8) and plerixafor (240 µg/kg/day for 3 days starting on day -6). The clinical outcomes in this study were compared to those in a historical dataset (n = 18). No patients had grade III/IV acute GVHD in either the study or the historical control group. Importantly, in the patients with WAS, there was no statistical significance in OS between those who underwent HSCT from haploidentical donors and those who underwent HSCT from MUDs (93.8% versus 88.5%; P = .612). All patients in the study group had full donor chimerism in whole blood and in the CD3+ compartments. The OS was 93.8%, and there were no cases of graft dysfunction. This study demonstrates the efficacy of adding G-CSF/plerixafor to the conditioning regimen before HSCT with TCRαß+/C D19+ graft depletion in patients with WAS.