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Memory B cells (MBCs) epitomize the adaptation of the immune system to the environment. We identify two MBC subsets in peripheral blood, CD27 dull and CD27 bright MBCs, whose frequency changes with age. Heavy chain variable region (VH) usage, somatic mutation frequency replacement-to-silent ratio, and CDR3 property changes, reflecting consecutive selection of highly antigen-specific, low cross-reactive antibody variants, all demonstrate that CD27 dull and CD27 bright MBCs represent sequential MBC developmental stages, and stringent antigen-driven pressure selects CD27 dull into the CD27 bright MBC pool. Dynamics of human MBCs are exploited in pregnancy, when 50% of maternal MBCs are lost and CD27 dull MBCs transit to the more differentiated CD27 bright stage. In the postpartum period, the maternal MBC pool is replenished by the expansion of persistent CD27 dull clones. Thus, the stability and flexibility of human B cell memory is ensured by CD27 dull MBCs that expand and differentiate in response to change., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Rituximab (RTX; anti-CD20 mAb) is a treatment option in children with refractory immune thrombocytopenia, autoimmune hemolytic anemia (AHA), and Evans syndrome (ES). Prevalence and clinical course of RTX-induced hypogammaglobulinemia in these patients are poorly known., Objective: To evaluate the prevalence and risk factors for persistent hypogammaglobulinemia (PH) after RTX use., Methods: Clinical and immunologic data from children treated with RTX for immune thrombocytopenia, AHA, and ES were collected from 16 Italian centers and 1 UK center at pre-RTX time point (0), +6 months, and yearly, up to 4 years post-RTX. Patients with previously diagnosed malignancy or primary immune deficiency (PID) were excluded., Results: We analyzed 53 children treated with RTX for immune thrombocytopenia (n = 36), AHA (n = 13), and ES (n = 4). Median follow-up was 30 months (range, 12-48). Thirty-two percent of patients (17 of 53) experienced PH, defined as IgG levels less than 2 SD for age at last follow-up (>12 months after RTX). Significantly delayed B-cell recovery was observed in children experiencing PH (hazard ratio, 0.55; P < .05), and 6 of 17 (35%) patients had unresolved B-cell lymphopenia at last follow-up. PH was associated with IgA and IgM deficiency, younger age at RTX use (51 vs 116 months; P < .01), a diagnosis of AHA/ES, and better response to RTX. Nine patients with PH (9 of 17 [53%]) were eventually diagnosed with a PID., Conclusions: Post-RTX PH is a frequent condition in children with autoimmune cytopenia; a sizable proportion of patients with post-RTX PH were eventually diagnosed with a PID. In-depth investigation for PID is therefore recommended in these patients., (Copyright © 2019 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Mutations affecting the non-canonical pathway of NF-κB were recently identified to underlie a form of common variable immunodeficiency strongly associated with autoimmunity. Although intrinsic B-cell abnormalities explain most of the humoral defects of this disease, detailed data on the impact of NFKB2 on follicular helper (Tfh) and regulatory (Tregs) T cells are scarce. Here, we show that Tfh, CXCR5 + , and CXCR5 - Treg cell subsets were significantly reduced in patients heterozygous for a truncating mutation of NFKB2. Plasma CXCL13 levels were reduced, underlining an important role for NFKB2 in regulating the germinal center (GC) response. Proinflammatory IFNγ, IL-17 and IL-10 cytokine production by CD4 T cells was lower in the mutated patients, but the production of IL-4 and IL-21 was not altered. Taken together, our findings show that NFKB2 influences the quality and efficiency of human GC reaction, by affecting not only the B cells but also GC-relevant T cell subsets., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hemophagocytic lymphohistiocytosis (HLH) is characterized by immune dysregulation due to inadequate restraint of overactivated immune cells and is associated with a variable clinical spectrum having overlap with more common pathophysiologies. HLH is difficult to diagnose and can be part of inflammatory syndromes. Here, we identify a novel hematological/autoinflammatory condition (NOCARH syndrome) in four unrelated patients with superimposable features, including neonatal-onset cytopenia with dyshematopoiesis, autoinflammation, rash, and HLH. Patients shared the same de novo CDC42 mutation (Chr1:22417990C>T, p.R186C) and altered hematopoietic compartment, immune dysregulation, and inflammation. CDC42 mutations had been associated with syndromic neurodevelopmental disorders. In vitro and in vivo assays documented unique effects of p.R186C on CDC42 localization and function, correlating with the distinctiveness of the trait. Emapalumab was critical to the survival of one patient, who underwent successful bone marrow transplantation. Early recognition of the disorder and establishment of treatment followed by bone marrow transplant are important to survival., (© 2019 Lam et al.)

Multiparametric flow cytometry (MFC) represents a rapid, highly reproducible, and sensitive diagnostic technology for primary immunodeficiencies (PIDs), which are characterized by a wide range of T cell perturbations and a broad clinical and genetic heterogeneity. MFC data from CD4+ and CD8+ T cell subsets were examined in 100 patients referred for Primary Immunodeficiencies to our center. Naïve, central memory, effector memory, and terminal effector memory cell differentiation stages were defined by the combined expression CD45RA/CD27 for CD4 and CD45RA/CCR7 for CD8. Principal component analysis (PCA), a non-hypothesis driven statistical analysis, was applied to analyze MFC data in order to distinguish the diverse PIDs. Among severe lymphopenic patients, those affected by severe combined and combined immunodeficiency (SCID and CID) segregated in a specific area, reflecting a homogenous, and a more severe T cell impairment, compared to other lymphopenic PID, such as thymectomized and partial DiGeorge syndrome patients. PID patients with predominantly antibody defects were distributed in a heterogeneous pattern, but unexpectedly PCA was able to cluster some patients' resembling CID, hence warning for additional and more extensive diagnostic tests and a diverse clinical management. In conclusion, PCA applied to T cell MFC data might help the physician to estimate the severity of specific PID and to diversify the clinical and diagnostic approach of the patients., (Copyright © 2019 Attardi, Di Cesare, Amodio, Giancotta, Cotugno, Cifaldi, Chiriaco, Palma, Finocchi, Di Matteo, Rossi and Cancrini.)

Background: Patients with 22q11.2 deletion syndrome (22q11.2DS) may develop severe thrombocytopenic purpura and hemolytic anemia. There are no reliable predictors for the development of hematologic autoimmunity (HA) in these patients., Objective: To describe the peculiar B and T subpopulation defects in patients with 22q11DS who have developed HA and test if these defects precede the development of HA., Methods: We performed a case-control multicenter study. Patients with HA were compared with a control population of 22q11.2DS without HA (non-HA). A complete immunological evaluation was performed at diagnosis and at the last follow-up including extensive T and B phenotypes., Results: Immunophenotype at the last follow-up was available in 23 HA and 45 non-HA patients. HA patients had significantly decreased percentage of naïve CD4 + cells (26.8% vs 43.2%, P = .003) and recent thymic emigrants (48.6% vs 80.5%, P = .046); decreased class-switched B cells (2.0% vs 5.9%, P = .04) and increased naive B cells (83.5% vs 71.4%, P = .02); increased CD16+/56+ both in absolute number (312 vs 199, P = .009) and percentage (20.0% vs 13.0%, P = .03). Immunophenotype was performed in 36 patients (11 HA and 25 non-HA) at diagnosis. Odds ratio (OR) of immune cytopenia were estimated for both CD4 naïve ≤30% (OR 14.0, P = .002) and switched memory B cells ≤2% (OR 44.0, P = .01). The estimated survival curves reached statistical significance, respectively, P = .0001 and P = .002., Conclusions: Among patients with 22q11.2DS, those with HA have characteristic lymphocyte anomalies that appear considerably before HA onset. Systematic immunophenotyping of patients with 22q11.2DS at diagnosis is advisable for early identification of patients at risk for this severe complication., (Copyright © 2019 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Severe combined immunodeficiencies (SCIDs) are a group of inborn errors of the immune system, usually associated with severe or life-threatening infections. Due to the variability of clinical phenotypes, the diagnostic complexity and the heterogeneity of the genetic basis, they are often difficult to recognize, leading to a significant diagnostic delay (DD). Aim of this study is to define presenting signs and natural history of SCID in a large cohort of patients, prior to hematopoietic stem cell or gene therapies. To this purpose, we conducted a 30-year retro-prospective multicenter study within the Italian Primary Immunodeficiency Network. One hundred eleven patients, diagnosed as typical or atypical SCID according to the European Society for Immune Deficiencies criteria, were included. Patients were subsequently classified based on the genetic alteration, pathogenic mechanism and immunological classification. A positive relationship between the age at onset and the DD was found. SCID patients with later onset were identified only in the last decade of observation. Syndromic SCIDs represented 28% of the cohort. Eight percent of the subjects were diagnosed in Intensive Care Units. Fifty-three percent had an atypical phenotype and most of them exhibited a discordant genotype-immunophenotype. Pre-treatment mortality was higher in atypical and syndromic patients. Our study broadens the knowledge of clinical and laboratory manifestations and genotype/phenotype correlation in patients with SCID and may facilitate the diagnosis of both typical and atypical forms of the disease in countries where newborn screening programs have not yet been implemented.

Natural Killer (NK) cells play a critical role in host defense against viral infections. The mechanisms of recognition and killing of virus-infected cells mediated by NK cells are still only partially defined. Several viruses induce, on the surface of target cells, the expression of molecules that are specifically recognized by NK cell-activating receptors. The main NK cell-activating receptors involved in the recognition and killing of virus-infected cells are NKG2D and DNAM-1. In particular, ligands for DNAM-1 are nectin/nectin-like molecules involved also in mechanisms allowing viral infection. Viruses adopt several immune evasion strategies, including those affecting NK cell-mediated immune surveillance, causing persistent viral infection and the development of virus-associated diseases. The virus's immune evasion efficacy depends on molecules differently expressed during the various phases of infection. In this review, we overview the molecular strategies adopted by viruses, specifically cytomegalovirus (CMV), human immunodeficiency virus (HIV-1), herpes virus (HSV), Epstein-Barr virus (EBV) and hepatitis C virus (HCV), aiming to evade NK cell-mediated surveillance, with a special focus on the modulation of DNAM-1 activating receptor and its ligands in various phases of the viral life cycle. The increasing understanding of mechanisms involved in the modulation of activating ligands, together with those mediating the viral immune evasion strategies, would provide critical tools leading to design novel NK cell-based immunotherapies aiming at viral infection control, thus improving cure strategies of virus-associated diseases.

Background: Although autoimmunity and hyperinflammation secondary to recombination activating gene (RAG) deficiency have been associated with delayed diagnosis and even death, our current understanding is limited primarily to small case series., Objective: Understand the frequency, severity, and treatment responsiveness of autoimmunity and hyperinflammation in RAG deficiency., Methods: In reviewing the literature and our own database, we identified 85 patients with RAG deficiency, reported between 2001 and 2016, and compiled the largest case series to date of 63 patients with prominent autoimmune and/or hyperinflammatory pathology., Results: Diagnosis of RAG deficiency was delayed a median of 5 years from the first clinical signs of immune dysregulation. Most patients (55.6%) presented with more than 1 autoimmune or hyperinflammatory complication, with the most common etiologies being cytopenias (84.1%), granulomas (23.8%), and inflammatory skin disorders (19.0%). Infections, including live viral vaccinations, closely preceded the onset of autoimmunity in 28.6% of cases. Autoimmune cytopenias had early onset (median, 1.9, 2.1, and 2.6 years for autoimmune hemolytic anemia, immune thrombocytopenia, and autoimmune neutropenia, respectively) and were refractory to intravenous immunoglobulin, steroids, and rituximab in most cases (64.7%, 73.7%, and 71.4% for autoimmune hemolytic anemia, immune thrombocytopenia, and autoimmune neutropenia, respectively). Evans syndrome specifically was associated with lack of response to first-line therapy. Treatment-refractory autoimmunity/hyperinflammation prompted hematopoietic stem cell transplantation in 20 patients., Conclusions: Autoimmunity/hyperinflammation can be a presenting sign of RAG deficiency and should prompt further evaluation. Multilineage cytopenias are often refractory to immunosuppressive treatment and may require hematopoietic cell transplantation for definitive management., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Mutations affecting the TMEM173 gene cause STING-associated vasculopathy with onset in infancy (SAVI). No standard immunosuppressive treatment approach is able to control disease progression in patients with SAVI. We studied the efficacy and safety of targeting type I IFN signaling with the Janus kinase inhibitor, ruxolitinib., Methods: We used DNA sequencing to identify mutations in TMEM173 in patients with peripheral blood type I IFN signature. The JAK1/2 inhibitor ruxolitinib was administered on an off-label basis., Results: We identified three patients with SAVI presenting with skin involvement and progressive severe interstitial lung disease. Indirect echocardiographic signs of pulmonary hypertension were present in one case. Following treatment with ruxolitinib, we observed improvements of respiratory function including increased forced vital capacity in two patients, with discontinuation of oxygen therapy and resolution of echocardiographic abnormalities in one case. Efficacy was persistent in one patient and only transitory in the other two patients. Clinical control of skin complications was obtained, and one patient discontinued steroid treatment. One patient, who presented with kidney involvement, showed resolution of hematuria. One patient experienced increased recurrence of severe viral respiratory infections. Monitoring of peripheral blood type I interferon signature during ruxolitinib treatment did not show a stable decrease., Conclusions: We conclude that targeting type I IFN receptor signaling may represent a promising therapeutic option for a subset of patients with SAVI syndrome and severe lung involvement. However, the occurrence of viral respiratory infection might represent an important cautionary note for the application of such form of treatment.

Background: Jagunal homolog 1 (JAGN1) gene was identified as a novel responsible for severe congenital neutropenia. The protein encoded by this gene is required for neutrophil differentiation, survival and function in microbial activity. JAGN1-deficient human neutrophils are characterized by alterations in trafficking within the endoplasmic reticulum and golgi compartments because of ultrastructural defects in endoplasmic reticulum and susceptibility to apoptosis., Observations: We report a patient exhibiting an intermittent neutropenia, for which a next-generation sequencing revealed a homozygous mutation in the JAGN1 gene., Conclusions: The patient extends the clinical variability associated to JAGN1 mutations, and this case highlights the importance of genetic investigations in patients with suspected neutropenia.

Background: Primary Immunodeficiencies (PIDs) are a heterogeneous group of genetic immune disorders. While some PIDs can manifest with more than one phenotype, signs, and symptoms of various PIDs overlap considerably. Recently, novel defects in immune-related genes and additional variants in previously reported genes responsible for PIDs have been successfully identified by Next Generation Sequencing (NGS), allowing the recognition of a broad spectrum of disorders. Objective: To evaluate the strength and weakness of targeted NGS sequencing using custom-made Ion Torrent and Haloplex (Agilent) panels for diagnostics and research purposes. Methods: Five different panels including known and candidate genes were used to screen 105 patients with distinct PID features divided in three main PID categories: T cell defects, Humoral defects and Other PIDs . The Ion Torrent sequencing platform was used in 73 patients. Among these, 18 selected patients without a molecular diagnosis and 32 additional patients were analyzed by Haloplex enrichment technology. Results: The complementary use of the two custom-made targeted sequencing approaches allowed the identification of causative variants in 28.6% ( n = 30) of patients. Twenty-two out of 73 (34.6%) patients were diagnosed by Ion Torrent. In this group 20 were included in the SCID/CID category. Eight out of 50 (16%) patients were diagnosed by Haloplex workflow. Ion Torrent method was highly successful for those cases with well-defined phenotypes for immunological and clinical presentation. The Haloplex approach was able to diagnose 4 SCID/CID patients and 4 additional patients with complex and extended phenotypes, embracing all three PID categories in which this approach was more efficient. Both technologies showed good gene coverage. Conclusions: NGS technology represents a powerful approach in the complex field of rare disorders but its different application should be weighted. A relatively small NGS target panel can be successfully applied for a robust diagnostic suspicion, while when the spectrum of clinical phenotypes overlaps more than one PID an in-depth NGS analysis is required, including also whole exome/genome sequencing to identify the causative gene.

Severe combined immunodeficiencies (SCID) comprise a group of genetic diseases characterized by abrogated development of T lymphocytes. In some case reports of atypical SCID patients elevated proportions of γδ T lymphocytes have been reported. However, it is unknown whether these γδ T cells modulate or reflect the patient's clinical phenotype. We investigated the frequency of elevated γδ T cell proportions and associations with clinical disease manifestations in a cohort of 76 atypical SCID patients. Increased proportions of γδ T lymphocytes were present in approximately 60% of these patients. Furthermore, we identified positive correlations between elevated proportions of γδ T cells and the occurrence of CMV infections and autoimmune cytopenias. We discuss that CMV infections might trigger an expansion of γδ T lymphocytes, which could drive the development of autoimmune cytopenias. We advocate that atypical SCID patients should be screened for elevated proportions of γδ T lymphocytes, CMV infection and autoimmune cytopenias., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Background: X-linked agammaglobulinemia is an inherited immunodeficiency recognized since 1952. In spite of seven decades of experience, there is still a limited understanding of regional differences in presentation and complications. This study was designed by the Primary Immunodeficiencies Committee of the World Allergy Organization to better understand regional needs, challenges and unique patient features., Methods: A survey instrument was designed by the Primary Immunodeficiencies Committee of the World Allergy Organization to collect both structured and semi-structured data on X-linked agammaglobulinemia. The survey was sent to 54 centers around the world chosen on the basis of World Allergy Organization participation and/or registration in the European Society for Immunodeficiencies. There were 40 centers that responded, comprising 32 countries., Results: This study reports on 783 patients from 40 centers around the world. Problems with diagnosis are highlighted by the reported delays in diagnosis>24 months in 34% of patients and the lack of genetic studies in 39% of centers Two infections exhibited regional variation. Vaccine-associated paralytic poliomyelitis was seen only in countries with live polio vaccination and two centers reported mycobacteria. High rates of morbidity were reported. Acute and chronic lung diseases accounted for 41% of the deaths. Unusual complications such as inflammatory bowel disease and large granular lymphocyte disease, among others were specifically enumerated, and while individually uncommon, they were collectively seen in 20.3% of patients. These data suggest that a broad range of both inflammatory, infectious, and autoimmune conditions can occur in patients. The breadth of complications and lack of data on management subsequently appeared as a significant challenge reported by centers. Survival above 20 years of age was lowest in Africa (22%) and reached above 70% in Australia, Europe and the Americas. Centers were asked to report their challenges and responses (n = 116) emphasized the difficulties in access to immunoglobulin products (16%) and reflected the ongoing need for education of both patients and referring physicians., Conclusions: This is the largest study of patients with X-linked agammaglobulinemia and emphasizes the continued morbidity and mortality of XLA despite progress in diagnosis and treatment. It presents a world view of the successes and challenges for patients and physicians alike. A pivotal finding is the need for education of physicians regarding typical symptoms suggesting a possible diagnosis of X-linked agammaglobulinemia and sharing of best practices for the less common complications.

The thymus plays a fundamental role in establishing and maintaining central and peripheral tolerance and defects in thymic architecture or AIRE expression result in the development of autoreactive lymphocytes. Patients with partial DiGeorge Syndrome (pDGS) and Down Syndrome (DS) present alterations in size and architecture of the thymus and higher risk to develop autoimmunity. We sought to evaluate thymic architecture and thymocyte development in DGS and DS patients and to determine the extent to which thymic defects result in immune dysregulation and T cell homeostasis perturbation in these patients. Thymi from pediatric patients and age-matched controls were obtained to evaluate cortex and medullary compartments, AIRE expression and thymocyte development. In the same patients we also characterized immunophenotype of peripheral T cells. Phenotypic and functional characterization of thymic and peripheral regulatory T (Treg) cells was finally assessed. Histologic analysis revealed peculiar alterations in thymic medulla size and maturation in DGS and DS patients. Perturbed distribution of thymocytes and altered thymic output was also observed. DGS patients showed lower mature CD4 + and CD8 + T cell frequency, associated with reduced proportion and function of Tregs both in thymus and peripheral blood. DS patients showed increased frequency of single positive (SP) thymocytes and thymic Treg cells. However, Tregs isolated both from thymus and peripheral blood of DS patients showed reduced suppressive ability. Our results provide novel insights on thymic defects associated with DGS and DS and their impact on peripheral immune dysregulation. Indeed, thymic abnormalities and defect in thymocyte development, in particular in Treg cell number and function could contribute in the pathogenesis of the immunodysregulation present in pDGS and in DS patients.

Background: Mutations in the Janus Kinase 3 (JAK3) gene cause an autosomal recessive form of severe combined immunodeficiency (SCID) usually characterized by the absence of both T and NK cells, but preserved numbers of B lymphocytes (T-B+NK-SCID). The detection of larger (>100 bp) genomic duplications or deletions can be more difficult to be detected by PCR-based methods or standard NGS protocols, and a broad range of mutation detection techniques are necessary., Methods: We report four unrelated Italian patients (two females and two males) with SCID phenotype. Protein expression, functional studies, molecular analysis by standard methods and NGS, and transcripts studies were performed to obtain a definitive diagnosis., Results: Here, we describe four JAK3-deficient patients from four unrelated families. The first patient is homozygous for the known c.1951 C>T mutation causing the amino acidic change p.R651W. The other two patients, originating from the same small Italian town, resulted compound heterozygotes for the same g.15410&#95;16542del deletion and two different novel mutations, g.13319&#95;13321delTTC and c.933T>G (p.F292V), respectively. The fourth patient was compound heterozygous for the novel mutations p.V599G and p.W709R. Defective STAT5 phosphorylation after IL2 or IL15 stimulation corroborated the mutation pathogenicity. Concerning g.15410&#95;16542del mutation, probably due to an unequal homologous recombination between Alu elements of JAK3 gene, microsatellites analysis revealed that both unrelated Pt2 and Pt3 and their carrier family members shared the same haplotype. These data support the hypothesis of a founder effect for the g.15410&#95;16542del mutation that might have inherited in both unrelated families from the same ancient progenitor., Conclusion: Different molecular techniques are still required to obtain a definitive diagnosis of AR-SCID particularly in all cases in which a monoallelic mutation is found by standard mutation scanning methods., (© 2018 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Activated phosphoinositide 3-kinase (PI3K) δ Syndrome (APDS), caused by autosomal dominant mutations in PIK3CD (APDS1) or PIK3R1 (APDS2), is a heterogeneous primary immunodeficiency. While initial cohort-descriptions summarized the spectrum of clinical and immunological manifestations, questions about long-term disease evolution and response to therapy remain. The prospective European Society for Immunodeficiencies (ESID)-APDS registry aims to characterize the disease course, identify outcome predictors, and evaluate treatment responses. So far, 77 patients have been recruited (51 APDS1, 26 APDS2). Analysis of disease evolution in the first 68 patients pinpoints the early occurrence of recurrent respiratory infections followed by chronic lymphoproliferation, gastrointestinal manifestations, and cytopenias. Although most manifestations occur by age 15, adult-onset and asymptomatic courses were documented. Bronchiectasis was observed in 24/40 APDS1 patients who received a CT-scan compared with 4/15 APDS2 patients. By age 20, half of the patients had received at least one immunosuppressant, but 2-3 lines of immunosuppressive therapy were not unusual before age 10. Response to rapamycin was rated by physician visual analog scale as good in 10, moderate in 9, and poor in 7. Lymphoproliferation showed the best response (8 complete, 11 partial, 6 no remission), while bowel inflammation (3 complete, 3 partial, 9 no remission) and cytopenia (3 complete, 2 partial, 9 no remission) responded less well. Hence, non-lymphoproliferative manifestations should be a key target for novel therapies. This report from the ESID-APDS registry provides comprehensive baseline documentation for a growing cohort that will be followed prospectively to establish prognostic factors and identify patients for treatment studies.

Background: Autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency (AD EDA-ID) is caused by heterozygous point mutations at or close to serine 32 and serine 36 or N-terminal truncations in IκBα that impair its phosphorylation and degradation and thus activation of the canonical nuclear factor κ light chain enhancer of activated B cells (NF-κB) pathway. The outcome of hematopoietic stem cell transplantation is poor in patients with AD EDA-ID despite achievement of chimerism. Mice heterozygous for the serine 32I mutation in IκBα have impaired noncanonical NF-κB activity and defective lymphorganogenesis., Objective: We sought to establish genotype-phenotype correlation in patients with AD EDA-ID., Methods: A disease severity scoring system was devised. Stability of IκBα mutants was examined in transfected cells. Immunologic, biochemical, and gene expression analyses were performed to evaluate canonical and noncanonical NF-κB signaling in skin-derived fibroblasts., Results: Disease severity was greater in patients with IκBα point mutations than in those with truncation mutations. IκBα point mutants were expressed at significantly higher levels in transfectants compared with truncation mutants. Canonical NF-κB-dependent IL-6 secretion and upregulation of the NF-κB subunit 2/p100 and RELB proto-oncogene, NF-κB subunit (RelB) components of the noncanonical NF-κB pathway were diminished significantly more in patients with point mutations compared with those with truncations. Noncanonical NF-κB-driven generation of the transcriptionally active p100 cleavage product p52 and upregulation of CCL20, intercellular adhesion molecule 1 (ICAM1), and vascular cell adhesion molecule 1 (VCAM1), which are important for lymphorganogenesis, were diminished significantly more in LPS plus α-lymphotoxin β receptor-stimulated fibroblasts from patients with point mutations compared with those with truncations., Conclusions: IκBα point mutants accumulate at higher levels compared with truncation mutants and are associated with more severe disease and greater impairment of canonical and noncanonical NF-κB activity in patients with AD EDA-ID., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Incontinentia pigmenti (IP) is a rare inherited genodermatosis that usually involves the skin, and also teeth, oral cavity, central nervous system, eyes, blood with eosinophilia, and rarely skeletal system, breast, heart, and lungs. Skin lesions usually appear early, at birth or within the first 2 weeks of life, with four different phases tending to follow Blaschko lines that may overlap., Case Report: We report a rare case of a neonate with transient reversible pulmonary hypertension that presented at day 9 of life. She manifested increasing dyspnea and deterioration of respiratory dynamics with a serious pulmonary hypertension without a primary pulmonary disease. Hence, oxygen therapy at high flows and nitric oxide have been administered with an initial response, but, subsequently, because of the worsening of the respiratory activity, she underwent sildenafil and bosentan treatment with respiratory dynamics improvement and progressive decrease of the pulmonary pressures., Conclusion: In literature only a few cases of cardiopulmonary anomalies in IP have been described with different outcomes, and these rare complications are probably underestimated by physicians. We could suppose that microangiopathic damages may have a critical role in endothelial alterations, and these processes are probably shared by multiple organs involved in IP and rarely by lungs and heart., (© 2017 The International Society of Dermatology.)

Monogenic defects in genes related to primary immunodeficiencies can be responsible for inflammatory bowel disease (IBD). Mutations in the X-linked inhibitor of apoptosis ( XIAP ) gene have been described in several patients suffering from IBD and, in particular, with very early-onset inflammatory bowel disease (VEOIBD) features. We report a VEOIBD child with a novel XIAP gene mutation characterized by a complicated disease course, which is unresponsive to several medical treatment options. A next-generation sequencing was performed and revealed a de novo hemizygous mutation in XIAP gene: c.565T>C p.L189P. After mutation discovery, we investigated the XIAP protein expression and nucleotide-binding oligomerization domain protein 2 (NOD2) signaling by western blotting. Flow-cytometry was used to analyze intracellular protein expression in different cell subsets and T cell apoptosis. We observed reduced protein expression in lymphocytes, granulocytes, monocytes, an Epstein-Barr virus-immortalized B cell line as well as increased apoptosis, and impairment in NOD2 signaling. The child was successfully treated with HLA-haploidentical hemapoietic stem cells transplant, acquired from his mother, after ex vivo elimination of α/β T cells and CD19 B cells. One year after the transplant, we repeated the analysis to appreciate the changes in his impairments. The recovery of XIAP protein expression, function, and normalization of apoptosis were observed. Our report emphasizes the important role of genetic analysis in the diagnosis of VEOIBD, illustrates the complete immunological and gastrointestinal recovery after transplant, and shows one of the few successful transplant cases of XIAP patients.

Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal hyperinflammatory condition. Variants in different genes have been associated with the familial forms of the syndrome (FHL), usually presenting within the first 2 years of life. Due to increasing awareness of the signs and symptoms of HLH and a better understanding of the genetic basis of the disease, FHL has been increasingly diagnosed in patients presenting beyond infancy. Here, we report on two brothers with atypical, late-onset HLH in which whole exome sequencing revealed a homozygous pathogenic UNC13D variant. In the first brother, the clinical phenotype was dominated by a massive lung involvement. In the second brother a progressive neurological deterioration was observed. In both cases, the clinical manifestations at symptom onset were misleading, making the diagnosis difficult to achieve. This report expands the spectrum of clinical presentations of FLH3. Moreover, it highlights the importance to warn clinicians to keep a high level of suspicion in patients presenting with fever, cytopenia, splenomegaly of unknown origin, and unresponsiveness to conventional treatment even beyond early childhood. Moreover, this report emphasizes that insidious neurologic symptoms may represent the initial or sole presenting sign of FHL, even in the absence of peripheral signs of activation.

Background: Chronic granulomatous disease (CGD) is a primary immunodeficiency of phagocytes, characterized by life-threatening infections and hyperinflammation. Due to survival improvement, inflammatory bowel disease (IBD) is becoming increasingly relevant. Here, we report our 20 year experience., Methods: We retrospectively analyzed clinic, endoscopic, and histologic features, as well as the management of CGD-IBD patients referred to the Bambino Gesù Children's Hospital in Rome, Italy., Results: Of 20 patients with CGD, 9 presented with CGD-IBD at diagnosis and/or during follow-up. Symptoms occurred at a median age of 16 years (range 3.2-42), with a median delay of 6 months for endoscopic confirmation. Patients mainly complained of nonspecific diarrhea (55%), with discrepancy between symptom paucity and severe endoscopic appearance, mainly represented by extensive colonic involvement (44%). Histology revealed at least 2 characteristic features (epithelioid granulomas, pigmented macrophages, and increased eosinophils) in 78% of patients. Eight of 9 patients received oral mesalamine, and 5 required systemic steroids. One patient received azathioprine due to steroid dependence. No patient required biological therapy or surgery. Clinical remission was obtained in all patients, but the majority complained of mild relapses. Two episodes of severe infection occurred early after steroid therapy., Conclusions: Penetrance of CGD-IBD increases with age. Clinical manifestations may be subtle, and clinicians should have a low threshold to recommend endoscopy. Treatment with NSAIDs and/or steroids achieves a good response, but relapses usually occur. Infection surveillance is mandatory during treatment, to prevent opportunistic infections. A close collaboration between pediatric immunologists and gastroenterologists is pivotal, including combined follow-up., (© 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

[This corrects the article on p. 798 in vol. 8, PMID: 28769923.].

Mutations of the recombinase-activating genes 1 and 2 ( RAG1 and RAG2 ) in humans are associated with a broad range of phenotypes. For patients with severe clinical presentation, hematopoietic stem cell transplantation (HSCT) represents the only curative treatment; however, high rates of graft failure and incomplete immune reconstitution have been observed, especially after unconditioned haploidentical transplantation. Studies in mice have shown that Rag -/- natural killer (NK) cells have a mature phenotype, reduced fitness, and increased cytotoxicity. We aimed to analyze NK cell phenotype and function in patients with mutations in RAG and in non-homologous end joining (NHEJ) genes. Here, we provide evidence that NK cells from these patients have an immature phenotype, with significant expansion of CD56 bright CD16 -/int CD57 - cells, yet increased degranulation and high perforin content. Correlation was observed between in vitro recombinase activity of the mutant proteins, NK cell abnormalities, and in vivo clinical phenotype. Addition of serotherapy in the conditioning regimen, with the aim of depleting the autologous NK cell compartment, may be important to facilitate engraftment and immune reconstitution in patients with RAG and NHEJ defects treated by HSCT.

Activated PI3-kinase delta syndrome (APDS) was recently reported as a novel primary immunodeficiency caused by heterozygous gain-of-function mutations in PIK3CD gene. Here we describe immunological studies in a 19year old APDS patient for whom genetic diagnosis was discovered by Whole Exome Sequencing (WES) analysis. In addition to the progressive lymphopenia and defective antibody production we showed that the ability of the patient's B cells to differentiate in vitro is severely reduced. An in depth analysis of the myeloid compartment showed an increased expression of CD83 activation marker on monocytes and mono-derived DC cells. Moreover, monocytes-derived macrophages (MDMs) failed to solve the Mycobacterium bovis bacillus Calmette Guèrin (BCG) infection in vitro. Selective p110δ inhibitor IC87114 restored the MDM capacity to kill BCG in vitro. Our data show that the constitutive activation of Akt-mTOR pathway induces important alterations also in the myeloid compartment providing new insights in order to improve the therapeutic approach in these patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Absent T-cell immunity resulting in life-threatening infections provides a clear rationale for hematopoetic stem cell transplantation (HSCT) in patients with severe combined immunodeficiency (SCID). Combined immunodeficiencies (CIDs) and "atypical" SCID show reduced, not absent T-cell immunity. If associated with infections or autoimmunity, they represent profound combined immunodeficiency (P-CID), for which outcome data are insufficient for unambiguous early transplant decisions., Objectives: We sought to compare natural histories of severity-matched patients with/without subsequent transplantation and to determine whether immunologic and/or clinical parameters may be predictive for outcome., Methods: In this prospective and retrospective observational study, we recruited nontransplanted patients with P-CID aged 1 to 16 years to compare natural histories of severity-matched patients with/without subsequent transplantation and to determine whether immunologic and/or clinical parameters may be predictive for outcome., Results: A total of 51 patients were recruited (median age, 9.6 years). Thirteen of 51 had a genetic diagnosis of "atypical" SCID and 14 of 51 of CID. About half of the patients had less than 10% naive T cells, reduced/absent T-cell proliferation, and at least 1 significant clinical event/year, demonstrating their profound immunodeficiency. Nineteen patients (37%) underwent transplantation within 1 year of enrolment, and 5 of 51 patients died. Analysis of the HSCT decisions revealed the anticipated heterogeneity, favoring an ongoing prospective matched-pair analysis of patients with similar disease severity with or without transplantation. Importantly, so far neither the genetic diagnosis nor basic measurements of T-cell immunity were good predictors of disease evolution., Conclusions: The P-CID study for the first time characterizes a group of patients with nontypical SCID T-cell deficiencies from a therapeutic perspective. Because genetic and basic T-cell parameters provide limited guidance, prospective data from this study will be a helpful resource for guiding the difficult HSCT decisions in patients with P-CID., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Switched and IgM memory B cells execute different and noninterchangeable functions. We studied memory B cells in children of different ages, in peripheral blood and spleen and compared them with those of children born asplenic or unable to build germinal centers. We show that, whereas switched memory B cells are mostly generated in the germinal centers at all ages, IgM memory B cells can be distinct in three types with different developmental history. Innate IgM memory B cells, the largest pool in infants, are generated in the spleen by a germinal center-independent mechanism. With age, if the spleen is present and germinal centers are functional, innate IgM memory B cells are remodelled and accumulate somatic mutations. The third type of IgM memory B cell is a by-product of the germinal center reaction. Our data suggest that the B-cell memory developmental program is implemented during the first 5-6 years of life., (© 2016 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Around 65% of primary immunodeficiencies are antibody deficiencies. Functional tests are useful tools to study B-cell functions in vitro. However, no accepted guidelines for performing and evaluating functional tests have been issued yet. Here, we report our experience on the study of B-cell functions in infancy and throughout childhood. We show that T-independent stimulation with CpG measures proliferation and differentiation potential of memory B cells. Switched memory B cells respond better than IgM memory B cells. On the other hand, CD40L, a T-dependent stimulus, does not induce plasma cell differentiation, but causes proliferation of naïve and memory B cells. During childhood, the production of plasmablasts in response to CpG increases with age mirroring the development of memory B cells. The response to CD40L does not change with age. In patients with selective IgA deficiency (SIgAD), we observed that switched memory B cells are reduced due to the absence of IgA memory B cells. In agreement, IgA plasma cells are not generated in response to CpG. Unexpectedly, B cells from SIgAD patients show a reduced proliferative response to CD40L. Our results demonstrate that functional tests are an important tool to assess the functions of the humoral immune system., (© 2016 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Primary immunodeficiency diseases (PIDDs) are clinically and genetically heterogeneous disorders thus far associated with mutations in more than 300 genes. The clinical phenotypes derived from distinct genotypes can overlap. Genetic etiology can be a prognostic indicator of disease severity and can influence treatment decisions., Objective: We sought to investigate the ability of whole-exome screening methods to detect disease-causing variants in patients with PIDDs., Methods: Patients with PIDDs from 278 families from 22 countries were investigated by using whole-exome sequencing. Computational copy number variant (CNV) prediction pipelines and an exome-tiling chromosomal microarray were also applied to identify intragenic CNVs. Analytic approaches initially focused on 475 known or candidate PIDD genes but were nonexclusive and further tailored based on clinical data, family history, and immunophenotyping., Results: A likely molecular diagnosis was achieved in 110 (40%) unrelated probands. Clinical diagnosis was revised in about half (60/110) and management was directly altered in nearly a quarter (26/110) of families based on molecular findings. Twelve PIDD-causing CNVs were detected, including 7 smaller than 30 Kb that would not have been detected with conventional diagnostic CNV arrays., Conclusion: This high-throughput genomic approach enabled detection of disease-related variants in unexpected genes; permitted detection of low-grade constitutional, somatic, and revertant mosaicism; and provided evidence of a mutational burden in mixed PIDD immunophenotypes., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Recombination-activating genes 1 and 2 ( RAG1 and RAG2 ) play a critical role in T and B cell development by initiating the recombination process that controls the expression of T cell receptor (TCR) and immunoglobulin genes. Mutations in the RAG1 and RAG2 genes in humans cause a broad spectrum of phenotypes, including severe combined immunodeficiency (SCID) with lack of T and B cells, Omenn syndrome, leaky SCID, and combined immunodeficiency with granulomas or autoimmunity (CID-G/AI). Using next-generation sequencing, we analyzed the TCR and B cell receptor (BCR) repertoire in 12 patients with RAG mutations presenting with Omenn syndrome ( n = 5), leaky SCID ( n = 3), or CID-G/AI ( n = 4). Restriction of repertoire diversity skewed usage of variable (V), diversity (D), and joining (J) segment genes, and abnormalities of CDR3 length distribution were progressively more prominent in patients with a more severe phenotype. Skewed usage of V, D, and J segment genes was present also within unique sequences, indicating a primary restriction of repertoire. Patients with Omenn syndrome had a high proportion of class-switched immunoglobulin heavy chain transcripts and increased somatic hypermutation rate, suggesting in vivo activation of these B cells. These data provide a framework to better understand the phenotypic heterogeneity of RAG deficiency., (Copyright © 2016, American Association for the Advancement of Science.)

NK cell cytotoxicity in Chédiak-Higashi syndrome (CHS) is strongly impaired as lytic granules are not released upon NK-target cell contact, contributing to several defects typical of this severe immunodeficiency. Correction of NK cell defects in CHS should improve the outcome of hematopoietic stem-cell transplantation, proposed as therapy. We investigated NK cell functions in a CHS patient before and after cord-blood transplantation, and the ability of in vitro IL-2 treatment to restore them. Before the transplant, the strong defect in NK cell-mediated natural and antibody-dependent cytotoxicity, as well as in IFN-γ production, could be restored up to normal levels by in vitro IL-2 treatment. This cytokine also caused the appearance of smaller lysosomal granules and their orientation towards the NK-target cell contact area, thus suggesting that IL-2 had a more general capacity to restore NK cell effector functions. Moreover after the transplant, although the successful engraftment, NK cell cytotoxicity resulted still partially impaired at one year, almost normal at ten years and, anyhow, fully recovered by in vitro IL-2 treatment. Taken together, our results indicate that IL-2 had a wide capacity to restore NK cell effector functions, being able to reverse the altered cytotoxic activity, lytic granule pattern, and cytokine production observed in the CHS patient., (Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Vaccine-preventable diseases are a significant cause of morbidity and mortality in solid organ transplant recipients who undergo immunosuppression after transplantation. Data on immune responses and long-term maintenance after vaccinations in such population are still limited.We cross-sectionally evaluated the maintenance of immune response to measles vaccine in kidney transplanted children on immunosuppressive therapy. Measles-specific enzyme-linked immunosorbent assay and B-cell enzyme-linked immunosorbent spot were performed in 74 kidney transplant patients (Tps) and in 23 healthy controls (HCs) previously vaccinated and tested for humoral protection against measles. The quality of measles antibody response was measured by avidity test. B-cell phenotype, investigated via flow cytometry, was further correlated to the ability of Tps to maintain protective humoral responses to measles over time.We observed the loss of vaccine-induced immunity against measles in 19% of Tps. Nonseroprotected children showed signs of impaired B-cell distribution as well as immune senescence and lower antibody avidity. We further reported as time elapsed between vaccination and transplantation, as well as the vaccine administration during dialysis are clinical factors affecting the maintenance of the immune memory response against measles.Tps present both quantitative and qualitative alterations in the maintenance of protective immunity to measles vaccine. Prospective studies are needed to optimize the vaccination schedules in kidney transplant recipients in order to increase the immunization coverage over time in this population.

The effects of an immunoadsorption procedure, specifically designed to remove immunoglobulin E (IgE), on food-induced anaphylaxis have never been evaluated. We evaluate the effects of IgE removal on the allergic thresholds to foods. A 6-year-old boy with anaphylaxis to multiple foods and steroid-resistant unstable allergic asthma displayed serum IgE levels of 2800 to 3500 kU/L. To lower IgE serum concentrations, which could be overridden by a high dose of omalizumab, 1.5 plasma volumes were exchanged in 8 apheresis sessions. During the procedure, serum IgE levels fell to 309 kU/L. After the procedure, the threshold of reactivity to baked milk increased from 0.125 to 5 g of milk protein (full tolerance) after the first session, and the threshold of reactivity to hazelnut increased from 0.037 to 0.142 g of protein after the first session, 0.377 g after the eighth, and 1.067 g (full tolerance) after the first administration of omalizumab. Immediately after the sixth IgE immunoadsorption, we started omalizumab therapy. In the next 40 days, the threshold of reactivity to hazelnut increased to 7.730 (full tolerance). Asthma control was obtained, treatment with montelukast was stopped, and fluticasone was tapered from 500 to 175 μg/day. The boy became partially or fully tolerant to all the tested foods, and quality of life was improved. IgE immunoadsorption, used to establish the starting basis for omalizumab administration, is able to increase the tolerance threshold to foods., (Copyright © 2015 by the American Academy of Pediatrics.)

Patients with mutations of the recombination-activating genes (RAG) present with diverse clinical phenotypes, including severe combined immune deficiency (SCID), autoimmunity, and inflammation. However, the incidence and extent of immune dysregulation in RAG-dependent immunodeficiency have not been studied in detail. Here, we have demonstrated that patients with hypomorphic RAG mutations, especially those with delayed-onset combined immune deficiency and granulomatous/autoimmune manifestations (CID-G/AI), produce a broad spectrum of autoantibodies. Neutralizing anti-IFN-α or anti-IFN-ω antibodies were present at detectable levels in patients with CID-G/AI who had a history of severe viral infections. As this autoantibody profile is not observed in a wide range of other primary immunodeficiencies, we hypothesized that recurrent or chronic viral infections may precipitate or aggravate immune dysregulation in RAG-deficient hosts. We repeatedly challenged Rag1S723C/S723C mice, which serve as a model of leaky SCID, with agonists of the virus-recognizing receptors TLR3/MDA5, TLR7/-8, and TLR9 and found that this treatment elicits autoantibody production. Altogether, our data demonstrate that immune dysregulation is an integral aspect of RAG-associated immunodeficiency and indicate that environmental triggers may modulate the phenotypic expression of autoimmune manifestations.

Purpose: Hematopoietic cell transplantation (HCT) provides a curative therapy for severe forms of primary immunodeficiencies (PID). While the timing and extent of T-cell reconstitution following transplant for PID has been studied in depth, less is known about the kinetics of B-cell development and long-term restoration of humoral functions, which been often reported to be suboptimal after HCT., Methods: We studied longitudinally B-cell development and function in a cohort of 13 PID patients transplanted between 1997 and 2010, with a follow-up ranging from 0.7 to 15 years. Flow cytometric analysis of naïve and antigen-experienced B-cell subsets and in vitro functional responses to CpG were compared with data from healthy children and correlated with the degree of B-cell chimerism and in vivo antibody production., Results: We found that total memory B-cells count remained below normal levels for the first 2 years of follow up and progressively normalized. Switched memory B-cells (CD19+CD27+IgD-IgM-) were restored early and better than IgM memory B-cells (CD19+CD27+IgD+IgM+), which remained significantly reduced long-term. The recovery of memory B-cells correlated with good in vivo humoral function and normalization of CpG-response. A complete B-cell reconstitution was usually associated with donor B-cells chimerism and pre-transplant conditioning. Donor source and the underlying genetic defect represented also important variables., Conclusion: Monitoring of phenotypic and functional changes on B-cells following HCT may prove clinically relevant to tailor patients' care. In particular the analysis of IgM memory and switched memory B-cells in addition to in vitro B-cells stimulation are recommended before Ig replacement therapy (IgRT) discontinuation.

A definitive understanding of survival and differentiation potential in humans of T cell subpopulations is of paramount importance for the development of effective T cell therapies. In particular, uncovering the dynamics in vivo in humans of the recently described T memory stem cells (TSCM) would be crucial for therapeutic approaches that aim at taking advantage of a stable cellular vehicle with precursor potential. We exploited data derived from two gene therapy clinical trials for an inherited immunodeficiency, using either retrovirally engineered hematopoietic stem cells or mature lymphocytes to trace individual T cell clones directly in vivo in humans. We compared healthy donors and bone marrow-transplanted patients, studied long-term in vivo T cell composition under different clinical conditions, and specifically examined TSCM contribution according to age, conditioning regimen, disease background, cell source, long-term reconstitution, and ex vivo gene correction processing. High-throughput sequencing of retroviral vector integration sites (ISs) allowed tracing the fate of more than 1700 individual T cell clones in gene therapy patients after infusion of gene-corrected hematopoietic stem cells or mature lymphocytes. We shed light on long-term in vivo clonal relationships among different T cell subtypes, and we unveiled that TSCM are able to persist and to preserve their precursor potential in humans for up to 12 years after infusion of gene-corrected lymphocytes. Overall, this work provides high-resolution tracking of T cell fate and activity and validates, in humans, the safe and functional decade-long survival of engineered TSCM, paving the way for their future application in clinical settings., (Copyright © 2015, American Association for the Advancement of Science.)

Autoimmunity can develop from an often undetermined interplay of genetic and environmental factors. Rare forms of autoimmune conditions may also result from single gene mutations as for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, an autosomal recessive disease associated with mutated forms of the autoimmune regulator gene. It was proposed that genetic variability in the autoimmune regulator locus, in particular heterozygous loss-of-function mutations, might favor the development of organ-specific autoimmunity by affecting the presentation of self-antigens in the thymus. Indeed, heterozygous mutations of the autoimmune regulator gene were reported in patients with organ-specific autoimmunity. Also, in primary immunodeficiencies, a breakdown in central/peripheral tolerance frequently produces association with autoimmunity. The causative link may involve a common genetic background and several gene defects have been identified as putative culprits. We report a unique patient, a 14 year old male from Lazio region, affected by common variable immunodeficiency associated with autoimmune manifestations (alopecia, onychodystrophy) and heterozygote for the S250C variant located in the SAND domain of the autoimmune regulator gene protein. To our knowledge this is the first report of the S250C variant in a patient bearing this unusual combination of autoimmunity and immunodeficiency. To obtain insights into the possible molecular effects of the S250C variant, we have carried out an in silico analysis of the SAND domain structure of the autoimmune regulator protein. In particular, homology modeling has allowed us to observe that the cysteine introduced by the S250C variant is surrounded by cationic residues, and by means of molecular dynamics simulations together with pKa calculations, we have shown that these residues remain stably proximal to cysteine-250 lowering its pKa and thus conferring high chemical reactivity to the mutated residue. We propose that the enhanced reactivity of cysteine-250, which is likely to impair the protein function but probably insufficient to produce alone a phenotype as a heterozygous S250C variant due to compensation mechanisms, might become manifest when combined with other genetic/environmental factors. These results can provide the rationale for the patient's unusual phenotype, shedding new light into the pathogenesis of the clinical association of autoimmunity and immunodeficiency., (Copyright © 2014 Elsevier B.V. All rights reserved.)