The human CD77- B cell population represents a heterogeneous subset of cells comprising centroblasts, centrocytes, and plasmablasts, prompting phenotypical revision

The process of becoming an Ig-producing plasma cell takes the mature B cell through the germinal center, where Ig genes are diversified through somatic hypermutation and class switch recombination. To more clearly define functional characteristics of the germinal center dark zone centroblasts and the light zone centrocytes, we have performed expression analysis of the CD77(+) and CD77(-) populations, because CD77 has been accepted as a discriminator of centroblasts and centrocytes. Our results demonstrated that the CD77(+) and the CD77(-) populations lack functional associated expression programs discriminating the two populations. Both populations are shown to be actively cycling and to share common features associated with cell cycle regulation and DNA maintenance. They are also shown to have an equally active DNA repair program, as well as components involved in somatic hypermutation and class switch recombination. Moreover, the data also demonstrated that the CD77(-) population comprises cells with an already initiated plasma cell differentiation program. Together this demonstrates that CD77 does not discriminate centroblasts and centrocytes and that the CD77(-) population represents a heterogeneous subset of cells, comprising centroblasts, centrocytes, and plasmablast.     

Modelling two possible mechanisms for the regulation of the germinal center dynamics

Research on the germinal center has tried to unravel the mechanisms that control its dynamics. In this study we focus on the termination of the germinal center reaction, which is still an open problem. We propose two hypothetical biological mechanisms that may be responsible for the control of germinal center dynamics and analyze them through mathematical models. The first one is based on the differentiation of follicular dendritic cells and/or T cells. Interaction of these cells in the differentiated state with germinal center B cells would promote B cell differentiation into memory B cells and Ab-forming cells, ending the germinal center reaction. The second mechanism applies only to a scenario without recycling and consists of the decay of a hypothetical proliferation signal for centroblasts that limits the number of cell divisions. Each of the models makes predictions that can be experimentally tested.     

A mathematical model for germinal centre kinetics and affinity maturation

We present a mathematical model which reproduces experimental data on the germinal centre (GC) kinetics of the primed primary immune response and on affinity maturation observed during the reaction. We show that antigen masking by antibodies which are produced by emerging plasma cells can drive affinity maturation and provide a feedback mechanism by which the reaction is stable against variations in the initial antigen amount over several orders of magnitude. This provides a possible answer to the long-standing question of the role of antigen reduction in driving affinity maturation. By comparing model predictions with experimental results, we propose that the selection probability of centrocytes and the recycling probability of selected centrocytes are not constant but vary during the GC reaction with respect to time. It is shown that the efficiency of affinity maturation is highest if clones with an affinity for the antigen well above the average affinity in the GC leave the GC for either the memory or plasma cell pool. It is further shown that termination of somatic hypermutation several days before the end of the germinal centre reaction is beneficial for affinity maturation. The impact on affinity maturation of simultaneous initiation of memory cell formation and somatic hypermutation vs. delayed initiation of memory cell formation is discussed.     

A mathematical model on germinal center kinetics and termination.

We devise a mathematical model to study germinal center (GC) kinetics. Earlier models for GC kinetics are extended by explicitly modeling 1) the cell division history of centroblasts, 2) the Ag uptake by centrocytes, and 3) T cell dynamics. Allowing for T cell kinetics and T-B cell interactions, we study the role of GC T cells in GC kinetics, GC termination, and B cell selection. We find that GC T cells play a major role in GC formation, but that the maintenance of established GC reactions requires very few T cells only. The results therefore suggest that the termination of a GC reaction is largely caused by lack of Ag on the follicular dendritic cells and is hardly influenced by Th cells. Ag consumption by centrocytes is the major factor determining the decay rate of the antigenic stimulus during a GC reaction. Investigating the effect of the Ag dose on GC kinetics, we find that both the total size of the GC and its duration are hardly influenced by the initial amount of Ag. In the model this is due to a buffering effect by competition for limited T cell help and/or competition between proliferating centroblasts.     

Distinct role of follicular dendritic cells and T cells in the proliferation, differentiation, and apoptosis of a centroblast cell line, L3055

Germinal center (GC) B cells undergo complex interactions with follicular dendritic cells (FDC) and T cells in the course of differentiation into memory B and plasma cells. To delineate the individual roles of FDC and T cells at each stage of GC B cell differentiation at the clonal level and to analyze the signals involved, we adopted a unique experimental model using an FDC line, HK, and a lymphoma cell line, L3055, that resembles centroblasts. A detailed phenotypic analysis revealed L3055 cells to be a clonal population originating from the GC. Like freshly isolated centroblasts, L3055 cells underwent spontaneous apoptosis when cultured in the absence of fresh FDC or HK cells. L3055 cells proliferated continuously in the presence of HK cells, while they differentiated into a population with the phenotype of centrocytes after stimulation with CD40 ligand (CD40L) and IL-4. The CD40L-stimulated L3055 cells underwent CD95-mediated apoptosis, which was reminiscent of the feature of CD40L-stimulated tonsillar GC B cells. In contrast to HK cells that did not protect L3055 cells from anti-Ig killing, CD40L plus IL-2, IL-4, and IL-10 prevented anti-Ig-induced apoptosis. These experimental results demonstrate a distinct function of FDC and activated T cells, in that FDC provide signals for rapid proliferation of centroblasts, whereas T cells confer signals for differentiation of centroblasts into centrocytes and resistance to B cell receptor-mediated apoptosis. T cells collaborate with FDC in the protection and expansion of the Ag-specific GC B cells.     

Cutting edge: polycomb gene expression patterns reflect distinct B cell differentiation stages in human germinal centers

Polycomb group (Pc-G) proteins regulate homeotic gene expression in Drosophila, mouse, and humans. Mouse Pc-G proteins are also essential for adult hematopoietic development and contribute to cell cycle regulation. We show that human Pc-G expression patterns correlate with different B cell differentiation stages and that they reflect germinal center (GC) architecture. The transition of resting mantle B cells to rapidly dividing Mib-1(Ki-67)+ follicular centroblasts coincides with loss of BMI-1 and RING1 Pc-G protein detection and appearance of ENX and EED Pc-G protein expression. By contrast, differentiation of centroblasts into centrocytes correlates with reappearance of BMI-1/RING1 and loss of ENX/EED and Mib-1 expression. The mutually exclusive expression of ENX/EED and BMI-1/RING1 reflects the differential composition of two distinct Pc-G complexes. The Pc-G expression profiles in various GC B cell differentiation stages suggest a role for Pc-G proteins in GC development.     

Molecular definition of the germinal centre stage of B-cell differentiation

Genomic-scale gene expression analysis provides views of biological processes as a whole that are difficult to obtain using traditional single-gene experimental approaches. In the case of differentiating systems, gene expression profiting can define a stage of differentiation by the characteristic expression of hundreds of genes. Using specialized DNA microarrays termed 'Lymphochips', gene expression during mature B-cell differentiation has been defined. Germinal centre B cells represent a stage of differentiation that can be defined by a gene expression signature that is not shared by other highly proliferative B-cell populations such as mitogenically activated peripheral blood B cells. The germinal centre gene expression signature is maintained to a significant degree in lymphoma cell lines derived from this stage of differentiation, demonstrating that this gene expression programme does not require ongoing interactions with other germinal centre cell types. Analysis of representative cDNA libraries prepared from resting and activated peripheral blood B cells, germinal centre centroblasts, centrocytes and tonsillar memory B cells has confirmed and extended the results of DNA microarray gene expression analysis.     

RAG1 and RAG2 expression by B cell subsets from human tonsil and peripheral blood

It has been suggested that B cells acquire the capacity for secondary V(D)J recombination during germinal center (GC) reactions. The nature of these B cells remains controversial. Subsets of tonsil and blood B cells and also individual B cells were examined for the expression of recombination-activating gene (RAG) mRNA. Semiquantitative analysis indicated that RAG1 mRNA was present in all tonsil B cell subsets, with the largest amount found in naive B cells. RAG2 mRNA was only found in tonsil naive B cells, centrocytes, and to a lesser extent in centroblasts. Neither RAG1 nor RAG2 mRNA was routinely found in normal peripheral blood B cells. In individual tonsil B cells, RAG1 and RAG2 mRNAs were found in 18% of naive B cells, 22% of GC founder cells, 0% of centroblasts, 13% of centrocytes, and 9% of memory B cells. Individual naive tonsil B cells containing both RAG1 and RAG2 mRNA were activated (CD69(+)). In normal peripheral blood approximately 5% of B cells expressed both RAG1 and RAG2. These cells were uniformly postswitch memory B cells as documented by the coexpression of IgG mRNA. These results indicate that coordinate RAG expression is not found in normal peripheral naive B cells but is up-regulated in naive B cells which are activated in the tonsil. With the exception of centroblasts, RAG1 and RAG2 expression can be found in all components of the GC, including postswitch memory B cells, some of which may circulate in the blood of normal subjects.     

Antigen-driven selection in germinal centers as reflected by the shape characteristics of immunoglobulin gene lineage trees: a large-scale simulation study

During the immune response, the generation of memory B lymphocytes in germinal centers involves affinity maturation of the cells’ antigen receptors, based on somatic hypermutation of receptor genes and antigen-driven selection of the resulting mutants. Affinity maturation is vital for immune protection, and is the basis of humoral immune learning and memory. Lineage trees of somatically hypermutated immunoglobulin genes often serve to qualitatively illustrate claims concerning the dynamics of affinity maturation in germinal centers. Here, we derive the quantitative relationships between parameters characterizing affinity maturation dynamics (proliferation, differentiation and mutation rates, initial affinity of the Ig to the antigen, and selection thresholds) and the mathematical properties of lineage trees, using a computer simulation which combines mathematical models for all mature B cell populations, stochastic models of hypermutation and selection, lineage tree generation and measurement of graphical tree characteristics. We identified seven key lineage tree properties, and found correlations of these with initial clone affinity and with the selection threshold. These two parameters were found to be the main factors affecting lineage tree shapes in both primary and secondary response trees. The results also confirm that recycling from centrocytes back to centroblasts is highly likely.     

Tissue distribution and ultrastructure of B lymphocytes reacting with the monoclonal antibody anti-Y29/55

The reactivity of normal tonsilar cells with the monoclonal antibody anti-Y29/55 is characterized at the tissue and ultrastructural cytological level. Using an indirect immuno-alkaline phosphatase method on frozen sections the antibody labels mantle zone and germinal center lymphocytes. This staining reaction is more generalized in B-lymphocyte areas than that obtained with antibodies to IgM and IgD. By indirect immunoperoxidase staining, as well as by an indirect rosetting procedure in cell suspensions, the reactive cell population were either small resting lymphocytes or activated lymphocytes corresponding to centrocytes, centroblasts, immunoblasts and plasmoblasts; some plasma cells were also labeled. These results characterize the monoclonal antibody anti-Y29/55 as a pan-B-marker antibody, useful for labeling resting and activated peripheral B-lymphocytes in frozen tissue sections and cell suspensions.     

Beraprost enhances the APC function of B cells by upregulating CD86 expression levels

Lipid mediators are emerging as important regulators of the immune system. Based on our previous result that shows strong expression of prostacyclin synthase in the germinal center, we investigated whether prostacyclin would regulate the APC function of B cells. Owing to the very short half-life of prostacyclin in experimental conditions, we used a more stable analog, beraprost. Beraprost increased the amounts of the costimulatory molecule CD86 but not CD80 on the surface of activated B cells in time- and dose-dependent manners. However, the enhancing effect of beraprost was not observed on memory B cells, centroblasts, and centrocytes. Beraprost required BCR and CD40 signals to upregulate CD86 expression levels. Other prostanoids such as PGE(2), 6-keto-PGF(1α), and PGF(2α) failed to alter CD86 expression levels, whereas other prostacyclin analogs were as potent as beraprost. Results carried out with receptor antagonists revealed that beraprost enhanced CD86 levels by binding to prostacyclin receptor IP and by increasing intracellular cAMP concentrations. Beraprost-treated B cells potently stimulated allogeneic T cells, which was significantly abolished by CD86 neutralization. Our data imply an unrecognized cellular and molecular mechanism about the germinal center reactions.     

B cell activation state-governed formation of germinal centers following viral infection

Germinal centers are structures that promote humoral memory cell formation and affinity maturation, but the triggers for their development are not entirely clear. Activated extrafollicular B cells can form IgM-producing plasmablasts or enter a germinal center reaction and differentiate into memory or plasma cells, mostly of the IgG isotype. Vesicular stomatitis virus (VSV) induces both types of response, allowing events that promote each of these pathways to be studied. In this work, extrafollicular vs germinal center responses were examined at a cellular level, analyzing VSV-specific B cells in infected mice. We show that VSV-specific germinal centers are transiently formed when insufficient proportions of specific T cell help is available and that strong B cell activation in cells expressing high levels of the VSV-specific BCR promoted their differentiation into early blasts, whereas moderate stimulation of B cells or interaction with Th cells restricted extrafollicular responses and promoted germinal center formation.     

CX3CR1 Is Expressed by Human B Lymphocytes and Meditates CX3CL1 Driven Chemotaxis of Tonsil Centrocytes

Background

Fractalkine/CX₃CL1, a surface chemokine, binds to CX₃CR1 expressed by different lymphocyte subsets. Since CX₃CL1 has been detected in the germinal centres of secondary lymphoid tissue, in this study we have investigated CX₃CR1 expression and function in human naïve, germinal centre and memory B cells isolated from tonsil or peripheral blood.

Methodology/Principal Findings

We demonstrate unambiguously that highly purified human B cells from tonsil and peripheral blood expressed CX₃CR1 at mRNA and protein levels as assessed by quantitative PCR, flow cytometry and competition binding assays. In particular, naïve, germinal centre and memory B cells expressed CX₃CR1 but only germinal centre B cells were attracted by soluble CX₃CL1 in a transwell assay. CX₃CL1 signalling in germinal centre B cells involved PI3K, Erk1/2, p38, and Src phosphorylation, as assessed by Western blot experiments. CX₃CR1⁺ germinal centre B cells were devoid of centroblasts and enriched for centrocytes that migrated to soluble CX₃CL1. ELISA assay showed that soluble CX₃CL1 was secreted constitutively by follicular dendritic cells and T follicular helper cells, two cell populations homing in the germinal centre light zone as centrocytes. At variance with that observed in humans, soluble CX₃CL1 did not attract spleen B cells from wild type mice. OVA immunized CX₃CR1⁻/⁻ or CX₃CL1⁻/⁻ mice showed significantly decreased specific IgG production compared to wild type mice.

Conclusion/Significance

We propose a model whereby human follicular dendritic cells and T follicular helper cells release in the light zone of germinal centre soluble CX₃CL1 that attracts centrocytes. The functional implications of these results warrant further investigation.     

Follicular dendritic cells catalyze hepatocyte growth factor (HGF) activation in the germinal center microenvironment by secreting the serine protease HGF activator

Ag-specific B cell differentiation, the process that gives rise to plasma cells and memory B cells, involves the formation of germinal centers (GC). Within the GC microenvironment, multiple steps of B cell proliferation, selection, and maturation take place, which are controlled by the BCR in concert with cytokines and contact-dependent signals from follicular dendritic cells (FDCs) and T cells. Signaling by the multifunctional cytokine hepatocyte growth factor (HGF) and its receptor MET has been shown to induce integrin-mediated adhesion of B cells to VCAM-1, which is expressed by FDCs. In the present study we have examined the expression of regulatory components of the HGF/MET pathway, including HGF activator (HGFA), within the secondary lymphoid organ microenvironment. We show that MET is expressed by both centroblasts and plasma cells, and that HGFA is expressed by plasma cells. Because we have shown that HGF is a potent growth and survival factor for malignant plasma cells, HGF may also serve as a survival factor for normal plasma cells. Furthermore, we demonstrate that FDCs are the major source for HGF and its activator within the GC microenvironment. Both HGF and HGFA are expressed by FDCs in the GC dark zone (CD21high/CD23low), but not in the light zone (CD21high/CD23high). These findings suggest that HGF and HGFA provided by dark zone FDCs help to regulate the proliferation, survival, and/or adhesion of MET-positive centroblasts.     

Cutting edge: back to "one-way" germinal centers

The present status of germinal center (GC) research is revisited using in silico simulations based on recent lymphocyte motility data in mice. The generally adopted view of several rounds of somatic hypermutations and positive selection is analyzed with special emphasis on the spatial organization of the GC reaction. We claim that the development of dark zones is not necessary for successful GC reactions to develop. We find that a recirculation of positively selected centrocytes to the dark zone is rather unlikely. Instead we propose a scenario that combines a multiple-step mutation and selection concept with a "one-way" GC in the sense of cell migration.     

Re-evaluating the recycling hypothesis in the germinal centre

Mathematical models have been used to study different aspects of the germinal centre reaction, in particular, affinity maturation of antibodies and the hypothesis of recycling. So far, interpretation of several theoretical and experimental results has pointed to the existence of recycling. However, theoretical models have seldom been compared with experimental data from specific immune responses and the potential relevance of recycling in the germinal centre is still an open problem. In this article, we propose a model without recycling that takes into account selection mechanisms that were previously uncovered experimentally. We apply the model to several experimental systems that use different Ag and compare the results with experimental data of affinity maturation whenever available. The results obtained for a primary immune response to the hapten (4-hydroxy-3-nitrophenyl)-acetyl show that recycling is not a necessary mechanism to achieve the level of affinity maturation observed in germinal centre reactions. Similar levels of affinity maturation are obtained for other responses, although for antibodies involving several affinity-enhancing mutations the affinity maturation obtained with the model is much lower. Interpretation of these results and consequences towards the concept of recycling are discussed.