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BACKGROUND: Pediatric endogenous Cushing syndrome is mainly caused by pituitary corticotropin-producing adenomas and most glucocorticoid-dependent effects progressively regress upon tumor removal. Endogenous Cushing syndrome reproduces long-term high-dose glucocorticoid therapy, representing a clean, natural and unbiased model in which to study glucocorticoid bona-fide effects on immunity. OBJECTIVES: To perform extensive immunologic studies in otherwise healthy pediatric patients with endogenous Cushing syndrome before and 6–13 months after tumor resection, as well as in in-vitro glucocorticoid-treated control cells. METHODS: Flow cytometry, immunoblotting, ELISA, qRT-PCR and RNA-seq techniques were used to characterize patients’ and in-vitro glucocorticoid treated cells. RESULTS: Reduced thymic output, decreased naïve T cells, diminished proliferation, and increased T-cell apoptosis were detected before surgery, and all these defects eventually normalized after tumor removal in patients. In-vitro studies also showed increased T-cell apoptosis, with correspondingly diminished NF-κB signaling and IL-21 levels. In this setting, IL-21 addition upregulated anti-apoptotic BCL2 expression and rescued T-cell apoptosis in a PI3K pathway-dependent manner. Similar and reproducible findings were confirmed in endogenous Cushing syndrome patient cells as well. CONCLUSIONS: We identified decreased thymic output and lymphocyte proliferation, together with increased apoptosis, as the underlying causes to T-cell lymphopenia in endogenous Cushing syndrome patients. IL-21 was decreased in both natural and in-vitro long-term high-dose glucocorticoid environments, and in-vitro addition of IL-21 counteracted the pro-apoptotic effects of glucocorticoid therapy. Thus, our results suggest that administration of IL-21 in patients receiving long-term high-dose glucocorticoid therapy may contribute to ameliorate lymphopenia and the complications associated to it.

BACKGROUND: CARD9 deficiency is an autosomal recessive primary immunodeficiency underlying increased susceptibility to fungal infection primarily presenting as invasive CNS Candida and/or cutaneous/invasive dermatophyte infections. More recently, a rare heterozygous dominant negative CARD9 variant c.1434 + 1G > C was reported to be protective from inflammatory bowel disease. OBJECTIVE: We studied two siblings carrying homozygous CARD9 variants (c.1434 + 1G > C) and born to heterozygous asymptomatic parents. One sibling was asymptomatic and the other presented with candida esophagitis, upper respiratory infections, hypogammaglobulinemia, and low class-switched memory B cells. METHODS AND RESULTS: The CARD9 c.1434 + 1G > C variant generated two mutant transcripts confirmed by mRNA and protein expression: an out-of-frame c.1358–1434 deletion/ ~ 55 kDa protein (CARD9Δex.11) and an in-frame c.1417–1434 deletion/ ~ 61 kDa protein (CARD9Δ18 nt.). Neither transcript was able to form a complete/functional CBM complex, which includes TRIM62. Based on the index patient’s CVID-like phenotype, CARD9 expression was tested and detected in lymphocytes and monocytes from humans and mice. The functional impact of different CARD9 mutations and gene dosage conditions was evaluated in heterozygous and homozygous c.1434 + 1 G > C members of the index family, and in WT (two WT alleles), haploinsufficiency (one WT, one null allele), and null (two null alleles) individuals. CARD9 gene dosage impacted lymphocyte and monocyte functions including cytokine generation, MAPK activation, T-helper commitment, transcription, plasmablast differentiation, and immunoglobulin production in a differential manner. CONCLUSIONS: CARD9 exon 11 integrity is critical to CBM complex function. CARD9 is expressed and affects particular T and B cell functions in a gene dosage-dependent manner, which in turn may contribute to the phenotype of CARD9 deficiency.

Background Diethylcarbamazine citrate (DEC) treatment of loiasis is complicated by adverse reactions that are correlated with the number of circulating microfilariae (mf). The cause of these reactions is unknown, but they are accompanied by a dramatic interleukin-5 (IL-5)-dependent increase in eosinophilia and evidence of eosinophil activation. Methods To explore the role of IL-5 driven eosinophilia in post-DEC reactions, 8 adults with confirmed loiasis and Results Baseline characteristics were comparable between the two groups. Single dose reslizumab lowered the AEC by 77% prior to initiation of DEC therapy (vs. 12% in the placebo group, P Conclusions In summary, although reslizumab was able to blunt peripheral eosinophilia post-DEC treatment in subjects with loiasis and had no effect on microfilarial clearance, the reduction in AEC appeared to have been insufficient to prevent post-treatment AEs.

Germline gain-of-function (GOF) mutation of the signal transducer and activator of transcription 3 (STAT3) gene causes a disease clinically characterized by a significant lymphoproliferation, including lymphadenopathy and/or hepatosplenomegaly, as well as childhood onset autoimmunity. Here we present an adult patient who, during his early years of life, presented recurrent infections, autoimmune hemolytic anemia and benign lymphoproliferative disease, characterized by hepatosplenomegaly and lymphadenopathy, being diagnosed with common variable immunodeficiency (CVID) at 13 years of age. He was diagnosed with lymphocytic interstitial pneumonia at the age of 20. When he was 40 years old, after a diagnostic review, it was decided to perform genetic studies. A heterozygous mutation in STAT3 NM_003150 c.2141CT, p.P714L was detected by whole exome sequencing and validated by Sanger. Previously published functional studies performed in two siblings showed that this mutation resulted in gain-of-function. They were initially diagnosed with autoimmune lymphoproliferative syndrome, and later with STAT3 GOF as a second genetic defect. Our patient developed severe pulmonary disease and died, without access to treatment targeted to his molecular defect due to the advanced nature of his pulmonary involvement and the fact that many of the therapies were still in development at that time. The diagnosis of STAT3 GOF mutations should be suspected in patients with early-onset of lymphoproliferative disease, autoimmunity and hypogammaglobulinemia. This must be considered especially in the group of CVID patients with these characteristics, in order to allow the implementation of treatments targeting the molecular defect (JAK inhibitors and Il-6 receptor antagonists) that could modify the disease evolution.Mutaciones en línea germinal con ganancia de función (GOF) del gen transductor de señales y activador de la transcripción 3 (STAT3) provocan una enfermedad caracterizada por importante linfoproliferación, incluyendo linfadenopatías y/o hepatoesplenomegalia, así como autoinmunidad de inicio en la infancia. Presentamos un paciente adulto que, durante sus primeros años de vida, presentó infecciones recurrentes, anemia hemolítica autoinmune y enfermedad linfoproliferativa benigna, caracterizada inicialmente por hepatoesplenomegalia y linfoadenopatías, diagnosticado de inmunodeficiencia común variable (IDCV) a los 13 años. A los 20 años, al ser estudiado por compromiso pulmonar, se diagnosticó neumonía intersticial linfocítica. A los 40 años, tras revisión diagnóstica se decidió realizar estudios genéticos. Por secuenciación del exoma completo se detectó una mutación heterocigota en STAT3 NM_003150 c.2141CT, p.P714L, que se validó por Sanger. Estudios funcionales previamente publicados realizados en dos hermanos con diagnóstico inicial de síndrome linfoproliferativo autoinmune, mostraron que esta mutación daba lugar a una ganancia de función. Nuestro paciente desarrolló enfermedad pulmonar grave y falleció a los 41 años, sin posibilidad de acceder a tratamiento dirigido a su defecto molecular por lo avanzado de su compromiso pulmonar y a que muchas de las terapias se encontraban en ese momento en desarrollo. El diagnóstico de mutaciones STAT3 GOF debe sospecharse en pacientes con enfermedad linfoproliferativa temprana, autoinmunidad e hipogammaglobulinemia. Esto debe ser considerado especialmente en pacientes con IDCV con estas carac terísticas, para permitir la implementación de tratamientos dirigidos al defecto molecular (inhibidores de JAK y antagonistas del receptor de Il-6) que podrían modificar la evolución de la enfermedad.

Sterile alpha motif (SAM) and Src homology-3 (SH3) domain-containing 3 (SASH3), also called SH3-containing lymphocyte protein (SLY1), is a putative adaptor protein that is postulated to play an important role in the organization of signaling complexes and propagation of signal transduction cascades in lymphocytes. The SASH3 gene is located on the X-chromosome. Here, we identified 3 novel SASH3 deleterious variants in 4 unrelated male patients with a history of combined immunodeficiency and immune dysregulation that manifested as recurrent sinopulmonary, cutaneous, and mucosal infections and refractory autoimmune cytopenias. Patients exhibited CD4+ T-cell lymphopenia, decreased T-cell proliferation, cell cycle progression, and increased T-cell apoptosis in response to mitogens. In vitro T-cell differentiation of CD34+ cells and molecular signatures of rearrangements at the T-cell receptor α (TRA) locus were indicative of impaired thymocyte survival. These patients also manifested neutropenia and B-cell and natural killer (NK)-cell lymphopenia. Lentivirus-mediated transfer of the SASH3 complementary DNA–corrected protein expression, in vitro proliferation, and signaling in SASH3-deficient Jurkat and patient-derived T cells. These findings define a new type of X-linked combined immunodeficiency in humans that recapitulates many of the abnormalities reported in mice with Sly1–/– and Sly1Δ/Δ mutations, highlighting an important role of SASH3 in human lymphocyte function and survival.

Interferon gamma receptor-1 (IFNγR1) deficiency is a rare inborn error of immunity. It can be inherited as an autosomal recessive (AR), partial or complete, or autosomal dominant (AD) trait and exhibits high allelic variability. Biallelic deleterious recessive variants of IFNGR1 that result in absent expression or defective downstream signaling of IFNγR1, cause failure to activate macrophages in response to interferon-gamma (IFN-γ), rendering innate immunity ineffective in clearing intracellular pathogens.

Patients with the monogenic immune dysregulatory syndrome autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), which is caused by loss-of-function mutations in the autoimmune regulator (AIRE) gene, uniformly carry neutralizing autoantibodies directed against type-I interferons (IFNs) and many develop autoimmune pneumonitis, both of which place them at high risk for life-threatening COVID-19 pneumonia. Bamlanivimab and etesevimab are monoclonal antibodies (mAbs) that target the SARS-CoV-2 spike protein and block entry of SARS-CoV-2 in host cells. The use of bamlanivimab and etesevimab early during infection was associated with reduced COVID-19–associated hospitalization and death in patients at high risk for progressing to severe disease, which led the US Food and Drug Administration to issue an emergency use authorization for their administration in non-hypoxemic, non-hospitalized high-risk patients. However, the safety and efficacy of these mAbs has not been evaluated in APECED patients. We enrolled two siblings with APECED on an IRB-approved protocol (NCT01386437) and admitted them prophylactically at the NIH Clinical Center for evaluation of mild-to-moderate COVID-19. We assessed the safety and clinical effects of early treatment with bamlanivimab and etesevimab. The administration of bamlanivimab and etesevimab was well tolerated and was associated with amelioration of COVID-19 symptoms and prevention of invasive ventilatory support, admission to the intensive care, and death in both patients without affecting the production of antibodies to the nucleocapsid protein of SARS-CoV-2. If given early in the course of COVID-19 infection, bamlanivimab and etesevimab may be beneficial in APECED and other high-risk patients with neutralizing autoantibodies directed against type-I IFNs.

The noncanonical NF-κB pathway is implicated in diverse biological and immunological processes. Monoallelic C-terminus loss-of-function and gain-of-function mutations of NFKB2 have been recently identified as a cause of immunodeficiency manifesting with common variable immunodeficiency (CVID) or combined immunodeficiency (CID) phenotypes. Herein we report a family carrying a heterozygous nonsense mutation in NFKB2 (c.809G A, p.W270*). This variant is associated with increased mRNA decay and no mutant NFKB2 protein expression, leading to NFKB2 haploinsufficiency. Our findings demonstrate that bona fide NFKB2 haploinsufficiency, likely caused by mutant mRNA decay and protein instability leading to the transcription and expression of only the wild-type allele, is associated with clinical immunodeficiency, although with incomplete clinical penetrance. Abnormal B cell development, hypogammaglobulinemia, poor antibody response, and abnormal noncanonical (but normal canonical) NF-κB pathway signaling are the immunologic hallmarks of this disease. This adds a third allelic variant to the pathophysiology of NFKB2-mediated immunodeficiency disorders.

Summary We describe the first case of MAN2B2 deficiency in a patient with immune dysregulation, developmental delay, and stroke. Altered mannosylation profile was restored in patient cells upon transduction of wild-type MAN2B2.

The noncanonical NF-κB pathway is implicated in diverse biological and immunological processes. Monoallelic C-terminus loss-of-function and gain-of-function mutations of NFKB2 have been recently identified as a cause of immunodeficiency manifesting with common variable immunodeficiency (CVID) or combined immunodeficiency (CID) phenotypes. Herein we report a family carrying a heterozygous nonsense mutation in NFKB2 (c.809G > A, p.W270*). This variant is associated with increased mRNA decay and no mutant NFKB2 protein expression, leading to NFKB2 haploinsufficiency. Our findings demonstrate that bona fide NFKB2 haploinsufficiency, likely caused by mutant mRNA decay and protein instability leading to the transcription and expression of only the wild-type allele, is associated with clinical immunodeficiency, although with incomplete clinical penetrance. Abnormal B cell development, hypogammaglobulinemia, poor antibody response, and abnormal noncanonical (but normal canonical) NF-κB pathway signaling are the immunologic hallmarks of this disease. This adds a third allelic variant to the pathophysiology of NFKB2-mediated immunodeficiency disorders.

Background Chronic active Epstein-Barr virus (CAEBV) presents with high levels of viral genomes in blood and tissue infiltration with Epstein-Barr virus (EBV)–positive lymphocytes. The pathogenesis of CAEBV is poorly understood. Methods We evaluated 2 patients with natural killer (NK) cell CAEBV and studied their NK cell phenotype and signaling pathways in cells. Results Both patients had increased numbers of NK cells, EBV predominantly in NK cells, and immature NK cells in the blood. Both patients had increased phosphorylation of Akt, S6, and STAT1 in NK cells, and increased total STAT1. Treatment of 1 patient with sirolimus reduced phosphorylation of S6 in T and B cells, but not in NK cells and did not reduce levels of NK cells or EBV DNA in the blood. Treatment of both patients’ cells with JAK inhibitors in vitro reduced phosphorylated STAT1 to normal. Patients with T- or B-cell CAEBV had increased phosphorylation of Akt and S6 in NK cells, but no increase in total STAT1. Conclusions The increase in phosphorylated Akt, S6, and STAT1, as well as immature NK cells describe a new phenotype for NK cell CAEBV. The reduction of STAT1 phosphorylation in their NK cells with JAK inhibitors suggests a novel approach to therapy.

NF-κB signaling through its NFKB1-dependent canonical and NFKB2-dependent noncanonical pathways plays distinctive roles in a diverse range of immune processes. Recently, mutations in these 2 genes have been associated with common variable immunodeficiency (CVID). While studying patients with genetically uncharacterized primary immunodeficiencies, we detected 2 novel nonsense gain-of-function (GOF) NFKB2 mutations (E418X and R635X) in 3 patients from 2 families, and a novel missense change (S866R) in another patient. Their immunophenotype was assessed by flow cytometry and protein expression; activation of canonical and noncanonical pathways was examined in peripheral blood mononuclear cells and transfected HEK293T cells through immunoblotting, immunohistochemistry, luciferase activity, real-time polymerase chain reaction, and multiplex assays. The S866R change disrupted a C-terminal NF-κΒ2 critical site affecting protein phosphorylation and nuclear translocation, resulting in CVID with adrenocorticotropic hormone deficiency, growth hormone deficiency, and mild ectodermal dysplasia as previously described. In contrast, the nonsense mutations E418X and R635X observed in 3 patients led to constitutive nuclear localization and activation of both canonical and noncanonical NF-κΒ pathways, resulting in a combined immunodeficiency (CID) without endocrine or ectodermal manifestations. These changes were also found in 2 asymptomatic relatives. Thus, these novel NFKB2 GOF mutations produce a nonfully penetrant CID phenotype through a different pathophysiologic mechanism than previously described for mutations in NFKB2.

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Through next-generation sequencing on a cohort of patients with severe atopic dermatitis with and without comorbid infections, we found eight individuals, from four families, with novel heterozygous mutations in CARD11, which encodes a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant CARD11 expression constructs into T cell lines demonstrated both loss-of-function and dominant-interfering activity upon antigen receptor-induced activation of nuclear factor-κB and mammalian target of rapamycin complex 1 (mTORC1). Patient T cells had similar defects, as well as low production of the cytokine interferon-γ (IFN-γ). The mTORC1 and IFN-γ production defects were partially rescued by supplementation with glutamine, which requires CARD11 for import into T cells. Our findings indicate that a single hypomorphic mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.

To the editor: Somatic, gain-of-function mutations in STAT3 and STAT5 have been described in leukemias and lymphomas[1][1][⇓][2][⇓][3][⇓][4][⇓][5]-[6][6] and, more recently, in CD3-CD4+ T cells from a patient with lymphocytic variant hypereosinophilic syndrome[7][7]; whereas germ line gain

Autoimmune lymphoproliferative syndrome (ALPS) is caused by germline or somatic loss of function FAS mutations resulting in impaired apoptosis and consequent expansion of T-lymphocytes causing organomegaly and autoimmune anemia, neutropenia and thrombocytopenia. Herein, we report on a case of disseminated varicella zoster infection after post-partum vaccination in a patient found to have CD4 lymphopenia and eventually diagnosed with ALPS caused by a novel germline missense mutation in FAS death-domain. A subsequent retrospective analysis of 169 patients of the NIH ALPS-FAS cohort, revealed that CD4-T-cells lymphopenia (< 300 cells/μl) may occur in 5% of ALPS-FAS patients irrespectively of the underlying genetic defect, organomegaly or immunosuppressive treatment. Although immunophenotyping did not show depletion of specific CD4-T-cells subpopulations, CD4-lymphopenic ALPS-FAS subjects had an expansion of a subset of circulating T-follicular-helper (cTfh) cells, associated with autoantibody production (CCR7lowPD-1high). Furthermore, autoantibodies binding on CD4-T-cells were detected in 50% of the CD4-lymphopenic ALPS-FAS patients and caused cytotoxicity in a natural killer (NK)-mediated antibody-dependent-cellular cytotoxicity assay. Such autoantibodies can therefore be associated with CD4-T-cell death, impaired activation induced proliferation or impaired trafficking. The expansion of autoreactive T-cells in ALPS-FAS is known to be associated with autoimmune clinical manifestations, however our study reveals that ALPS-FAS can also be associated with a paradoxical depletion of CD4-T-cells due to the presence of autoantibodies on the surface of CD4-T-cells which can in turn result in increased susceptibility to opportunistic infections. These novel findings have implications for the diagnosis, clinical monitoring, and management of patients with ALPS-FAS.

Background: HOIP is the catalytic subunit of the linear ubiquitination chain assembly complex (LUBAC) that is essential for NF-κB signaling and thus proper innate and adaptive immunity. To date only one patient with HOIP deficiency has been reported with clinical characteristics that include autoinflammation, immunodeficiency, amylopectinosis, and systemic lymphangiectasia. Case: We sought to identify a genetic cause of a disease for an 8 year-old girl who presented with early-onset immune deficiency and autoinflammation. Methods: Targeted next generation sequencing of 352 immune-related genes was performed. Functional studies included transcriptome analysis, cytokine profiling, and protein analysis in patients' primary cells. Results: We identified biallelic variants in close proximity to splice sites (c.1197G>C and c.1737+3A>G) in the RNF31 gene. RNA extracted from patient cells showed alternatively spliced transcripts not present in control cells. Protein expression of HOIP and LUBAC was reduced in primary cells as shown by western blotting. Patient-derived fibroblasts demonstrated attenuated IL-6 production, while PBMCs showed higher TNF production after stimulation with proinflammatory cytokines. RNA sequencing of whole blood RNA and PBMCs demonstrated a marked transcriptome wide change including differential expression of type I interferon regulated genes. Conclusion: We report the second case of HOIP deficiency with novel compound heterozygous mutations in RNF31 and distinct clinical and molecular features. Our results expand on the clinical spectrum of HOIP deficiency and molecular signatures associated with LUBAC deficiency.

Severe combined immunodeficiency (SCID) represents one of the most severe forms of primary immunodeficiency (PID) disorders characterized by impaired cellular and humoral immune responses. Here, we report the clinical, immunological, and molecular findings in 57 patients diagnosed with SCID from India. Majority of our patients (89%) presented within 6 months of age. The most common clinical manifestations observed were recurrent pneumonia (66%), failure to thrive (60%), chronic diarrhea (35%), gastrointestinal infection (21%), and oral candidiasis (21%). Hematopoietic Stem Cell Transplantation (HSCT) is the only curative therapy available for treating these patients. Four patients underwent HSCT in our cohort but had a poor survival outcome. Lymphopenia (absolute lymphocyte counts/μL

Phosphoinositide 3-kinase (PI3K) plays an integral role in lymphocyte function. Mutations in PIK3CD and PIK3R1, encoding the PI3K p110δ and p85α subunits, respectively, cause increased PI3K activity and result in immunodeficiency with immune dysregulation. We describe here the first cases of disseminated and congenital toxoplasmosis in a mother and child who share a pathogenic mutation in PIK3R1 and review the mechanisms underlying susceptibility to severe Toxoplasma gondii infection in activated PI3Kδ syndrome (APDS) and in other forms of primary immunodeficiency.

International audience; Ikaros/IKZF1 is an essential transcription factor expressed throughout hematopoiesis. IKZF1 is implicated in lymphocyte and myeloid differentiation and negative regulation of cell proliferation. In humans, somatic mutations in IKZF1 have been linked to the development of B cell acute lymphoblastic leukemia (ALL) in children and adults. Recently, heterozygous germline IKZF1 mutations have been identified in patients with a B cell immune deficiency mimicking common variable immunodeficiency. These mutations demonstrated incomplete penetrance and led to haploinsufficiency. Herein, we report 7 unrelated patients with a novel early-onset combined immunodeficiency associated with de novo germline IKZF1 heterozygous mutations affecting amino acid N159 located in the DNA-binding domain of IKZF1. Different bacterial and viral infections were diagnosed, but Pneumocystis jirovecii pneumonia was reported in all patients. One patient developed a T cell ALL. This immunodeficiency was characterized by innate and adaptive immune defects, including low numbers of B cells, neutrophils, eosinophils, and myeloid dendritic cells, as well as T cell and monocyte dysfunctions. Notably, most T cells exhibited a naive phenotype and were unable to evolve into effector memory cells. Functional studies indicated these mutations act as dominant negative. This defect expands the clinical spectrum of human IKZF1-associated diseases from somatic to germline, from haploinsufficient to dominant negative.

Severe chronic active Epstein-Barr virus (CAEBV) disease is defined as a severe progressive illness lasting 6 months or longer with infiltration of tissues with EBV-positive lymphocytes, markedly elevated levels of EBV DNA in the blood, and no known immunodeficiency such as HIV. These patients usually have fever, splenomegaly, lymphadenopathy, and may have markedly elevated EBV antibody titers to viral capsid antigen. Although the cause of most cases of severe CAEBV is unknown, one well-documented case was associated with compound heterozygous mutations in PRF1 (perforin 1). Here we report a patient with prolonged severe CAEBV who underwent bone marrow transplant for his disease and subsequently was found to have compound heterozygous mutations in STXBP2 (MUNC18-2) as well as a heterozygous mutation in PRF1 (perforin 1).

Idiopathic CD4+ lymphopenia (ICL) predisposes to opportunistic infections (OIs) but can often remain asymptomatic and does not have a strong association with monogenic mutations. Likewise, cryptococcal meningoencephalitis, the most common OI in ICL, is not strongly associated with monogenic mutations. In this study, we describe 2 patients with ICL plus an additional immune defect: one from an E57K genetic mutation in the nuclear factor-κβ essential modulator, and the other with acquired autoantibodies to granulocyte-macrophage colony-stimulating factor. Thus, these cases may exemplify a “multi-hit model” in patients with ICL who acquire OIs.

Gain of function (GOF) mutation in the p110δ catalytic subunit of the phosphatidylinositol-3-OH kinase (PIK3CD) is the cause of a primary immunodeficiency (PID) characterized by recurrent sinopulmonary infections and lymphoproliferation. We describe a family of two adults and three children with GOF mutation in PIK3CD, all with recurrent sinopulmonary infections and varied infectious and non-infectious complications. The two adults have primary sclerosing cholangitis (PSC) without evidence of Cryptosporidium parvum infection and have required liver transplantation. PSC is a novel phenotype of GOF mutation in PI3CKD.

Beware of T cells that don't know how to stop During an infection, T cells divide extensively and secrete proteins that can severely damage tissues. But T cells know when to stop—they express proteins on their surface such as CTLA4, which put on the brakes. Kuehn et al. now report genetic evidence of the importance of CTLA4 in humans (see the Perspective by Rieux-Laucat and Casanova). They identified six patients with mutations in one copy of CTLA4 . Patients presented with symptoms of an overzealous immune response, with immune cells infiltrating their organs. The findings support the idea that CTLA4 tells the immune system when enough is enough. Science , this issue p. 1623 ; see also p. 1560

Genetic defects in MOGS, the gene encoding mannosyl-oligosaccharide glucosidase (the first enzyme in the processing pathway of N-linked oligosaccharide), cause the rare congenital disorder of glycosylation type IIb (CDG-IIb), also known as MOGS-CDG. MOGS is expressed in the endoplasmic reticulum and is involved in the trimming of N-glycans. We evaluated two siblings with CDG-IIb who presented with multiple neurologic complications and a paradoxical immunologic phenotype characterized by severe hypogammaglobulinemia but limited clinical evidence of an infectious diathesis. A shortened immunoglobulin half-life was determined to be the mechanism underlying the hypogammaglobulinemia. Impaired viral replication and cellular entry may explain a decreased susceptibility to infections.

Autosomal dominant gain of function mutations in the gene encoding PI3K p110δ were recently associated with a novel combined immune deficiency characterized by recurrent sinopulmonary infections, CD4 lymphopenia, reduced class-switched memory B cells, lymphadenopathy, CMV and/or EBV viremia and EBV-related lymphoma. A subset of affected patients also had elevated serum IgM. Here we describe three patients in two families who were diagnosed with HIGM at a young age and were recently found to carry heterozygous mutations in PIK3CD. These patients had an abnormal circulating B cell distribution featuring a preponderance of early transitional (T1) B cells and plasmablasts. When stimulated in vitro, PIK3CD mutated B cells were able to secrete class-switched immunoglobulins. This finding implies that the patients' elevated serum IgM levels were unlikely a product of an intrinsic B cell functional inability to class switch. All three patients developed malignant lymphoproliferative syndromes that were not associated with EBV. Thus, we identified a novel subset of patients with PIK3CD mutations associated with HIGM, despite indications of preserved in vitro B cell class switch recombination, as well as susceptibility to non-EBV-associated malignancies.

The p110δ subunit of phosphatidylinositol-3-OH kinase (PI(3)K) is selectively expressed in leukocytes and is critical for lymphocyte biology. Here we report fourteen patients from seven families who were heterozygous for three different germline, gain-of-function mutations in PIK3CD (which encodes p110δ). These patients presented with sinopulmonary infections, lymphadenopathy, nodular lymphoid hyperplasia and viremia due to cytomegalovirus (CMV) and/or Epstein-Barr virus (EBV). Strikingly, they had a substantial deficiency in naive T cells but an over-representation of senescent effector T cells. In vitro, T cells from patients exhibited increased phosphorylation of the kinase Akt and hyperactivation of the metabolic checkpoint kinase mTOR, enhanced glucose uptake and terminal effector differentiation. Notably, treatment with rapamycin to inhibit mTOR activity in vivo partially restored the abundance of naive T cells, largely 'rescued' the in vitro T cell defects and improved the clinical course.

Memory B cells, unlike naive B cells, require a reduced level of STAT3 activation to differentiate into antibody-secreting plasmablasts in response to IL-10 and IL-21; however, this process requires IL-21R expression in both naive and memory cells., Long-lived antibody memory is mediated by the combined effects of long-lived plasma cells (PCs) and memory B cells generated in response to T cell–dependent antigens (Ags). IL-10 and IL-21 can activate multiple signaling pathways, including STAT1, STAT3, and STAT5; ERK; PI3K/Akt, and potently promote human B cell differentiation. We previously showed that loss-of-function mutations in STAT3, but not STAT1, abrogate IL-10– and IL-21–mediated differentiation of human naive B cells into plasmablasts. We report here that, in contrast to naive B cells, STAT3-deficient memory B cells responded to these STAT3-activating cytokines, differentiating into plasmablasts and secreting high levels of IgM, IgG, and IgA, as well as Ag-specific IgG. This was associated with the induction of the molecular machinery necessary for PC formation. Mutations in IL21R, however, abolished IL-21–induced responses of both naive and memory human B cells and compromised memory B cell formation in vivo. These findings reveal a key role for IL-21R/STAT3 signaling in regulating human B cell function. Furthermore, our results indicate that the threshold of STAT3 activation required for differentiation is lower in memory compared with naive B cells, thereby identifying an intrinsic difference in the mechanism underlying differentiation of naive versus memory B cells.

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Via next generation sequencing on a cohort of patients with severe atopic dermatitis, some with comorbid infections, we found 8 individuals from 4 families with novel heterozygous mutations in CARD11, a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant expression constructs into T cell lines demonstrated both loss of function and dominant interfering activity upon antigen receptor-induced NF-κB and mTORC1 activation. Patient T-cells had similar defects, as well as diminished IFN-γ cytokine production. The mTORC1 and IFN-γ production defects could be partially rescued by supplementing with glutamine, which requires CARD11 for import into T cells. Our findings indicate a single hypomorphic gene mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.

Episodic angioedema with eosinophilia (Gleich syndrome) is a rare disorder characterized by episodes of angioedema and eosinophilia that occur at monthly intervals and resolve spontaneously without therapy. Despite the striking periodicity of this disorder, its similarity to other cyclic hematopoietic disorders with multilineage involvement has not been assessed. To characterize the involvement of cell lineages in the etiology and pathogenesis of episodic angioedema with eosinophilia, four subjects were evaluated by blood counts and other analyses over the course of 1-2 months. Surface marker expression was assessed on T cells by flow cytometry and clonality by polymerase chain reaction. Intracellular cytokine evaluation, bone marrow and skin biopsies were performed during different parts of the cycle. Cycling of multiple cell lineages, including neutrophils, lymphocytes and eosinophils, was observed in the four subjects with the disorder with a periodicity of 25-35 days. An aberrant CD3(-)CD4(+) T-cell population was detected in all four subjects, and T-cell receptor rearrangement studies showed a clonal pattern in three subjects. A peak of type II cytokines was detected in the serum of subjects prior to the onset of symptoms and eosinophil cycling and corresponded to ex-vivo type II cytokines detected intracellularly in CD3(+)CD4(+)CD154(+) T cells. Although the etiology of episodic angioedema with eosinophilia is not yet known, multiple lineages, including lymphocytes, neutrophils and mast cells, are involved and may be related to disease pathogenesis. Whether these cells act directly or promote eosinophilia and eosinophil activation remains to be elucidated. All subjects gave informed consent and were evaluated under an Institutional Review Board-approved protocol (NCT00001406).

Background Follicular helper T (T FH ) cells underpin T cell–dependent humoral immunity and the success of most vaccines. T FH cells also contribute to human immune disorders, such as autoimmunity, immunodeficiency, and malignancy. Understanding the molecular requirements for the generation and function of T FH cells will provide strategies for targeting these cells to modulate their behavior in the setting of these immunologic abnormalities. Objective We sought to determine the signaling pathways and cellular interactions required for the development and function of T FH cells in human subjects. Methods Human primary immunodeficiencies (PIDs) resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Circulating follicular helper T (cT FH ) cell subsets, memory B cells, and serum immunoglobulin levels were quantified and functionally assessed in healthy control subjects, as well as in patients with PIDs resulting from mutations in STAT3 , STAT1 , TYK2 , IL21 , IL21R , IL10R , IFNGR1/2 , IL12RB1 , CD40LG , NEMO , ICOS , or BTK . Results Loss-of-function (LOF) mutations in STAT3 , IL10R , CD40LG , NEMO , ICOS , or BTK reduced cT FH cell frequencies. STAT3 and IL21/R LOF and STAT1 gain-of-function mutations skewed cT FH cell differentiation toward a phenotype characterized by overexpression of IFN-γ and programmed death 1. IFN-γ inhibited cT FH cell function in vitro and in vivo , as corroborated by hypergammaglobulinemia in patients with IFNGR1/2 , STAT1 , and IL12RB1 LOF mutations. Conclusion Specific mutations affect the quantity and quality of cT FH cells, highlighting the need to assess T FH cells in patients by using multiple criteria, including phenotype and function. Furthermore, IFN-γ functions in vivo to restrain T FH cell–induced B-cell differentiation. These findings shed new light on T FH cell biology and the integrated signaling pathways required for their generation, maintenance, and effector function and explain the compromised humoral immunity seen in patients with some PIDs.

Effective management of pain often requires the use of opioid analgesics (American Pain Society 1999, 2002; World Health Organization 2000; American Pain Society 2004). Because of their potential for abuse, opioid analgesics are regulated under federal narcotics and controlled substances laws (Joranson et al. 2000). The Controlled Substances Act of 1970 authorizes the Drug Enforcement Administration to supervise the manufacturing and distribution of legal narcotics and places all substances regulated under existing federal law into one of five schedules. Schedule II is reserved for drugs or substances with (a) high potential for abuse, (b) currently accepted medical use in treatment in the United States, and (c) potential for severe psychological or physical dependence if abused. Currently, 11 oral opioid analgesics have a Schedule II assignment (Drug Facts 2002). In addition to federal regulations, many states have enacted programs to monitor the use of opioid analgesics. Although details vary by state, prescription monitoring programs typically collect prescribing and dispensing data from pharmacies, review and analyze the data, and disseminate information to appropriate law enforcement and regulatory authorities (Joranson et al. 2000). Recent analyses suggest that the use of opioid analgesics has grown considerably over the last decade. From 1990 to 1996, there were steady increases in the use of morphine, fentanyl, oxycodone, and hydromorphone (Joranson et al. 2000). The same pattern persisted from 1997 to 2002, with marked increases in the use of fentanyl and oxycodone (Gilson et al. 2004). Considerable attention has been given to the use and abuse of controlled-release oxycodone hydrochloride (Clancy 2000; Gold 2000; Graettinger 2000; Ordway 2000; Tough 2001). Notably, abuse and diversion of controlled-release oxycodone has been concentrated in certain geographic areas, with abuse in rural Maine, Kentucky, Virginia, and West Virginia bringing national attention to the problem (Clines and Meier 2001; Rogers 2001; Rosenberg 2001; Drug Enforcement Administration 2002). Geographic variations in the use of other prescription medications have been previously examined. In particular, significant geographic variation has been documented in the use of stimulant medication in children (Zito et al. 1997; Wennberg and Wennberg 2000; Cox et al. 2003), antihypertensive medications in the Veterans Affairs health system (Lopez et al. 2004), and lipid lowering drugs, proton pump inhibitors, antianxiety drugs, and antihistamines among adults in Michigan (Wennberg and Wennberg 2000). By contrast, an analysis of medication use for five conditions (depression, asthma, congestive heart failure, rheumatoid/osteoarthritis, and upper respiratory infection) in 11 California regions found relatively little geographic variation (DuBois, Batchlor, and Wade 2002). To date, no study has explored geographic variation in the use of opioid analgesics. Examining geographic variation in the use of opioid analgesics is particularly important given the presence of state policies that may limit the prescription of these drugs. That is, geographic variation may yield important insights about the effects of these state policies. Evidence from the 1989 National Ambulatory Medical Care Survey (NAMCS) suggests that physicians in states with multiple-copy prescription programs are significantly less likely to prescribe opioid analgesics during an office visit (Wastila and Bishop 1996). Although a nationally representative sample, the observed NAMCS sample visits that occurred in states with multiple-copy prescription programs were likely heavily weighted toward states with especially large populations. Consequently, the generalizability of the findings to other states is unclear. Prior work has also identified other factors related to the medical and nonmedical use of abusable prescription drugs. A study using the 1987 National Medical Expenditure Survey found that female gender, age less than 35 years, socioeconomic status, and diagnosis were independently and positively associated with the probability of narcotic analgesic use (Simoni-Wastila 2000). Using the 1991 National Household Survey on Drug Abuse (NHSDA), Simoni-Wastila, Ritter, and Strickler (2004) identified female gender, age less than 35 years, annual income greater than $40,000, poor health status, and use of illicit drugs in the previous year as independent predictors of nonmedical use of prescription drugs. Simoni-Wastila and Strickler (2004) found that female gender and single marital status were positively and independently associated with problem use of narcotic analgesics, whereas age less than 25 years and illicit drug use in the previous year were negatively associated. In the present study, we used a large, outpatient pharmaceutical claims database of commercially insured individuals to build upon prior work in two ways. First, we examined state-level prevalence of and geographic variations in the use of Schedule II oral opioid analgesics. Second, we investigated the influence of prescription monitoring programs and a variety of other factors on county-level claim rates for all opioid analgesics and for controlled-release oxycodone alone. Based on prior work, we hypothesized that the presence of a prescription monitoring program would be negatively and independently associated with claim rates for opioid analgesics, whereas female gender, age less than 35 years, and prior use of illicit drugs would be positively and independently associated.

Background: Primary immunodeficiencies (PIDs) are a diverse group of disorders caused by multiple genetic defects. Obtaining a molecular diagnosis for PID patients using a phenotype-based approach is often complex, expensive, and not always successful. Next-generation sequencing (NGS) methods offer an unbiased genotype-based approach, which can facilitate molecular diagnostics. Objective: To develop an efficient NGS method to identify variants in PID-related genes. Methods: We performed HaloPlex custom target enrichment and NGS using the Ion Torrent PGM to screen 173 genes in 11 healthy controls, 13 PID patients previously evaluated with either an identified mutation or SNP, and 120 patients with undiagnosed PIDs. Sensitivity and specificity were determined by comparing NGS and Sanger sequencing results for 33 patients. Run metrics and coverage analyses were done to identify systematic deficiencies. Results: A molecular diagnosis was identified for 18 of 120 patients who previously lacked a genetic diagnosis, including 9 who had atypical presentations and extensive previous genetic and functional studies. Our NGS method detected variants with 98.1% sensitivity and >99.9% specificity. Uniformity was variable (72–89%), and we were not able to reliably sequence 45 regions (45/2455 or 1.8% of total regions) due to low (

Defective lymphocyte apoptosis results in chronic lymphadenopathy and/or splenomegaly associated with autoimmune phenomena. The prototype for human apoptosis disorders is the autoimmune lymphoproliferative syndrome (ALPS), which is caused by mutations in the FAS apoptotic pathway. Recently, patients with an ALPS-like disease called RAS-associated autoimmune leukoproliferative disorder, in which somatic mutations in NRAS or KRAS are found, also were described. Despite this progress, many patients with ALPS-like disease remain undefined genetically. We identified a homozygous, loss-of-function mutation in PRKCD (PKCδ) in a patient who presented with chronic lymphadenopathy, splenomegaly, autoantibodies, elevated immunoglobulins and natural killer dysfunction associated with chronic, low-grade Epstein-Barr virus infection. This mutation markedly decreased protein expression and resulted in ex vivo B-cell hyperproliferation, a phenotype similar to that of the PKCδ knockout mouse. Lymph nodes showed intense follicular hyperplasia, also mirroring the mouse model. Immunophenotyping of circulating lymphocytes demonstrated expansion of CD5+CD20+ B cells. Knockdown of PKCδ in normal mononuclear cells recapitulated the B-cell hyperproliferative phenotype in vitro. Reconstitution of PKCδ in patient-derived EBV-transformed B-cell lines partially restored phorbol-12-myristate-13-acetate–induced cell death. In summary, homozygous PRKCD mutation results in B-cell hyperproliferation and defective apoptosis with consequent lymphocyte accumulation and autoantibody production in humans, and disrupts natural killer cell function.

In this issue of HSR, we report findings from our analysis of geographic variations in the prescription of Schedule II opioid analgesics in the United States (Curtis et al. 2006). We write now to inform HSR readers of inaccuracies in the report. It has come to our attention that the “total claims” field in the county-level data set we received was mislabeled. Instead of total claims, the field reflected the number of subjects in each county according to an alternate set of inclusion and exclusion criteria. Believing that the total claims field was accurately labeled, we reported a 12-fold variation in claim rates for Schedule II oral opioid analgesics at the state level, and variation of a similar magnitude in claim rates for controlled-release oxycodone. We also reported that the presence of a state-based prescription monitoring program was associated with lower claim rates at the county level. We now know that the denominator used in those calculations was not claims, but rather a measure of the number of subjects. Because of a corporate acquisition since the time of the analysis, the raw data file is no longer available. As a consequence, it is not possible to recalculate the claim rates as claims per 1000 total claims. Nevertheless, using the known subject population as the denominator, we recalculated the claim rates and updated Table 2 and Figure 1 accordingly. We also corrected the title and row headings of Table 3 to reflect that the denominator of the dependent variable is subjects, not claims. The recalculated data support our original conclusion that considerable variation exists in the prescription of oral opioid analgesics, including controlled-release oxycodone. However, because the denominator refers to subjects rather than claims, we cannot eliminate the possibility that the geographic variations we observed reflect geographic variations in overall prescription drug use. In other words, if claims were the denominator, geographic variations in the use of oral opioid analgesics could be distinguished from geographic variations in the use of total prescription drugs. However, with subjects as the denominator, we cannot account for the variations due to different intensity of overall prescription drug use by region. Figure 1 (Revised): Claim Rates by State for All Opioid Analgesics and Controlled-Release Oxycodone. The figure also displays the year in which state-based Schedule II prescriptionmonitoring programs were first enacted in the states that have such a program. Table 2 (Revised). Claims for Opioid Analgesics by Location* Table 3 (Revised). Multivariable Regression Models for County-Level Opioid Analgesic Prescription Claim Rates per 1000 Subjects*

Background Although most patients with hypereosinophilic syndromes (HES) present with clinical signs and symptoms attributable to eosinophilic tissue infiltration, some untreated patients remain asymptomatic or have signs and symptoms, such as allergic rhinitis, for which the relationship to peripheral eosinophilia is unclear (hypereosinophilia of unknown significance [HE US ]). Objective To identify and characterize subjects with HE US of 5 years duration or more as compared to untreated patients with symptomatic HES and healthy normal volunteers. Methods All subjects with eosinophilia underwent yearly evaluation, including a standardized clinical evaluation, whole blood flow cytometry to assess lymphocyte subsets and eosinophil activation, and serum collection. Peripheral blood mononuclear cells were cultured overnight with and without phorbol 12-myristate 13-acetate/ionomycin. Cytokines and chemokines were measured in serum and cell supernatants, and mRNA expression was assessed by using quantitative real-time PCR. Results Eight of the 210 subjects referred for the evaluation of eosinophilia (absolute eosinophil count [AEC] > 1500/μL) met the criteria for HE US of 5 years duration or more (range, 7-29 years). Peak eosinophil count and surface expression of eosinophil activation markers were similar in subjects with HE US and in untreated subjects with platelet-derived growth factor alpha–negative HES (n = 28). Aberrant or clonal T-cell populations were identified in 50% of the subjects with HE US as compared to 29% of the subjects with HES ( P = .12). Increased levels of IL-5, GM-CSF, IL-9, and IL-17A were also comparable in subjects with HE US and HES. Serum levels of IgE and IL-13 were significantly increased only in subjects with HES. Conclusions A small number of patients with persistent peripheral eosinophilia (AEC > 1500/μL) appear to have clinically benign disease.

Systemic mastocytosis (SM) is a clonal myeloproliferative disease with variable clinical manifestations. The D816V mutation in the c-kit gene, present in over 90% of adult patients with SM, results in constitutive activation of the receptor tyrosine kinase and is believed to be related to disease pathogenesis. Although the majority of patients with SM lack peripheral blood eosinophilia, a subgroup exists and is classified as SM with eosinophilia (SM-eo). Recently, several reports of patients with SM-eo described the presence of either the Kit D816V mutation or the FIP1L1/PDGFRA fusion oncogene. Numerous similarities between patients with FIP1L1/PDGFRA and KIT D816V-associated peripheral blood eosinophilia have caused confusion about the management and specifically the role of imatinib in the treatment of these patients. It is of paramount importance to distinguish these two groups with pathologically similar, but molecularly and clinically distinct diseases. We thus compared the clinical, laboratory, and molecular features of 12 patients who met WHO criteria for SM (including presence of the D816V kit mutation) and had associated peripheral eosinophilia with those of 17 patients with peripheral eosinophilia and the FIP1L1/PDGFRA fusion oncogene (diagnosed with HES and evaluated at the same institution) and to the published reports of FIP1L1/PDGFRA-HES patients. Based on these comparisons, a number of clinical features appeared to be of potential use in distinguishing these two disorders. The presence of cardiac symptoms, a total serum tryptase under 60 ng/ml or the presence of either scattered mast cells or loose aggregates was found to be suggestive of FIP1L1/PDGFRA-associated disease. The presence of urticaria pigmentosa, a total serum tryptase over 150 ng/ml, the presence of dense mast cell aggregates and female sex were suggestive of Kit D816V-associated disease. To confirm and standardize this clinical classification, statistical methods were employed to test 21 possible risk factors for their ability to distinguish Kit and FIP1L1/PDGFRA-associated disease. Calculated risk factor scores were developed based on this analysis. Applying this risk factor based system, 16/17 FIP1L1/PDGFRA patients were classified correctly, with one patient neutral and all 12 Kit D816V SM-eo patients were classified correctly. Thirty four FIP1L1/PDGFRA patients in the literature were available for analysis, although all risk factors to create the score were not available for all patients. Despite this, 25/34 FIP1L1/PDGFRA patients were correctly predicted as FIP1L1/PDGFRA, 4/34 patients were neutral and 5/34 were misclassified as Kit D816V-associated SM-eo. These data suggest that the risk factor-based system presented in this study is useful in distinguishing imatinib-sensitive FIP1L1/PDGFRA-associated disease from imatinib-resistant Kit D816V-associated disease. | Parameter | SM-eo | FIP1L1/PDGFRA HES | |:---------------------------:| ----- | ----------------- | | Number patients | 12 | 17 | | Male/Female | 7/5 | 17/0 | | Cardiac symptoms | 0/12 | 6/15 | | UP | 7/12 | 0/15 | | Mean serum tryptase (ng/ml) | 229 | 28 (n=13) | | Dense marrow aggregates | 12/12 | 1/10