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.     

Maturation of the immune response in germinal centers.

Germinal centers develop in peripheral lymphatic tissue during the primary immune response and may play a crucial role in affinity maturation. We have compared the diversification of the antigen-specific repertoire of B cells, both from within and from outside the germinal centers, during the murine response to 2-phenyloxazolone (phOx). By sequencing V kappa Ox1 L-chains characteristic of phOx-specific antibodies, we show that somatic mutations accumulate in germinal center B cells and that a mutation conferring high affinity binding is found with increasing frequency. An analysis of V/D/J rearrangements suggests that this mutation occurred independently in many B cells, which were then preferentially expanded. We conclude that, although the hypermutation mechanism may be activated before germinal centers develop, affinity maturation by hypermutation and selection takes place in the germinal centers.     

B-cell memory: are subsets necessary?

B-cell memory is provided by populations of quiescent memory B cells and long-lived plasma cells. Whereas it is clear that both of these cell populations arise from germinal centres, the signals and circumstances that trigger germinal-centre B cells to enter and then persist in memory compartments are poorly defined. Here, I propose that germinal centres produce memory B cells and plasma cells throughout the immune response and that memory B cells arise by the emigration of B cells that are chosen at random from the pool available in the germinal centre. The ability of such emigrants to survive as memory B cells depends on their germinal-centre 'history', with the persistence of high-affinity B-cell variants being favoured.     

Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes.

BACKGROUND: The developmental stage from which stems the malignant B cell population in Burkitt's lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rear-ranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells and their descendents. MATERIALS AND METHODS: Rearranged V kappa region genes from 10 kappa-expressing sporadic and endemic BL-derived cell lines (9 IgM and 1 IgG positive) and three kappa-expressing endemic BL biopsy specimens were amplified by polymerase chain reaction and sequenced. In addition, VH region gene sequences from these cell lines were determined. RESULTS: All BL cell lines and the three biopsy specimens carried somatically mutated V region genes. The average mutation frequency of rearranged V kappa genes from eight BL cell lines established from sporadic BL was 1.8%. A higher frequency (6%) was found in five endemic cases (three biopsy specimens and two BL cell lines). CONCLUSIONS: The detection of somatic mutations in the rearranged V region genes suggests that both sporadic and endemic BL represent a B-cell malignancy originating from germinal center B cells or their descendants. Interestingly, the mutation frequency detected in sporadic BL is in a range similar to that characteristic for IgM-expressing B cells in the human peripheral blood and for mu chain-expressing germinal center B cells, whereas the mutation frequency found in endemic BL is significantly higher.     

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.     

Latent murine gamma-herpesvirus infection is established in activated B cells, dendritic cells, and macrophages

Intranasal infection of mice with the murine gamma-herpesvirus MHV-68 results in an acute lytic infection in the lung, followed by the establishment of lifelong latency. Development of an infectious mononucleosis-like syndrome correlates with the establishment of latency and is characterized by splenomegaly and the appearance of activated CD8+ T cells in the peripheral blood. Interestingly, a large population of activated CD8+ T cells in the peripheral blood expresses the V beta 4+ element in their TCR. In this report we show that MHV-68 latency in the spleen after intranasal infection is harbored in three APC types: B cells, macrophages, and dendritic cells. Surprisingly, since latency has not previously been described in dendritic cells, these cells harbored the highest frequency of latent virus. Among B cells, latency was preferentially associated with activated B cells expressing the phenotype of germinal center B cells, thus formally linking the previously reported association of latency gene expression and germinal centers to germinal center B cells. Germinal center formation, however, was not required for the establishment of latency. Significantly, although three cell types were latently infected, the ability to stimulate V beta 4+CD8+ T cell hybridomas was limited to latently infected, activated B cells.     

Axon growth and guidance genes identify T-dependent germinal centre B cells

Selection of B cells subjected to hypermutation in germinal centres (GC) during T cell-dependent (TD) antibody responses yields memory cells and long-lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self-antigens. GC also form in T-independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T-dependent and TI germinal centre B cells. We compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC. Significantly, the largest cluster comprises genes involved in growth and guidance of neuron axons such as Plexin B2, Basp1, Nelf, Shh, Sc4mol and Sult4alpha. This is consistent with formation of long neurite (axon and dendrite)-like structures by mouse and human GC B cells, which may facilitate T:B cell interactions within GC, affinity maturation and B cell memory formation. Expression of BASP1 and PLEXIN B2 protein is very low or undetectable in resting and TI GC B cells, but markedly upregulated in GC B cells induced in the presence of T cell help. Finally we show some of the axon growth genes upregulated in TD-GC B cells including Basp1, Shh, Sult4alpha, Sc4mol are also preferentially expressed in post-GC B cell neoplasms.     

Germinal center cells are a major IL-5-responsive B cell population in peripheral lymph nodes engaged in the immune response

Germinal center formation and the development of B cell memory in lymphoid tissue is a T cell-dependent process. The specific B cell-T cell interactions, and/or cytokines, resulting in germinal center cell growth have not yet been identified. Germinal center B cells were separated from other lymph node (LN) B cells by panning on peanut agglutinin (PNA)-coated dishes. Resulting fractions enriched for PNA+ (germinal center) B cells, and the PNA- (other) LN B cells from immune SJL mice were assayed for proliferation in the presence of cytokines. PNA+ and PNA- B cells responded equally to IL-4 in the anti-mu co-stimulator assay. In contrast, PNA+ B cells responded to murine (r)IL-5 or human B cell growth factor in the dextran sulfate (DxS) co-stimulator assay, to a much greater degree than did PNA- B cells. The same results were obtained with PNA+ and PNA- cells from LAF1 mice. Unfractionated LN B cells from nonimmunized SJL or BALB/c mice did not respond to IL-5 with or without DxS. B cell populations from BALB/c mice such as from spleen and peritoneal cavity, which are known to be high in Ly-1+B cells, responded to IL-5 alone, and more dramatically, to IL-5 as a co-stimulator with DxS. Such populations of cells from SJL mice, which are known to contain low numbers of Ly-1+B cells, responded markedly less. These results are consistent with those of others which show that in nonimmunized mice, Ly-1+B cells are a major IL-5 responsive subpopulation. IL-1 enhanced the proliferation of PNA+ cells in response to rIL-5 and had no effect on PNA- cells. IL-4 and IL-5 did not enhance each other's effects as co-stimulators of proliferation. In contrast to PNA+ B cells from immune LN, B cells activated by Escherichia coli endotoxin exhibited no responses to rIL-5. The present results indicate that in immune LN, PNA+, germinal center B cells constitute a prominent IL-5-responsive population.     

Evolution in miniature: selection, survival and distribution of antigen reactive cells in the germinal centre

Productive immune responses to T-cell-dependent antigens ultimately generate two long-lived compartments: memory B cells and bone marrow-resident plasma cells, which both arise from within germinal centres. The ability of a B-cell clone to populate these effector compartments requires its descendents to outcompete those of other clones participating in the response. Selection occurs at several stages of the response and the criteria differ at these different stages. While affinity predominates as the key, underlying driving force of selection, there is a distinction made at the point at which germinal centre cells initiate entry into the plasma cell and memory B-cell compartments. Becoming a plasma cell requires high affinity and cannot be subverted by blocking cell death, while becoming a memory B cell is dependent on survival alone. While such survival is typically mediated by affinity within the GC, the distinction has important mechanistic implications.     

Molecular programming of B cell memory

The development of high-affinity B cell memory is regulated through three separable phases, each involving antigen recognition by specific B cells and cognate T helper cells. Initially, antigen-primed B cells require cognate T cell help to gain entry into the germinal centre pathway to memory. Once in the germinal centre, B cells with variant B cell receptors must access antigens and present them to germinal centre T helper cells to enter long-lived memory B cell compartments. Following antigen recall, memory B cells require T cell help to proliferate and differentiate into plasma cells. A recent surge of information - resulting from dynamic B cell imaging in vivo and the elucidation of T follicular helper cell programmes - has reshaped the conceptual landscape surrounding the generation of memory B cells. In this Review, we integrate this new information about each phase of antigen-specific B cell development to describe the newly unravelled molecular dynamics of memory B cell programming.     

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.     

Immunohistochemical identification of T- and B-lymphocytes delineated by the unlabelled antibody enzyme method

Summary T-lymphocytes and B-lymphocytes are identified in tissue sections of human tonsils by applying the unlabelled antibody enzyme method. The epithelium of the tonsils contains a majority of immunoglobulin-positive cells and fewer T-lymphocytes. In the subepithelial zones, areas composed of B-cells predominate, however, regions containing T-lymphocytes are also present. The latter are mainly arranged in the lamina propria around high-endothelial venules and often include plasma cells containing immunoglobulin. Follicles containing germinal centres display a complex structure which changes during development. The lymphocytic cap consists of densely packed lymphocytes, labelled heavily by anti-IgM and anti-IGD, and of individual T-lymphocytes. Germinal centres show a framework of immunoglobulin-positive dendritic reticular cells; they contain some heavily labelled lymphoid cells and several cells weakly labelled by anti-IgM and anti-IgA, as well as a small number of T-lymphocytes. Furthermore, the total areas of T- and B-lymphocytes measured by planimetry may differ considerably between different tonsils. Especially total areas of germinal centres vary to a great extent. The quantitative data on amounts of T- and B-cells achieved by planimetry are comparable to those reported in cellular suspensions of tonsils.     

Th17 Effector Cells Support B Cell Responses Outside of Germinal Centres

Th17 cells are pro-inflammatory CD4⁺T cells, which are important in immune responses against fungal pathogens and extracellular bacteria and have also been implicated in various autoimmune syndromes. However, their role in supporting B cell responses in these scenarios remains unclear, representing a significant lapse in our understanding of the role Th17 play in vaccine responses and the regulation of autoimmunity. We employed T cell and B cell receptor transgenic mice specific for model antigens, and adoptive transfer approaches that allowed the tracking of cognate B and T cells in situ and ex vivo using immunological methods. We have found that T cells activated under Th17 polarising conditions have a greater capacity to provide cognate B cell help compared with Th1 polarised populations, supporting higher expansion of antigen specific B cells and enhanced antibody titres. This advantage is associated with the increased persistence of Th17 polarised cells in areas of the lymph nodes where they can provide help (i.e. the B cell follicles). Also the Th17 cells are characterised by their higher expression of ICOS, a costimulatory molecule important for B cell help. Surprisingly, contrary to published reports, Th17 cells were not detected inside germinal centres, although they were found in close proximity to cognate B cells in the follicle early in the genesis of the humoral immune response. These data indicate that, Th17 cells have a more significant role earlier in the initiation/development of the germinal centre response and/or germinal centre-independent events, consistent with their early effector status.     

Why do B cells produce CD40 ligand?

The CD40-CD40 ligand (CD40L) interaction is one of the most important receptor-ligand interactions that occurs during a T dependent immune response. However, while CD40L is expressed on a range of cell types including activated T cells and B cells, dendritic cells granulocytes, macrophages and platelets, only CD40L on T cells is considered by most immunologists when planning experiments or analysing data. The current theory professes that T cells expressing CD40L can provide signals to B cells that induce proliferation, immunoglobulin class switching, antibody secretion, rescue from apoptosis at different times during the life of a B cell and also has a role in the development of germinal centres and the survival of memory B cells. However, the whole story is more complex than presently understood as human and mouse B cells express CD40L on their surface following activation and can release a soluble form of the ligand. This paper hypothesizes how CD40L on B cells may regulate antibody responses and the development of germinal centres.     

Follicular dendritic cells and apoptosis: Life and death in the germinal centre

Summary The germinal centre forms a specialized microenvironment thought to play a key role in the induction of antibody synthesis, affinity maturation of B cells and memory B cell formation. Clonal-expanded follicular B lymphocytes with mutated antigen receptors (centrocytes) have to be selected on the basis of their capacity to compete for binding to antigen held in limited amounts on the follicular dendritic cells. In this way, only high-affinity B cells are selected. Binding to a follicular dendritic cell is an unconditional prerequisite for centrocytes to survive. Cells that do not succeed in binding to a follicular dendritic cell die rapidly by apoptosis. Apoptosis is a common form of cell death characterized by the activation of an endonuclease culminating in nuclear destruction. The pathway by which apoptosis is triggered varies from cell type to cell type. However, for germinal centre B cells this process is still poorly understood.     

Antigen-dependent B cell differentiation in the synovial tissue of a patient with reactive arthritis.

BACKGROUND: Reactive arthritis (ReA) can develop as a consequence of a bacterial infection with organisms such as Chlamydia trachomata, Shigella flexneri, or Yersinia enterocolitica. Although the mechanism underlying the induction of a chronic synovitis is unknown, the expression of HLA-B27 seems to play a crucial role in the etiology of the disease. Bacterial antigens induce a humoral immune response, but little is know about the impact of B cells on the inflammatory processes developing in the synovial membrane. MATERIALS AND METHODS: Cryostat sections were prepared from the synovial tissue (ST) of patients with ReA and stained with antibodies specific for T, B, and follicular dendritic cells. Lymphoid infiltrates were directly isolated by microdisection and DNA was prepared from them. The rearranged V genes were amplified by polymerase chain reaction (PCR), cloned, and sequenced. RESULTS: Histological staining showed that germinal, center-like structures develop in the ST of patients with ReA. B cells with a heterogenous repertoire were isolated from these lymphoid infiltrates. The majority of V regions carried somatic mutations indicating that sequences are derived from memory B cells. Genealogical trees demonstrate clonal expansion and diversification of the B cell repertoire in the ST. CONCLUSIONS: The finding of local V-region diversification suggests that in the ST of patients with ReA, an antigen-driven, T cell-dependent differentiation of B cells occurs. This local B cell response may contribute to the progress of the disease. Whether B cells are specific for the bacteria inducing the synovitis or for self-determinants present in the ST remains to be determined.     

Somatic hypermutation and selection of B cells in thymic germinal centers responding to acetylcholine receptor in myasthenia gravis

The muscle weakness in myasthenia gravis (MG) is mediated by autoantibodies against the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. Production of these pathogenic autoantibodies is believed to be associated with germinal centers (GC) and anti-AChR-secreting plasma cells in the hyperplastic thymus of patients with early onset MG (EOMG). Here, we describe the repertoire of rearranged heavy chain V genes and their clonal origins in GC from a typical EOMG patient. Three hundred fifteen rearranged Ig V(H) genes were amplified, cloned, and sequenced from sections of four thymic GC containing AChR-specific B cells. We found that thymic GC contain a remarkably heterogeneous population of B cells. Both naive and circulating memory B cells undergo Ag-driven clonal proliferation, somatic hypermutation, and selection. Numerous B cell clones were present, with no individual clone dominating the response. Comparisons of B cell clonal sequences from different GC and known anti-AChR Abs from other patients showed convergent mutations in the complementarity determining regions. These results are consistent with AChR driving an ongoing GC response in the thymus of EOMG patients. This is the first detailed analysis of B cell clones in human GC responding to a defined protein Ag, and the response we observed may reflect the effects of chronic stimulation by autoantigen.     

The function and origin of follicular dendritic cells

Follicular dendritic cells (dendritic reticular cells) in germinal centres bind antigen-antibody complexes via C3 receptors and retain the complexes at their surface for long periods of time. The follicular dendritic cells (FDC) are distinct from macrophages and from dendritic cells found in T-dependent areas, and are not derived from bone marrow stem cells. On histological evidence it has been proposed that they are derived from reticulum cells. Complexes are probably transported to FDC by a subpopulation of B cells in the marginal zone. Binding of complexes to FDC causes germinal centre enlargement and is a very efficient, and possibly essential stimulus to the generation of B memory cells which recognize epitopes on antigen or antibody in the complexes. An hypothesis is discussed which draws together these observations and suggests that antigen on FDC plays a central role in control of humoral immunity.     

Germinal centers and the B-cell system

Summary Germinal centers of the rabbit appendix were studied for the presence of complement receptors, immunoglobulin and alkaline phosphatase. In popliteal lymph nodes, de novo-developing germinal centers were studied with respect to these markers up to 21 days after sheep red blood cell (SRBC)-stimulation. In addition, the possible presence of antigen (SRBC) receptor-bearing cells in these germinal centers was investigated.The results may be summarized as follows: 1) Germinal centers in the appendix as well as those in popliteal lymph nodes were rich in complement receptor-bearing cells. Complement-receptor density did not significantly change during a germinal-center reaction. 2) Immunoglobulins were present only at very low densities on the surface of lymphoid cells in the densely populated area of germinal centers. In germinal centers of popliteal lymph nodes lymphoid cells in the thinly populated area again showed higher densities. Immunoglobulins possibly complexed with antigen on the surface of follicular dendritic cells were not observed in the initial phase of a germinal center reaction. In contrast, in germinal centers of the appendix, immunoglobulin was present in excessive amounts throughout the thinly populated area, possibly complexed with antigen, which is also abundantly present. 3) Reticular staining patterns of alkaline phosphatase were present in the densely populated area, but absent in the thinly populated area of germinal centers in both appendix and popliteal lymph nodes. Primary follicles and young germinal centers were alkaline phosphatase-negative. 4) Antigen receptor-bearing cells were detected in germinal centers of popliteal lymph nodes as early as 5 days after SRBC-stimulation, reaching a maximum at day 10.In conclusion, with the present experimental approach, microenvironmental differences were shown between the densely populated area and the thinly populated area of germinal centers. However, no indication was obtained for a postulated maturation event of the lymphocytes within germinal centers, or for functional differences that may exist between germinal centers in the appendix and popliteal lymph nodes.