Aydan Ikinciogullari

Defects along the TH17 differentiation pathway underlie genetically distinct forms of the hyper IgE syndrome

BACKGROUND The hyper IgE syndrome (HIES) is characterized by abscesses, eczema, recurrent infections, skeletal and connective tissue abnormalities, elevated serum IgE, and diminished inflammatory responses. It exists as autosomal-dominant and autosomal-recessive forms that manifest common and distinguishing clinical features. A majority of those with autosomal-dominant HIES have heterozygous mutations in signal transducer and activator of transcription (STAT)-3 and impaired T(H)17 differentiation. OBJECTIVE To elucidate mechanisms underlying different forms of HIES. METHODS A cohort of 25 Turkish children diagnosed with HIES were examined for STAT3 mutations by DNA sequencing. Activation of STAT3 by IL-6 and IL-21 and STAT1 by IFN-alpha was assessed by intracellular staining with anti-phospho (p)STAT3 and -pSTAT1 antibodies. T(H)17 and T(H)1 cell differentiation was assessed by measuring the production of IL-17 and IFN-gamma, respectively. RESULTS Six subjects had STAT3 mutations affecting the DNA binding, Src homology 2, and transactivation domains, including 3 novel ones. Mutation-positive but not mutation-negative subjects with HIES exhibited reduced phosphorylation of STAT3 in response to cytokine stimulation, whereas pSTAT1 activation was unaffected. Both patient groups exhibited impaired T(H)17 responses, but whereas STAT3 mutations abrogated early steps in T(H)17 differentiation, the defects in patients with HIES with normal STAT3 affected more distal steps. CONCLUSION In this cohort of Turkish children with HIES, a majority had normal STAT3, implicating other targets in disease pathogenesis. Impaired T(H)17 responses were evident irrespective of the STAT3 mutation status, indicating that different genetic forms of HIES share a common functional outcome.

Inherited IL-17RC deficiency in patients with chronic mucocutaneous candidiasis

Autosomal-recessive IL-17RA, IL-17RC, and ACT1 deficiencies and autosomal-dominant IL-17F deficiency in humans underlie susceptibility to chronic mucocutaneous candidiasis.

Monogenic mutations differentially affect the quantity and quality of T follicular helper cells in patients with human primary immunodeficiencies

BACKGROUND Follicular helper T (TFH) cells underpin T cell-dependent humoral immunity and the success of most vaccines. TFH cells also contribute to human immune disorders, such as autoimmunity, immunodeficiency, and malignancy. Understanding the molecular requirements for the generation and function of TFH 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 TFH 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 (cTFH) 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 cTFH cell frequencies. STAT3 and IL21/R LOF and STAT1 gain-of-function mutations skewed cTFH cell differentiation toward a phenotype characterized by overexpression of IFN-γ and programmed death 1. IFN-γ inhibited cTFH 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 cTFH cells, highlighting the need to assess TFH cells in patients by using multiple criteria, including phenotype and function. Furthermore, IFN-γ functions in vivo to restrain TFH cell-induced B-cell differentiation. These findings shed new light on TFH 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.

Early-onset inflammatory bowel disease and common variable immunodeficiency–like disease caused by IL-21 deficiency

BACKGROUND Alterations of immune homeostasis in the gut can result in development of inflammatory bowel disease (IBD). Recently, Mendelian forms of IBD have been discovered, as exemplified by deficiency of IL-10 or its receptor subunits. In addition, other types of primary immunodeficiency disorders might be associated with intestinal inflammation as one of their leading clinical presentations. OBJECTIVE We investigated a large consanguineous family with 3 children who presented with early-onset IBD within the first year of life, leading to death in infancy in 2 of them. METHODS Homozygosity mapping combined with exome sequencing was performed to identify the molecular cause of the disorder. Functional experiments were performed to assess the effect of IL-21 on the immune system. RESULTS A homozygous mutation in IL21 was discovered that showed perfect segregation with the disease. Deficiency of IL-21 resulted in reduced numbers of circulating CD19(+) B cells, including IgM(+) naive and class-switched IgG memory B cells, with a concomitant increase in transitional B-cell numbers. In vitro assays demonstrated that mutant IL-21(Leu49Pro) did not induce signal transducer and activator of transcription 3 phosphorylation and immunoglobulin class-switch recombination. CONCLUSION Our study uncovers IL-21 deficiency as a novel cause of early-onset IBD in human subjects accompanied by defects in B-cell development similar to those found in patients with common variable immunodeficiency. IBD might mask an underlying primary immunodeficiency, as illustrated here with IL-21 deficiency.

Biallelic loss-of-function mutation in NIK causes a primary immunodeficiency with multifaceted aberrant lymphoid immunity

Primary immunodeficiency disorders enable identification of genes with crucial roles in the human immune system. Here we study patients suffering from recurrent bacterial, viral and Cryptosporidium infections, and identify a biallelic mutation in the MAP3K14 gene encoding NIK (NF-κB-inducing kinase). Loss of kinase activity of mutant NIK, predicted by in silico analysis and confirmed by functional assays, leads to defective activation of both canonical and non-canonical NF-κB signalling. Patients with mutated NIK exhibit B-cell lymphopenia, decreased frequencies of class-switched memory B cells and hypogammaglobulinemia due to impaired B-cell survival, and impaired ICOSL expression. Although overall T-cell numbers are normal, both follicular helper and memory T cells are perturbed. Natural killer (NK) cells are decreased and exhibit defective activation, leading to impaired formation of NK-cell immunological synapses. Collectively, our data illustrate the non-redundant role for NIK in human immune responses, demonstrating that loss-of-function mutations in NIK can cause multiple aberrations of lymphoid immunity.

Genetic, immunological, and clinical features of patients with bacterial and fungal infections due to inherited IL-17RA deficiency

Significance Chronic mucocutaneous candidiasis (CMC) is defined as persistent or recurrent infections of the skin and/or mucosae by commensal fungi of the Candida genus. It is often seen in patients with T-cell deficiencies, whether inherited or acquired, who typically suffer from multiple infectious diseases. Rare patients are otherwise healthy and display isolated CMC, which often segregates as a Mendelian trait. In 2011, we described the first genetic cause of isolated CMC, with autosomal recessive (AR), complete IL-17 receptor A (IL-17RA) deficiency, in a single patient. We report here 21 patients from 12 unrelated kindreds, homozygous for 12 different mutant alleles that underlie AR IL-17RA deficiency. All patients have isolated CMC and their cells do not respond to IL-17A, -17F, and -17E/IL-25. Chronic mucocutaneous candidiasis (CMC) is defined as recurrent or persistent infection of the skin, nails, and/or mucosae with commensal Candida species. The first genetic etiology of isolated CMC—autosomal recessive (AR) IL-17 receptor A (IL-17RA) deficiency—was reported in 2011, in a single patient. We report here 21 patients with complete AR IL-17RA deficiency, including this first patient. Each patient is homozygous for 1 of 12 different IL-17RA alleles, 8 of which create a premature stop codon upstream from the transmembrane domain and have been predicted and/or shown to prevent expression of the receptor on the surface of circulating leukocytes and dermal fibroblasts. Three other mutant alleles create a premature stop codon downstream from the transmembrane domain, one of which encodes a surface-expressed receptor. Finally, the only known missense allele (p.D387N) also encodes a surface-expressed receptor. All of the alleles tested abolish cellular responses to IL-17A and -17F homodimers and heterodimers in fibroblasts and to IL-17E/IL-25 in leukocytes. The patients are currently aged from 2 to 35 y and originate from 12 unrelated kindreds. All had their first CMC episode by 6 mo of age. Fourteen patients presented various forms of staphylococcal skin disease. Eight were also prone to various bacterial infections of the respiratory tract. Human IL-17RA is, thus, essential for mucocutaneous immunity to Candida and Staphylococcus, but otherwise largely redundant. A diagnosis of AR IL-17RA deficiency should be considered in children or adults with CMC, cutaneous staphylococcal disease, or both, even if IL-17RA is detected on the cell surface.

Upregulation of CD63 or CD203c Alone or in Combination Is Not Sensitive in the Diagnosis of Nonsteroidal Anti-Inflammatory Drug Intolerance

Background: The oral aspirin (ASA) provocation test is considered to be the gold standard in the diagnosis of ASA sensitivity. However, since it may be associated with severe adverse reactions, safer alternatives would be highly desirable. The basophil activation test has been proposed as such an alternative, but there is limited information about its usefulness. Our aim was to evaluate the clinical usefulness of flow cytometric basophil activation in the diagnosis of ASA sensitivity. Methods: Patients with ASA sensitivity (n = 18), patients with ASA tolerance (n = 12) and healthy volunteers (n = 12) were included in the study. A 2-day single-blind placebo-controlled oral ASA provocation test was performed on all patients. Basophil activation after lysine-ASA and diclofenac stimulation was measured by Flow-CASTTM (Bühlmann Laboratories) for CD63 and an allergenicity kit (Beckman Coulter) for CD203c. The results of CD63 and CD203c were compared within groups, and sensitivity and specificity of the assay were measured against oral ASA provocation. Results: The highest sensitivity and specificity of CD63 were 33.3 and 79.2%, respectively, and of CD203c were 16.7 and 100%, respectively, for ASA. The highest sensitivity and specificity of CD63 were 16.7 and 91.7%, respectively, and of CD203c were 22.2 and 100%, respectively, fordiclofenac. Neither the addition of CD203c to CD63 nor the addition of diclofenac improves the overall sensitivity and specificity of CD63 to ASA. Conclusion: At present, basophil activation using CD63 and CD203c does not seem to be optimally sensitive for the diagnosis of ASA sensitivity.

Combined immunodeficiency and Epstein-Barr virus?induced B cell malignancy in humans with inherited CD70 deficiency

In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)–related diseases. Three patients presented with EBV-associated Hodgkin’s lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8+ T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro–generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8+ T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70–CD27 interactions therefore play a nonredundant role in T and B cell–mediated immunity, especially for protection against EBV and humoral immunity.

Is immune system influenced by adenotonsillectomy in children

OBJECTIVE Tonsils and adenoids are lymphoid tissues that are located in the pharynx and play an important role against invading antigens of the upper respiratory tract. The present study analyses serum immunoglobulin levels and peripheral blood (PB) lymphocyte subsets in children, 24-48 h prior to and 4-6 weeks after adenotonsillectomy, in order to determine early effects of adenotonsillectomy on the immune system. METHODS The study population consists of 15 children (aged 4-10 years) who underwent adenotonsillectomy because of adenoidal hypertrophy and chronic tonsillitis and 15 age-matched healthy children without a history of adenotonsillectomy. Serum IgG, IgA and IgM levels were measured by nephelometry. PB lymphocyte subsets were analysed by using monoclonal antibodies and flow cytometry. RESULTS Children with chronic tonsillitis have increased levels of CD19+ B lymphocytes compared to healthy controls in the pre-operative period. The percentage of B lymphocytes bearing CD23 was found to be significantly higher in patients, most likely representing in vivo B lymphocyte activation due to chronic antigenic stimulation. After the adenotonsillectomy, despite ongoing B lymphocyte activation, CD8+ T lymphocyte levels increased and B cell levels returned to normal. A slight decrease in serum IgG, IgA and IgM levels was detected in the post-operative period compared to prior levels. CONCLUSION Adenotonsillectomy performed in children leads to alterations that may reflect a compensatory response of the developing immune system after the removal of the lymphoid tissue in the setting of chronic antigenic stimulation. However, these changes do not cause significant immune deficiency.

Granulocyte Transfusions in Children With Chronic Granulomatous Disease and Invasive Aspergillosis

Abstract:  The transfusion of granulocytes to restore host defenses in severely granulocytopenic patients or in patients with defective granulocyte functions has been studied for more than 60 years. However, inadequate dosage of cells and inconsistent efficacy has limited the usage of these transfusions. Recently, the use of mobilizing agents such as granulocyte colony stimulating factors and dexamethasone has renewed interest in these treatment modalities. The present study is conducted to determine an appropriate method of enriched granulocyte collection with Fresenius AS.TEC.204 cell separator (Fresenius, Bad Homburg, Germany) and to evaluate the preliminary clinical results of granulocyte transfusion therapy in patients with chronic granulomatous disease and invasive Aspergillosis in parallel with in vitro granulocyte function. Three patients who have been treated for chronic granulomatous disease and invasive Aspergillosis received a total of 20 granulocyte transfusions. To mobilize granulocytes, healthy donors were given 450 µg of granulocyte colony‐stimulating factor (G‐CSF) subcutaneously and 8 mg of dexamethasone orally approximately 12 h before collection. Five µg/kg/day of G‐CSF was also subcutaneously administered prior to granulocyte transfusions. The first patient received 4; the second, 14 and the third, 2 transfusions. The granulocyte count given to these patients ranged between 0.4 and 3.0 × 109/kg. Most transfusions were well tolerated. The nitroblue tetrazolium (NBT) tests that were done 16–24 h after the transfusion showed 14–46% dye reduction. Two of the three patients survived the infection. Granulocyte transfusions from G‐CSF and dexamethasone stimulated donors could be a choice of treatment in chronic granulomatous disease patients, especially with disseminated invasive Aspergillosis.