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Abstract RAG2-SCID is a primary immunodeficiency caused by mutations in Recombination-activating gene 2 (RAG2), a gene intimately involved in the process of lymphocyte maturation and function. ex-vivo manipulation of a patient’s own hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9/rAAV6 gene editing could provide a therapeutic alternative to the only current treatment, allogeneic hematopoietic stem cell transplantation (HSCT). Here we show an innovative RAG2 correction strategy that replaces the entire endogenous coding sequence (CDS) for the purpose of preserving the critical endogenous spatiotemporal gene regulation and locus architecture. Expression of the corrective transgene leads to successful development into CD3+TCRαβ+ and CD3+TCRγδ+ T cells and promotes the establishment of highly diverse TRB and TRG repertoires in an in-vitro T-cell differentiation platform. Thus, our proof-of-concept study holds promise for safer gene therapy techniques of tightly regulated genes.

Abstract We describe the first cases of germline biallelic null mutations in ARPC5, part of the Arp2/3 actin nucleator complex, in two unrelated patients presenting with recurrent and severe infections, early-onset autoimmunity, inflammation, and dysmorphisms. This defect compromises multiple cell lineages and functions, and when protein expression is reestablished in-vitro, the Arp2/3 complex conformation and functions are rescued. As part of the pathophysiological evaluation, we also show that interleukin (IL)−6 signaling is distinctively impacted in this syndrome. Disruption of IL-6 classical but not trans-signaling highlights their differential roles in the disease and offers perspectives for therapeutic molecular targets.

Severe combined immunodeficiency (SCID) is a group of disorders caused by mutations in genes involved in the process of lymphocyte maturation and function. CRISPR-Cas9 gene editing of the patient’s own hematopoietic stem and progenitor cells (HSPCs) ex vivo could provide a therapeutic alternative to allogeneic hematopoietic stem cell transplantation, the current gold standard for treatment of SCID. To eliminate the need for scarce patient samples, we engineered genotypes in healthy donor (HD)-derived CD34+ HSPCs using CRISPR-Cas9/rAAV6 gene-editing, to model both SCID and the therapeutic outcomes of gene-editing therapies for SCID via multiplexed homology-directed repair (HDR). First, we developed a SCID disease model via biallelic knockout of genes critical to the development of lymphocytes; and second, we established a knockin/knockout strategy to develop a proof-of-concept single-allelic gene correction. Based on these results, we performed gene correction of RAG2-SCID patient-derived CD34+ HSPCs that successfully developed into CD3+ T cells with diverse TCR repertoires in an in vitro T cell differentiation platform. In summary, we present a strategy to determine the optimal configuration for CRISPR-Cas9 gene correction of SCID using HD-derived CD34+ HSPCs, and the feasibility of translating this gene correction approach in patient-derived CD34+ HSPCs.

PurposePatients with X-linked agammaglobulinemia (XLA) are characterized by humoral impairment and are routinely treated with intravenous immunoglobulin (IVIG). In this study, we aimed to investigate the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in IVIG preparations harvested globally and evaluate the transfer of SARS-CoV-2 antibodies to the XLA patient.MethodsA single-center, prospective cohort study was conducted in the period of November 2020 to November 2022. Clinical and laboratory data, specifically, SARS-CoV-2 spike IgG levels from the serum of 115 IVIG preparations given to 5 XLA patient were collected. Concurrently, SARS-CoV-2 spike IgG levels from the serum of the 5 XLA was collected monthly.ResultsFive XLA patients were evaluated within the study period. All were treated monthly with commercial IVIG preparations. A total of 115 IVIG treatments were given over the study period. The origin country and the date of IVIG harvesting was obtained for 111 (96%) of the treatments. Fifty-four IVIG preparations (49%) were harvested during the COVID-19 pandemic of which 76% were positive (>50AU/mL) for SARS-CoV-2 spike antibodies which were subsequently transmitted to the XLA patients in an approximate 10-fold reduction. SARS-CoV2 spike IgG was first detected in IVIG batches that completed their harvest date by September 2021. Positive products were harvested from origin countries with a documented prevalence over 2,000 per 100,000 population.ConclusionAs the prevalence of COVID-19 infections rises, detection of SARS-CoV-2 spike IgG in commercial IVIG products increases and is then transmitted to the patient. Future studies are needed to investigate the neutralizing capabilities of SARS-CoV-2 IgG and whether titer levels in IVIG remain consistent as the incidence of infection and vaccination rates in the population changes.

BackgroundPatients with primary immunodeficiency disorders (PIDs) often suffer from recurrent infections because of their inappropriate immune response to both common and less common pathogens. These patients may present with unique and severe cutaneous infectious manifestations that are not common in healthy individuals and may be more challenging to diagnose and treat.ObjectiveTo describe a cohort of patients with PIDs with atypical presentations of skin infections, who posed a diagnostic and/or therapeutic challenge.MethodsThis is a retrospective study of pediatric patients with PID with atypical presentations of infections, who were treated at the immunodeficiency specialty clinic and the pediatric dermatology clinic at the Sheba Medical Center between September 2012 and August 2022. Epidemiologic data, PID diagnosis, infectious etiology, presentation, course, and treatment were recorded.ResultsEight children with a diagnosis of PID were included, five of whom were boys. The average age at PID diagnosis was 1.7 (±SD 3.2) years. The average age of cutaneous infection was 6.9 (±SD 5.9) years. Three patients were born to consanguineous parents. The PIDs included the following: common variable immunodeficiency, severe combined immunodeficiency, DOCK8 deficiency, ataxia telangiectasia, CARD11 deficiency, MALT1 deficiency, chronic granulomatous disease, and a combined cellular and humoral immunodeficiency syndrome of unknown etiology. The infections included the following: ulcerative-hemorrhagic varicella-zoster virus (two cases) atypical fungal and bacterial infections, resistant Norwegian scabies, giant perianal verrucae (two cases), and diffuse molluscum contagiosum.ConclusionsIn this case series, we present unusual manifestations of infectious skin diseases in pediatric patients with PID. In some of the cases, recognition of the infectious process prompted life-saving treatment. Increasing familiarity with these dermatological manifestations, as well as keeping a high index of suspicion, is important to enabling early diagnosis of cutaneous infections in PIDs and initiation of prompt suitable treatment.

BackgroundAutosomal dominant hyper-IgE syndrome (AD-HIES) caused by dominant negative (DN) variants in the signal transducer and activator of transcription 3 gene (STAT3) is characterized by recurrent Staphylococcal abscesses, severe eczema, chronic mucocutaneous candidiasis (CMC), and non-immunological facial and skeletal features.ObjectivesTo describe our experience with the diagnosis and treatment of adult patients with AD-HIES induced by DN-STAT3 variants.MethodsThe medical records of adult patients (>18 years) treated at the Allergy and Clinical Immunology Clinic of Hadassah Medical Center, Jerusalem, Israel, were retrospectively analyzed. Immune and genetic workups were used to confirm diagnosis.ResultsThree adult patients (2 males; age 29-41 years) were diagnosed with DN-STAT3 variants. All patients had non-immunological features, including coarse faces and osteopenia. Serious bacterial infections were noted in all patients, including recurrent abscesses, recurrent pneumonia, and bronchiectasis. CMC and diffuse dermatophytosis were noted in two patients. Two patients had severe atopic dermatitis refractory to topical steroids and phototherapy. Immune workup revealed elevated IgE in three patients and eosinophilia in two patients. Whole exome sequencing revealed DN-STAT3 variants (c.1166C>T; p.Thr389Ile in two patients and c.1268G>A; p. Arg423Gln in one patient). Variants were located in DNA-binding domain (DBD) and did not hamper STAT3 phosphorylation Treatment included antimicrobial prophylaxis with trimethoprim/sulfamethoxazole (n=2) and amoxycillin-clavulanic acid (n=1), and anti-fungal treatment with fluconazole (n=2) and voriconazole (n=1). Two patients who had severe atopic dermatitis, were treated with dupilumab with complete resolution of their rash. No adverse responses were noted in the dupilumab-treated patients.DiscussionDupilumab can be used safely as a biotherapy for atopic dermatitis in these patients as it can effectively alleviate eczema-related symptoms. Immunologists and dermatologists treating AD-HIES adult patients should be aware of demodicosis as a possible manifestation. DN-STAT3 variants in DBD do not hamper STAT3 phosphorylation.

PurposeReceptor-interacting serine/threonine-protein kinase 1 (RIPK1) is an important regulator of necroptosis and inflammatory responses. We present the clinical features, genetic analysis and immune work-up of two patients with infantile-onset inflammatory bowel disease (IBD) resulting from RIPK1 mutations.MethodsWhole exome and Sanger sequencing was performed in two IBD patients. Mass cytometry time of flight (CyTOF) was conducted for in-depth immunophenotyping on one of the patient’s peripheral blood mononuclear cells, and compared to control subjects and patients with Crohn’s disease.ResultsThe patients presented with severe colitis and perianal fistulas in the first months of life, without severe/atypical infections. Genetic studies identified pathogenic genetic variants in RIPK1 (Patient 1, A c.1934C>T missense mutation in Exon 11; Patient 2, c.580G>A missense mutation residing in Exon 4). Protein modeling demonstrated that the mutation in Patient 1 displaces a water molecule, potentially disrupting the local environment, and the mutation in Patient 2 may lead to disruption of the packing and conformation of the kinase domain. Immunofluorescence RIPK1 staining in rectal biopsies demonstrated no expression for Patient 1 and minimal expression for Patient 2, compared to controls and patients with active Crohn’s disease. Using CyTOF unbiased clustering analysis, we identified peripheral immune dysregulation in one of these patients, characterized by an increase in IFNγ CD8+ T cells along with a decrease in monocytes, dendritic cells and B cells. Moreover, RIPK1-deficient patient’s immune cells exhibited decreased IL-6 production in response to lipopolysaccharide (LPS) across multiple cell types including T cells, B cells and innate immune cells.ConclusionsMutations in RIPK1 should be considered in very young patients presenting with colitis and perianal fistulas. Given RIPK1’s role in inflammasome activation, but also in epithelial cells, it is unclear whether IL1 blockade or allogeneic hematopoietic stem cell transplantation can suppress or cure the hyper-inflammatory response in these patients. Additional studies in humans are required to better define the role of RIPK1 in regulating intestinal immune responses, and how treatment can be optimized for patients with RIPK1 deficiency.

Background and aimAnti-TNFα is measurable in infants exposed in utero up to 12 months of age. Data about the exposure effect on the infant’s adaptive immunity are limited. We aimed to prospectively evaluate the distribution and function of T and B cells, in infants of females with inflammatory bowel disease, in utero exposed to anti-TNFα or azathioprine.MethodsA prospective multi-center study conducted 2014–2017. Anti-TNFα levels were measured in cord blood, and at 3 and 12 months. T-cell repertoire and function were analyzed at 3 and 12 months by flow-cytometry, expression of diverse T cell receptors (TCR) and T-cell receptor excision circles (TREC) quantification assay. Serum immunoglobulins and antibodies for inactivated vaccines were measured at 12 months. Baseline clinical data were retrieved, and 2-monthly telephonic interviews were performed regarding child infections and growth.Results24 pregnant females, age 30.6 (IQR 26.5–34.5) years were recruited, 20 with anti-TNFα (infliximab 8, adalimumab 12), and 4 with azathioprine treatment. Cord blood anti-TNFα was higher than maternal blood levels [4.3 (IQR 2.3–9.2) vs. 2.5 (IQR 1.3–9.7) mcg/ml], declining at 3 and 12 months. All infants had normal number of B-cells (n = 17), adequate levels of immunoglobulins (n = 14), and protecting antibody levels to Tetanus, Diphtheria, Hemophilus influenza-B and hepatitis B (n = 17). All had normal CD4+, CD8+ T-cells, and TREC numbers. TCR repertoire was polyclonal in 18/20 and slightly skewed in 2/20 infants. No serious infections requiring hospitalization were recorded.ConclusionWe found that T-cell and B-cell immunity is fully mature and immune function is normal in infants exposed in utero to anti-TNFα, as in those exposed to azathioprine. Untreated controls and large-scale studies are needed to confirm these results.

BackgroundDuring the process of generating diverse T and B cell receptor (TCR and BCR, respectively) repertoires, double-strand DNA breaks are produced. Subsequently, these breaks are corrected by a complex system led by the non-homologous end-joining (NHEJ). Pathogenic variants in genes involved in this process, such as the NHEJ1 gene, cause severe combined immunodeficiency syndrome (SCID) along with neurodevelopmental disease and sensitivity to ionizing radiation.ObjectiveTo provide new clinical and immunological insights on NHEJ1 deficiency arising from a newly diagnosed patient with severe immunodeficiency.Materials and methodsA male infant, born to consanguineous parents, suspected of having primary immunodeficiency underwent immunological and genetic workup. This included a thorough assessment of T cell phenotyping and lymphocyte activation by mitogen stimulation tests, whole-exome sequencing (WES), TCR repertoire Vβ repertoire via flow cytometry analysis, and TCR and BCR repertoire analysis via next-generation sequencing (NGS).ResultsClinical findings included microcephaly, recurrent pneumonia, and failure to thrive. An immune workup revealed lymphopenia, reduced T cell function, and hypogammaglobulinemia. Skewed TCR Vβ repertoire, TCR gamma (TRG) repertoire, and BCR repertoire were determined in the patient. Genetic analysis identified a novel homozygous missense pathogenic variant in XLF/Cernunnos: c.A580Ins.T; p.M194fs. The patient underwent a successful hematopoietic stem cell transplantation (HSCT).ConclusionA novel NHEJ1 pathogenic variant is reported in a patient who presented with SCID phenotype that displayed clonally expanded T and B cells. An adjusted HSCT was safe to ensure full T cell immune reconstitution.

The transcription factor GATA2 plays a key role in the survival and self-renewal of hematopoietic stem and progenitor cells. Autosomal dominant variants in GATA2 cause a broad spectrum of heterogeneous phenotypes. Here, we present our experience with GATA2 deficiency in a retrospective multicenter analysis of computerized medical records of adult patients (age ≥18 years) treated between 2018 and 2022 at Shaare Zedek Medical Center in Jerusalem and Sheba Tel-Hashomer Medical Center in Ramat Gan, Israel. Two male and two female patients with GATA2 deficiency were identified. Three of the patients presented with symptoms in adult life and all patients were diagnosed as adults. Age at presentation was 10.5-36 years and age at diagnosis 24-47 years. Diagnosis was delayed in all patients by 1-24.5 years. The phenotypic diversity was notable. Patients presented with myelodysplastic syndrome (n=2), pulmonary alveolar proteinosis (n=1), and recurrent viral (n=1), bacterial (n=3), and mycobacterial (n=1) infections. Bone marrow biopsy revealed cytogenetic abnormalities in one patient (monosomy 7). Patients were diagnosed by exome sequencing (n=3) and Sanger sequencing of the coding exons in GATA2 (n=1). Novel heterozygous GATA2 variants (c.177C>A, p.Y59* and c.610dup, p.R204Pfs*78) were identified in two patients. Immune workup revealed B cell lymphopenia and monocytopenia in all tested patients. One patient died from overwhelming sepsis despite all patients being treated with antibiotics and anti-mycobacterials. Our cohort highlights the phenotypic diversity, late presentation, and delayed diagnosis of GATA2 deficiency. Increased awareness of this primary immune deficiency presenting in adult life is needed and should involve a high index of suspicion.

FCH domain only 1 (FCHO1) is a key molecule involved in clathrin-mediated endocytosis (CME). Here, the authors report homozygous FCHO1 mutations in individuals with variable T and B cell lymphopenia, which are associated with loss-of-function of FCHO1 and impaired formation of clathrin-coated pits in T cells.

Abstract Background Systemic lupus erythematosus (SLE) comprise a diverse range of clinical manifestations. To date, more than 30 single gene causes of lupus/lupus like syndromes in humans have been identified. In the clinical setting, identifying the underlying molecular diagnosis is challenging due to phenotypic and genetic heterogeneity. Methods We employed whole exome sequencing (WES) in patients presenting with childhood-onset lupus with severe and/or atypical presentations to identify cases that are explained by a single-gene (monogenic) cause. Results From January 2015 to June 2018 15 new cases of childhood-onset SLE were diagnosed in Edmond and Lily Safra Children’s Hospital. By WES we identified causative mutations in four subjects in five different genes: C1QC, SLC7A7, MAN2B1, PTEN and STAT1. No molecular diagnoses were established on clinical grounds prior to genetic testing. Conclusions We identified a significant fraction of monogenic SLE etiologies using WES and confirm the genetic locus heterogeneity in childhood-onset lupus. These results highlight the importance of establishing a genetic diagnosis for children with severe or atypical lupus by providing accurate and early etiology-based diagnoses and improving subsequent clinical management.

This is the first report of persistent oropharyngeal mucosal infection with type 2 poliovirus (iVDPV2) in a primary immune deficient patient (PID) after wild type 2 poliovirus eradication. The iVDPV2 also established persistence in the gut. iVDPV2 at both loci evolved independently. Persistent oral infections present a potential risk for oral-oral as well as fecal-oral poliovirus transmission during transition to a poliovirus 2-free world. Keywords: Primary immune deficiency (PID), Type 2 vaccine-derived poliovirus (iVDPV2), Stools, Oropharyngeal mucosa, Contact isolation, Droplet isolation

In the last few months the world has witnessed a global pandemic due to severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection causing coronavirus disease 2019 (COVID-19). Obviously, this pandemic affected individuals differently, with a significant impact on populations considered to be at high-risk. One such population, was assumed to be patients with primary genetic defect involving components or pathways of the immune system. While human immunity against COVID-19 is not fully understood, it is, so far, well documented, that both adaptive and innate cells have a critical role in protection against SARS-CoV-2. Here, we aimed to summarize the clinical and laboratory data on primary immunodeficiency (PID) patients in Israel, who were tested positive for SARS-CoV-2, in order to estimate the impact of COVID-19 on such patients. Data was collected from mid-February to end-September. During this time Israel experienced two “waves” of COVID-19 diseases; the first, from mid-February to mid-May and the second from mid-June and still ongoing at the end of data collection. A total of 20 PID patients, aged 4 months to 60 years, were tested positive for SARS-CoV-2, all but one, were detected during the second wave. Fourteen of the patients were on routine monthly IVIG replacement therapy at the time of virus detection. None of the patients displayed severe illness and none required hospitalization; moreover, 7/20 patients were completely asymptomatic. Possible explanations for the minimal clinical impact of COVID-19 pandemic observed in our PID patients include high level of awareness, extra-precautions, and even self-isolation. It is also possible that only specific immune pathways (e.g. type I interferon signaling), may increase the risk for a more severe course of disease and these are not affected in many of the PID patients. In some cases, lack of an immune response actually may be a protective measure against the development of COVID-19 sequelae.

Background: More than 50 different monogenic disorders have been identified as directly causing inflammatory bowel diseases, typically manifesting in the first years of life. We present the clinical course and immunological work-up of an adult patient who presented in adolescent years with an atypical gastrointestinal phenotype and was diagnosed more than two decades later with a monogenic disorder with important therapeutic implications.Methods: Whole exome sequencing was performed in a 37-years-old patient with a history of diarrhea since adolescence. Sanger sequencing was used to validate the suspected variant. Mass cytometry (CyTOF) and flow cytometry were conducted on peripheral blood mononuclear cells for deep immunophenotyping. Next-generation sequencing of the TCRB and IgH was performed for global immune repertoire analysis of circulating lymphocytes.Results: We identified a novel deleterious c.1455C>A (p.Y485X) mutation in LRBA. CyTOF studies demonstrated significant changes in immune landscape in the LRBA-deficient patient, including an increase in myeloid derived suppressor cells and double-negative T cells, decreased B cells, low ratio of naïve:memory T cells, and reduced capacity of T cells to secrete various cytokines following stimulation, including tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). In addition, this patient exhibited low frequency of regulatory T cells, with a reduction in their CTLA4 expression and interleukin (IL)-10 secretion. Finally, we show marked oligoclonal expansion of specific B- and T-cell clones in the peripheral blood of the LRBA-deficient patient.Conclusions: LRBA deficiency is characterized by marked immunological changes in innate and adaptive immune cells. This case highlights the importance of advanced genetic studies in patients with a unique phenotype, regardless of their age at presentation.

Patients with loss-of-function mutations in IL10 or IL10 receptor (IL10R) genes develop severe, medical-refractory, infantile-onset inflammatory bowel disease (IBD). We have previously reported significant alterations in innate and adaptive immune responses in these patients. Next generation sequencing platforms enable a comprehensive assessment of T cell receptor (TCR) and B cell receptor (BCR) repertoire patterns. We aimed to characterize TCR and BCR features in peripheral blood of patients with deleterious IL10 signaling defects. DNA was isolated from blood of seven patients with IL10R mutations and one with an IL10 mutation, along with eight controls, and subjected to next generation sequencing of TRB and IgH loci. A significant increase in clonality was observed in both TCR and BCR repertoires in circulating lymphocytes of IL10/IL10R-deficient patients, but to a much greater extent in T cells. Furthermore, short CDR3β length and altered hydrophobicity were demonstrated in T cells of patients, but not in B cells, secondary to lower rates of insertions of nucleotides, but not deletions, at the V-, D-, or J-junctions. We were unable to observe specific T or B clones that were limited only to the patients or among controls. Moreover, the expanded T cells clones were unique to each patient. In conclusion, next generation sequencing of the TCR and BCR is a powerful tool for characterizing the adaptive immune cell phenotype and function in immune-mediated disorders. The oligoclonality observed among IL10/IL10R-deficient patients may suggest specialization of unique clones that likely have a role in mediating tissue damage. Nevertheless, the lack of shared clones between patients provides another piece of evidence that the adaptive immune response in IBD is not triggered against common antigens. Additional studies are required to define the specific antigens that interact with the expanded IL10/IL10R-deficient clones.

Abstract Background Ataxia telangiectasia (AT) is a rare, multi-systemic, genetic disorder. Mutations in the ATM gene cause dysfunction in cell-cycle, apoptosis and V (D) J recombination leading to neurodegeneration, cellular, humoral immunodeficiencies and predisposition to malignancies. Previous studies have suggested that a sub-group of AT patients with elevated IgM levels have a distinct and more severe phenotype. In the current study we aimed to better characterize this group of patients. Methods We performed a retrospective review of 46 patient records, followed from January 1986 to January 2015 at the Israeli National AT Center. Demographic, clinical, radiological, laboratory data was reviewed and compared between AT patients with elevated IgM levels (EIgM) and patients with normal IgM levels (NIgM). Results 15/46(32.6%) patients had significantly elevated IgM levels. This group had a unique phenotype characterized mainly by increased risk of infection and early mortality. Colonization of lower respiratory tract with Mycobacterium gordonae and Pseudomonas aeruginosa as well as viral skin infections were more frequent in EIgM patients. Patients with NIgM had a significantly longer survival as compared to patients with EIgM but had an increased incidence of fatty liver or cirrhosis. T-cell recombination excision circles and kappa-deleting element recombination circle levels were significantly lower in the EIgM group, suggesting an abnormal class switching in this group. Conclusions EIgM in AT patients are indicative of a more severe phenotype that probably results from a specific immune dysfunction. EIgM in AT should be considered a unique AT phenotype that may require different management.

The alpha subunit of IL-7 receptor (IL7R7α) is critical for the differentiation of T cells, specifically for the development and maintenance of γδT cells. Mutations in IL7RA are associated with Severe Combined Immunodeficiency (SCID). Infants with IL7RA deficiency can be identified through newborn screening program. We aimed at defining the immunological and genetic parameters that are directly affected by the IL7RA mutation on the immune system of five unrelated patients which were identified by our newborn screening program for SCID. The patients were found to have a novel identical homozygote mutation in IL7RA (n.c.120 C>G; p.F40L). Both surface expression of IL7Rα and functionality of IL-7 signaling were impaired in patients compared to controls. Structural modeling demonstrated instability of the protein structure due to the mutation. Lastly the TRG immune repertoire of the patients showed reduced diversity, increased clonality and differential CDR3 characteristics. Interestingly, the patients displayed significant different clinical outcome with two displaying severe clinical picture of immunodeficiency and three had spontaneous recovery. Our data supports that the presented IL7RA mutation affects the IL-7 signaling and shaping of the TRG repertoire, reinforcing the role of IL7RA in the immune system, while non-genetic factors may exist that attribute to the ultimate clinical presentation and disease progression.

Immunoglobulin replacement therapy is a mainstay therapy for patients with primary immunodeficiency (PID). The content of these preparations was studied extensively. Nevertheless, data regarding the effective specific antibodies content (especially in the nadir period), and, in different groups of PID patients is limited. We studied trough IgG concentrations as well as anti-Pneumococcus, anti-Haemophilus influenzae b, anti-Tetanus, and anti-Measles antibody concentrations in 17 PID patients receiving intravenous immunoglobulin (IVIg) compared with healthy controls matched for age and ethnicity. We also analyzed these results according to the specific PID diagnosis: X-linked agammaglobulinemia (XLA), combined immunodeficiency (CID), and ataxia telangiectasia (AT). We recorded a higher concentration of anti-pneumococcal polysaccharide antibodies in healthy controls compared to the entire group of PID patients. We also found significantly higher anti-tetanus toxoid antibody concentrations in the XLA patients, compared to CID patients. Anti-Haemophilus Influenzae b antibody titers were overall similar between all the groups. Interestingly, there were overall low titers of anti-Measles antibodies below protective cutoff antibody concentrations in most patients as well as in healthy controls. We conclude that relying on total IgG trough levels is not necessarily a reflection of effective specific antibodies in the patient’s serum. This is especially relevant to CID patients who may have production of nonspecific antibodies. In such patients, a higher target trough IgG concentration should be considered. Another aspect worth considering is that the use of plasma from adult donors with a waning immunity for certain pathogens probably affects the concentrations of specific antibodies in IVIg preparations.

Hyaline fibromatosis syndrome (HFS), resulting from ANTXR2 mutations, is an ultra-rare disease that causes intestinal lymphangiectasia and protein-losing enteropathy (PLE). The mechanisms leading to the gastrointestinal phenotype in these patients are not well defined. We present two patients with congenital diarrhea, severe PLE and unique clinical features resulting from deleterious ANTXR2 mutations. Intestinal organoids were generated from one of the patients, along with CRISPR-Cas9 ANTXR2 knockout, and compared with organoids from two healthy controls. The ANTXR2-deficient organoids displayed normal growth and polarity, compared to controls. Using an anthrax-toxin assay we showed that the c.155C>T mutation causes loss-of-function of ANTXR2 protein. An intrinsic defect of monolayer formation in patient-derived or ANTXR2KO organoids was not apparent, suggesting normal epithelial function. However, electron microscopy and second harmonic generation imaging showed abnormal collagen deposition in duodenal samples of these patients. Specifically, collagen VI, which is known to bind ANTXR2, was highly expressed in the duodenum of these patients. In conclusion, despite resistance to anthrax-toxin, epithelial cell function, and specifically monolayer formation, is intact in patients with HFS. Nevertheless, loss of ANTXR2-mediated signaling leads to collagen VI accumulation in the duodenum and abnormal extracellular matrix composition, which likely plays a role in development of PLE.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Severe combined immunodeficiency (SCID), the most severe form of T cell immunodeficiency, is detectable through quantification of T cell receptor excision circles (TRECs) in dried blood spots obtained at birth. Herein, we describe the results of the first year of the Israeli SCID newborn screening (NBS) program. This important, life-saving screening test is available at no cost for every newborn in Israel. Eight SCID patients were diagnosed through the NBS program in its first year, revealing an incidence of 1:22,500 births in the Israeli population. Consanguine marriages and Muslim ethnic origin were found to be a risk factor in affected newborns, and a founder effect was detected for both IL7Rα and DCLRE1C deficiency SCID. Lymphocyte subset analysis and TREC quantification in the peripheral blood appear to be sufficient for confirmation of typical and leaky SCID and ruling out false positive (FP) results. Detection of secondary targets (infants with non-SCID lymphopenia) did not significantly affect the management or outcomes of these infants in our cohort. In the general, non-immunodeficient population, TREC rises along with gestational age and birth weight, and is significantly higher in females and the firstborn of twin pairs. Low TREC correlates with both gestational age and birth weight in extremely premature newborns. Additionally, the rate of TREC increase per week consistently accelerates with gestational age. Together, these findings mandate a lower cutoff or a more lenient screening algorithm for extremely premature infants, in order to reduce the high rate of FPs within this group. A significant surge in TREC values was observed between 28 and 30 weeks of gestation, where median TREC copy numbers rise by 50% over 2 weeks. These findings suggest a maturational step in T cell development around week 29 gestation, and imply moderate to late preterms should be screened with the same cutoff as term infants. The SCID NBS program is still in its infancy, but is already bearing fruit in the early detection and improved outcomes of children with SCID in Israel and other countries.

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 CD56bright 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.

Immunodeficiency-associated vaccine-derived polioviruses (iVDPVs) have been isolated from primary immunodeficiency (PID) patients exposed to oral poliovirus vaccine (OPV). Patients may excrete poliovirus strains for months or years; the excreted viruses are frequently highly divergent from the parental OPV and have been shown to be as neurovirulent as wild virus. Thus, these patients represent a potential reservoir for transmission of neurovirulent polioviruses in the post-eradication era. In support of WHO recommendations to better estimate the prevalence of poliovirus excreters among PIDs and characterize genetic evolution of these strains, 635 patients including 570 with primary antibody deficiencies and 65 combined immunodeficiencies were studied from 13 OPV-using countries. Two stool samples were collected over 4 days, tested for enterovirus, and the poliovirus positive samples were sequenced. Thirteen patients (2%) excreted polioviruses, most for less than 2 months following identification of infection. Five (0.8%) were classified as iVDPVs (only in combined immunodeficiencies and mostly poliovirus serotype 2). Non-polio enteroviruses were detected in 30 patients (4.7%). Patients with combined immunodeficiencies had increased risk of delayed poliovirus clearance compared to primary antibody deficiencies. Usually, iVDPV was detected in subjects with combined immunodeficiencies in a short period of time after OPV exposure, most for less than 6 months. Surveillance for poliovirus excretion among PID patients should be reinforced until polio eradication is certified and the use of OPV is stopped. Survival rates among PID patients are improving in lower and middle income countries, and iVDPV excreters are identified more frequently. Antivirals or enhanced immunotherapies presently in development represent the only potential means to manage the treatment of prolonged excreters and the risk they present to the polio endgame.

Background . Hashimoto’s thyroiditis usually presents with nonspecific systemic symptoms. The purpose of our study was to characterize the various properties of severe ongoing hypothyroidism and the rate of normalization following treatment. Methods . An adolescent girl with severe primary hypothyroidism was studied. Clinical evaluation, laboratory testing, brain magnetic resonance imaging, resting metabolic rate (RMR) testing, electroencephalogram, and visual field examination were performed at baseline and following treatment with levothyroxine. Results . At baseline, a significant psychomotor retardation was observed, serum thyroid-stimulating hormone concentration was 1088.4 mIU/mL. Magnetic resonance imaging showed a large intrasellar mass. Electroencephalogram was abnormal, and RMR was significantly reduced. Restoration of neurocognitive function and normalization of RMR, electroencephalogram, and laboratory tests occurred rapidly, alongside vanishing of the pituitary mass within 4 weeks of treatment. Conclusions . The various signs and symptoms of severe prolonged hypothyroidism may resolve rapidly with treatment, including the disappearance of a large pituitary mass.

Newborn screening (NBS) programs for severe combined immunodeficiency (SCID), the most severe type of primary immunodeficiency, are being implemented in more and more countries with every passing year. Since October 2015, SCID screening via T cell receptor excision circle (TREC) quantification in dried blood spots (DBS) has been part of the Israeli NBS program. As an NBS program in its infancy, SCID screening is still evolving, making gathering input from the various programs crucial for compiling an ideal screening algorithm. The relatively high rate of consanguineous marriages in Israel, especially among non-Jews, correlates with an increased incidence of SCID. The Israeli algorithm uses a commercial kit and consists of a two-Guthrie card confirmation system prior to referral to a national immunology center. Preliminary data from the first year and a half of SCID screening in Israel has identified a surprisingly high prevalence of DNA cross-link repair protein 1c (DCLRE1C; ARTEMIS) mutations as the cause of SCID in Israel. The clinically unbiased nature of SCID screening helps unearth mild/leaky SCID phenotypes, resulting in a better understanding of true SCID prevalence and etiology.

Quantification of the T cell receptor excision circles (TRECs) has recently emerged as a useful non-invasive clinical and research tool to investigate thymic activity. It allows the identification of T cell production by the thymus. Quantification of TREC copies has recently been implemented as the preferred test to screen neonates with severe combined immunodeficiency (SCID) or significant lymphopenia. Neonatal genetic screening for SCID is highly important in countries with high rates of consanguinous marriages, such as Israel, and can be used for early diagnosis, enabling prompt therapeutic intervention that will save lives and improve the outcome of these patients. TREC measurement is also applicable in clinical settings where T cell immunity is involved, including any T cell immunodeficiencies, HIV infection, the aging process, autoimmune diseases, and immune reconstitution after bone marrow transplantation.

The kinetics of T and B cell immune recovery after bone marrow transplantation (BMT) is affected by many pre- and post-transplant factors. Because of the profoundly depleted baseline T and B cell immunity in recombination activating gene 2 (RAG-2)-deficient severe combined immunodeficiency (SCID) patients, some of these factors are eliminated, and the immune recovery after BMT can then be clearly assessed. This process was followed in ten SCID patients in parallel to their associated transplant-related complications. Early peripheral presence of T and B cells was observed in 8 and 4 patients, respectively. The latter correlated with pre-transplant conditioning therapy. Cells from these patients carried mainly signal joint DNA episomes, indicative of newly derived B and T cells. They were present before the normalization of the T cell receptor (TCR) and the B cell receptor (BCR) repertoire. Early presentation of the ordered TCR gene rearrangements after BMT occurred simultaneously, but this pattern was heterogeneous over time, suggesting different and individual thymic recovery processes. Our findings early after transplant could suggest the long-term patients' clinical outcome. Early peripheral presence of newly produced B and T lymphocytes from their production and maturation sites after BMT suggests donor stem cell origin rather than peripheral expansion, and is indicative of successful outcome. Peripheral detection of TCR excision circles and kappa-deleting recombination excision circles in RAG-2-deficient SCID post-BMT are early markers of T and B cell reconstitution, and can be used to monitor outcome and tailor specific therapy for patients undergoing BMT.

Introduction. Patients with severe combined immunodeficiency (SCID) may present with residual circulating T cells. While all cells are functionally deficient, resulting in high susceptibility to infections, only some of these cells are causing autoimmune symptoms. Methods. Here we compared T-cell functions including the number of circulating CD3+ T cells, in vitro responses to mitogens, T-cell receptor (TCR) repertoire, TCR excision circles (TREC) levels, and regulatory T cells (Tregs) enumeration in several immunodeficinecy subtypes, clinically presenting with nonreactive residual cells (MHC-II deficiency) or reactive cells. The latter includes patients with autoreactive clonal expanded T cell and patients with alloreactive transplacentally maternal T cells. Results. MHC-II deficient patients had slightly reduced T-cell function, normal TRECs, TCR repertoires, and normal Tregs enumeration. In contrast, patients with reactive T cells exhibited poor T-cell differentiation and activity. While the autoreactive cells displayed significantly reduced Tregs numbers, the alloreactive transplacentally acquired maternal lymphocytes had high functional Tregs. Conclusion. SCID patients presenting with circulating T cells show different patterns of T-cell activity and regulatory T cells enumeration that dictates the immunodeficient and autoimmune manifestations. We suggest that a high-tolerance capacity of the alloreactive transplacentally acquired maternal lymphocytes represents a toleration advantage, yet still associated with severe immunodeficiency.

BackgroundLeukocyte adhesion deficiency 1 (LAD1) is an inherited disorder of neutrophil function. Nonsense mutations in the affected CD18 (ITB2) gene have rarely been described. In other genes containing such mutations, treatments with aminoglycoside types of antibiotics (e.g., gentamicin) were reported to partially correct the premature protein termination, by induction of readthrough mechanism.Methodology/principal findingsGenetic analysis was performed on 2 LAD1 patients. Expression, functional and immunofluorescence assays of CD18 in the patients were used to determine the in-vivo and in-vitro effects of gentamicin-induced readthrough. A theoretical modeling of the corrected CD18 protein was developed to predict the protein function.ResultsWe found a novel premature termination codon, C562T (R188X), in exon 6 of the CD18 gene that caused a severe LAD1 phenotype in two unrelated Palestinian children. In-vivo studies on these patients' cells after gentamicin treatment showed abnormal adhesion and chemotactic functions, while in-vitro studies showed mislocalization of the corrected protein to the cytoplasm and not to the cell surface. A theoretical modeling of the corrected CD18 protein suggested that the replacement of the wild type arginine by gentamicin induced tryptophan at the position of the nonsense mutation, although enabled the expression of the entire CD18 protein, this was not sufficient to stabilize the CD18/11 heterodimer at the cell surface.ConclusionA novel nonsense mutation in the CD18 gene causing a complete absence of CD18 protein and severe LAD1 clinical phenotype is reported. Both in vivo and in vitro treatments with gentamicin resulted in the expression of a corrected full-length dysfunctional or mislocalized CD18 protein. However, while the use of gentamicin increased the expression of CD18, it did not improve leukocyte adhesion and chemotaxis. Moreover, the integrity of the CD18/CD11 complex at the cell surface was impaired, due to abnormal CD18 protein and possibly lack of CD11a expression.