Wiskott-Aldrich syndrome: a study of 577 patients defines the genotype as a biomarker for disease severity and survival.

ABSTRACT: Wiskott-Aldrich syndrome (WAS) is a multifaceted monogenic disorder with a broad disease spectrum and variable disease severity and a variety of treatment options including allogeneic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT). No reliable biomarker exists to predict disease course and outcome for individual patients. A total of 577 patients with a WAS variant from 26 countries and a median follow-up of 8.9 years (range, 0.3-71.1), totaling 6118 patient-years, were included in this international retrospective study. Overall survival (OS) of the cohort (censored at HSCT or GT) was 82% (95% confidence interval, 78-87) at age 15 years and 70% (61-80) at 30 years. The type of variant was predictive of outcome: patients with a missense variant in exons 1 or 2 or with the intronic hot spot variant c.559+5G>A (class I variants) had a 15-year OS of 93% (89-98) and a 30-year OS of 91% (86-97), compared with 71% (62-81) and 48% (34-68) in patients with any other variant (class II; P < .0001). The cumulative incidence rates of disease-related complications such as severe bleeding (P = .007), life-threatening infection (P < .0001), and autoimmunity (P = .004) occurred significantly later in patients with a class I variant. The cumulative incidence of malignancy (P = .6) was not different between classes I and II. It confirms the spectrum of disease severity and quantifies the risk for specific disease-related complications. The class of the variant is a biomarker to predict the outcome for patients with WAS.

The lack of either IRF9, or STAT2, has surprisingly little effect on human natural killer cell development and function.

Analysis of genetically defined immunodeficient patients allows study of the effect of the absence of specific proteins on human immune function in real-world conditions. Here we have addressed the importance of type I interferon signalling for human NK cell development by studying the phenotype and function of circulating NK cells isolated from patients suffering primary immunodeficiency disease due to mutation of either the human interferon regulatory factor 9 (IRF9) or the signal transducer and activator of transcription 2 (STAT2) genes. IRF9, together with phosphorylated STAT1 and STAT2, form a heterotrimer called interferon stimulated gene factor 3 (ISGF3) which promotes the expression of hundreds of IFN-stimulated genes that mediate antiviral function triggered by exposure to type I interferons. IRF9- and STAT2-deficient patients are unable to respond efficiently to stimulation by type I interferons and so our experiments provide insights into the importance of type I interferon signalling and the consequences of its impairment on human NK cell biology. Surprisingly, the NK cells of these patients display essentially normal phenotype and function.

A Validated Highly Sensitive Microsatellite Instability Assay Accurately Identifies Individuals Harboring Biallelic Germline PMS2 Pathogenic Variants in Constitutional Mismatch Repair Deficiency.

BACKGROUND: Constitutional mismatch repair deficiency (CMMRD) is a rare and extraordinarily penetrant childhood-onset cancer predisposition syndrome. Genetic diagnosis is often hampered by the identification of mismatch repair (MMR) variants of unknown significance and difficulties in PMS2 analysis, the most frequently mutated gene in CMMRD. We present the validation of a robust functional tool for CMMRD diagnosis and the characterization of microsatellite instability (MSI) patterns in blood and tumors. METHODS: The highly sensitive assessment of MSI (hs-MSI) was tested on a blinded cohort of 66 blood samples and 24 CMMRD tumor samples. Hs-MSI scores were compared with low-pass genomic instability scores (LOGIC/MMRDness). The correlation of hs-MSI scores in blood with age of cancer onset and the distribution of insertion-deletion (indel) variants in microsatellites were analyzed in a series of 169 individuals (n = 68 CMMRD, n = 124 non-CMMRD). RESULTS: Hs-MSI achieved high accuracy in the identification of CMMRD in blood (sensitivity 98.5% and specificity 100%) and detected MSI in CMMRD-associated tumors. Hs-MSI had a strong positive correlation with whole low-pass genomic instability LOGIC scores (r = 0.89, P = 2.2e-15 in blood and r = 0.82, P = 7e-3 in tumors). Indel distribution identified PMS2 pathogenic variant (PV) carriers from other biallelic MMR gene PV carriers with an accuracy of 0.997. Higher hs-MSI scores correlated with younger age at diagnosis of the first tumor (r = -0.43, P = 0.011). CONCLUSIONS: Our study confirms the accuracy of the hs-MSI assay as ancillary testing for CMMRD diagnosis, which can also characterize MSI patterns in CMMRD-associated cancers. Hs-MSI is a powerful tool to pinpoint PMS2 as the affected germline gene and thus potentially personalize cancer risk.

Enrichment of Immune Dysregulation Disorders in Adult Patients with Human Inborn Errors of Immunity.

Human inborn errors of immunity (IEI) comprise a group of diseases resulting from molecular variants that compromise innate and adaptive immunity. Clinical features of IEI patients are dominated by susceptibility to a spectrum of infectious diseases, as well as autoimmune, autoinflammatory, allergic, and malignant phenotypes that usually appear in childhood, which is when the diagnosis is typically made. However, some IEI patients are identified in adulthood due to symptomatic delay of the disease or other reasons that prevent the request for a molecular study. The application of next-generation sequencing (NGS) as a diagnostic technique has given rise to an ever-increasing identification of IEI-monogenic causes, thus improving the diagnostic yield and facilitating the possibility of personalized treatment. This work was a retrospective study of 173 adults with IEI suspicion that were sequenced between 2005 and 2023. Sanger, targeted gene-panel, and whole exome sequencing were used for molecular diagnosis. Disease-causing variants were identified in 44 of 173 (25.43%) patients. The clinical phenotype of these 44 patients was mostly related to infection susceptibility (63.64%). An enrichment of immune dysregulation diseases was found when cohorts with molecular diagnosis were compared to those without. Immune dysregulation disorders, group 4 from the International Union of Immunological Societies Expert Committee (IUIS), were the most prevalent among these adult patients. Immune dysregulation as a new item in the Jeffrey Model Foundation warning signs for adults significantly increases the sensitivity for the identification of patients with an IEI-producing molecular defect.

Non-apoptotic FAS signaling controls mTOR activation and extrafollicular maturation in human B cells.

Defective FAS (CD95/Apo-1/TNFRSF6) signaling causes autoimmune lymphoproliferative syndrome (ALPS). Hypergammaglobulinemia is a common feature in ALPS with FAS mutations (ALPS-FAS), but paradoxically, fewer conventional memory cells differentiate from FAS-expressing germinal center (GC) B cells. Resistance to FAS-induced apoptosis does not explain this phenotype. We tested the hypothesis that defective non-apoptotic FAS signaling may contribute to impaired B cell differentiation in ALPS. We analyzed secondary lymphoid organs of patients with ALPS-FAS and found low numbers of memory B cells, fewer GC B cells, and an expanded extrafollicular (EF) B cell response. Enhanced mTOR activity has been shown to favor EF versus GC fate decision, and we found enhanced PI3K/mTOR and BCR signaling in ALPS-FAS splenic B cells. Modeling initial T-dependent B cell activation with CD40L in vitro, we showed that FAS competent cells with transient FAS ligation showed specifically decreased mTOR axis activation without apoptosis. Mechanistically, transient FAS engagement with involvement of caspase-8 induced nuclear exclusion of PTEN, leading to mTOR inhibition. In addition, FASL-dependent PTEN nuclear exclusion and mTOR modulation were defective in patients with ALPS-FAS. In the early phase of activation, FAS stimulation promoted expression of genes related to GC initiation at the expense of processes related to the EF response. Hence, our data suggest that non-apoptotic FAS signaling acts as molecular switch between EF versus GC fate decisions via regulation of the mTOR axis and transcription. The defect of this modulatory circuit may explain the observed hypergammaglobulinemia and low memory B cell numbers in ALPS.

JAK inhibitor treatment for inborn errors of JAK/STAT signaling: An ESID/EBMT-IEWP retrospective study.

BACKGROUND: Inborn errors of immunity (IEI) with dysregulated JAK/STAT signaling present with variable manifestations of immune dysregulation and infections. Hematopoietic stem cell transplantation (HSCT) is potentially curative, but initially reported outcomes were poor. JAK inhibitors (JAKi) offer a targeted treatment option that may be an alternative or bridge to HSCT. However, data on their current use, treatment efficacy and adverse events are limited. OBJECTIVE: We evaluated the current off-label JAKi treatment experience for JAK/STAT inborn errors of immunity (IEI) among European Society for Immunodeficiencies (ESID)/European Society for Blood and Marrow Transplantation (EBMT) Inborn Errors Working Party (IEWP) centers. METHODS: We conducted a multicenter retrospective study on patients with a genetic disorder of hyperactive JAK/STAT signaling who received JAKi treatment for at least 3 months. RESULTS: Sixty-nine patients (72% children) were evaluated (45 STAT1 gain of function [GOF], 21 STAT3-GOF, 1 STAT5B-GOF, 1 suppressor of cytokine signaling 1 [aka SOCS1] loss of function, 1 JAK1-GOF). Ruxolitinib was the predominantly prescribed JAKi (80%). Overall, treatment resulted in improvement (partial or complete remission) of clinical symptoms in 87% of STAT1-GOF and in 90% of STAT3-GOF patients. We documented highly heterogeneous dosing and monitoring regimens. The response rate and time to response varied across different diseases and manifestations. Adverse events including infection and weight gain were frequent (38% of patients) but were mild (grade I-II) and transient in most patients. At last follow-up, 52 (74%) of 69 patients were still receiving JAKi treatment, and 11 patients eventually underwent HSCT after receipt of previous JAKi bridging therapy, with 91% overall survival. CONCLUSIONS: Our study suggests that JAKi may be highly effective to treat symptomatic JAK/STAT IEI patients. Prospective studies to define optimal JAKi dosing for the variable clinical presentations and age ranges should be pursued.

Unbiased assessment of genome integrity and purging of adverse outcomes at the target locus upon editing of CD4+ T-cells for the treatment of Hyper IgM1.

Hyper IgM1 is an X-linked combined immunodeficiency caused by CD40LG mutations, potentially treatable with CD4+ T-cell gene editing with Cas9 and a "one-size-fits-most" corrective template. Contrary to established gene therapies, there is limited data on the genomic alterations following long-range gene editing, and no consensus on the relevant assays. We developed drop-off digital PCR assays for unbiased detection of large on-target deletions and found them at high frequency upon editing. Large deletions were also common upon editing different loci and cell types and using alternative Cas9 and template delivery methods. In CD40LG edited T cells, on-target deletions were counter-selected in culture and further purged by enrichment for edited cells using a selector coupled to gene correction. We then validated the sensitivity of optical genome mapping for unbiased detection of genome wide rearrangements and uncovered on-target trapping of one or more vector copies, which do not compromise functionality, upon editing using an integrase defective lentiviral donor template. No other recurring events were detected. Edited patient cells showed faithful reconstitution of CD40LG regulated expression and function with a satisfactory safety profile. Large deletions and donor template integrations should be anticipated and accounted for when designing and testing similar gene editing strategies.

Scalable GMP-compliant gene correction of CD4+ T cells with IDLV template functionally validated in vitro and in vivo.

Hyper-IgM1 is a rare X-linked combined immunodeficiency caused by mutations in the CD40 ligand (CD40LG) gene with a median survival of 25 years, potentially treatable with in situ CD4+ T cell gene editing with Cas9 and a one-size-fits-most corrective donor template. Here, starting from our research-grade editing protocol, we pursued the development of a good manufacturing practice (GMP)-compliant, scalable process that allows for correction, selection and expansion of edited cells, using an integrase defective lentiviral vector as donor template. After systematic optimization of reagents and conditions we proved maintenance of stem and central memory phenotypes and expression and function of CD40LG in edited healthy donor and patient cells recapitulating the physiological CD40LG regulation. We then documented the preserved fitness of edited cells by xenotransplantation into immunodeficient mice. Finally, we transitioned to large-scale manufacturing, and developed a panel of quality control assays. Overall, our GMP-compliant process takes long-range gene editing one step closer to clinical application with a reassuring safety profile.

TCF3 haploinsufficiency defined by immune, clinical, gene-dosage, and murine studies.

BACKGROUND: TCF3 is a transcription factor contributing to early lymphocyte differentiation. Germline monoallelic dominant negative and biallelic loss-of-function (LOF) null TCF3 mutations cause a fully penetrant severe immunodeficiency. We identified 8 individuals from 7 unrelated families with monoallelic LOF TCF3 variants presenting with immunodeficiency with incomplete clinical penetrance. OBJECTIVE: We sought to define TCF3 haploinsufficiency (HI) biology and its association with immunodeficiency. METHODS: Patient clinical data and blood samples were analyzed. Flow cytometry, Western blot analysis, plasmablast differentiation, immunoglobulin secretion, and transcriptional activity studies were conducted on individuals carrying TCF3 variants. Mice with a heterozygous Tcf3 deletion were analyzed for lymphocyte development and phenotyping. RESULTS: Individuals carrying monoallelic LOF TCF3 variants showed B-cell defects (eg, reduced total, class-switched memory, and/or plasmablasts) and reduced serum immunoglobulin levels; most but not all presented with recurrent but nonsevere infections. These TCF3 LOF variants were either not transcribed or translated, resulting in reduced wild-type TCF3 protein expression, strongly suggesting HI pathophysiology for the disease. Targeted RNA sequencing analysis of T-cell blasts from TCF3-null, dominant negative, or HI individuals clustered away from healthy donors, implying that 2 WT copies of TCF3 are needed to sustain a tightly regulated TCF3 gene-dosage effect. Murine TCF3 HI resulted in a reduction of circulating B cells but overall normal humoral immune responses. CONCLUSION: Monoallelic LOF TCF3 mutations cause a gene-dosage-dependent reduction in wild-type protein expression, B-cell defects, and a dysregulated transcriptome, resulting in immunodeficiency. Tcf3+/- mice partially recapitulate the human phenotype, underscoring the differences between TCF3 in humans and mice.

Dominant-negative heterozygous mutations in AIRE confer diverse autoimmune phenotypes.

Autoimmune polyendocrine syndrome type 1 (APS-1) is an autosomal recessive disease characterized by severe and childhood onset organ-specific autoimmunity caused by mutations in the autoimmune regulator (AIRE) gene. More recently, dominant-negative mutations within the PHD1, PHD2, and SAND domains have been associated with an incompletely penetrant milder phenotype with later onset familial clustering, often masquerading as organ-specific autoimmunity. Patients with immunodeficiencies or autoimmunity where genetic analyses revealed heterozygous AIRE mutations were included in the study and the dominant-negative effects of the AIRE mutations were functionally assessed in vitro. We here report additional families with phenotypes ranging from immunodeficiency, enteropathy, and vitiligo to asymptomatic carrier status. APS-1-specific autoantibodies can hint to the presence of these pathogenic AIRE variants although their absence does not rule out their presence. Our findings suggest functional studies of heterozygous AIRE variants and close follow-up of identified individuals and their families.

Inborn Error of STAT2-Dependent IFN-I Immunity in a Patient Presented with Hemophagocytic Lymphohistiocytosis and Multisystem Inflammatory Syndrome in Children.

Human inborn errors of immunity (IEI) affecting the type I interferon (IFN-I) induction pathway have been associated with predisposition to severe viral infections. Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening systemic hyperinflammatory syndrome that has been increasingly associated with inborn errors of IFN-I-mediated innate immunity. Here is reported a novel case of complete deficiency of STAT2 in a 3-year-old child that presented with typical features of HLH after mumps, measles, and rubella vaccination at the age of 12 months. Due to the life-threatening risk of viral infection, she received SARS-CoV-2 mRNA vaccination. Unfortunately, she developed multisystem inflammatory syndrome in children (MIS-C) after SARS-CoV-2 infection, 4 months after the last dose. Functional studies showed an impaired IFN-I-induced response and a defective IFNα expression at later stages of STAT2 pathway induction. These results suggest a possible more complex mechanism for hyperinflammatory reactions in this type of patients involving a possible defect in the IFN-I production. Understanding the cellular and molecular links between IFN-I-induced signaling and hyperinflammatory syndromes can be critical for the diagnosis and tailored management of these patients with predisposition to severe viral infection.

Revisiting autoimmune lymphoproliferative syndrome caused by Fas ligand mutations.

BACKGROUND: Fas ligand (FasL) is expressed by activated T cells and induces death in target cells upon binding to Fas. Loss-of-function FAS or FASLG mutations cause autoimmune-lymphoproliferative syndrome (ALPS) characterized by expanded double-negative T cells (DNT) and elevated serum biomarkers. While most ALPS patients carry heterozygous FAS mutations, FASLG mutations are rare and usually biallelic. Only 2 heterozygous variants were reported, associated with an atypical clinical phenotype. OBJECTIVE: We revisited the significance of heterozygous FASLG mutations as a cause of ALPS. METHODS: Clinical features and biomarkers were analyzed in 24 individuals with homozygous or heterozygous FASLG variants predicted to be deleterious. Cytotoxicity assays were performed with patient T cells and biochemical assays with recombinant FasL. RESULTS: Homozygous FASLG variants abrogated cytotoxicity and resulted in early-onset severe ALPS with elevated DNT, raised vitamin B12, and usually no soluble FasL. In contrast, heterozygous variants affected FasL function by reducing expression, impairing trimerization, or preventing Fas binding. However, they were not associated with elevated DNT and vitamin B12, and they did not affect FasL-mediated cytotoxicity. The dominant-negative effects of previously published variants could not be confirmed. Even Y166C, causing loss of Fas binding with a dominant-negative effect in biochemical assays, did not impair cellular cytotoxicity or cause vitamin B12 and DNT elevation. CONCLUSION: Heterozygous loss-of-function mutations are better tolerated for FASLG than for FAS, which may explain the low frequency of ALPS-FASLG.

Case report: Novel treatment regimen for enterovirus encephalitis in SCID.

Most non-polio enterovirus infections in immunocompetent individuals are acute and self-limiting in nature; however, infection can be severe, chronic and have devastating outcomes in immunocompromised hosts. Therapeutic strategies have predominantly involved supportive care, with the lack of approved antiviral treatments proving challenging for management. We report a case of an 8-month-old child who presented with severe enterovirus encephalitis following gene therapy for X-linked severe combined immunodeficiency (X-SCID) and who demonstrated clinical and microbiological improvement after a novel regimen of favipiravir, fluoxetine, and high-dose intravenous immunoglobulin (IVIg). The patient presented 6 weeks post-gene therapy with rapid neurological deterioration in the context of incomplete immune reconstitution, with microbiological and radiological evidence confirming enterovirus encephalitis. His neurologic examination stabilised 8 weeks after treatment, and he subsequently demonstrated excellent immune recovery. This is the first case report of combined therapy with favipiravir, fluoxetine, and high-dose IVIg in the context of severe enterovirus encephalitis in an immunocompromised host. This case highlights the importance of considering enterovirus encephalitis in immunocompromised patients presenting with both acute and chronic neurological signs, as well as developmental regression. The demonstrated treatment success and the associated low risk of toxicity warrant further investigation of this therapeutic regimen.

Autoantibodies against type I IFNs in patients with critical influenza pneumonia.

Autoantibodies neutralizing type I interferons (IFNs) can underlie critical COVID-19 pneumonia and yellow fever vaccine disease. We report here on 13 patients harboring autoantibodies neutralizing IFN-α2 alone (five patients) or with IFN-ω (eight patients) from a cohort of 279 patients (4.7%) aged 6-73 yr with critical influenza pneumonia. Nine and four patients had antibodies neutralizing high and low concentrations, respectively, of IFN-α2, and six and two patients had antibodies neutralizing high and low concentrations, respectively, of IFN-ω. The patients' autoantibodies increased influenza A virus replication in both A549 cells and reconstituted human airway epithelia. The prevalence of these antibodies was significantly higher than that in the general population for patients <70 yr of age (5.7 vs. 1.1%, P = 2.2 × 10-5), but not >70 yr of age (3.1 vs. 4.4%, P = 0.68). The risk of critical influenza was highest in patients with antibodies neutralizing high concentrations of both IFN-α2 and IFN-ω (OR = 11.7, P = 1.3 × 10-5), especially those <70 yr old (OR = 139.9, P = 3.1 × 10-10). We also identified 10 patients in additional influenza patient cohorts. Autoantibodies neutralizing type I IFNs account for ∼5% of cases of life-threatening influenza pneumonia in patients <70 yr old.

Regulatory B cells in patients suffering from inborn errors of immunity with severe immune dysregulation.

BACKGROUND: Immune dysregulation as a result of an inborn error of immunity (IEI) leads to the complicated symptoms of refractory multi-organ immune dysregulation. B lymphocytes with immune regulatory capacity (Breg) are activated by environmental triggers and act as regulators of the immune response as observed in several autoimmune diseases. OBJECTIVE: We sought to investigate the Breg profile and the CD21low expressing B cells of patients with LRBA deficiency (N = 6) and non-LRBA deficiency IEI (N = 13) with overlapping clinical symptoms of immune dysregulation. Normal values for Breg subpopulations were obtained from patients age-matched healthy cohorts (N = 48). Furthermore, we investigated the impact of abatacept treatment in LRBA deficient patients receiving biweekly abatacept (N = 5). METHODS: Using a flow cytometric approach with a pre-formulated antibody panel in peripheral blood samples, Breg subsets including plasmablasts (CD27+CD38hi), transitional B cells (CD24hiCD38hi), and B10 cells (CD24hiCD27+), and additionally the CD21low B cells (CD21lowCD38low) were analyzed. Breg function was assessed by the interleukin-10 expression within the CD19+ population. Additionally, B cell cytokines were measured in cell culture supernatants. RESULTS: We observe significant alterations of B cell/Breg subpopulations in the LRBA deficient cohort including a severe lack of memory B cells (P = 0.031) and B10 cells (P = 0.031) as well as a tendency towards higher CD21low B cells (P = 0.063). Within the non-LRBA deficient cohort, we observe a significant expansion of the plasmablasts (P = 0.012), and a tendency towards elevated levels of CD21low expressing B cells (P = 0.063). The treatment with abatacept ameliorated disease symptoms in the LRBA deficient cohort and led to an effective decrease in CD21low B cells over time (P = 0.021). Furthermore, there was a significantly increased level of B cell-activating factor (BAFF; P = 0.02) and lower IL-12p70 secretion upon stimulation (P = 0.020) in the LRBA cohort. CONCLUSION: Aberrant maturation of Breg subsets and the pathological expansion of CD21low B cells in patients with IEI may have therapeutic implications. Patients suffering from LRBA deficiency show a lack of memory B cells, insufficient expansion of B10 cells, increased BAFF levels as well as an increase in circulating CD21low B cells. Abatacept treatment results in a steady decrease in CD21low B cells.

Impact of cytomegalovirus infection prior to hematopoietic stem cell transplantation in children with inborn errors of immunity.

The presence of active viral infections has an impact on the prognosis of patients undergoing hematopoietic stem cell transplantation (HSCT). Nevertheless, the number of reports of cytomegalovirus infection in patients with inborn errors of immunity (IEI) who undergo HSCT is relatively low. To analyze the effect of cytomegalovirus infection acquired prior to curative treatment on patient survival in 123 children with IEI. An observational and retrospective study was performed with patients younger than 18 years diagnosed with IEI who were candidates for HSCT, gene therapy, or thymus transplantation at five hospitals in Spain between 2008 and 2019. We included 123 children, 25 infected by cytomegalovirus prior to undergoing curative treatment (20.3%). At IEI diagnosis, 24 of the patients were already infected, 21 of whom had symptomatic cytomegalovirus disease (87%), while the other three patients developed disease before undergoing curative treatment. The patients with cytomegalovirus infection had higher mortality than those without (p = 0.006). Fourteen patients developed refractory cytomegalovirus infection (56%), all of whom died, while no patients with non-refractory infection died (p = 0.001) All deaths that occurred before curative treatment and three of the five after the treatment were attributed to cytomegalovirus. Patients with refractory cytomegalovirus disease had the highest pre-HSCT mortality rate (64.3%), compared with the non-infected children and those with non-refractory cytomegalovirus disease (10.1%) (p < 0.0001). CONCLUSION: Prevention and prompt control of cytomegalovirus infection, together with early HSCT/gene therapy, are crucial for improving the prognosis in children with IEI. WHAT IS KNOWN: • Cytomegalovirus is the most frequent viral infection in children with inborn errors of immunity who are candidates to hematopoietic stem cell transplantation (HSCT). • Active viral infections at the time of HSCT lead to worse prognosis. WHAT IS NEW: • In children with inborn errors of immunity and indication of HSCT, refractory cytomegalovirus disease is associated with a very high mortality rate, compared with non-infected children and those with non-refractory cytomegalovirus disease. • In patients with novel transplantation indications, the presence and treatment response of CMV infection should be considered to decide the best possible moment for HSCT.

Disease Progression of WHIM Syndrome in an International Cohort of 66 Pediatric and Adult Patients.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome (WS) is a combined immunodeficiency caused by gain-of-function mutations in the C-X-C chemokine receptor type 4 (CXCR4) gene. We characterize a unique international cohort of 66 patients, including 57 (86%) cases previously unreported, with variable clinical phenotypes. Of 17 distinct CXCR4 genetic variants within our cohort, 11 were novel pathogenic variants affecting 15 individuals (23%). All variants affect the same CXCR4 region and impair CXCR4 internalization resulting in hyperactive signaling. The median age of diagnosis in our cohort (5.5 years) indicates WHIM syndrome can commonly present in childhood, although some patients are not diagnosed until adulthood. The prevalence and mean age of recognition and/or onset of clinical manifestations within our cohort were infections 88%/1.6 years, neutropenia 98%/3.8 years, lymphopenia 88%/5.0 years, and warts 40%/12.1 years. However, we report greater prevalence and variety of autoimmune complications of WHIM syndrome (21.2%) than reported previously. Patients with versus without family history of WHIM syndrome were diagnosed earlier (22%, average age 1.3 years versus 78%, average age 5 years, respectively). Patients with a family history of WHIM syndrome also received earlier treatment, experienced less hospitalization, and had less end-organ damage. This observation reinforces previous reports that early treatment for WHIM syndrome improves outcomes. Only one patient died; death was attributed to complications of hematopoietic stem cell transplantation. The variable expressivity of WHIM syndrome in pediatric patients delays their diagnosis and therapy. Early-onset bacterial infections with severe neutropenia and/or lymphopenia should prompt genetic testing for WHIM syndrome, even in the absence of warts.

Respiratory syncytial virus infections requiring hospitalization in patients with primary immunodeficiency.

INTRODUCTION: The aim of the study was to assess the incidence of hospital admission due to severe acute respiratory infection by respiratory syncytial virus (RSV-ARI) in children with primary immunodeficiencies (PIDs) and the severity of RSV-ARI in these patients. METHODS: We conducted a nationwide cross-sectional retrospective and prospective multicentre study in the 2011-2017 period. The study was performed in 15 Spanish hospitals and included children with PID who required hospital admission due to RSV-ARI. RESULTS: Out of 439 patients with PID followed up at participating hospitals, 13 (3%) required hospital admission due to RSV-ARI. The median age of admitted patients was 1.6 years (interquartile range, 0.5-2.2), and 7 were male. The types of PID most frequently associated with admission due to RSV-ARI were combined immunodeficiency (CID; 4/71; 6%) and CID with associated or syndromic features (CIDwASF; 6/147; 4%). Two of the 13 patients were receiving palivizumab for RSV prophylaxis, and 3 received potentially active therapies against RSV during the hospital stay. Viral coinfection was detected in 6 patients, 5 (39%) developed complications, and 4 (31%) required admission to the paediatric intensive care unit. There were no documented RSV-related deaths. CONCLUSIONS: In the group of patients with PID, severe RSV infection requiring hospitalization is more frequent in patients with CID and CIDwASF, in whom special efforts should be made to prevent RSV infection. Further studies are needed to confirm these results.

Altered CXCR4 dynamics at the cell membrane impairs directed cell migration in WHIM syndrome patients.

Chemokine receptor nanoscale organization at the cell membrane is orchestrated by the actin cytoskeleton and influences cell responses. Using single-particle tracking analysis we show that CXCR4R334X, a truncated mutant chemokine receptor linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis), fails to nanoclusterize after CXCL12 stimulation, and alters the lateral mobility and spatial organization of CXCR4 when coexpressed. These findings correlate with multiple phalloidin-positive protrusions in cells expressing CXCR4R334X, and their inability to correctly sense chemokine gradients. The underlying mechanisms involve inappropriate actin cytoskeleton remodeling due to the inadequate ß-arrestin1 activation by CXCR4R334X, which disrupts the equilibrium between activated and deactivated cofilin. Overall, we provide insights into the molecular mechanisms governing CXCR4 nanoclustering, signaling and cell function, and highlight the essential scaffold role of ß-arrestin1 to support CXCL12-mediated actin reorganization and receptor clustering. These defects associated with CXCR4R334X expression might contribute to the severe immunological symptoms associated with WHIM syndrome.