Maria Carmina Castiello

Autoimmunity in wiskott-Aldrich syndrome: an unsolved enigma.

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.

Wiskott-Aldrich Syndrome protein deficiency perturbs the homeostasis of B-cell compartment in humans

Wiskott–Aldrich Syndrome protein (WASp) regulates the cytoskeleton in hematopoietic cells and mutations in its gene cause the Wiskott–Aldrich Syndrome (WAS), a primary immunodeficiency with microthrombocytopenia, eczema and a higher susceptibility to develop tumors. Autoimmune manifestations, frequently observed in WAS patients, are associated with an increased risk of mortality and still represent an unsolved aspect of the disease. B cells play a crucial role both in immune competence and self-tolerance and defects in their development and function result in immunodeficiency and/or autoimmunity. We performed a phenotypical and molecular analysis of central and peripheral B-cell compartments in WAS pediatric patients. We found a decreased proportion of immature B cells in the bone marrow correlating with an increased presence of transitional B cells in the periphery. These results could be explained by the defective migratory response of WAS B cells to SDF-1α, essential for the retention of immature B cells in the BM. In the periphery, we observed an unusual expansion of CD21low B-cell population and increased plasma BAFF levels that may contribute to the high susceptibility to develop autoimmune manifestations in WAS patients. WAS memory B cells were characterized by a reduced in vivo proliferation, decreased somatic hypermutation and preferential usage of IGHV4-34, an immunoglobulin gene commonly found in autoreactive B cells. In conclusion, our findings demonstrate that WASp-deficiency perturbs B-cell homeostasis thus adding a new layer of immune dysregulation concurring to the increased susceptibility to develop autoimmunity in WAS patients.

Lentiviral-mediated gene therapy leads to improvement of B-cell functionality in a murine model of Wiskott-Aldrich syndrome

BACKGROUND Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency characterized by thrombocytopenia, eczema, infections, autoimmunity, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical donors is curative, but it is not available to all patients. We have developed a gene therapy (GT) approach for WAS by using a lentiviral vector encoding for human WAS promoter/cDNA (w1.6W) and demonstrated its preclinical efficacy and safety. OBJECTIVE To evaluate B-cell reconstitution and correction of B-cell phenotype in GT-treated mice. METHODS We transplanted Was(-/-) mice sublethally irradiated (700 rads) with lineage marker-depleted bone marrow wild-type cells, Was(-/-) cells untransduced or transduced with the w1.6W lentiviral vector and analyzed B-cell reconstitution in bone marrow, spleen, and peritoneum. RESULTS Here we show that WAS protein(+) B cells were present in central and peripheral B-cell compartments from GT-treated mice and displayed the strongest selective advantage in the splenic marginal zone and peritoneal B1 cell subsets. After GT, splenic architecture was improved and B-cell functions were restored, as demonstrated by the improved antibody response to pneumococcal antigens and the reduction of serum IgG autoantibodies. CONCLUSION WAS GT leads to improvement of B-cell functions, even in the presence of a mixed chimerism, further validating the clinical application of the w1.6W lentiviral vector.

Wiskott-Aldrich syndrome protein–mediated actin dynamics control type-I interferon production in plasmacytoid dendritic cells

Wiskott-Aldrich Syndrome protein (WASp)–mediated actin polymerization controls intracellular trafficking and compartmentalization of TLR9 ligands in plasmacytoid dendritic cells.

Revertant T lymphocytes in a patient with Wiskott-Aldrich syndrome: Analysis of function and distribution in lymphoid organs

BACKGROUND The Wiskott-Aldrich syndrome (WAS) is a rare genetic disease characterized by thrombocytopenia, immunodeficiency, autoimmunity, and hematologic malignancies. Secondary mutations leading to re-expression of WAS protein (WASP) are relatively frequent in patients with WAS. OBJECTIVE The tissue distribution and function of revertant cells were investigated in a novel case of WAS gene secondary mutation. METHODS A vast combination of approaches was used to characterize the second-site mutation, to investigate revertant cell function, and to track their distribution over a 18-year clinical follow-up. RESULTS The WAS gene secondary mutation was a 4-nucleotide insertion, 4 nucleotides downstream of the original deletion. This somatic mutation allowed the T-cell-restricted expression of a stable, full-length WASP with a 3-amino acid change compared with the wild-type protein. WASP(+) T cells appeared early in the spleen (age 10 years) and were highly enriched in a mesenteric lymph node at a later time (age 23 years). Revertant T cells had a diversified T-cell-receptor repertoire and displayed in vitro and in vivo selective advantage. They proliferated and produced cytokines normally on T-cell-receptor stimulation. Consistently, the revertant WASP correctly localized to the immunologic synapse and to the leading edge of migrating T cells. CONCLUSION Despite the high proportion of functional revertant T cells, the patient still has severe infections and autoimmune disorders, suggesting that re-expression of WASP in T cells is not sufficient to normalize immune functions fully in patients with WAS.

B-cell reconstitution after lentiviral vector–mediated gene therapy in patients with Wiskott-Aldrich syndrome

Background Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, and susceptibility to autoimmunity and lymphomas. Hematopoietic stem cell transplantation is the treatment of choice; however, administration of WAS gene–corrected autologous hematopoietic stem cells has been demonstrated as a feasible alternative therapeutic approach. Objective Because B-cell homeostasis is perturbed in patients with WAS and restoration of immune competence is one of the main therapeutic goals, we have evaluated reconstitution of the B-cell compartment in 4 patients who received autologous hematopoietic stem cells transduced with lentiviral vector after a reduced-intensity conditioning regimen combined with anti-CD20 administration. Methods We evaluated B-cell counts, B-cell subset distribution, B cell–activating factor and immunoglobulin levels, and autoantibody production before and after gene therapy (GT). WAS gene transfer in B cells was assessed by measuring vector copy numbers and expression of Wiskott-Aldrich syndrome protein. Results After lentiviral vector-mediated GT, the number of transduced B cells progressively increased in the peripheral blood of all patients. Lentiviral vector-transduced progenitor cells were able to repopulate the B-cell compartment with a normal distribution of B-cell subsets both in bone marrow and the periphery, showing a WAS protein expression profile similar to that of healthy donors. In addition, after GT, we observed a normalized frequency of autoimmune-associated CD19+CD21−CD35− and CD21low B cells and a reduction in B cell–activating factor levels. Immunoglobulin serum levels and autoantibody production improved in all treated patients. Conclusions We provide evidence that lentiviral vector-mediated GT induces transgene expression in the B-cell compartment, resulting in ameliorated B-cell development and functionality and contributing to immunologic improvement in patients with WAS.

Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1

Preclinical studies establish the conditions for safe and effective correction of SCID-X1 by targeted gene editing of hematopoietic stem cells. Gene correction, one step at a time Although gene therapy has been proposed for a variety of genetic disorders, including severe combined immunodeficiency, it has not yet found routine use in the clinic, in part because of potential complications. To help pave the way for safer translation of such gene therapy, Schiroli et al. studied potential approaches to it in mouse models of severe combined immunodeficiency. The authors systematically analyzed the outcomes of using different approaches to conditioning, different numbers of gene-edited cells, different techniques for editing the faulty gene, and other aspects of the technology to find the safest and most effective method. Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all IL2RG (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the IL2RG-edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of IL2RG editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases.

Intestinal microbiota sustains inflammation and autoimmunity induced by hypomorphic RAG defects

Rigoni et al. report that hypomorphic Rag2R229Q mutation is associated with altered microbiota composition and defects in the gut–blood barrier and suggest that intestinal microbes may play a critical role in the distinctive immune dysregulation of Omenn syndrome.

Lentiviral-mediated gene therapy restores B cell tolerance in Wiskott-Aldrich syndrome patients

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.

Wiskott–Aldrich syndrome protein deficiency in natural killer and dendritic cells affects antitumor immunity

Wiskott–Aldrich syndrome (WAS) is a primary immunodeficiency caused by reduced or absent expression of the WAS protein (WASP). WAS patients are affected by microthrombocytopenia, recurrent infections, eczema, autoimmune diseases, and malignancies. Although immune deficiency has been proposed to play a role in tumor pathogenesis, there is little evidence on the correlation between immune cell defects and tumor susceptibility. Taking advantage of a tumor‐prone model, we show that the lack of WASP induces early tumor onset because of defective immune surveillance. Consistently, the B16 melanoma model shows that tumor growth and the number of lung metastases are increased in the absence of WASP. We then investigated the in vivo contribution of Was−/− NK cells and DCs in controlling B16 melanoma development. We found fewer B16 metastases developed in the lungs of Was−/− mice that had received WT NK cells as compared with mice bearing Was−/− NK cells. Furthermore, we demonstrated that Was−/− DCs were less efficient in inducing NK‐cell activation in vitro and in vivo. In summary, for the first time, we demonstrate in in vivo models that WASP deficiency affects resistance to tumor and causes impairment in the antitumor capacity of NK cells and DCs.