Sylvia Gutenberger

B-cell activating factor receptor deficiency is associated with an adult-onset antibody deficiency syndrome in humans

B-cell survival depends on signals induced by B-cell activating factor (BAFF) binding to its receptor (BAFF-R). In mice, mutations in BAFF or BAFF-R cause B-cell lymphopenia and antibody deficiency. Analyzing BAFF-R expression and BAFF-binding to B cells in common variable immunodeficiency (CVID) patients, we identified two siblings carrying a homozygous deletion in the BAFF-R gene. Removing most of the BAFF-R transmembrane part, the deletion precludes BAFF-R expression. Without BAFF-R, B-cell development is arrested at the stage of transitional B cells and the numbers of all subsequent B-cell stages are severely reduced. Both siblings have lower IgG and IgM serum levels but, unlike most CVID patients, normal IgA concentrations. They also did not mount a T-independent immune response against pneumococcal cell wall polysaccharides but only one BAFF-R-deficient sibling developed recurrent infections. Therefore, deletion of the BAFF-R gene in humans causes a characteristic immunological phenotype but it does not necessarily lead to a clinically manifest immunodeficiency.

Circulating CD21low B cells in common variable immunodeficiency resemble tissue homing, innate-like B cells.

The homeostasis of circulating B cell subsets in the peripheral blood of healthy adults is well regulated, but in disease it can be severely disturbed. Thus, a subgroup of patients with common variable immunodeficiency (CVID) presents with an extraordinary expansion of an unusual B cell population characterized by the low expression of CD21. CD21low B cells are polyclonal, unmutated IgM+IgD+ B cells but carry a highly distinct gene expression profile which differs from conventional naïve B cells. Interestingly, while clearly not representing a memory population, they do share several features with the recently defined memory-like tissue, Fc receptor-like 4 positive B cell population in the tonsils of healthy donors. CD21low B cells show signs of previous activation and proliferation in vivo, while exhibiting defective calcium signaling and poor proliferation in response to B cell receptor stimulation. CD21low B cells express decreased amounts of homeostatic but increased levels of inflammatory chemokine receptors. This might explain their preferential homing to peripheral tissues like the bronchoalveolar space of CVID or the synovium of rheumatoid arthritis patients. Therefore, as a result of the close resemblance to the gene expression profile, phenotype, function and preferential tissue homing of murine B1 B cells, we suggest that CD21low B cells represent a human innate-like B cell population.

Genetic CD21 deficiency is associated with hypogammaglobulinemia

BACKGROUND Complement receptor 2 (CR2/CD21) is part of the B-cell coreceptor and expressed by mature B cells and follicular dendritic cells. CD21 is a receptor for C3d-opsonized immune complexes and enhances antigen-specific B-cell responses. OBJECTIVE Genetic inactivation of the murine CR2 locus results in impaired humoral immune responses. Here we report the first case of a genetic CD21 deficiency in human subjects. METHODS CD21 protein expression was analyzed by means of flow cytometry and Western blotting. CD21 transcripts were quantified by using real-time PCR. The CD21 gene was sequenced. Wild-type and mutant CD21 cDNA expression was studied after transfection of 293T cells. Binding of EBV-gp350 or C3d-containing immune complexes and induction of calcium flux in CD21-deficient B cells were analyzed by means of flow cytometry. Antibody responses to protein and polysaccharide vaccines were measured. RESULTS A 28-year-old man presented with recurrent infections, reduced class-switched memory B cells, and hypogammaglobulinemia. CD21 receptor expression was undetectable. Binding of C3d-containing immune complexes and EBV-gp350 to B cells was severely reduced. Sequence analysis revealed a compound heterozygous deleterious mutation in the CD21 gene. Functional studies with anti-immunoglobulin- and C3d-containing immune complexes showed a complete loss of costimulatory activity of C3d in enhancing suboptimal B-cell receptor stimulation. Vaccination responses to protein antigens were normal, but the response to pneumococcal polysaccharide vaccination was moderately impaired. CONCLUSIONS Genetic CD21 deficiency adds to the molecular defects observed in human subjects with hypogammaglobulinemia.

B Cell Receptor-Mediated Calcium Signaling Is Impaired in B Lymphocytes of Type Ia Patients with Common Variable Immunodeficiency

Several lines of evidence have demonstrated B cell intrinsic activation defects in patients with common variable immunodeficiency (CVID). The rapid increase of intracellular free calcium concentrations after engagement of the BCR represents one crucial element in this activation process. The analysis of 53 patients with CVID for BCR-induced calcium flux identified a subgroup of patients with significantly reduced Ca2+ signals in primary B cells. This subgroup strongly corresponded to the class Ia of the Freiburg classification. Comparison at the level of defined B cell subpopulations revealed reduced Ca2+ signals in all mature B cell populations of patients with CVID class Ia when compared with healthy individuals and other groups of patients with CVID but not in circulating transitional B cells. BCR-induced Ca2+ responses were the lowest in CD21low B cells in patients as well as healthy donors, indicating an additional cell-specific mechanism inhibiting the Ca2+ flux. Although proximal BCR signaling events are unperturbed in patients’ B cells, including normal phospholipase Cγ2 phosphorylation and Ca2+ release from intracellular stores, Ca2+ influx from the extracellular space is significantly impaired. CD22, a negative regulator of calcium signals in B cells, is highly expressed on CD21low B cells from patients with CVID Ia and might be involved in the attenuated Ca2+ response of this B cell subpopulation. These data from patients with CVID suggest that a defect leading to impaired BCR-induced calcium signaling is associated with the expansion of CD21low B cells, hypogammaglobulinemia, autoimmune dysregulation, and lymphadenopathy.

CD21low B cells in common variable immunodeficiency do not show defects in receptor editing, but resemble tissue-like memory B cells.

To the editor: We read with great interest the article by Isnardi et al[1][1] wherein the authors showed very similar phenotypic and genotypic features of CD21−/lo B cells as previously reported.[2][2] In view of the polyreactivity, increased autoreactivity, and strongly reduced calcium response

B-cell signaling in persistent polyclonal B lymphocytosis (PPBL).

Persistent polyclonal B lymphocytosis (PPBL) is a benign hematological disorder characterized by a selective expansion of circulating polyclonal marginal zone (MZ)‐like B cells. Previous reports demonstrated that cases of PPBL showed poor activation, proliferation and survival of B cells in vitro, yet the underlying defect remains unknown. Here we report for the first time an attenuated activation of the canonical NF‐κB (nuclear factor of kappa light polypeptide gene enhancer in B cells) and mitogen‐activated protein kinase/extracellular signal‐regulated kinase pathway after CD40 stimulation. This defect was selective, as alternative NF‐κB signaling after CD40 stimulation and both B‐cell receptor‐ and Toll‐like receptor 9‐mediated activation remained unaffected. Reduced canonical NF‐κB activation resulted in decreased IκBα and CD40 expression in resting cells. In PPBL patients, expression of Bcl‐xL in MZ‐like B cells did not increase upon activation, consistent with the high apoptosis rates of PPBL‐derived B cells that were observed in vitro. The B‐cell phenotype of mice with selective knockouts of early components of the CD40 signaling pathway resembles PPBL, but sequencing corresponding genes in sorted MZ‐like B cells of PPBL patients did not reveal relevant genetic alterations. Nevertheless, the frequently observed mutations in early signaling components of the NF‐κB pathway in MZ lymphomas underline the relevance of our findings for the pathogenesis of PPBL.

Recurrence of persistent polyclonal B lymphocytosis (PPBL) after rituximab treatment

Dear Editor, Persistent polyclonal B lymphocytosis (PPBL) is a rare entity with a polyclonal increase of marginal zone-like B lymphocytes in the peripheral blood [1, 2], most prevalent in smoking middle-aged women [3]. Patients display elevated immunoglobulin M (IgM) and sometimes decreased IgG levels in the serum [4–6]. Splenomegaly and/or polyclonal lymphocytic infiltration of the bone marrow may be present [7, 8]. The disease has been associated with HLA-DR7 positivity and chromosomal abnormalities [6], but its etiology remains elusive. Herein, we report its recurrence after rituximab treatment, revealing interesting aspects of its putative pathophysiology. A 44-year-old Caucasian womanwith a history of Sudeck’s dystrophy and hysterectomy presented with epigastric pain. The patient was an active smoker. Awork-up revealed splenomegaly (3.6×16.5 cm) in the absence of enlarged lymph nodes. A differential blood count showed normal leukocyte count (9440/μl) with lymphocytosis (4863/μl, normal 1000– 2800/μl), and blood smear showed atypical, agranular large lymphocytes with bilobated nuclei (Fig. 1a). IgM was elevated (3.18 g/l, normal 0.4–2.3 g/l, Fig. 1b) without monoclonal bands in immunofixation, and IgG was reduced (4.09 g/l, normal 7–16 g/l). A bone marrow biopsy showed normal hematopoiesis and mild lymphocytosis without blasts or light chain restriction. Relative lymphocytosis in the peripheral blood was traced back to a B lymphocytosis (3457/μl, normal 100–500/μl, Fig. 1c) with no detectablemonoclonality in flow cytometry and IgH clonality analysis (Fig. 1d). The patient was HLA-DR7 negative. Albeit a watch-and-wait strategy is generally indicated in this situation, the patient was treated four times with 375 mg/ m rituximab resulting in the normalization of her spleen size (3.7×10.8 cm) and IgM level (2.1 g/l), as well as an eradication of her peripheral blood B lymphocytes (0/μl). Eighteen months after rituximab treatment, her B cell counts were back to normal (307/μl) and a mild polyclonal IgM elevation had returned (2.47 g/l). B cell numbers continued to rise over the next months (Fig. 1c), lymphocytes with bilobated nuclei reappeared, and B cell subpopulation analysis 2 years after the rituximab treatment showed an expansion of marginal zone-like B lymphocytes in the peripheral blood (52.1 % of B cells, normal <30.8 %) indicating recurrence of PPBL. However, her spleen size was still normal. FISH analysis revealed one or two 3q gains in 3.5 % of B cells, respectively (Fig. 1e, cutoffs for non-B cells 0.5–1.0 %). The recurrence of the PPBL phenotype after eradication of the recirculating B cell pool is remarkable and gives rise to various speculations on the etiology of PPBL. Given the high cellular burden in PPBL, an insufficient eradication and re-expansion of affected B cells in peripheral tissues is a possible explanation; however, the eradication from peripheral blood was complete. Alternatively, as CD20 expression is C. Wehr : L. Houet : S. Gutenberger :K. Warnatz (*) Center for Chronic Immunodeficiency (CCI), University Medical Center Freiburg, University of Freiburg, Breisacherstrasse 117, 79106 Freiburg, Germany e-mail: klaus.warnatz@uniklinik-freiburg.de