Zelm, M.C. van

Antibody deficiency in patients with ataxia telangiectasia is caused by disturbed B- and T-cell homeostasis and reduced immune repertoire diversity (Article)

2013-04-08T00:00:00Z

Background: Ataxia telangiectasia (AT) is a multisystem DNA-repair disorder caused by mutations in the ataxia telangiectasia mutated (ATM) gene. Patients with AT have reduced B- and T-cell numbers and a highly variable immunodeficiency. ATM is important for V(D)J recombination and immunoglobulin class-switch recombination (CSR); however, little is known about the mechanisms resulting in antibody deficiency severity. Objective: We sought to examine the immunologic mechanisms responsible for antibody deficiency heterogeneity in patients with AT. Methods: In this study we included patients with classical AT plus early-onset hypogammaglobulinemia (n = 3), classical AT (n = 8), and variant AT (late onset, n = 4). We studied peripheral B- and T-cell subsets, B-cell subset replication history, somatic hypermutation frequencies, CSR patterns, B-cell repertoire, and ATM kinase activity. Results: Patients with classical AT lacked ATM kinase activity, whereas patients with variant AT showed residual function. Most patients had disturbed naive B-cell and T-cell homeostasis, as evidenced by low cell numbers, increased proliferation, a large proportion CD21lowCD38lowanergic B cells, and decreased antigen receptor repertoire diversity. Impaired formation of T cell-dependent memory B cells was predominantly found in patients with AT plus hypogammaglobulinemia. These patients had extremely low naive CD4+T-cell counts, which were more severely reduced compared with those seen in patients with classical AT without hypogammaglobulinemia. Finally, AT deficiency resulted in defective CSR to distal constant regions that might reflect an impaired ability of B cells to undergo multiple germinal center reactions. Conclusion: The severity of the antibody deficiency in patients with AT correlates with disturbances in B- and T-cell homeostasis resulting in reduced immune repertoire diversity, which consequently affects the chance of successful antigen-dependent cognate B-T interaction.

Perigranuloma localization and abnormal maturation of B cells emerging key players in sarcoidosis? (Article)

2013-02-15T00:00:00Z

Rationale: Recent observations of abnormal immunoglobulin responses and case reports describing successful B-cell ablative therapy suggest involvement ofBcells in the pathogenesis of sarcoidosis. Objectives: To investigate how abnormal B-cell maturation and function in patients with sarcoidosis contribute to disease. Methods: Patients with sarcoidosis (n = 32) were included for detailed analysis by immunohistochemistry of tissue, flow cytometry of blood B-cell subsets, andserumimmunoglobulin levels.Vaccinationresponses in patients with sarcoidosis to influenza virus and encapsulated bacteria andmolecular analysis ofimmunoglobulin heavy chain transcriptswere studied for functional analysis of immunoglobulin responses. Measurements and Main Results: Perigranuloma localization of IgAproducing plasma cells and numerous B cells were found in affected tissues. Total blood B-cell numbers were normal, CD271 memory B cells were significantly reduced, and CD272IgA1 B cells were significantly increased; the results are normalized in patients treated with TNF-ablockers.Despitethis,patientshadnormal serumimmunoglobulin levels and normal antigen-specific immunoglobulin responses. IgAand IgGtranscripts, however, showed highfrequencies of somatic hypermutations and increased usage of downstream IgG subclasses, suggestive for prolonged or repetitive responses. Conclusions: The large B-cell infiltrates in granulomatous tissue and increased molecular signs of antibody maturation are indicative of direct involvement of B cells in local inflammatory processes in patients with sarcoidosis. Moreover, CD272IgA1 B cells could be a marker for treatment with TNF-a blockers. These findings of B cells as emerging key players provide a rationale for a systematic study on B-cell ablative therapy in patients with sarcoidosis. Copyright

New frontiers of primary antibody deficiencies (Article)

2012-01-01T00:00:00Z

Primary antibody deficiencies (PAD) form the largest group of inherited disorders of the immune system. They are characterized by a marked reduction or absence of serum immunoglobulins (Ig) due to disturbed B cell differentiation and by a poor response to vaccination. PAD can be divided into agammaglobulinemia, Ig class switch recombination deficiencies, and idiopathic hypogammaglobulinemia. Over the past 20 years, defects have been identified in 18 different genes, but in many PAD patients the underlying gene defects have not been found. Diagnosis of known PAD and discovery of new PAD is important for good patient care. In this review, we present the effects of genetic defects in the context of normal B cell differentiation, and we discuss how new technical developments can support understanding and discovering new genetic defects in PAD.

B-cell replication history and somatic hypermutation status identify distinct pathophysiologic backgrounds in common variable immunodeficiency (Article)

2011-12-22T00:00:00Z

Common variable immunodeficiency disorder (CVID) is the most prevalent form of primary idiopathic hypogammaglobulinemia. Identification of genetic defects in CVID is hampered by clinical and immunologic heterogeneity. By flow cytometric immunophenotyping and cell sorting of peripheral B-cell subsets of 37 CVID patients, we studied the B-cell compartment at the B-cell subset level using the κ-deleting recombination excision circle assay to determine the replication history and the Igκ-restriction enzyme hot-spot mutation assay to assess the somatic hypermutation status. Using this approach, 5 B-cell patterns were identified, which delineated groups with unique replication and somatic hypermutation characteristics. Each B-cell pattern reflected an immunologically homogenous patient group for which we proposed a different pathophysiology: (1) a B-cell production defect (n ∇ 8, 18%), (2) an early peripheral B-cell maturation or survival defect (n ∇ 4, 11%), (3) a B-cell activation and proliferation defect (n ∇ 12, 32%), (4) a germinal center defect (n ∇ 7, 19%), and (5) a postgerminal center defect (n ∇ 6, 16%). The results of the present study provide for the first time insight into the underlying pathophysiologic background in 5 immunologically homogenous groups of CVID patients. Moreover, this study forms the basis for larger cohort studies with the defined homogenous patient groups and will facilitate the identification of underlying genetic defects in CVID.

The nature of circulating CD27 +CD43 + B cells (Article)

2011-12-19T00:00:00Z

Checkpoints of B cell differentiation: Visualizing Ig-centric processes (Article)

2011-12-01T00:00:00Z

The generation of antibody responses and B cell memory can only take place following multiple steps of differentiation. Key molecular processes during precursor B cell differentiation in bone marrow generate unique antibodies. These antibodies are further optimized via molecular modifications during immune responses in peripheral lymphoid organs. Multiple checkpoints ensure proper differentiation of precursor and mature B lymphocytes. Many of these checkpoints have been found disrupted in patients with a primary immunodeficiency. Based on studies in these patients and in mouse models, new insights have been generated in B cell differentiation and antibody responses. Still, in many patients with impaired antibody formation, it remains unclear how B cells are affected. In this perspective, we present 11 critical processes in B cell differentiation. We discuss how defects in these processes can result in impaired checkpoint selection and how they can be visualized in healthy subjects and patients with immunodeficiency or other immunological disease.

Artemis splice defects cause atypical SCID and can be restored in vitro by an antisense oligonucleotide (Article)

2011-09-01T00:00:00Z

Artemis deficiency is known to result in classical TB severe combined immunodeficiency (SCID) in case of Artemis null mutations, or Omenn's syndrome in case of hypomorphic mutations in the Artemis gene. We describe two unrelated patients with a relatively mild clinical TB SCID phenotype, caused by different homozygous Artemis splice-site mutations. The splice-site mutations concern either dysfunction of a 5′ splice-site or an intronic point mutation creating a novel 3′ splice-site, resulting in mutated Artemis protein with residual activity or low levels of wild type (WT) Artemis transcripts. During the first 10 years of life, the patients suffered from recurrent infections necessitating antibiotic prophylaxis and intravenous immunoglobulins. Both mutations resulted in increased ionizing radiation sensitivity and insufficient variable, diversity and joining (V(D)J) recombination, causing B-lymphopenia and exhaustion of the naive T-cell compartment. The patient with the novel 3′ splice-site had progressive granulomatous skin lesions, which disappeared after stem cell transplantation (SCT). We showed that an alternative approach to SCT can, in principle, be used in this case; an antisense oligonucleotide (AON) covering the intronic mutation restored WT Artemis transcript levels and non-homologous end-joining pathway activity in the patient fibroblasts.

Human memory B cells originate from three distinct germinal center-dependent and -independent maturation pathways (Article)

2011-08-25T00:00:00Z

Multiple distinct memory B-cell subsets have been identified in humans, but it remains unclear how their phenotypic diversity corresponds to the type of responses from which they originate. Especially, the contribution of germinal center-independent responses in humans remains controversial. We defined 6 memory B-cell subsets based on their antigen-experienced phenotype and differential expression of CD27 and IgH isotypes. Molecular characterization of their replication history, Ig somatic hypermutation, and class-switch profiles demonstrated their origin from 3 different pathways. CD27-IgG+and CD27+IgM+B cells are derived from primary germinal center reactions, and CD27+IgA+and CD27+IgG+B cells are from consecutive germinal center responses (pathway 1). In contrast, natural effector and CD27-IgA+memory B cells have limited proliferation and are also present in CD40L-deficient patients, reflecting a germinal center-independent origin. Natural effector cells at least in part originate from systemic responses in the splenic marginal zone (pathway 2). CD27-IgA+cells share low replication history and dominant Igλ and IgA2 use with gut lamina propria IgA+ B cells, suggesting their common origin from local germinal center-independent responses (pathway 3). Our findings shed light on human germinal center-dependent and -independent B-cell memory formation and provide new opportunities to study these processes in immunologic diseases.

IL-7R expression and IL-7 signaling confer a distinct phenotype on developing human B-lineage cells (Article)

2011-08-25T00:00:00Z

IL-7 is an important cytokine for lymphocyte differentiation. Similar to what occurs in vivo, human CD19+cells developing in human/murine xenogeneic cultures show differential expression of the IL-7 receptor α (IL-7Rα) chain (CD127). We now describe the relationship between CD127 expression/signaling and Ig gene rearrangement. In the present study, < 10% of CD19+CD127+and CD19+CD127-populations had complete VDJH rearrangements. IGH locus conformation measurements by 3D FISH revealed that CD127+and CD127-cells were less contracted than pediatric BM pro-B cells that actively rearrange the IGH locus. Complete IGH rearrangements in CD127+and CD127-cells had smaller CDR3 lengths and fewer N-nucleotide insertions than pediatric BM B-lineage cells. Despite the paucity of VDJH rearrangements, microarray analysis indicated that CD127+cells resembled large pre-B cells, which is consistent with their low level of Ig lightchain rearrangements. Unexpectedly, CD127-cells showed extensive Ig light-chain rearrangements in the absence of IGH rearrangements and resembled small pre-B cells. Neutralization of IL-7 in xenogeneic cultures led to an increase in Ig light-chain rearrangements in CD127+cells, but no change in complete IGH rearrangements. We conclude that IL-7-mediated suppression of premature Ig light-chain rearrangement is the most definitive function yet described for IL-7 in human B-cell development.

Genetic analysis of contiguous X-chromosome deletion syndrome encompassing the BTK and TIMM8A genes (Article)

2011-08-01T00:00:00Z

Patients with X-linked agammaglobulinemia (XLA) can present with sensorineural deafness. This can result from a gross deletion that not only involved the Bruton's tyrosine kinase (BTK) gene, but also TIMM8A, mutations in which underlie the Mohr-Tranebjærg syndrome (MTS). We analyzed the genomic break points observed in three XLA-MTS patients and compared these with deletions break points from XLA patients. Patient 1 had a 63-kb deletion with break points in intron 15 of BTK and 4 kb upstream of TAF7L. Patients 2 and 3 had 149.7 and 196 kb deletions comprising BTK, TIMM8A, TAF7L and DRP2. The break points in patients 1 and 3 were located in Alu and endogenous retrovirus (ERV) repeats, whereas the break points in patient 2 did not show involvement of transposable elements. Comparison of gross deletion sizes and involvement of transposable elements in XLA and XLA-MTS patients from the literature showed preferential involvement of Alu elements in smaller deletions (<10 kb). These results show further insights into the molecular mechanisms underlying gross deletions in patients with primary immunodeficiency.

Antibody deficiency due to a missense mutation in CD19 demonstrates the importance of the conserved tryptophan 41 in immunoglobulin superfamily domain formation (Article)

2011-05-01T00:00:00Z

Immunoglobulin superfamily (IgSF) domains are conserved structures present in many proteins in eukaryotes and prokaryotes. These domains are well-capable of facilitating sequence variation, which is most clearly illustrated by the variable regions in immunoglobulins (Igs) and T cell receptors (TRs). We studied an antibody-deficient patient suffering from recurrent respiratory infections and with impaired antibody responses to vaccinations. Patient's B cells showed impaired Ca2+influx upon stimulation with anti-IgM and lacked detectable CD19 membrane expression. CD19 sequence analysis revealed a homozygous missense mutation resulting in a tryptophan to cystein (W52C) amino acid change. The affected tryptophan is CONSERVED-TRP 41 located on the C-strand of the first extracellular IgSF domain of CD19 and was found to be highly conserved, not only in mammalian CD19 proteins, but in nearly all characterized IgSF domains. Furthermore, the tryptophan is present in all variable domains in Ig and TR and was not mutated in 117 Ig class-switched transcripts of B cells from controls, despite an overall 10% amino acid change frequency. In vitro complementation studies and CD19 western blotting of patient's B cells demonstrated that the mutated protein remained immaturely glycosylated. This first missense mutation resulting in a CD19 deficiency demonstrates the crucial role of a highly conserved tryptophan in proper folding or stability of IgSF domains.

Estimating human age from T-cell DNA rearrangements (Article)

2010-11-23T00:00:00Z

Predicting human phenotypes from genotypes is a newly emerging field with relevance for personalized medicine [1] and forensics [2]. However, only a few phenotypic traits can currently be identified from DNA information with accuracies sufficient for practical applications [1], most notably human eye (iris) color [3]. It could be expected that individual age is too biologically complex to allow a simple and accurate molecular estimation from biological materials. Indeed, previously proposed genetic methods for human age estimation, based on the accumulation of mitochondrial DNA deletions or on telomere shortening, show low accuracies and various technical problems, and are therefore not suitable for practical applications [4]. Proposed biochemical methods, such as those based on the accumulation of D-aspartic acid, involve the destructive analysis of specific body parts (such as bones, teeth and ligaments), and suffer from technical issues and bio-degradation [4]. In the present study, we demonstrate that human individual age can be estimated accurately and reliably from blood using T-cell DNA rearrangements, and we provide a robust and sensitive real-time quantitative PCR protocol for application in various areas of bioscience.

B-cell maturation and antibody responses in individuals carrying a mutated CD19 allele (Article)

2010-10-01T00:00:00Z

Homozygous CD19 mutations lead to an antibody deficiency due to disruption of the CD19 complex and consequent impaired signaling by the B-cell antigen receptor. We studied the effects of heterozygous CD19 mutations on peripheral B-cell development and antibody responses in a large family with multiple consanguineous marriages. Sequence analysis of 96 family members revealed 30 carriers of the CD19 mutation. Lymphocyte subset counts were not significantly different between carriers and noncarriers in three different age groups (0-10 years; 11-18 years; adults). B cells of carriers had reduced CD19 and CD21 median expression levels, and had reduced proportions of transitional (0-10 years) and CD5+ B cells (adults). CD19 carriers did not show clinical signs of immunodeficiency; they were well capable to produce normal serum Ig levels and had normal responses to primary and booster vaccinations. The frequency of mutated Vκ alleles was not affected. Heterozygous loss of CD19 causes some changes in the naive B-cell compartment, but overall in vivo B-cell maturation or humoral immunity is not affected. Many antibody deficiencies are not monogenetic, but likely caused by a combination of multiple genetic variations. Therefore, functional analyses of immune cell function should be carried out to show whether heterozygous mutations contribute to disease.Genes and Immunity advance online publication, 6 May 2010; doi:10.1038/gene.2010.22.

Human peripheral blood B-Cell compartments: A crossroad in B-cell traffic (Article)

2010-09-20T00:00:00Z

A relatively high number of different subsets of B-cells are generated through the differentiation of early B-cell precursors into mature B-lymphocytes in the bone marrow (BM) and antigen-triggered maturation of germinal center B-cells into memory B-lymphocytes and plasmablasts in lymphoid tissues. These B-cell subpopulations, which are produced in the BM and lymphoid tissues, recirculate through peripheral blood (PB), into different tissues including mucosa and the BM, where long-living plasma cells produce antibodies. These circulating PB B-cells can be classified according to their maturation stage into i) immature/transitional, ii) na?̈ve, and iii) memory B-lymphocytes, and iv) plasmablasts/plasma cells. Additionally, unique subsets of memory B-lymphocytes and plasmablasts/plasma cells can be identified based on their differential expression of unique Ig-heavy chain isotypes (e.g.: IgM, IgD, IgG, IgA). In the present paper, we review recent data reported in the literature about the distribution, immunophenotypic and functional characteristics of these cell subpopulations, as well as their distribution in PB according to age and seasonal changes. Additional information is also provided in this regard based on the study of a population-based cohort of 600 healthy adults aged from 20 to 80 years, recruited in the Salamanca area in western Spain. Detailed knowledge of the distribution and traffic of B-cell subsets through PB mirrors the immune status of an individual subject and it may also contribute to a better understanding of B-cell disorders related to B-cell biology and homeostasis, such as monoclonal B-cell lymphocytosis (MBL).

An Artemis polymorphic variant reduces Artemis activity and confers cellular radiosensitivity (Article)

2010-09-01T00:00:00Z

Artemis is required for V(D)J recombination and the repair of a subset of radiation-induced DNA double strand breaks (DSBs). Artemis-null patients display radiosensitivity (RS) and severe combined immunodeficiency (SCID), classified as RS-SCID. Strongly impacting hypomorphic Artemis mutations confer marked infant immunodeficiency and a predisposition for EBV-associated lymphomas. Here, we provide evidence that a polymorphic Artemis variant (c.512C >G: p.171P >R), which has a world-wide prevalence of 15%, is functionally impacting. The c.512C >G mutation causes an ∼3-fold decrease in Artemis endonuclease activity in vitro. Cells derived from a patient who expressed a single Artemis allele with the polymorphic mutational change, showed radiosensitivity and a DSB repair defect in G2 phase, with Artemis cDNA expression rescuing both phenotypes. The c.512C >G change has an additive impact on Artemis function when combined with a novel C-terminal truncating mutation (p.436C >X), which also partially inactivates Artemis activity. Collectively, our findings provide strong evidence that monoallelic expression of the c.512C >G variant impairs Artemis function causing significant radiosensitivity and a G2 phase DSB repair defect. The patient exhibiting monoallelic c.512C >G-Artemis expression showed immunodeficiency only in adulthood, developed bilateral carcinoma of the nipple and myelodysplasia raising the possibility that modestly decreased Artemis function can impact clinically.

CD81 gene defect in humans disrupts CD19 complex formation and leads to antibody deficiency (Article)

2010-04-01T00:00:00Z

Antibody deficiencies constitute the largest group of symptomatic primary immunodeficiency diseases. In several patients, mutations in CD19 have been found to underlie disease, demonstrating the critical role for the protein encoded by this gene in antibody responses; CD19 functions in a complex with CD21, CD81, and CD225 to signal with the B cell receptor upon antigen recognition. We report here a patient with severe nephropathy and profound hypogammaglobulinemia. The immunodeficiency was characterized by decreased memory B cell numbers, impaired specific antibody responses, and an absence of CD19 expression on B cells. The patient had normal CD19 alleles but carried a homozygous CD81 mutation resulting in a complete lack of CD81 expression on blood leukocytes. Retroviral transduction and glycosylation experiments on EBV-transformed B cells from the patient revealed that CD19 membrane expression critically depended on CD81. Similar to CD19-deficient patients, CD81-deficient patients had B cells that showed impaired activation upon stimulation via the B cell antigen receptor but no overt T cell subset or function defects. In this study, we present what we believe to be the first antibody deficiency syndrome caused by a mutation in the CD81 gene and consequent disruption of the CD19 complex on B cells. These findings may contribute to unraveling the genetic basis of antibody deficiency syndromes and the nonredundant functions of CD81 in humans.

Chromatin Architecture and the Generation of Antigen Receptor Diversity (Article)

2009-08-07T00:00:00Z

The adaptive immune system generates a specific response to a vast spectrum of antigens. This remarkable property is achieved by lymphocytes that each express single and unique antigen receptors. During lymphocyte development, antigen receptor coding elements are assembled from widely dispersed gene segments. The assembly of antigen receptors is controlled at multiple levels, including epigenetic marking, nuclear location, and chromatin topology. Here, we review recently uncovered mechanisms that underpin long-range genomic interactions and the generation of antigen receptor diversity.

Molecular diagnostics of primary immunodeficiencies: benefits and future challenges. (Article)

2009-01-01T00:00:00Z

The 3D-structure of the Immunoglobulin Heavy Chain Locus: implications for long-range genomic interactions (Article)

2008-04-18T00:00:00Z

The immunoglobulin heavy-chain (Igh) locus is organized into distinct regions that contain multiple variable (V(H)), diversity (D(H)), joining (J(H)) and constant (C(H)) coding elements. How the Igh locus is structured in 3D space is unknown. To probe the topography of the Igh locus, spatial distance distributions were determined between 12 genomic markers that span the entire Igh locus. Comparison of the distance distributions to computer simulations of alternative chromatin arrangements predicted that the Igh locus is organized into compartments containing clusters of loops separated by linkers. Trilateration and triple-point angle measurements indicated the mean relative 3D positions of the V(H), D(H), J(H), and C(H) elements, showed compartmentalization and striking conformational changes involving V(H) and D(H)-J(H) elements during early B cell development. In pro-B cells, the entire repertoire of V(H) regions (2 Mbp) appeared to have merged and juxtaposed to the D(H) elements, mechanistically permitting long-range genomic interactions to occur with relatively high frequency.

Gross Deletions Involving IGHM, BTK, or Artemis: A Model for Genomic Lesions Mediated by Transposable Elements (Article)

2008-02-08T00:00:00Z

Most genetic disruptions underlying human disease are microlesions, whereas gross lesions are rare with gross deletions being most frequently found (6%). Similar observations have been made in primary immunodeficiency genes, such as BTK, but for unknown reasons the IGHM and DCLRE1C (Artemis) gene defects frequently represent gross deletions (∼60%). We characterized the gross deletion breakpoints in IGHM-, BTK-, and Artemis-deficient patients. The IGHM deletion breakpoints did not show involvement of recombination signal sequences or immunoglobulin switch regions. Instead, five IGHM, eight BTK, and five unique Artemis breakpoints were located in or near sequences derived from transposable elements (TE). The breakpoints of four out of five disrupted Artemis alleles were located in highly homologous regions, similar to Ig subclass deficiencies and Vh deletion polymorphisms. Nevertheless, these observations suggest a role for TEs in mediating gross deletions. The identified gross deletion breakpoints were mostly located in TE subclasses that were specifically overrepresented in the involved gene as compared to the average in the human genome. This concerned both long (LINE1) and short (Alu, MIR) interspersed elements, as well as LTR retrotransposons (ERV). Furthermore, a high total TE content (>40%) was associated with an increased frequency of gross deletions. Both findings were further investigated and confirmed in a total set of 20 genes disrupted in human disease. Thus, to our knowledge for the first time, we provide evidence that a high TE content, irrespective of the type of element, results in the increased incidence of gross deletions as gene disruption underlying human disease.

The 3D-structure of the Immunoglobulin Heavy Chain Locus: implications for long-range genomic interactions [supplemental data] (Miscellaneous)

2008-01-01T00:00:00Z

The immunoglobulin heavy-chain (Igh) locus is organized into distinct regions that contain multiple variable (VH), diversity (DH), joining (JH) and constant (CH) coding elements. How the Igh locus is structured in 3D space is unknown. To probe the topography of the Igh locus, spatial distance distributions were determined between 12 genomic markers that span the entire Igh locus. Comparison of the distance distributions to computer simulations of alternative chromatin arrangements predicted that the Igh locus is organized into compartments containing clusters of loops separated by linkers. Trilateration and triplepoint angle measurements indicated the mean relative 3D positions of the VH, DH, JH, and CH elements, showed compartmentalization and striking conformational changes involving VH and DH-JH elements during early B cell development. In pro-B cells, the entire repertoire of VH regions (2 Mbp) appeared to have merged and juxtaposed to the DH elements, mechanistically permitting long-range genomic interactions to occur with relatively high frequency.

Novel mutations in a Japanese patient with CD19 deficiency (Article)

2007-12-01T00:00:00Z

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by recurrent bacterial infections, hypogammaglobulinemia and low to normal numbers of circulating B cells. Mutations in the ICOS, TACI and CD19 genes have recently been identified in <10% of CVID patients. We, herein, describe two novel CD19 gene disruptions in an 8-year-old Japanese boy, who had been clinically diagnosed as having CVID at the age of 5 years. Flow-cytometric analysis demonstrated absence of CD19 and reduced CD21 expression on CD20-postive peripheral blood B cells. Mutation analysis of CD19 revealed a mutation in the splice acceptor site of intron 5 (IVS5-1G>T) of the maternal allele, resulting in skipping of exon 6, and a truncated protein product. The paternal allele was disrupted by a gross deletion encompassing at least the ATP2A1, CD19 and NFATC2IP genes. The patient had a small number of IgD-CD27+memory B cells, in which somatic mutation were detected. His B cells showed substantial proliferation upon stimulation, but reduced IgG and IgA production in vitro. These findings extend the mutation spectrum of the CD19 deficiency to four, and confirm the homogeneity of the CD19 deficiency as a unique type of CVID.

B-cell Development and Primary Antibody Deficiencies (Doctoral Thesis)

2007-06-20T00:00:00Z

B lymphocytes are generated throughout life from hematopoietic stem cells in bone marrow, and contribute to the immune system by the production of antigen-specific antibodies (immunoglobulins; Ig). Two distinct phase of B-cell development can be distinguished: 1) antigen-independent precursor-B-cell differentiation in bone marrow, and 2) antigendependent B-cell maturation in peripheral lymphoid organs. The aim of precursor-B-cell differentiation is to generate a functional Ig receptor by V(D)J recombination of the genes encoding the Ig heavy (IgH) and Ig light (Ig. or Ig.) chains. When a precursor-B-cell succeeds in creating a functional Ig receptor, it will migrate to the periphery and become part of the naive B-cell pool. Because every precursor-B-cell creates a unique Ig receptor, the peripheral B-cell pool bears a diverse repertoire of specific receptors for antigen. Naive B lymphocytes are short-lived cells and because new cells are continuously generated, there is a high turnover. However, once a naive mature B-lymphocyte recognizes antigen with its specific Ig receptor, it will undergo clonal proliferation and differentiation, thereby generating a large number of plasma cells that produce and secrete antigen-specific Igs. During this response, the B-cell initiates additional molecular mechanisms to adapt and optimize the antigen-binding affinity and the effector function of its Ig molecule. Generation of a large repertoire of B lymphocytes and the response of one of these B lymphocytes to antigen is a multi-step process for which multiple proteins are required. The lack of one of these proteins leads to a problem with the humoral immunity of the individual, which is seen in patients with primary antibody deficiency diseases. These children or young adults carry rare inherited disorders and are subject to multiple, recurrent (mainly) bacterial infections. Several genetic defects have been identified in patients with a primary antibody deficiency. In many patients, however, the B-cell defect is not well understood. The studies in this thesis address several aspects of B-cell development and defects that lead to primary antibody deficiency diseases.

Replication history of B lymphocytes reveals homeostatic proliferation and extensive antigen-induced B cell expansion (Article)

2007-03-19T00:00:00Z

The contribution of proliferation to B lymphocyte homeostasis and antigen responses is largely unknown. We quantified the replication history of mouse and human B lymphocyte subsets by calculating the ratio between genomic coding joints and signal joints on kappa-deleting recombination excision circles (KREC) of the IGK-deleting rearrangement. This approach was validated with in vitro proliferation studies. We demonstrate that naive mature B lymphocytes, but not transitional B lymphocytes, undergo in vivo homeostatic proliferation in the absence of somatic mutations in the periphery. T cell-dependent B cell proliferation was substantially higher and showed higher frequencies of somatic hypermutation than T cell-independent responses, fitting with the robustness and high affinity of T cell-dependent antibody responses. More extensive proliferation and somatic hypermutation in antigen-experienced B lymphocytes from human adults compared to children indicated consecutive responses upon additional antigen exposures. Our combined observations unravel the contribution of proliferation to both B lymphocyte homeostasis and antigeninduced B cell expansion. We propose an important role for both processes in humoral immunity. These new insights will support the understanding of peripheral B cell regeneration after hematopoietic stem cell transplantation or B cell-directed antibody therapy, and the identification of defects in homeostatic or antigen-induced B cell proliferation in patients with common variable immunodeficiency or another antibody deficiency. JEM