Magdalena Berkowska

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

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.

Disorders of pitch production in tone deafness.

Singing is as natural as speaking for the majority of people. Yet some individuals (i.e., 10–15%) are poor singers, typically performing or imitating pitches and melodies inaccurately. This condition, commonly referred to as “tone deafness,” has been observed both in the presence and absence of deficient pitch perception. In this article we review the existing literature concerning normal singing, poor-pitch singing, and, briefly, the sources of this condition. Considering that pitch plays a prominent role in the structure of both music and speech we also focus on the possibility that speech production (or imitation) is similarly impaired in poor-pitch singers. Preliminary evidence from our laboratory suggests that pitch imitation may be selectively inaccurate in the music domain without being affected in speech. This finding points to separability of mechanisms subserving pitch production in music and language.

Singing Proficiency in the Majority: Normality and Phenotypes of Poor Singing

Recent evidence indicates that the majority of occasional singers can carry a tune. For example, when asked to sing a well‐known song (e.g., “Happy Birthday”), nonmusicians performing at a slow tempo are as proficient as professional singers. Yet, some occasional singers are poor singers, mostly in the pitch domain, and sometimes despite not having impoverished perception. Poor singing is not a monolithic deficit, but is likely to be characterized by a diversity of singing “phenotypes.” Here we systematically examined singing proficiency in a group of occasional singers, with the goal of characterizing the different patterns of poor singing. Participants sang three well‐known melodies (e.g., “Jingle Bells”) at a natural tempo and at a slow tempo, as indicated by a metronome. For each rendition, we computed objective measures of pitch and time accuracy with an acoustical method. The results confirmed previous observations that the majority of occasional singers can sing in tune and in time. Moreover, singing at a slow tempo after the target melody to be imitated was presented with a metronome improved pitch and time accuracy. In general, poor singers were mostly impaired on the pitch dimension, although various patterns of impairment emerged. Pitch accuracy or time accuracy could be selectively impaired; moreover, absolute measures of singing proficiency (pitch or tempo transposition) dissociated from relative measures of proficiency (pitch intervals, relative duration). These patterns of dissociations point to a multicomponent system underlying proficient singing that fractionates as a result of a developmental anomaly.

Human IgE+ B cells are derived from T cell–dependent and T cell–independent pathways

BACKGROUND The prevalence of IgE-mediated diseases has been increasing worldwide, yet IgE-expressing B cells are poorly characterized, mainly because of their scarcity and low membrane IgE levels. OBJECTIVE We sought to study the immunobiology of human IgE-expressing B cells in healthy subjects and patients with allergic disease. METHODS We used a stepwise approach for flow cytometric detection and purification of human IgE-expressing B cells in control subjects, CD40 ligand-deficient patients, and patients with atopic dermatitis. Molecular analysis of replication histories, somatic hypermutation (SHM), and immunoglobulin class-switching was performed. RESULTS Using multicolor flow cytometry, we reliably detected IgE-expressing plasma cells and 2 IgE-expressing memory B-cell subsets. These IgE-expressing cells showed molecular and phenotypic signs of antigen responses. The replication history and SHM levels of IgE(+) plasma cells and CD27(+)IgE(+) memory B cells fitted with a germinal center (GC)-dependent pathway, often through an IgG intermediate, as evidenced from Sγ remnants in Sμ-Sε switch regions. CD27(-)IgE(+) cells showed limited proliferation and SHM and were present in CD40 ligand-deficient patients, indicating a GC-independent origin. Patients with atopic dermatitis had normal numbers of blood IgE(+) plasma cells and CD27(+)IgE(+) memory B cells but increased numbers of CD27(-)IgE(+) memory B cells with high SHM loads compared with those seen in healthy control subjects and patients with psoriasis. CONCLUSIONS We delineated GC-dependent and GC-independent IgE(+) B-cell responses in healthy subjects and indicated involvement of the GC-independent pathway in a human IgE-mediated disease. These findings provide new insights into the pathogenesis of IgE-mediated diseases and might contribute to accurate monitoring of IgE(+) B cells in patients with severe disease undergoing anti-IgE treatment.

Reducing Linguistic Information Enhances Singing Proficiency in Occasional Singers

In this study we examined the effect of reducing linguistic information on singing proficiency in occasional singers. Thirty‐nine occasional singers were asked to sing from memory and to imitate three familiar melodies with lyrics and on the syllable /la/. Performances were analyzed with an acoustically based method yielding objective measures of pitch and temporal accuracy. Results obtained in production and imitation tasks revealed increased accuracy (e.g., fewer pitch interval errors and contour errors) when occasional singers produced melodies on a syllable as compared to singing with lyrics. This effect may be the result of reduced memory load and/or motor entrainment.

Circulating Human CD27−IgA+ Memory B Cells Recognize Bacteria with Polyreactive Igs

The vast majority of IgA production occurs in mucosal tissue following T cell–dependent and T cell–independent Ag responses. To study the nature of each of these responses, we analyzed the gene-expression and Ig-reactivity profiles of T cell–dependent CD27+IgA+ and T cell–independent CD27−IgA+ circulating memory B cells. Gene-expression profiles of IgA+ subsets were highly similar to each other and to IgG+ memory B cell subsets, with typical upregulation of activation markers and downregulation of inhibitory receptors. However, we identified the mucosa-associated CCR9 and RUNX2 genes to be specifically upregulated in CD27−IgA+ B cells. We also found that CD27−IgA+ B cells expressed Abs with distinct Ig repertoire and reactivity compared with those from CD27+IgA+ B cells. Indeed, Abs from CD27−IgA+ B cells were weakly mutated, often used Igλ chain, and were enriched in polyreactive clones recognizing various bacterial species. Hence, T cell–independent IgA responses are likely involved in the maintenance of gut homeostasis through the production of polyreactive mutated IgA Abs with cross-reactive anti-commensal reactivity.

Loss of juxtaposition of RAG-induced immunoglobulin DNA ends is implicated in the precursor B-cell differentiation defect in NBS patients

The Nijmegen breakage syndrome (NBS) is a rare inherited condition, characterized by microcephaly, radiation hypersensitivity, chromosomal instability, an increased incidence of (mostly) lymphoid malignancies, and immunodeficiency. NBS is caused by hypomorphic mutations in the NBN gene (8q21). The NBN protein is a subunit of the MRN (Mre11-Rad50-NBN) nuclear protein complex, which associates with double-strand breaks. The immunodeficiency in NBS patients can partly be explained by strongly reduced absolute numbers of B lymphocytes and T lymphocytes. We show that NBS patients have a disturbed precursor B-cell differentiation pattern and significant disturbances in the resolution of recombination activating gene-induced IGH breaks. However, the composition of the junctional regions as well as the gene segment usage of the reduced number of successful immunoglobulin gene rearrangements were highly similar to healthy controls. This indicates that the NBN defect leads to a quantitative defect in V(D)J recombination through loss of juxtaposition of recombination activating gene-induced DNA ends. The resulting reduction in bone marrow B-cell efflux appeared to be partly compensated by significantly increased proliferation of mature B cells. Based on these observations, we conclude that the quantitative defect will affect the B-cell receptor repertoire, thus contributing to the observed immunodeficiency in NBS patients.

The Majority of Human Memory B Cells Recognizing RhD and Tetanus Resides in IgM+ B Cells

B cell memory to T cell–dependent (TD) Ags are considered to largely reside in class-switched CD27+ cells. However, we previously observed that anti-RhD (D) Igs cloned from two donors, hyperimmunized with D+ erythrocytes, were predominantly of the IgM isotype. We therefore analyzed in this study the phenotype and frequency of D- and tetanus toxoid–specific B cells by culturing B cells in limiting dilution upon irradiated CD40L-expressing EL4.B5 cells and testing the culture supernatant. Most Ag-specific B cells for both TD Ags were found to reside in the IgM-expressing B cells, including CD27− B cells, in both hyperimmunized donors and nonhyperimmunized volunteers. Only shortly after immunization a sharp increase in Ag-specific CD27+IgG+ B cells was observed. Next, B cells were enriched with D+ erythrocyte ghosts and sorted as single cells. Sequencing of IGHV, IGLV, IGKV, and BCL6 genes from these D-specific B cell clones demonstrated that both CD27−IgM+ and CD27+IgM+ B cells harbored somatic mutations, documenting their Ag-selected nature. Furthermore, sequencing revealed a clonal relationship between the CD27−IgM+, CD27+IgM+, and CD27+IgG+ B cell subsets. These data strongly support the recently described multiple layers of memory B cells to TD Ags in mice, where IgM+ B cells represent a memory reservoir which can re-enter the germinal center and ensure replenishment of class-switched memory CD27+ B cells from Ag-experienced precursors.

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

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 VDJ(H) 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 VDJ(H) rearrangements, microarray analysis indicated that CD127⁺ cells resembled large pre-B cells, which is consistent with their low level of Ig light-chain 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.

Checkpoints of B cell differentiation: visualizing Ig-centric processes.

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.