Berkowska, M.A.

Generation of an immunocompetent B-cell Repertoire (Doctoral Thesis)

2012-11-28T00:00:00Z

The immune system of higher vertebrates, such as humans, has multiple lines of defense against invading microorganisms. The first line is a physical and chemical barrier formed by the skin, mucous membranes and their secretions to prevent the entrance of microorganisms into the body.1-3 The second line consists of proteins (e.g. complement)4 and immune cells (e.g. macrophages, granulocytes, natural killer cells) which are the components of the innate immune system.5-6 Finally, the third line concerns the adaptive immune system with T and B lymphocytes specifically interacting with the invading pathogen. These cells do not only recognize pathogen with a specific antigen receptor, they also adapt their response during an infection to improve recognition of the pathogen and they generate long-term immunological memory.

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.

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.

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

2010-06-01T00:00:00Z

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.