Giacomo Tampella

Deleterious Mutations in LRBA Are Associated with a Syndrome of Immune Deficiency and Autoimmunity

Most autosomal genetic causes of childhood-onset hypogammaglobulinemia are currently not well understood. Most affected individuals are simplex cases, but both autosomal-dominant and autosomal-recessive inheritance have been described. We performed genetic linkage analysis in consanguineous families affected by hypogammaglobulinemia. Four consanguineous families with childhood-onset humoral immune deficiency and features of autoimmunity shared genotype evidence for a linkage interval on chromosome 4q. Sequencing of positional candidate genes revealed that in each family, affected individuals had a distinct homozygous mutation in LRBA (lipopolysaccharide responsive beige-like anchor protein). All LRBA mutations segregated with the disease because homozygous individuals showed hypogammaglobulinemia and autoimmunity, whereas heterozygous individuals were healthy. These mutations were absent in healthy controls. Individuals with homozygous LRBA mutations had no LRBA, had disturbed B cell development, defective in vitro B cell activation, plasmablast formation, and immunoglobulin secretion, and had low proliferative responses. We conclude that mutations in LRBA cause an immune deficiency characterized by defects in B cell activation and autophagy and by susceptibility to apoptosis, all of which are associated with a clinical phenotype of hypogammaglobulinemia and autoimmunity.

Bruton tyrosine kinase mediates TLR9-dependent human dendritic cell activation

BACKGROUND Bruton tyrosine kinase (BTK) plays an essential role in various biologic functions of different cell types. Mutations in BTK lead to X-linked agammaglobulinemia (XLA) in humans. BTK was recently linked to the innate immune system, in particular, the Toll-like receptor (TLR) pathway; however, the TLR9 pathway has never been studied in dendritic cells (DCs) of patients with XLA. OBJECTIVE The aim of this study was to investigate the role of BTK in human DC activation upon TLR9 stimulation. METHODS DCs of patients with XLA and healthy donors were stimulated via TLR4/9 and evaluated for cell activation and cytokine production. RESULTS We showed that BTK plays an essential role in DC responses to unmethylated CpG oligodeoxynucleotide: although responses to lipopolysaccaride/TLR4 induce normal DC activation in terms of upregulation of specific markers (CD86, CD83, CD80, HLA-DR), the CpG/TLR9 pathway is completely impaired in patients with XLA. Furthermore, cytokine production upon TLR9 activation in patients with XLA is radically impaired in terms of IL-6, IL-12, TNF-α, and IL-10 production. Interestingly, BTK mediated STAT1/3 upregulation in a TLR9-dependent manner. The important role of BTK in human DC activation was confirmed after incubation of healthy DCs with ibrutinib, the specific BTK inhibitor, which resulted in impairment of TLR9 responses as seen in patients with XLA. CONCLUSION Analysis of these data suggests that BTK regulates human DC responses upon TLR9 engagement in terms of activation, cytokine production, and STAT1/3 upregulation. These findings may be of important significance for better understanding and managing different clinical conditions, such as agammaglobulinemia and lymphoid malignancies.

Allele*1 of HS1,2 enhancer associates with selective IgA deficiency and IgM concentration

Selective IgA deficiency (IGAD) is the most common primary immunodeficiency, yet its pathogenesis is elusive. The IG (heavy) H chain human 3′ Regulatory Region harbors three enhancers and has an important role in Ig synthesis. HS1.2 is the only polymorphic enhancer of the 3′RRs. We therefore evaluated HS1.2 allelic frequencies in 88 IGAD patients and 101 controls. Our data show that IGAD patients have a highly significant increase of homozygousity of the allele *1 (39% in the IGAD patients and 15% in controls), with an increase of 2.6-fold. Allele *4 has a similar trend of allele *2, both showing a significant decrease of frequency in IGAD. No relationship was observed between allele *1 frequencies and serum levels of IgG. However, allele *1 was associated in IGAD patients with relatively low IgM levels (within the 30th lowest percentile of patients). The HS1.2 polymorphism influences Ig seric production, but not IgG switch, in fact 30th lowest or highest percentile of IgG in patients did not associate to different frequencies of HS1.2 alleles. The control on normal healthy subjects did not correlate high or low levels of IgM or IgG with HS1.2 allelic frequence variation. Overall our candidate gene approach confirms that the study of polymorphisms in human diseases is a valid tool to investigate the function of these Regulatory Regions that confers multiple immune features.

NFKB1 regulates human NK cell maturation and effector functions.

NFKB1, a component of the canonical NF-κB pathway, was recently reported to be mutated in a limited number of CVID patients. CVID-associated mutations in NFKB2 (non-canonical pathway) have previously been shown to impair NK cell cytotoxic activity. Although a biological function of NFKB1 in non-human NK cells has been reported, the role of NFKB1 mutations for human NK cell biology and disease has not been investigated yet. We decided therefore to evaluate the role of monoallelic NFKB1 mutations in human NK cell maturation and functions. We show that NFKB1 mutated NK cells present impaired maturation, defective cytotoxicity and reduced IFN-γ production upon in vitro stimulation. Furthermore, human IL-2 activated NFKB1 mutated NK cells fail to up-regulate the expression of the activating marker NKp44 and show reduced proliferative capacity. These data suggest that NFKB1 plays an essential novel role for human NK cell maturation and effector functions.

The Genetic Heterogeneity of Common Variable Immunodeficiency (CVID): An Update

B cells are generated in the bone marrow and then enter the periphery, where the maturation process takes place leading to the formation of an effective humoral immune response. Defects in this highly regulated process in the periphery have been considered to be responsible for the pathogenesis of Common Variable Immunodeficiency (CVID) for more than 6 decades. CVID is traditionally characterized by low immunoglobulin serum levels and defective antibody response in the presence of normal peripheral B cell numbers. The clinical spectrum of CVID is highly variable, including recurrent infections, autoimmune complications and increased susceptibility to cancer and lymphomas. However, only in the last decade, the genetic defects underlying this maturational B cell defect have been partially elucidated in a small percentage of affected patients. This review will focus on the current state of art regarding the known genetic alterations associated with the pathogenesis of CVID.

Correlation of bone marrow abnormalities, peripheral lymphocyte subsets and clinical features in uncomplicated common variable immunodeficiency (CVID) patients

B cell developmental defects in CVID were recently described in a limited number of cases. To date, a detailed correlation between this maturational defect and the clinical presentation of affected patients has not been reported. In this study, we correlated bone marrow B cell evaluation, peripheral B and T lymphocyte subsets and clinical findings in 15 CVID patients. Early B cell developmental defects were observed in one third of patients. Combined bone marrow and peripheral lymphocytes evaluation allowed to further subdivide CVID patients in three groups with shared clinical features at diagnosis and during follow-up. These data broaden the number of CVID patients with early B cell developmental defects and, together with the peripheral lymphocytes evaluation, offer insight into the related clinical features in affected patients.

BAFF-R mutations in Good's syndrome.

Goods syndrome (GS) is a rare disorder characterized by thymoma and immunodeficiency. Typically the immunological presentation of affected patients includes low B cells, hypogammaglobulinemia and altered T cell function in the presence of normal or elevated T cell counts [1]. Opportunistic infections may accompany the clinical history. The pathogenesis of this disorder is still unknown and no definite genetic studies have been reported so far. Recently in this journal, mutations in TACI were identified in two affected patients [2,3]. The rationale for analyzing TACI in patients with GS was provided by the fact that TACI is mutated in patients affected with Common Variable Immunodeficiency (CVID) [4] and that the immunological phenotype of GS may resemble Common Variable Immunodeficiency (CVID), although with certain differences: for example, GS, unlike CVID, is characterized by B cell deficiency. TACI, together with BAFF-R and BCMA, are members of the Tumor Necrosis Factor Receptor (TNFR) superfamily, and are expressed on B cells. Their binding to specific ligands (all three bind to BAFF while only TACI and BCMA bind to APRIL) is important for B cell maturation and homeostasis. BAFF-R in particular, is essential for B cell development both in mice and humans [4,5]. The animal knockout model for BAFF-R results in peripheral B cell deficiency [4]. Humans affected with BAFF-R deficiency and hypogammaglobulinemia show low peripheral B cell percentages as well (1.1%–3.2%) [5]. Human TACI mutations on the other hand are not typically associated with reduced peripheral B cells [4].

B Cell Responses to CpG Correlate with CXCL16 Expression Levels in Common Variable Immunodeficiency

Broad Toll-like receptor 9 (TLR9) signalling defects after CpG in vitro stimulation have been described in common variable immunodeficiency (CVID). CXCL16, a surface receptor, was recently shown to influence cell responses to CpG. We evaluated the expression and function of CXCL16 on B cells from healthy controls and CVID patients. We report that CXCL16 is normally expressed on B cells throughout peripheral maturation. Decreased B cell expression of CXCL16 was observed in a subgroup of CVID patients that correlated with defective in vitro responses to CpG (such as upregulation of CD69, CD86, AICDA, IL-6, and TLR9). Our data suggest that expression levels of a surface receptor, namely, CXCL16, correlate with B cell responses mediated by TLR9 in common variable immunodeficiency.

Autosomal Recessive Agammaglobulinemia: The Third Case of Igβ Deficiency Due to a Novel Non-sense Mutation

This study describes the third case worldwide of autosomal recessive agammaglobulinemia due to a novel non-sense mutation in Igβ presenting with neutropenia, ecthyma and mild respiratory infections.

Profound T‐cell defects in Dubowitz syndrome

To the Editor, Dubowitz syndrome is a rare autosomal recessive disorder characterized by short stature, microcephaly, low platelet counts, dysmorphic features, eczema, and mild to severe mental retardation. Less than 150 cases have been reported so far in the literature, and diagnosis is mainly based on key clinical findings (1–5). Clinical presentation is rather variable and the only core clinical feature seems to be a characteristic face (3). While the neurologic/behavioral features have been deeply investigated, the immunologic characterization of this syndrome remains rather incomplete, apart from anecdotal reports of selective IgA deficiency, hypogammaglobulinemia and hyper-IgE-like syndrome in index cases (6–9). We report on the first extensive immunologic evaluation of a male patient with Dubowitz syndrome revealing profound T-cell defects resembling combined immunodeficiency. The patient was born to consanguineous parents of Pakistani origin. He came to our attention at the age of 8 for immunologic evaluation due to partial IgA deficiency. His clinical history included short stature (height: 120 cm <3°; weight: 18.5 kg <3°, cranial circumference: 49 cm <3°), delay in mental development and speech, recurrent episodes of thrombocytopenia (with normal bone marrow aspirate). Key clinical features included high and receding forehead, sparse lateral eyebrows, blepharoptosis, abnormal ear conformation, broad and flat nose bridge, clinodactyly of the fifth finger of the hand and cutaneous syndactyly between the fourth and fifth fingers of the feet (Fig. 1a). Skin inspection revealed eczema in folds, keratosis pilaris on the back of arms and legs, and numerous molluscum contagiosum lesions (Fig. S1). Based on these clinical findings, diagnosis of Dubowitz syndrome was made. The patient’s infectious history was particularly severe: sepsis at 6 yr of age, osteomyelitis at 8 yr of age and recurrent molluscum contagiosum skin infections from the age of 6. Asymptomatic low platelet counts were confirmed (ranging from 42.000 to 66.000/mm), while the rest of the differential blood count was normal. Immunoglobulin serum levels were normal, with the exception of IgA serum levels at the lower range for age: IgG: 1370 mg/dl (normal range for age: 633– 1916), IgA: 31 mg/dl (normal range for age: 41–315), IgM: 64 mg/dl (normal range for age: 56–261). Biochemical features such as cholesterol, LDL, HDL levels were within the normal range (cholesterol 160 mg/dl, LDL: 98 mg/dl, HDL 44 mg/dl). The patient carried a normal karyogram. No chromosomal instability, both spontaneous and bleomycin induced, was observed. Recently, the NSUN2 gene was reported to be