Case Report: Susceptibility to viral infections and secondary hemophagocytic lymphohistiocytosis responsive to intravenous immunoglobulin as primary manifestations of adenosine deaminase 2 deficiency.

Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive disease associated with a highly variable clinical presentation, including systemic vasculitis, immunodeficiency, and cytopenia. We report a case of a 16-year-old girl affected by recurrent viral infections [including cytomegalovirus (CMV)-related hepatitis and measles vaccine virus-associated manifestations] and persistent inflammation, which occurred after Parvovirus infection and complicated by secondary hemophagocytic lymphohistiocytosis (HLH). HLH's first episode presented at 6 years of age and was preceded by persistent fever and arthralgia with evidence of Parvovirus B19 infection. The episode responded to intravenous steroids but relapsed during steroids tapering. High-dose intravenous immunoglobulin (IVIG) helped manage her clinical symptoms and systemic inflammation. The frequency of IVIG administration and the dosage were progressively reduced. At the age of 9, she experienced varicella zoster virus (VZV) reactivation followed by the recurrence of the inflammatory phenotype complicated by HLH with neurological involvement. Again, high-dose steroids and monthly IVIG resulted in a quick response. Targeted next-generation sequencing (NGS) for autoinflammatory diseases and immunodeficiencies revealed the homozygous Leu183Pro ADA2 mutation, which was confirmed by Sanger analysis. ADA2 enzymatic test showed a complete loss of ADA2 activity. For about 3 years, IVIG alone was completely effective in preventing flares of inflammation and neurological manifestations. Anti-TNF treatment was started at the age of 13 for the appearance of recurrent genital ulcers, with a complete response. This case further expands the clinical spectrum of DADA2 and emphasizes the importance of extensive genetic testing in clinical phenotypes characterized by persistent unspecific inflammatory syndromes. The use of high doses of IVIG might represent a possible effective immune modulator, especially in combination with anti-TNF treatment.

Adenosine Deaminase 2 Deficiency (DADA2): A Crosstalk Between Innate and Adaptive Immunity.

Deficiency of Adenosine deaminase 2 (DADA2) is a monogenic autoinflammatory disorder presenting with a broad spectrum of clinical manifestations, including immunodeficiency, vasculopathy and hematologic disease. Biallelic mutations in ADA2 gene have been associated with a decreased ADA2 activity, leading to reduction in deamination of adenosine and deoxyadenosine into inosine and deoxyinosine and subsequent accumulation of extracellular adenosine. In the early reports, the pivotal role of innate immunity in DADA2 pathogenic mechanism has been underlined, showing a skewed polarization from the M2 macrophage subtype to the proinflammatory M1 subtype, with an increased production of inflammatory cytokines such as TNF-α. Subsequently, a dysregulation of NETosis, triggered by the excess of extracellular Adenosine, has been implicated in the pathogenesis of DADA2. In the last few years, evidence is piling up that adaptive immunity is profoundly altered in DADA2 patients, encompassing both T and B branches, with a disrupted homeostasis in T-cell subsets and a B-cell skewing defect. Type I/type II IFN pathway upregulation has been proposed as a possible core signature in DADA2 T cells and monocytes but also an increased IFN-ß secretion directly from endothelial cells has been described. So far, a unifying clear pathophysiological explanation for the coexistence of systemic inflammation, immunedysregulation and hematological defects is lacking. In this review, we will explore thoroughly the latest understanding regarding DADA2 pathophysiological process, with a particular focus on dysregulation of both innate and adaptive immunity and their interacting role in the development of the disease.

Case Report: Deficiency of Adenosine Deaminase 2 Presenting With Overlapping Features of Autoimmune Lymphoproliferative Syndrome and Bone Marrow Failure.

Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive disease associated with a highly variable clinical presentation, such as vasculitis, inflammation, and hematologic manifestations. Some associations of clinical features can mimic autoimmune lymphoproliferative syndrome (ALPS). We report a case of a female patient who fulfilled the 2009 National Institute of Health revised criteria for ALPS and received a delayed diagnosis of DADA2. During her childhood, she suffered from autoimmune hemolytic anemia, immune thrombocytopenia, and chronic lymphoproliferation, which partially responded to multiple lines of treatments and were followed, at 25 years of age, by pulmonary embolism, septic shock, and bone marrow failure with myelodysplastic evolution. The patient died from the progression of pulmonary disease and multiorgan failure. Two previously unreported variants of gene ADA2/CECR1 were found through next-generation sequencing analysis, and a pathogenic role was demonstrated through a functional study. A single somatic STAT3 mutation was also found. Clinical phenotypes encompassing immune dysregulation and marrow failure should be evaluated at the early stage of diagnostic work-up with an extended molecular evaluation. A correct genetic diagnosis may lead to a precision medicine approach consisting of the use of targeted treatments or early hematopoietic stem cell transplantation.

A Novel LC-MS/MS-Based Method for the Diagnosis of ADA2 Deficiency from Dried Plasma Spot.

Adenosine Deaminase 2 Deficiency (DADA2) (OMIM: 607575) is a monogenic, autoinflammatory disease caused by the loss of functional homozygous or heterozygous mutations in the ADA 2 gene (previously CECR1, Cat Eye Syndrome Chromosome Region 1). A timely diagnosis is crucial to start Anti-TNF therapies that are efficacious in controlling the disease. The confirmation of DADA2 is based on DNA sequencing and enzymatic assay. It is, thus, very important to have robust and reliable assays that can be rapidly utilized in specialized laboratories that can centralize samples from other centers. In this paper, we show a novel enzymatic assay based on liquid chromatography-tandem mass spectrometry that allows the accurate determination of the ADA2 enzyme activity starting from very small amounts of plasma spotted on filter paper (dried plasma spot). The method allows significantly distinguishing healthy controls from affected patients and carriers and could be of help in implementing the diagnostic workflow of DADA2.

Lentiviral correction of enzymatic activity restrains macrophage inflammation in adenosine deaminase 2 deficiency.

Adenosine deaminase 2 deficiency (DADA2) is a rare inherited disorder that is caused by autosomal recessive mutations in the ADA2 gene. Clinical manifestations include early-onset lacunar strokes, vasculitis/vasculopathy, systemic inflammation, immunodeficiency, and hematologic defects. Anti-tumor necrosis factor therapy reduces strokes and systemic inflammation. Allogeneic hematopoietic stem/progenitor cell (HSPC) transplantation can ameliorate most disease manifestations, but patients are at risk for complications. Autologous HSPC gene therapy may be an alternative curative option for patients with DADA2. We designed a lentiviral vector encoding ADA2 (LV-ADA2) to genetically correct HSPCs. Lentiviral transduction allowed efficient delivery of the functional ADA2 enzyme into HSPCs from healthy donors. Supranormal ADA2 expression in human and mouse HSPCs did not affect their multipotency and engraftment potential in vivo. The LV-ADA2 induced stable ADA2 expression and corrected the enzymatic defect in HSPCs derived from DADA2 patients. Patients' HSPCs re-expressing ADA2 retained their potential to differentiate into erythroid and myeloid cells. Delivery of ADA2 enzymatic activity in patients' macrophages led to a complete rescue of the exaggerated inflammatory cytokine production. Our data indicate that HSPCs ectopically expressing ADA2 retain their multipotent differentiation ability, leading to functional correction of macrophage defects. Altogether, these findings support the implementation of HSPC gene therapy for DADA2.

Type I interferon activation in RAS-associated autoimmune leukoproliferative disease (RALD).

RAS-associated autoimmune leukoproliferative disease (RALD) is a rare immune dysregulation syndrome caused by somatic gain-of-function mutations of either NRAS or KRAS gene in hematopoietic cells. We describe a 27-year-old patient presenting at 5 months of age with recurrent infections and generalized lymphadenopathy who developed a complex multi-organ autoimmune syndrome with hypogammaglobulinemia, partially controlled with oral steroids, hydroxichloroquine, mofetil mycophenolate and IVIG prophylaxis. Activation of type I interferon pathway was observed in peripheral blood. Since 18 years of age, the patient developed regenerative nodular hyperplasia of the liver evolving into hepatopulmonary syndrome. Whole exome sequencing analysis of the peripheral blood DNA showed the NRAS p.Gly13Asp mutation validated as somatic. Our report highlights the possibility of detecting somatic NRAS gene mutations in patients with inflammatory immune dysregulation and type I interferon activation.

Dysregulation in B-cell responses and T follicular helper cell function in ADA2 deficiency patients.

Adenosine deaminase 2 deficiency (DADA2) is an autoinflammatory disease characterized by inflammatory vasculopathy, early strokes associated often with hypogammaglobulinemia. Pure red cell aplasia, thrombocytopenia, and neutropenia have been reported. The defect is due to biallelic loss of function of ADA2 gene, coding for a protein known to regulate the catabolism of extracellular adenosine. We therefore investigated immune phenotype and B- and T-cell responses in 14 DADA2 patients to address if ADA2 mutation affects B- and T-cell function. Here, we show a significant decrease in memory B cells, in particular class switch memory, and an expansion of CD21low B cells in DADA2 patients. In vitro stimulated B lymphocytes were able to secrete nonfunctional ADA2 protein, suggesting a cell intrinsic defect resulting in an impairment of B-cell proliferation and differentiation. Moreover, CD4+ and CD8+ T cells were diminished; however, the frequency of circulating T follicular helper cells was significantly increased but they had an impairment in IL-21 production possibly contributing to an impaired B cell help. Our findings suggest that ADA2 mutation could lead to a B-cell intrinsic defect but also to a defective Tfh cell function, which could contribute to the immunodeficient phenotype reported in DADA2 patients.

A novel knock-in mouse model of cryopyrin-associated periodic syndromes with development of amyloidosis: Therapeutic efficacy of proton pump inhibitors.

BACKGROUND: Cryopyrin-associated periodic syndromes (CAPS) are a group of autoinflammatory diseases linked to gain-of-function mutations in the NOD-like receptor family, pyrin domain containing 3 (NLRP3) gene, which cause uncontrolled IL-1ß secretion. Proton pump inhibitors (PPIs), which are commonly used as inhibitors of gastric acid production, also have anti-inflammatory properties, protect mice from sepsis, and prevent IL-1ß secretion by monocytes from patients with CAPS. OBJECTIVE: We sought to develop a novel Nlrp3 knock-in (KI) mouse model of CAPS to study amyloidosis, a severe CAPS complication, and test novel therapeutic approaches. METHODS: We generated KI mice by engineering the N475K mutation, which is associated with the CAPS phenotype, into the mouse Nlrp3 gene. KI and wild-type mice received PPIs or PBS intraperitoneally and were analyzed for survival, inflammation, cytokine secretion, and amyloidosis development. RESULTS: Mutant Nlrp3 KI mice displayed features that recapitulate the immunologic and clinical phenotype of CAPS. They showed systemic inflammation with high levels of serum proinflammatory cytokines, inflammatory infiltrates in various organs, and amyloid deposits in the spleen, liver, and kidneys. Toll-like receptor stimulated macrophages from KI mice secreted high levels of IL-1ß, IL-18, and IL-1α but low amounts of IL-1 receptor antagonist. Treatment of KI mice with PPIs had a clear clinical effect, showing a reduction in inflammatory manifestations, regression of amyloid deposits, and normalization of proinflammatory and anti-inflammatory cytokine production by macrophages. CONCLUSION: Nlrp3 KI mice displayed a CAPS phenotype with many characteristics of autoinflammation, including amyloidosis. The therapeutic effectiveness of PPIs associated with a lack of toxicity indicates that these drugs could represent relevant adjuvants to the anti-IL-1 drugs in patients with CAPS and other IL-1-driven diseases.

Mesenchymal Stromal Cell-Seeded Biomimetic Scaffolds as a Factory of Soluble RANKL in Rankl-Deficient Osteopetrosis.

Biomimetic scaffolds are extremely versatile in terms of chemical composition and physical properties, which can be defined to accomplish specific applications. One property that can be added is the production/release of bioactive soluble factors, either directly from the biomaterial, or from cells embedded within the biomaterial. We reasoned that pursuing this strategy would be appropriate to setup a cell-based therapy for RANKL-deficient autosomal recessive osteopetrosis, a very rare skeletal genetic disease in which lack of the essential osteoclastogenic factor RANKL impedes osteoclast formation. The exogenously administered RANKL cytokine is effective in achieving osteoclast formation and function in vitro and in vivo, thus, we produced murine Rankl-/- mesenchymal stromal cells (MSCs) overexpressing human soluble RANKL (hsRL) following lentiviral transduction (LVhsRL). Here, we described a three-dimensional (3D) culture system based on a magnesium-doped hydroxyapatite/collagen I (MgHA/Col) biocompatible scaffold closely reproducing bone physicochemical properties. MgHA/Col-seeded murine MSCs showed improved properties, as compared to two-dimensional (2D) culture, in terms of proliferation and hsRL production, with respect to LVhsRL-transduced cells. When implanted subcutaneously in Rankl-/- mice, these cell constructs were well tolerated, colonized by host cells, and intensely vascularized. Of note, in the bone of Rankl-/- mice that carried scaffolds with either WT or LVhsRL-transduced Rankl-/- MSCs, we specifically observed formation of TRAP+ cells, likely due to sRL released from the scaffolds into circulation. Thus, our strategy proved to have the potential to elicit an effect on the bone; further work is required to maximize these benefits and achieve improvements of the skeletal pathology in the treated Rankl-/- mice. Stem Cells Translational Medicine 2019;8:22-34.

In Vivo Chronic Stimulation Unveils Autoreactive Potential of Wiskott-Aldrich Syndrome Protein-Deficient B Cells.

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency caused by mutations in the gene encoding the hematopoietic-specific WAS protein (WASp). WAS is frequently associated with autoimmunity, indicating a critical role of WASp in maintenance of tolerance. The role of B cells in the induction of autoreactive immune responses in WAS has been investigated in several settings, but the mechanisms leading to the development of autoimmune manifestations have been difficult to evaluate in the mouse models of the disease that do not spontaneously develop autoimmunity. We performed an extensive characterization of Was-/- mice that provided evidence of the potential alteration in B cell selection, because of the presence of autoantibodies against double-stranded DNA, platelets, and tissue antigens. To uncover the mechanisms leading to the activation of the potentially autoreactive B cells in Was-/- mice, we performed in vivo chronic stimulations with toll-like receptors agonists (LPS and CpG) and apoptotic cells or infection with lymphocytic choriomeningitis virus. All treatments led to increased production of autoantibodies, increased proteinuria, and kidney tissue damage in Was-/- mice. These findings demonstrate that a lower clearance of pathogens and/or self-antigens and the resulting chronic inflammatory state could cause B cell tolerance breakdown leading to autoimmunity in WAS.

ADA2 deficiency (DADA2) as an unrecognised cause of early onset polyarteritis nodosa and stroke: a multicentre national study.

OBJECTIVES: To analyse the prevalence of CECR1 mutations in patients diagnosed with early onset livedo reticularis and/or haemorrhagic/ischaemic strokes in the context of inflammation or polyarteritis nodosa (PAN). Forty-eight patients from 43 families were included in the study. METHODS: Direct sequencing of CECR1 was performed by Sanger analysis. Adenosine deaminase 2 (ADA2) enzymatic activity was analysed in monocyte isolated from patients and healthy controls incubated with adenosine and with or without an ADA1 inhibitor. RESULTS: Biallelic homozygous or compound heterozygous CECR1 mutations were detected in 15/48 patients. A heterozygous disease-associated mutation (p.G47V) was observed in two affected brothers. The mean age of onset of the genetically positive patients was 24 months (6 months to 7 years). Ten patients displayed one or more cerebral strokes during their disease course. Low immunoglobulin levels were detected in six patients. Thalidomide and anti-TNF (tumour necrosis factor) blockers were the most effective drugs. Patients without CECR1 mutations had a later age at disease onset, a lower prevalence of neurological and skin manifestations; one of these patients displayed all the clinical features of adenosine deaminase 2deficiency (DADA2) and a defective enzymatic activity suggesting the presence of a missed mutation or a synthesis defect. CONCLUSIONS: DADA2 accounts for paediatric patients diagnosed with PAN-like disease and strokes and might explain an unrecognised condition in patients followed by adult rheumatologist. Timely diagnosis and treatment with anti-TNF agents are crucial for the prevention of severe complications of the disease. Functional assay to measure ADA2 activity should complement genetic testing in patients with non-confirming genotypes.

Murine Rankl-/- Mesenchymal Stromal Cells Display an Osteogenic Differentiation Defect Improved by a RANKL-Expressing Lentiviral Vector.

Autosomal recessive osteopetrosis (ARO) is a severe bone disease characterized by increased bone density due to impairment in osteoclast resorptive function or differentiation. Hematopoietic stem cell transplantation is the only available treatment; however, this therapy is not effective in RANKL-dependent ARO, since in bone this gene is mainly expressed by cells of mesenchymal origin. Of note, whether lack of RANKL production might cause a defect also in the bone marrow (BM) stromal compartment, possibly contributing to the pathology, is unknown. To verify this possibility, we generated and characterized BM mesenchymal stromal cell (BM-MSC) lines from wild type and Rankl-/- mice, and found that Rankl-/- BM-MSCs displayed reduced clonogenicity and osteogenic capacity. The differentiation defect was significantly improved by lentiviral transduction of Rankl-/- BM-MSCs with a vector stably expressing human soluble RANKL (hsRANKL). Expression of Rankl receptor, Rank, on the cytoplasmic membrane of BM-MSCs pointed to the existence of an autocrine loop possibly activated by the secreted cytokine. Based on the close resemblance of RANKL-defective osteopetrosis in humans and mice, we expect that our results are also relevant for RANKL-dependent ARO patients. Data obtained in vitro after transduction with a lentiviral vector expressing hsRANKL would suggest that restoration of RANKL production might not only rescue the defective osteoclastogenesis of this ARO form, but also improve a less obvious defect in the osteoblast lineage, thus possibly achieving higher benefit for the patients, when the approach is translated to clinics. Stem Cells 2017;35:1365-1377.

Monogenic polyarteritis: the lesson of ADA2 deficiency.

The deficiency of Adenosine Deaminase 2 (DADA2) is a new autoinflammatory disease characterised by an early onset vasculopathy with livedoid skin rash associated with systemic manifestations, CNS involvement and mild immunodeficiency.This condition is secondary to autosomal recessive mutations of CECR1 (Cat Eye Syndrome Chromosome Region 1) gene, mapped to chromosome 22q11.1, that encodes for the enzymatic protein adenosine deaminase 2 (ADA2). By now 19 different mutations in CECR1 gene have been detected.The pathogenetic mechanism of DADA2 is still unclear. ADA2 in a secreted protein mainly expressed by cells of the myeloid lineage; its enzymatic activity is higher in conditions of hypoxia, inflammation and oncogenesis. Moreover ADA2 is able to induce macrophages proliferation and differentiation; it's deficiency is in fact associated with a reduction of anti-inflammatory macrophages (M2). The deficiency of ADA2 is also associated with an up-regulation of neutrophils-expressed genes and an increased secretion of pro-inflammatory cytokines. The mild immunodeficiency detected in many DADA2 patients suggests a role of this protein in the adaptive immune response; an increased mortality of B cells and a reduction in the number of memory B cells, terminally differentiated B cells and plasmacells has been described in many patients. The lack of the protein is associated with endothelium damage; however the function of this protein in the endothelial homeostasis is still unknown.From the clinical point of view, this disease is characterized by a wide spectrum of severity. Chronic or recurrent systemic inflammation with fever, elevation of acute phase reactants and skin manifestations (mainly represented by livedo reticularis) is the typical clinical picture. While in some patients the disease is mild and skin-limited, others present a severe, even lethal, disease with multi-organ involvement; the CNS involvement is rather common with ischemic or hemorrhagic strokes. In many patients not only the clinical picture but also the histopathologic features are undistinguishable from those of systemic polyarteritis nodosa (PAN). Of note, patients with an unusual phenotype, mainly dominated by clinical manifestations suggestive for an immune-disrective condition, have been described.Due to its rarity, the response to treatment of DADA2 is still anecdotal. While steroids can control the disease's manifestations at high dosage, none of the common immunosuppressive drugs turned out to be effective. Biologic drugs have been used only in few patients, without a clear effectiveness; anti-TNF drugs are those associated to a better clinical response. Hematopoietic stem cells transplantation was effective in patients with a severe phenotype.

Single amino acid charge switch defines clinically distinct proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1)-associated inflammatory diseases.

BACKGROUND: Hyperzincemia and hypercalprotectinemia (Hz/Hc) is a distinct autoinflammatory entity involving extremely high serum concentrations of the proinflammatory alarmin myeloid-related protein (MRP) 8/14 (S100A8/S100A9 and calprotectin). OBJECTIVE: We sought to characterize the genetic cause and clinical spectrum of Hz/Hc. METHODS: Proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) gene sequencing was performed in 14 patients with Hz/Hc, and their clinical phenotype was compared with that of 11 patients with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome. PSTPIP1-pyrin interactions were analyzed by means of immunoprecipitation and Western blotting. A structural model of the PSTPIP1 dimer was generated. Cytokine profiles were analyzed by using the multiplex immunoassay, and MRP8/14 serum concentrations were analyzed by using an ELISA. RESULTS: Thirteen patients were heterozygous for a missense mutation in the PSTPIP1 gene, resulting in a p.E250K mutation, and 1 carried a mutation resulting in p.E257K. Both mutations substantially alter the electrostatic potential of the PSTPIP1 dimer model in a region critical for protein-protein interaction. Patients with Hz/Hc have extremely high MRP8/14 concentrations (2045 ± 1300 µg/mL) compared with those with PAPA syndrome (116 ± 74 µg/mL) and have a distinct clinical phenotype. A specific cytokine profile is associated with Hz/Hc. Hz/Hc mutations altered protein binding of PSTPIP1, increasing interaction with pyrin through phosphorylation of PSTPIP1. CONCLUSION: Mutations resulting in charge reversal in the y-domain of PSTPIP1 (E→K) and increased interaction with pyrin cause a distinct autoinflammatory disorder defined by clinical and biochemical features not found in patients with PAPA syndrome, indicating a unique genotype-phenotype correlation for mutations in the PSTPIP1 gene. This is the first inborn autoinflammatory syndrome in which inflammation is driven by uncontrolled release of members of the alarmin family.

IL-10 critically modulates B cell responsiveness in Rankl-/- mice.

The immune and the skeletal system are tightly interconnected, and B lymphocytes are uniquely endowed with osteo-interactive properties. In this context, receptor activator of NF-κB (RANK) ligand (RANKL) plays a pivotal role in lymphoid tissue formation and bone homeostasis. Although murine models lacking RANK or RANKL show defects in B cell number, the role of the RANKL-RANK axis on B physiology is still a matter of debate. In this study, we have characterized in detail B cell compartment in Rankl(-/-) mice, finding a relative expansion of marginal zone B cells, B1 cells, and plasma cells associated with increased Ig serum levels, spontaneous germinal center formation, and hyperresponse to CD40 triggering. Such abnormalities were associated with an increased frequency of regulatory B cells and augmented B cell-derived IL-10 production. Remarkably, in vivo IL-10-R blockade reduced T cell-triggered plasma cell differentiation and restrained the expansion of regulatory B cells. These data point to a novel role of the RANKL-RANK axis in the regulation of B cell homeostasis and highlight an unexpected link between IL-10 CD40 signaling and the RANKL pathway.

Dependence of immunoglobulin class switch recombination in B cells on vesicular release of ATP and CD73 ectonucleotidase activity.

Immunoglobulin (Ig) isotype diversification by class switch recombination (CSR) is an essential process for mounting a protective humoral immune response. Ig CSR deficiencies in humans can result from an intrinsic B cell defect; however, most of these deficiencies are still molecularly undefined and diagnosed as common variable immunodeficiency (CVID). Here, we show that extracellular adenosine critically contributes to CSR in human naive and IgM memory B cells. In these cells, coordinate stimulation of B cell receptor and toll-like receptors results in the release of ATP stored in Ca(2+)-sensitive secretory vesicles. Plasma membrane ectonucleoside triphosphate diphosphohydrolase 1 CD39 and ecto-5'-nucleotidase CD73 hydrolyze ATP to adenosine, which induces CSR in B cells in an autonomous fashion. Notably, CVID patients with impaired class-switched antibody responses are selectively deficient in CD73 expression in B cells, suggesting that CD73-dependent adenosine generation contributes to the pathogenesis of this disease.

Osteopetrosis rescue upon RANKL administration to Rankl(-/-) mice: a new therapy for human RANKL-dependent ARO.

In the last decades the molecular basis of monogenic diseases has been largely unraveled, although their treatment has often remained unsatisfactory. Autosomal recessive osteopetrosis (ARO) belongs to the small group of genetic diseases that are usually treated with hematopoietic stem cell transplantation (HSCT). However, this approach is not effective in the recently identified form carrying mutations in the receptor activator of NF-κB ligand (RANKL) gene. In this subset, therapy replacement approach based on RANKL delivery has a strong rationale. Here we demonstrate that the systematic administration of RANKL for 1 month to Rankl(-/-) mice, which closely resemble the human disease, significantly improves the bone phenotype and has beneficial effects on bone marrow, spleen and thymus; major adverse effects arise only when mice are clearly overtreated. Overall, we provide evidence that the pharmacological administration of RANKL represents the appropriate treatment option for RANKL-deficient ARO patients, to be validated in a pilot clinical trial.

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.

Homeostatic expansion of autoreactive immunoglobulin-secreting cells in the Rag2 mouse model of Omenn syndrome.

Hypomorphic RAG mutations, leading to limited V(D)J rearrangements, cause Omenn syndrome (OS), a peculiar severe combined immunodeficiency associated with autoimmune-like manifestations. Whether B cells play a role in OS pathogenesis is so far unexplored. Here we report the detection of plasma cells in lymphoid organs of OS patients, in which circulating B cells are undetectable. Hypomorphic Rag2(R229Q) knock-in mice, which recapitulate OS, revealed, beyond severe B cell developmental arrest, a normal or even enlarged compartment of immunoglobulin-secreting cells (ISC). The size of this ISC compartment correlated with increased expression of Blimp1 and Xbp1, and these ISC were sustained by elevated levels of T cell derived homeostatic and effector cytokines. The detection of high affinity pathogenic autoantibodies toward target organs indicated defaults in B cell selection and tolerance induction. We hypothesize that impaired B cell receptor (BCR) editing and a serum B cell activating factor (BAFF) abundance might contribute toward the development of a pathogenic B cell repertoire in hypomorphic Rag2(R229Q) knock-in mice. BAFF-R blockade reduced serum levels of nucleic acid-specific autoantibodies and significantly ameliorated inflammatory tissue damage. These findings highlight a role for B cells in OS pathogenesis.