Publications by authors named "Chaim M Roifman"

113 Publications

Disruption of exon-bridging interactions between the minor and major spliceosomes results in alternative splicing around minor introns.

Nucleic Acids Res 2021 04;49(6):3524-3545

Physiology and Neurobiology Department, University of Connecticut, 75 N. Eagleville Road, Storrs, CT 06269, USA.

Vertebrate genomes contain major (>99.5%) and minor (<0.5%) introns that are spliced by the major and minor spliceosomes, respectively. Major intron splicing follows the exon-definition model, whereby major spliceosome components first assemble across exons. However, since most genes with minor introns predominately consist of major introns, formation of exon-definition complexes in these genes would require interaction between the major and minor spliceosomes. Here, we report that minor spliceosome protein U11-59K binds to the major spliceosome U2AF complex, thereby supporting a model in which the minor spliceosome interacts with the major spliceosome across an exon to regulate the splicing of minor introns. Inhibition of minor spliceosome snRNAs and U11-59K disrupted exon-bridging interactions, leading to exon skipping by the major spliceosome. The resulting aberrant isoforms contained a premature stop codon, yet were not subjected to nonsense-mediated decay, but rather bound to polysomes. Importantly, we detected elevated levels of these alternatively spliced transcripts in individuals with minor spliceosome-related diseases such as Roifman syndrome, Lowry-Wood syndrome and early-onset cerebellar ataxia. In all, we report that the minor spliceosome informs splicing by the major spliceosome through exon-definition interactions and show that minor spliceosome inhibition results in aberrant alternative splicing in disease.
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http://dx.doi.org/10.1093/nar/gkab118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034651PMC
April 2021

Combined immunodeficiency caused by a novel homozygous NFKB1 mutation.

J Allergy Clin Immunol 2021 Feb 25;147(2):727-733.e2. Epub 2020 Sep 25.

Canadian Centre for Primary Immunodeficiency and the Jeffrey Modell Research Laboratory for the Diagnosis of Primary Immunodeficiency, Toronto, Ontario, Canada; Division of Immunology and Allergy, Department of Paediatrics, Hospital for Sick Children, Toronto, Ontario, Canada; Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada; Department of Immunology, University of Toronto, Toronto, Ontario, Canada. Electronic address:

Background: Genetic faults in several components of the nuclear factor-κB pathway cause immunodeficiency. Most defects lead to combined immunodeficiency with a range of severity. Heterozygous mutations in NFKB1 were associated with common variable immunodeficiency, however, homozygous mutations have not been described.

Objective: We studied the molecular basis of combined immunodeficiency in a patient who presented with failure to thrive, persistent EBV viremia and hepatitis, pneumocystis jirovecii pneumonitis, and generalized lymphadenopathy.

Methods: Whole genome and exome sequencing followed by Sanger confirmation were performed to identify the genetic defect. Molecular and cellular techniques were used to assess the variant impact on the nuclear factor-κB pathway and lymphocyte function.

Results: Genetic analysis revealed a novel homozygous mutation in NFKB1, c.2878G>A, p.Gly960Arg (G960R). This affected p105 phosphorylation and p50 formation on antigen and cytokine stimulation, as well as attenuating nuclear signal transmission. As a result, both T- and B-cell maturation and function were perturbed. The number of memory CD4 T cells were reduced, while CD8 T cells consisted predominately of expanded differentiated populations. The function of T cells were diminished as shown by reduced responses to mitogens as well as diminished cytokine secretion. B-cell maturation was also affected, with decreased IgDCD27 memory B cells while transitional B cells were increased, likely contributing to the reduced ability to produce specific antibodies.

Conclusion: Homozygous G960R mutation in NFKB1 leads to a severe clinical presentation of combined immunodeficiency. This was associated with blockade of nuclear factor-κB pathway signaling, resulting in aberrations in T- and B-cell maturation and function.
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http://dx.doi.org/10.1016/j.jaci.2020.08.040DOI Listing
February 2021

Characterization of the clinical and immunologic phenotype and management of 157 individuals with 56 distinct heterozygous NFKB1 mutations.

J Allergy Clin Immunol 2020 10 9;146(4):901-911. Epub 2020 Apr 9.

Primary Immunodeficiencies Unit, Hospital Dona Estefania, Centro Hospitalar de Lisboa Central, Lisbon, Portugal.

Background: An increasing number of NFKB1 variants are being identified in patients with heterogeneous immunologic phenotypes.

Objective: To characterize the clinical and cellular phenotype as well as the management of patients with heterozygous NFKB1 mutations.

Methods: In a worldwide collaborative effort, we evaluated 231 individuals harboring 105 distinct heterozygous NFKB1 variants. To provide evidence for pathogenicity, each variant was assessed in silico; in addition, 32 variants were assessed by functional in vitro testing of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) signaling.

Results: We classified 56 of the 105 distinct NFKB1 variants in 157 individuals from 68 unrelated families as pathogenic. Incomplete clinical penetrance (70%) and age-dependent severity of NFKB1-related phenotypes were observed. The phenotype included hypogammaglobulinemia (88.9%), reduced switched memory B cells (60.3%), and respiratory (83%) and gastrointestinal (28.6%) infections, thus characterizing the disorder as primary immunodeficiency. However, the high frequency of autoimmunity (57.4%), lymphoproliferation (52.4%), noninfectious enteropathy (23.1%), opportunistic infections (15.7%), autoinflammation (29.6%), and malignancy (16.8%) identified NF-κB1-related disease as an inborn error of immunity with immune dysregulation, rather than a mere primary immunodeficiency. Current treatment includes immunoglobulin replacement and immunosuppressive agents.

Conclusions: We present a comprehensive clinical overview of the NF-κB1-related phenotype, which includes immunodeficiency, autoimmunity, autoinflammation, and cancer. Because of its multisystem involvement, clinicians from each and every medical discipline need to be made aware of this autosomal-dominant disease. Hematopoietic stem cell transplantation and NF-κB1 pathway-targeted therapeutic strategies should be considered in the future.
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http://dx.doi.org/10.1016/j.jaci.2019.11.051DOI Listing
October 2020

Prevalence and Clinical Features of Inflammatory Bowel Diseases Associated With Monogenic Variants, Identified by Whole-Exome Sequencing in 1000 Children at a Single Center.

Gastroenterology 2020 06 19;158(8):2208-2220. Epub 2020 Feb 19.

SickKids Inflammatory Bowel Disease Center, The Hospital for Sick Children, Toronto, Ontario, Canada.

Background & Aims: A proportion of infants and young children with inflammatory bowel diseases (IBDs) have subtypes associated with a single gene variant (monogenic IBD). We aimed to determine the prevalence of monogenic disease in a cohort of pediatric patients with IBD.

Methods: We performed whole-exome sequencing analyses of blood samples from an unselected cohort of 1005 children with IBD, aged 0-18 years (median age at diagnosis, 11.96 years) at a single center in Canada and their family members (2305 samples total). Variants believed to cause IBD were validated using Sanger sequencing. Biopsies from patients were analyzed by immunofluorescence and histochemical analyses.

Results: We identified 40 rare variants associated with 21 monogenic genes among 31 of the 1005 children with IBD (including 5 variants in XIAP, 3 in DOCK8, and 2 each in FOXP3, GUCY2C, and LRBA). These variants occurred in 7.8% of children younger than 6 years and 2.3% of children aged 6-18 years. Of the 17 patients with monogenic Crohn's disease, 35% had abdominal pain, 24% had nonbloody loose stool, 18% had vomiting, 18% had weight loss, and 5% had intermittent bloody loose stool. The 14 patients with monogenic ulcerative colitis or IBD-unclassified received their diagnosis at a younger age, and their most predominant feature was bloody loose stool (78%). Features associated with monogenic IBD, compared to cases of IBD not associated with a single variant, were age of onset younger than 2 years (odds ratio [OR], 6.30; P = .020), family history of autoimmune disease (OR, 5.12; P = .002), extra-intestinal manifestations (OR, 15.36; P < .0001), and surgery (OR, 3.42; P = .042). Seventeen patients had variants in genes that could be corrected with allogeneic hematopoietic stem cell transplantation.

Conclusions: In whole-exome sequencing analyses of more than 1000 children with IBD at a single center, we found that 3% had rare variants in genes previously associated with pediatric IBD. These were associated with different IBD phenotypes, and 1% of the patients had variants that could be potentially corrected with allogeneic hematopoietic stem cell transplantation. Monogenic IBD is rare, but should be considered in analysis of all patients with pediatric onset of IBD.
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http://dx.doi.org/10.1053/j.gastro.2020.02.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283012PMC
June 2020

Ataxia Telangiectasia Diagnosed on Newborn Screening-Case Cohort of 5 Years' Experience.

Front Immunol 2019 20;10:2940. Epub 2019 Dec 20.

Division of Immunology and Allergy, Department of Paediatrics, The Hospital for Sick Children and the University of Toronto, Toronto, ON, Canada.

Ataxia telangiectasia (AT) is a genetic condition caused by mutations involving (Ataxia Telangiectasia Mutated). This gene is responsible for the expression of a DNA double stranded break repair kinase, the ATM protein kinase. The syndrome encompasses combined immunodeficiency and various degrees of neurological abnormalities and increased risk of malignancy. Typically, patients present early in life with delay in neurological milestones, but very infrequently, with life threatening infections typical of a profound T cell deficiency. It would therefore be unexpected to identify this condition immediately after birth using T cell receptor excision circle (TREC)-based newborn screening (NBS) for SCID. We sought to evaluate the frequency of AT detected by NBS, and to assess immunity as well as the genetic aberrations associated with this early presentation. Here, we describe the clinical, laboratory, and genetic features of patients diagnosed with AT through the Ontario NBS program for SCID, and followed in our center since its inception in 2013. Four patients were diagnosed with AT as a result of low TRECs on NBS. In each case, whole exome sequencing was diagnostic. All of our patients had compound heterozygous mutations involving the FRAP-ATM-TRRAP (FAT) domain of the gene, which appears critical for kinase activity and is highly sensitive to mutagenesis. Our patients presented with profound lymphopenia involving both B and T cells. The ratio of naïve/memory CD45+RA/RO T cells population was variable. T cell repertoire showed decreased T cell diversity. Two out of four patients had decreased specific antibody response to vaccination and hypogammaglobulinemia requiring IVIG replacement. In two patients, profound decreased responses to phytohemagglutinin stimulation was observed. In the other two patients, the initial robust response declined with time. In summary, the rate of detection of AT through NBS had been surprisingly high at our center. One case was identified per year, while the total rate for SCID has been five new cases per year. This early detection may allow for better prospective evaluation of AT shortly after birth, and may assist in formulating early and more effective interventions both for the neurological as well as the immune abnormalities in this syndrome.
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http://dx.doi.org/10.3389/fimmu.2019.02940DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6932992PMC
November 2020

Morbidity in an adenosine deaminase-deficient patient during 27 years of enzyme replacement therapy.

Clin Immunol 2020 02 5;211:108321. Epub 2019 Dec 5.

Division of Immunology and Allergy, Department of Pediatrics, Hospital for Sick Children, Toronto, ON, Canada; University of Toronto, Toronto, ON, Canada. Electronic address:

Introduction: Adenosine deaminase (ADA) deficiency causes severe immunodeficiency that is lethal in infancy. Enzyme replacement therapy (ERT) can improve the metabolic, immune and non-immune abnormalities in patients prior to transplantation, however, its benefits over extended periods are not well characterized. We describe a 28-year-old female who received ERT for 27 years. She suffered from EBV negative B cell lymphoma of the hip at 14 years of age and Guillian-Barre Syndrome 2 years later. At 22 years of age, she experienced a gastrointestinal infection with Mycobacterium genavense. At 26 years of age, lymphoma reoccurred with multiple liver lesions followed by Mycobacterium genavense infection with dissemination to the brain. Throughout this period, ADA activity in the plasma was within the therapeutic range. Repeated evaluations demonstrated very low lymphocyte counts and impaired T cell function.

Conclusions: ERT might be insufficient to maintain normal immunity over extended periods in some ADA-deficient patients.
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http://dx.doi.org/10.1016/j.clim.2019.108321DOI Listing
February 2020

Defining the biological responses of IL-6 by the study of a novel IL-6 receptor chain immunodeficiency.

J Allergy Clin Immunol 2020 03 26;145(3):1011-1015.e6. Epub 2019 Nov 26.

Canadian Centre for Primary Immunodeficiency and the Jeffrey Modell Research Laboratory for the Diagnosis of Primary Immunodeficiency, the Hospital for Sick Children, Toronto, Ontario, Canada; Division of Immunology and Allergy, Department of Paediatrics, the Hospital for Sick Children and the University of Toronto, Toronto, Ontario, Canada. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2019.11.015DOI Listing
March 2020

Time-dependent decline of T-cell receptor excision circle levels in ZAP-70 deficiency.

J Allergy Clin Immunol Pract 2020 02 23;8(2):806-808.e2. Epub 2019 Aug 23.

Division of Immunology and Allergy, Department of Pediatrics, The Hospital for Sick Children and the University of Toronto, Toronto, ON, Canada; The Canadian Centre for Primary Immunodeficiency and The Jeffrey Modell Research Laboratory for the Diagnosis of Primary Immunodeficiency, The Hospital for Sick Children, Toronto, ON, Canada. Electronic address:

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http://dx.doi.org/10.1016/j.jaip.2019.08.018DOI Listing
February 2020

New insights into minor splicing-a transcriptomic analysis of cells derived from TALS patients.

RNA 2019 09 7;25(9):1130-1149. Epub 2019 Jun 7.

INRIA Erable, CNRS LBBE UMR 5558, University Lyon 1, University of Lyon, F-69622 Villeurbanne, France.

Minor intron splicing plays a central role in human embryonic development and survival. Indeed, biallelic mutations in , transcribed into the minor spliceosomal U4atac snRNA, are responsible for three rare autosomal recessive multimalformation disorders named Taybi-Linder (TALS/MOPD1), Roifman (RFMN), and Lowry-Wood (LWS) syndromes, which associate numerous overlapping signs of varying severity. Although RNA-seq experiments have been conducted on a few RFMN patient cells, none have been performed in TALS, and more generally no in-depth transcriptomic analysis of the ∼700 human genes containing a minor (U12-type) intron had been published as yet. We thus sequenced RNA from cells derived from five skin, three amniotic fluid, and one blood biosamples obtained from seven unrelated TALS cases and from age- and sex-matched controls. This allowed us to describe for the first time the mRNA expression and splicing profile of genes containing U12-type introns, in the context of a functional minor spliceosome. Concerning -mutated patients, we show that as expected, they display distinct U12-type intron splicing profiles compared to controls, but that rather unexpectedly mRNA expression levels are mostly unchanged. Furthermore, although U12-type intron missplicing concerns most of the expressed U12 genes, the level of U12-type intron retention is surprisingly low in fibroblasts and amniocytes, and much more pronounced in blood cells. Interestingly, we found several occurrences of introns that can be spliced using either U2, U12, or a combination of both types of splice site consensus sequences, with a shift towards splicing using preferentially U2 sites in TALS patients' cells compared to controls.
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http://dx.doi.org/10.1261/rna.071423.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800510PMC
September 2019

NF-κB pathway and the Goldilocks principle: Lessons from human disorders of immunity and inflammation.

J Allergy Clin Immunol 2019 05 30;143(5):1688-1701. Epub 2019 Mar 30.

Division of Immunology and Allergy, Department of Pediatrics, The Hospital for Sick Children and the University of Toronto, Toronto, Ontario, Canada; Canadian Centre for Primary Immunodeficiency and the Jeffrey Modell Research Laboratory for the Diagnosis of Primary Immunodeficiency, The Hospital for Sick Children. Electronic address:

Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) signaling pathways play a key role in various cell processes related to host immunity. The last few years have seen an explosion of disorders associated with NF-κB components from core members of the canonical and noncanonical cascades to adaptor protein and ubiquitination-related enzymes. Disease phenotypes have extended beyond susceptibility to infections and include autoimmunity, lymphoproliferation, atopy, and inflammation. Concurrently, studies are unveiling a tightly regulated system marked by extensive cross-talk between the canonical and noncanonical pathways, as well as among the NF-κB and other signaling pathways. As the rate of discovery in the realm of NF-κB defects accelerates, this review presents a timely summary of major known defects causing human disease, as well as diagnostic, therapeutic, and research challenges and opportunities.
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http://dx.doi.org/10.1016/j.jaci.2019.03.016DOI Listing
May 2019

Two different STAT1 gain-of-function mutations lead to diverse IFN-γ-mediated gene expression.

NPJ Genom Med 2018 20;3:23. Epub 2018 Aug 20.

1Division of Immunology and Allergy, Department of Paediatrics, The Hospital for Sick Children and The University of Toronto, Toronto, ON Canada.

Signal transducer and activator of transcription 1 (STAT1) regulates multiple biological processes downstream of a variety of cytokine receptors in many cell types. Heterozygous gain-of-function (GOF) mutations in STAT1 have been associated with a diverse phenotype encompassing chronic mucocutaneous candidiasis (CMCC) and declining immunity. There is no clear correlation between STAT1 domain-specific mutations and phenotype, and it remains unclear why GOF mutations in STAT1 result in such a wide spectrum of clinical presentations. To begin exploring this dilemma, we have studied the patterns of gene expression mediated by two different GOF mutations. Analysis of IFN-γ response elements using RNA microarrays in cells transfected with the rare H629Y mutant or the common R274G mutant showed distinct patterns of gene expression. We show here that the impact of GOF mutations in STAT1 is variant-specific. This difference in gene expression may explain the diversity in clinical manifestations experienced by these patients.
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http://dx.doi.org/10.1038/s41525-018-0063-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102291PMC
August 2018

A homozygous mutation in the stem II domain of causes typical Roifman syndrome.

NPJ Genom Med 2017 10;2:23. Epub 2017 Jul 10.

Division of Immunology and Allergy, Department of Pediatrics, The Hospital for Sick Children and the University of Toronto, Toronto, ON Canada.

Roifman syndrome (OMIM# 616651) is a complex syndrome encompassing skeletal dysplasia, immunodeficiency, retinal dystrophy and developmental delay, and is caused by compound heterozygous mutations involving the Stem II region and one of the other domains of the gene. This small nuclear RNA gene is essential for minor intron splicing. The Canadian Centre for Primary Immunodeficiency Registry and Repository were used to derive patient information as well as tissues. Utilising RNA sequencing methodologies, we analysed samples from patients with Roifman syndrome and assessed intron retention. We demonstrate that a homozygous mutation in Stem II is sufficient to cause the full spectrum of features associated with typical Roifman syndrome. Further, we demonstrate the same pattern of aberration in minor intron retention as found in cases with compound heterozygous mutations.
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http://dx.doi.org/10.1038/s41525-017-0024-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5677950PMC
July 2017

Newborn screening for severe combined immunodeficiency: a primer for clinicians.

CMAJ 2017 12;189(50):E1551-E1557

Department of Pediatrics (Biggs, Turvey), British Columbia Children's Hospital, University of British Columbia, Vancouver, BC; Departments of Pediatrics, and Microbiology, Infection and Immunology (Haddad), University of Montreal, CHU Sainte-Justine, Montréal, Que.; Department of Pediatrics (Issekutz), IWK Health Centre, Dalhousie University, Halifax, NS; Division of Immunology and Allergy (Roifman), Hospital for Sick Children; Department of Pediatrics (Roifman), University of Toronto, Toronto, Ont.

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http://dx.doi.org/10.1503/cmaj.170561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738248PMC
December 2017

Dual loss of p110δ PI3-kinase and SKAP (KNSTRN) expression leads to combined immunodeficiency and multisystem syndromic features.

J Allergy Clin Immunol 2018 08 26;142(2):618-629. Epub 2017 Nov 26.

Canadian Centre for Primary Immunodeficiency, Immunogenomic Laboratory, Division of Immunology & Allergy, Department of Pediatrics, Hospital for Sick Children, Toronto, Ontario, Canada. Electronic address:

Background: We previously reported a novel syndrome characterized by combined immunodeficiency associated with severe developmental defects-subsequently known as Roifman-Chitayat syndrome (RCS; OMIM 613328). Linkage analysis identified 2 disease-associated loci.

Objectives: We sought to identify the genetic defect in these patients and characterize their immunologic cellular abnormalities.

Methods: Genetic, immunologic, protein, and cellular functional analyses were used to identify and characterize patient genetic deficiencies and aberrant patient cell behavior.

Results: Deleterious variants were found at both loci identified by linkage analysis: a homozygous stop codon in PI3-kinase p110δ (PIK3CD) and a homozygous frame shift mutation in SKAP (KNSTRN), both ablating protein expression. Patients with RCS display aberrant B-cell development, similar to p110δ-deficient mice, but also aberrant T-cell spreading, cell-cell interaction, and migration. Patients also display significant developmental abnormalities not seen in p110δ knockouts (eg, optic nerve atrophy and skeletal anomalies) that we ascribe to loss of SKAP. Aberrant SKAP expression can prolong anaphase and this may contribute to developmental defects. However, we also identified microtubule-associated protein 4 microtubule-binding protein as a novel SKAP-binding partner and show that it undergoes relocalization in patient T cells, with associated areas of aberrant microtubule hyperstabilization, likely contributing not only to the altered properties of RCS lymphoid cells but also to developmental defects.

Conclusions: The complex RCS presentation, with combined developmental and immunologic defects, is associated with a combined deficiency of 2 genes products, PI3-kinase p110δ and SKAP, both of which appear to play a significant role in the disease.
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http://dx.doi.org/10.1016/j.jaci.2017.10.033DOI Listing
August 2018

Long-term immune reconstitution after matched unrelated hematopoietic stem cell transplantation for immunodeficiency.

J Allergy Clin Immunol 2018 03 8;141(3):1154-1157.e3. Epub 2017 Nov 8.

Division of Immunology & Allergy, Department of Paediatrics, The Hospital for Sick Children, Toronto, Ontario, Canada; Canadian Centre for Primary Immunodeficiency, Toronto, Ontario, Canada; University of Toronto, Toronto, Ontario, Canada. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2017.10.015DOI Listing
March 2018

Combined immunodeficiency and atopy caused by a dominant negative mutation in caspase activation and recruitment domain family member 11 (CARD11).

J Allergy Clin Immunol 2018 05 19;141(5):1818-1830.e2. Epub 2017 Aug 19.

Division of Immunology and Allergy, Department of Pediatrics, Hospital for Sick Children and the University of Toronto, Toronto, Ontario, Canada; Canadian Centre for Primary Immunodeficiency and the Jeffrey Modell Research Laboratory for the Diagnosis of Primary Immunodeficiency, Hospital for Sick Children, Toronto, Ontario, Canada. Electronic address:

Background: Combined immunodeficiency (CID) is a T-cell defect frequently presenting with recurrent infections, as well as associated immune dysregulation manifesting as autoimmunity or allergic inflammation.

Objective: We sought to identify the genetic aberration in 4 related patients with CID, early-onset asthma, eczema, and food allergies, as well as autoimmunity.

Methods: We performed whole-exome sequencing, followed by Sanger confirmation, assessment of the genetic variant effect on cell signaling, and evaluation of the resultant immune function.

Results: A heterozygous novel c.C88T 1-bp substitution resulting in amino acid change R30W in caspase activation and recruitment domain family member 11 (CARD11) was identified by using whole-exome sequencing and segregated perfectly to family members with severe atopy only but was not found in healthy subjects. We demonstrate that the R30W mutation results in loss of function while also exerting a dominant negative effect on wild-type CARD11. The CARD11 defect altered the classical nuclear factor κB pathway, resulting in poor in vitro T-cell responses to mitogens and antigens caused by reduced secretion of IFN-γ and IL-2.

Conclusion: Unlike patients with biallelic mutations in CARD11 causing severe CID, the R30W defect results in a less profound yet prominent susceptibility to infections, as well as multiorgan atopy and autoimmunity.
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http://dx.doi.org/10.1016/j.jaci.2017.06.047DOI Listing
May 2018

Novel Combined Immune Deficiency and Radiation Sensitivity Blended Phenotype in an Adult with Biallelic Variations in and .

Front Immunol 2017 26;8:576. Epub 2017 May 26.

Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA.

With the advent of high-throughput genomic sequencing techniques, novel genetic etiologies are being uncovered for previously unexplained Mendelian phenotypes, and the underlying genetic architecture of disease is being unraveled. Although most of these "mendelizing" disease traits represent phenotypes caused by single-gene defects, a percentage of patients have blended phenotypes caused by pathogenic variants in multiple genes. We describe an adult patient with susceptibility to bacterial, herpesviral, and fungal infections. Immunologic defects included CD8 T cell lymphopenia, decreased T cell proliferative responses to mitogens, hypogammaglobulinemia, and radiation sensitivity. Whole-exome sequencing revealed compound heterozygous variants in . Biallelic mutations in are known to produce a spectrum of immune deficiency that includes the T cell abnormalities observed in this patient. Analyses for variants in genes associated with radiation sensitivity identified the presence of a homozygous variant of unknown significance. deficiency causes radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties syndrome and may account for the radiation sensitivity. Thus, the patient was found to have a novel blended phenotype associated with multilocus genomic variation: i.e., separate and distinct genetic defects. These findings further illustrate the clinical utility of applying genomic testing in patients with primary immunodeficiency diseases.
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http://dx.doi.org/10.3389/fimmu.2017.00576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445153PMC
May 2017

Hematopoietic stem cell transplantation in patients with gain-of-function signal transducer and activator of transcription 1 mutations.

J Allergy Clin Immunol 2018 02 7;141(2):704-717.e5. Epub 2017 Jun 7.

Canadian Center for Primary Immunodeficiency, Hospital for Sick Children, Toronto, Ontario, Canada.

Background: Gain-of-function (GOF) mutations in signal transducer and activator of transcription 1 (STAT1) cause susceptibility to a range of infections, autoimmunity, immune dysregulation, and combined immunodeficiency. Disease manifestations can be mild or severe and life-threatening. Hematopoietic stem cell transplantation (HSCT) has been used in some patients with more severe symptoms to treat and cure the disorder. However, the outcome of HSCT for this disorder is not well established.

Objective: We sought to aggregate the worldwide experience of HSCT in patients with GOF-STAT1 mutations and to assess outcomes, including donor engraftment, overall survival, graft-versus-host disease, and transplant-related complications.

Methods: Data were collected from an international cohort of 15 patients with GOF-STAT1 mutations who had undergone HSCT using a variety of conditioning regimens and donor sources. Retrospective data collection allowed the outcome of transplantation to be assessed. In vitro functional testing was performed to confirm that each of the identified STAT1 variants was in fact a GOF mutation.

Results: Primary donor engraftment in this cohort of 15 patients with GOF-STAT1 mutations was 74%, and overall survival was only 40%. Secondary graft failure was common (50%), and posttransplantation event-free survival was poor (10% by 100 days). A subset of patients had hemophagocytic lymphohistiocytosis before transplant, contributing to their poor outcomes.

Conclusion: Our data indicate that HSCT for patients with GOF-STAT1 mutations is curative but has significant risk of secondary graft failure and death.
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http://dx.doi.org/10.1016/j.jaci.2017.03.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7802430PMC
February 2018

Hematopoietic stem cell transplantation for RelB deficiency.

J Allergy Clin Immunol 2017 10 26;140(4):1199-1201.e3. Epub 2017 May 26.

Division of Immunology and Allergy, Department of Paediatrics, the Hospital for Sick Children and the University of Toronto, Toronto, Ontario, Canada; Canadian Centre for Primary Immunodeficiency and the Jeffrey Modell Research Laboratory for the Diagnosis of Primary Immunodeficiency, the Hospital for Sick Children and the University of Toronto, Toronto, Ontario, Canada. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2017.05.007DOI Listing
October 2017

Loss of the Arp2/3 complex component ARPC1B causes platelet abnormalities and predisposes to inflammatory disease.

Nat Commun 2017 04 3;8:14816. Epub 2017 Apr 3.

Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 0A4.

Human actin-related protein 2/3 complex (Arp2/3), required for actin filament branching, has two ARPC1 component isoforms, with ARPC1B prominently expressed in blood cells. Here we show in a child with microthrombocytopenia, eosinophilia and inflammatory disease, a homozygous frameshift mutation in ARPC1B (p.Val91Trpfs*30). Platelet lysates reveal no ARPC1B protein and greatly reduced Arp2/3 complex. Missense ARPC1B mutations are identified in an unrelated patient with similar symptoms and ARPC1B deficiency. ARPC1B-deficient platelets are microthrombocytes similar to those seen in Wiskott-Aldrich syndrome that show aberrant spreading consistent with loss of Arp2/3 function. Knockout of ARPC1B in megakaryocytic cells results in decreased proplatelet formation, and as observed in platelets from patients, increased ARPC1A expression. Thus loss of ARPC1B produces a unique set of platelet abnormalities, and is associated with haematopoietic/immune symptoms affecting cell lineages where this isoform predominates. In agreement with recent experimental studies, our findings suggest that ARPC1 isoforms are not functionally interchangeable.
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http://dx.doi.org/10.1038/ncomms14816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382316PMC
April 2017

Long-term outcomes of 176 patients with X-linked hyper-IgM syndrome treated with or without hematopoietic cell transplantation.

J Allergy Clin Immunol 2017 Apr 30;139(4):1282-1292. Epub 2016 Sep 30.

Royal Free Hospital, London, United Kingdom.

Background: X-linked hyper-IgM syndrome (XHIGM) is a primary immunodeficiency with high morbidity and mortality compared with those seen in healthy subjects. Hematopoietic cell transplantation (HCT) has been considered a curative therapy, but the procedure has inherent complications and might not be available for all patients.

Objectives: We sought to collect data on the clinical presentation, treatment, and follow-up of a large sample of patients with XHIGM to (1) compare long-term overall survival and general well-being of patients treated with or without HCT along with clinical factors associated with mortality and (2) summarize clinical practice and risk factors in the subgroup of patients treated with HCT.

Methods: Physicians caring for patients with primary immunodeficiency diseases were identified through the Jeffrey Modell Foundation, United States Immunodeficiency Network, Latin American Society for Immunodeficiency, and Primary Immune Deficiency Treatment Consortium. Data were collected with a Research Electronic Data Capture Web application. Survival from time of diagnosis or transplantation was estimated by using the Kaplan-Meier method compared with log-rank tests and modeled by using proportional hazards regression.

Results: Twenty-eight clinical sites provided data on 189 patients given a diagnosis of XHIGM between 1964 and 2013; 176 had valid follow-up and vital status information. Sixty-seven (38%) patients received HCT. The average follow-up time was 8.5 ± 7.2 years (range, 0.1-36.2 years). No difference in overall survival was observed between patients treated with or without HCT (P = .671). However, risk associated with HCT decreased for diagnosis years 1987-1995; the hazard ratio was significantly less than 1 for diagnosis years 1995-1999. Liver disease was a significant predictor of overall survival (hazard ratio, 4.9; 95% confidence limits, 2.2-10.8; P < .001). Among survivors, those treated with HCT had higher median Karnofsky/Lansky scores than those treated without HCT (P < .001). Among patients receiving HCT, 27 (40%) had graft-versus-host disease, and most deaths occurred within 1 year of transplantation.

Conclusion: No difference in survival was observed between patients treated with or without HCT across all diagnosis years (1964-2013). However, survivors treated with HCT experienced somewhat greater well-being, and hazards associated with HCT decreased, reaching levels of significantly less risk in the late 1990s. Among patients treated with HCT, treatment at an early age is associated with improved survival. Optimism remains guarded as additional evidence accumulates.
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http://dx.doi.org/10.1016/j.jaci.2016.07.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5374029PMC
April 2017

Bone marrow transplantation for monoallelic signal transducer and activator of transcription 1 deficiency.

J Allergy Clin Immunol 2016 08 6;138(2):612-615.e1. Epub 2016 Apr 6.

Division of Immunology and Allergy, Department of Pediatrics, Toronto, Ontario, Canada; Hospital for Sick Children and the University of Toronto, Toronto, Ontario, Canada.

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http://dx.doi.org/10.1016/j.jaci.2016.02.009DOI Listing
August 2016

Variants in TRIM22 That Affect NOD2 Signaling Are Associated With Very-Early-Onset Inflammatory Bowel Disease.

Gastroenterology 2016 05 4;150(5):1196-1207. Epub 2016 Feb 4.

Gastroenterology Department, The Children's Hospital at Westmead, Westmead, New South Wales, Australia; The James Fairfax Institute of Paediatric Nutrition, University of Sydney, New South Wales, Australia.

Background & Aims: Severe forms of inflammatory bowel disease (IBD) that develop in very young children can be caused by variants in a single gene. We performed whole-exome sequence (WES) analysis to identify genetic factors that might cause granulomatous colitis and severe perianal disease, with recurrent bacterial and viral infections, in an infant of consanguineous parents.

Methods: We performed targeted WES analysis of DNA collected from the patient and her parents. We validated our findings by a similar analysis of DNA from 150 patients with very-early-onset IBD not associated with known genetic factors analyzed in Toronto, Oxford, and Munich. We compared gene expression signatures in inflamed vs noninflamed intestinal and rectal tissues collected from patients with treatment-resistant Crohn's disease who participated in a trial of ustekinumab. We performed functional studies of identified variants in primary cells from patients and cell culture.

Results: We identified a homozygous variant in the tripartite motif containing 22 gene (TRIM22) of the patient, as well as in 2 patients with a disease similar phenotype. Functional studies showed that the variant disrupted the ability of TRIM22 to regulate nucleotide binding oligomerization domain containing 2 (NOD2)-dependent activation of interferon-beta signaling and nuclear factor-κB. Computational studies demonstrated a correlation between the TRIM22-NOD2 network and signaling pathways and genetic factors associated very early onset and adult-onset IBD. TRIM22 is also associated with antiviral and mycobacterial effectors and markers of inflammation, such as fecal calprotectin, C-reactive protein, and Crohn's disease activity index scores.

Conclusions: In WES and targeted exome sequence analyses of an infant with severe IBD characterized by granulomatous colitis and severe perianal disease, we identified a homozygous variant of TRIM22 that affects the ability of its product to regulate NOD2. Combined computational and functional studies showed that the TRIM22-NOD2 network regulates antiviral and antibacterial signaling pathways that contribute to inflammation. Further study of this network could lead to new disease markers and therapeutic targets for patients with very early and adult-onset IBD.
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http://dx.doi.org/10.1053/j.gastro.2016.01.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4842103PMC
May 2016

Compound heterozygous mutations in the noncoding RNU4ATAC cause Roifman Syndrome by disrupting minor intron splicing.

Nat Commun 2015 Nov 2;6:8718. Epub 2015 Nov 2.

The Centre for Applied Genomics (TCAG), Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Ontario, Canada M5G 0A4.

Roifman Syndrome is a rare congenital disorder characterized by growth retardation, cognitive delay, spondyloepiphyseal dysplasia and antibody deficiency. Here we utilize whole-genome sequencing of Roifman Syndrome patients to reveal compound heterozygous rare variants that disrupt highly conserved positions of the RNU4ATAC small nuclear RNA gene, a minor spliceosome component that is essential for minor intron splicing. Targeted sequencing confirms allele segregation in six cases from four unrelated families. RNU4ATAC rare variants have been recently reported to cause microcephalic osteodysplastic primordial dwarfism, type I (MOPD1), whose phenotype is distinct from Roifman Syndrome. Strikingly, all six of the Roifman Syndrome cases have one variant that overlaps MOPD1-implicated structural elements, while the other variant overlaps a highly conserved structural element not previously implicated in disease. RNA-seq analysis confirms extensive and specific defects of minor intron splicing. Available allele frequency data suggest that recessive genetic disorders caused by RNU4ATAC rare variants may be more prevalent than previously reported.
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http://dx.doi.org/10.1038/ncomms9718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4667643PMC
November 2015

The effects of RelB deficiency on lymphocyte development and function.

J Autoimmun 2015 Dec 15;65:90-100. Epub 2015 Sep 15.

The Canadian Centre for Primary Immunodeficiency, Immunogenomic Laboratory, Division of Immunology/Allergy, Department of Pediatrics, The Hospital for Sick Children and the University of Toronto, 555 University Ave., Toronto, On M5G 1X8, Canada. Electronic address:

Multiple receptors that control cell growth and inflammation activate the NFκB pathway that comprises of two pathways. Dysfunction of the classical pathway leads to impaired adaptive and innate immunity in humans. In contrast the exact role of the alternative NFκB pathway mediated by RelB in humans remains largely elusive. We have recently identified deleterious mutations in RelB in patients with combined immunodeficiency and autoimmunity. We studied here the biological effects of RelB deficiency on the immune system. We show that the thymus in this patient is dysplastic and consequently new thymus emigrants are rare and there is an accumulation of CD45 RO(+) T cells with an increase in CD62L(+) central memory cells. The TCR repertoire of these cells appears skewed with selective clonal expansion. In vitro responses to T cell mitogens were markedly depressed and so were PHA induced IL2 and IFNγ production. In addition, the TH1 promoting T bet and STAT1 were reduced. In contrast, hyper-activation was seen in response to anti-CD3 and CD28. T cell dependent antibody responses were low to absent in all patients. We found that BAFF-R was reduced and CD40 signaling aberrant. Critically, CD27(+) memory cells were absent. We have shown here for the first time the role of RelB on lymphocyte development in humans. In the absence of RelB, B cells development is arrested, resulting in poor production of immunoglobulins and specific antibodies. T cell maturation in the thymus appears altered with reduced output and production of a skewed T cell repertoire with expansion of clones which are likely the cause of the autoimmune features observed in these patients.
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http://dx.doi.org/10.1016/j.jaut.2015.09.001DOI Listing
December 2015

Mutations in Plasmalemma Vesicle Associated Protein Result in Sieving Protein-Losing Enteropathy Characterized by Hypoproteinemia, Hypoalbuminemia, and Hypertriglyceridemia.

Cell Mol Gastroenterol Hepatol 2015 Jul;1(4):381-394.e7

SickKids Inflammatory Bowel Disease Center and Cell Biology Program, Research Institute, Hospital for Sick Children, Toronto, ON, Canada ; Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of Toronto, Hospital for Sick Children, Toronto, ON, Canada ; Institute of Medical Science, University of Toronto, Toronto, ON, Canada ; Department of Biochemistry, University of Toronto, Toronto, ON, Canada.

Background & Aims Methods: Severe intestinal diseases observed in very young children are often the result of monogenic defects. We used whole exome sequencing (WES) to examine the genetic cause in a patient with a distinct severe form of protein losing enteropathy (PLE) characterized by hypoproteinemia, hypoalbuminemia, and hypertriglyceridemia.

Methods: WES was performed at the Centre for Applied Genomics, Hospital for Sick Children, Toronto, Canada. Exome library preparation was performed using the Ion Torrent AmpliSeq RDY Exome Kit. Functional studies were carried out based on the identified mutation.

Results: Using whole exome sequencing we identified a homozygous nonsense mutation (1072C>T; p.Arg358*) in the (plasmalemma vesicle associated protein) gene in an infant from consanguineous parents who died at five months of age of severe protein losing enteropathy. Functional studies determined that the mutated PLVAP mRNA and protein were not expressed in the patient biopsy tissues, presumably secondary to nonsense-mediated mRNA decay. Pathological analysis showed that the loss of PLVAP resulted in disruption of endothelial fenestrated diaphragms.

Conclusions: PLVAP p.Arg358* mutation resulted in loss of PLVAP expression with subsequent deletion of the diaphragms of endothelial fenestrae leading to plasma protein extravasation, protein-losing enteropathy and ultimately death.
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http://dx.doi.org/10.1016/j.jcmgh.2015.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4507283PMC
July 2015

Patients with T⁺/low NK⁺ IL-2 receptor γ chain deficiency have differentially-impaired cytokine signaling resulting in severe combined immunodeficiency.

Eur J Immunol 2014 Oct 28;44(10):3129-40. Epub 2014 Aug 28.

Center for Chronic Immunodeficiency (CCI), University Medical Center and University of Freiburg, Freiburg, Germany; Faculty of Biology, University of Freiburg, Freiburg, Germany.

X-linked severe combined immunodeficiency (X-SCID) leads to a T(-) NK(-) B(+) immunophenotype and is caused by mutations in the gene encoding the IL-2 receptor γ-chain (IL2RG). IL2RG(R222C) leads to atypical SCID with a severe early onset phenotype despite largely normal NK- and T-cell numbers. To address this discrepancy, we performed a detailed analysis of T, B, and NK cells, including quantitative STAT phosphorylation and functional responses to the cytokines IL-2, IL-4, IL-15, and IL-21 in a patient with the IL2RG(R222C) mutation. Moreover, we identified nine additional unpublished patients with the same mutations, all with a full SCID phenotype, and confirmed selected immunological observations. T-cell development was variably affected, but led to borderline T-cell receptor excision circle (TREC) levels and a normal repertoire. T cells showed moderately reduced proliferation, failing enhancement by IL-2. While NK-cell development was normal, IL-2 enhancement of NK-cell degranulation and IL-15-induced cytokine production were absent. IL-2 or IL-21 failed to enhance B-cell proliferation and plasmablast differentiation. These functional alterations were reflected by a differential impact of IL2RG(R222C) on cytokine signal transduction, with a gradient IL-4
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http://dx.doi.org/10.1002/eji.201444689DOI Listing
October 2014

Pulmonary alveolar proteinosis in adenosine deaminase-deficient mice.

J Allergy Clin Immunol 2014 May 15;133(5):1467-71, 1471.e1-4. Epub 2014 Jan 15.

Developmental & Stem Cell Biology Program, Hospital for Sick Children, Toronto, Ontario, Canada; Division of Immunology and Allergy, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2013.11.029DOI Listing
May 2014

Fatal combined immunodeficiency associated with heterozygous mutation in STAT1.

J Allergy Clin Immunol 2014 Mar 13;133(3):807-17. Epub 2013 Nov 13.

Division of Immunology and Allergy, the Canadian Centre for Primary Immunodeficiency, the Jeffrey Modell Research Laboratory for the Diagnosis of Primary Immunodeficiency, the Hospital for Sick Children and the University of Toronto, Toronto, Canada; Canada-Israel Immunodeficiency Research Alliance, Toronto, Ontario, Canada. Electronic address:

Background: Mutations in the gene for the signal transducer and activator of transcription 1, STAT1, have been shown to be associated with death at an early age due to overwhelming viral infection (complete STAT1 deficiency) or, more commonly, selective deficiencies to mycobacterial or fungal infection (typically heterozygous STAT1 mutations).

Objectives: To define the molecular basis of progressive combined immunodeficiency in a group of patients with fatal infections.

Methods: We studied a group of unrelated patients who displayed an unusual progressive form of combined immunodeficiency. Whole exome sequencing assisted in confirming a common genetic defect in this group, which consisted of a heterozygous mutation of the STAT1 gene. STAT1 protein level as well as function was assessed, and a detailed evaluation of the immune system, including analysis of thymus tissue, was performed.

Results: Patients were found to carry de novo heterozygous mutations in STAT1 encoding T385A, I294T, or C284R amino acid substitutions. STAT1 expression appeared significantly decreased as a result of these changes but not completely absent, with diminished signaling responses. This group display progressive loss in lymphocyte number and function accompanied by increasing autoimmune features as well as severe, fatal infections.

Conclusions: These findings show that some heterozygous aberrations of STAT1 can be associated with progressive combined immunodeficiency, quite distinct from the limited susceptibilities to infection previously reported for heterozygous STAT1 mutations. These mutations were not inherited, rather, arose de novo in each case. Accompanied by significant patient mortality, this finding suggests that this class of STAT1 mutation is ultimately fatal due to overwhelming infection.
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http://dx.doi.org/10.1016/j.jaci.2013.09.032DOI Listing
March 2014

IL-21 signalling via STAT3 primes human naive B cells to respond to IL-2 to enhance their differentiation into plasmablasts.

Blood 2013 Dec 24;122(24):3940-50. Epub 2013 Oct 24.

Immunology Research Program, Garvan Institute of Medical Research, Darlinghurst, NSW, Australia;

B-cell responses are guided by the integration of signals through the B-cell receptor (BCR), CD40, and cytokine receptors. The common γ chain (γc)-binding cytokine interleukin (IL)-21 drives humoral immune responses via STAT3-dependent induction of transcription factors required for plasma cell generation. We investigated additional mechanisms by which IL-21/STAT3 signaling modulates human B-cell responses by studying patients with STAT3 mutations. IL-21 strongly induced CD25 (IL-2Rα) in normal, but not STAT3-deficient, CD40L-stimulated naïve B cells. Chromatin immunoprecipitation confirmed IL2RA as a direct target of STAT3. IL-21-induced CD25 expression was also impaired on B cells from patients with IL2RG or IL21R mutations, confirming a requirement for intact IL-21R signaling in this process. IL-2 increased plasmablast generation and immunoglobulin secretion from normal, but not CD25-deficient, naïve B cells stimulated with CD40L/IL-21. IL-2 and IL-21 were produced by T follicular helper cells, and neutralizing both cytokines abolished the B-cell helper capacity of these cells. Our results demonstrate that IL-21, via STAT3, sensitizes B cells to the stimulatory effects of IL-2. Thus, IL-2 may play an adjunctive role in IL-21-induced B-cell differentiation. Lack of this secondary effect of IL-21 may amplify the humoral immunodeficiency in patients with mutations in STAT3, IL2RG, or IL21R due to impaired responsiveness to IL-21.
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http://dx.doi.org/10.1182/blood-2013-06-506865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3854113PMC
December 2013