During a T-dependent immune response, activated B cells enter structures called germinal centers (GC) in the follicles of secondary lymphoid organs. GC B cells proliferate and undergo diversification of their Ig through somatic hypermutation and class-switch recombination. These Ig diversifications require the activity of the enzyme activation-induced deaminase (AID). Clonal selection within GCs selects GC B cells with the highest affinities for antigen to mature into plasma cells and memory B cells. GCs are underpinned by stromal cells called follicular dendritic cells (FDC). FDC functions include secretion of B cell chemokines and the retention of Ag complexes that allow GC B cells to test the affinity of their Ig. FDCs require constitutive signaling through lymphotoxin beta receptor (LTβR) from lymphotoxin alpha-beta (LTαβ) on the surface of B cells to maintain their functions. In these studies, I investigated the properties of GCs using two primary experimental models. First, I employed genetic and pharmacological ablation of LTβR signaling to investigate the expression of AID and the function of GCs in the absence of FDCs. I determined that FDCs are not required for GC formation or the expression of AID in lymph nodes, but that FDCs are crucial for affinity maturation. Second, I examined the competition between AID-/- and WT B cells in the GCs of mixed BM chimeras to investigate the role of affinity maturation during clonal selection. I found that AID increases GC B cell apoptosis, likely by inducing DNA damage, and that this is important in regulating GC size. I also found that AID-/- B cells accumulate at the centrocyte stage of the GC reaction and that this is due to a partial block in plasma cell maturation.

No description available.

In this study, we have analyzed the steady-state kinetics of B lymphocytes in mouse and rat bone marrow using previously published experimental data. Over many years, Prof D.G. Osmond and his colleagues have built up a scheme of B cell development in mouse bone marrow based on the sequential expression of markers associated with the B lineage. The earliest precursor B cells comprise three populations of proliferating pro-B cells, i.e. early, intermediate, and late pro-B cells. The subsequent populations comprise pre-B cells that give rise to nondividing B lymphocytes expressing surface IgM. / In our analysis, we have checked the available published data for consistency with the proliferation of precursor B cells and their death via apoptosis at certain stages of cell development. We made an extensive summary of the existing data on the various B cell precursors and organized it into a comprehensible framework. We built a mathematical model for the proliferation and differentiation of mammalian B lymphocytes in laboratory mice and rats and estimated all of the parameters to explain the existing steady state data. In this context, mathematical modeling acts as a useful tool to analyze hypotheses and experimental results concerning the steady state numbers of B lymphocytes.

Graduation date: 1930

Tumour necrosis factor receptor (TNFR) family members are widely expressed in cells of the immune system and are essential for the development and function of many immune cell types. The TNFR associated factor (TRAF) family are signal adapter molecules that are recruited to various members of the TNFR family and are important for the transduction of signals downstream of these receptors. In these studies, gene targeting was used to create a mouse capable of undergoing conditional inactivation of the Traf3 gene. These mice were studied alongside previously generated mice that were similarly genetically modified with respect to the Traf2 gene. The mice produced lacked expression of either TRAF2 or TRAF3 in either B or T cells. In resting B cells TRAF2 and TRAF3 were shown to cooperate to negatively regulate the signalling of B cell activating factor of the TNF family (BAFF) and its receptor (BAFF-R), the TNF ligand and receptor pair that provide obligate survival signals to B cells. Thus, TRAF2- and TRAF3-deficient B cells displayed hyperactive NF-kB2 signalling, an increased ability to survive, and almost identical gene expression profiles, emphasizing the cooperative nature of their roles in resting B cells. Importantly, the survival of these B cells was completely independent of BAFF. In normal B cells, BAFF signalling was shown to lift the negative regulation of survival mediated by TRAF2 and TRAF3, by depleting TRAF3 from the cell. This process was shown to require TRAF2. T cells deficient in TRAF2 or TRAF3 also displayed hyperactivity of the NF-kB2 pathway, but they did not accumulate in vivo or show extended survival in vitro. Mice lacking TRAF2 or TRAF3 in their T cells did however display a decrease in the number of memory phenotype CD8+ T cells. These studies indicate that some of the roles of TRAF2 and TRAF3 are common between B and T cells. However, the consequences of loss of TRAF2 or TRAF3 in B and T cells differs considerably, presumably due to the differential TNFR expression and usage by each cell type.

Humoral immune responses arise when B lymphocytes respond to activation signals, enter mitosis and proliferate rapidly. Concurrent differentiation to antibody secreting and isotype switched effector cells is tightly linked to cell division, such that the degree of proliferation strongly influences the nature of the response that is mounted. Previous versions of a quantitative model of lymphocyte proliferation based on inherent variation in the time cells take to divide or die were able to accurately describe the entry of naïve, resting cells into division and subsequent population expansion. In the work described here, the model was tested and extended by investigating the proliferation cessation and population contraction phases of in vitro B cell responses. Experiments designed to assess the distribution of times to die of cells that had ceased proliferating revealed that the number of divisions achieved by individual cells is stochastically distributed in the population and varied in response to different stimuli. Both the concentration and duration of stimulation regulate the number of divisions undergone. A cell that stops dividing is described as having reached its division destiny. Further investigation revealed that cells reach a maximum division destiny even during repeated high-dose stimulation. This limit is dictated by cellular progression through divisions, and is not dependent on the survival capacity of the cells or time. Incorporation of division destiny in the quantitative model allows proliferation cessation to be described and the distribution of times to die after this point to be assessed. This extended model can describe the full course of in vitro lymphocyte proliferative responses to various different stimuli. (For complete abstract open document)

Group B streptococcus (GBS, S. agalactiae) and human ureaplasmas (U. parvumand U. urealyticum) are two clinically and phylogenetically related, potential perinatal pathogens. Their relationships between genotypes and pathogenesis of GBS and ureaplasma infection were still not well understood, one of the reason is that both of them are still short of a very practical genotyping system. In the study, to solve the above problem we developed genotyping systems for the organisms (the second section). For human ureaplasmas, based on four genes/gene clusters (rRNAgene clusters, the elongation factor Tu genes, urease gene complexes and multiplebanded antigen genes), we designed many primer pairs suitable for developing species identification assays for the two newly established human ureaplasma species (U. parvum and U. urealyticum). Further, based on the heterogeneity of ureaplasma multiple banded antigen gene (which contains species- and serovar-specific regions), we developed genotyping methods for each ureaplasma species.For GBS, based on three sets of molecular markers (capsular polysaccharidesynthesis gene clusters, surface protein antigen genes and mobile genetic elements),we developed a genotyping system. The primary evaluation of the genotyping systems showed that the genotyping systems were practical alternative assays for the conventional serotyping and they will be useful to further explore the relationships between genotypes and pathogenesis of GBS and ureaplasma infection. In the study, we introduced novel data and tools into GBS and ureaplasma studies especially from genomic- and bioinformatics-based molecular microbiology(the third section). For two newly established human ureaplasma species, based on the U. parvum serovar-3 genome, and using the above four important genes/geneclusters, we exposed some interesting problems in the understanding of newureaplasma taxonomy especially in the post genomic era. For GBS, we studied the two published full genomes and exposed some new problems or possible future new research fields. In particular we found the two finished and one ongoing GBS genomes were all non-typical and suggest that future genomic project had better have genetic population structure viewpoint. Finally, we suggested that integrated studies of the two potential or conditional perinatal pathogens, from the viewpoint of evolution, would provide a new understanding angle of the pathogenesis of the two organisms. Studies suggested that during coevolution, human ureaplasmas(especially U. parvum) became friendlier than their ancestors to their human host (by losing most of its virulence genes); however, GBS tried to increase its invasive abilities (by getting more virulence genes) to fight against the human host attack.

Thesis (Ph. D.)--University of Washington, 1989. / Vita. Includes bibliographical references (leaves [439]-452).

Proefschrift Universiteit van Amsterdam. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Universiẗat, Diss., 2006--Giessen.