Epstein-Barr virus and the B cell: a secret romance

Infection by the Epstein-Barr virus (EBV) in immunocompetent individuals seems mainly confined to antigen-experienced memory B cells. However, a recent report shows that EBV(+) post-transplant lymphoproliferative disease might arise not only from memory B cells but also from nai;ve and germinal center (GC) B cells. Intriguingly, some of the EBV-positive B-cell clones seem to carry non-functional Ig-V-region genes as a result of deleterious somatic mutations acquired during the GC reaction. Given that such GC B cells are destined to die by apoptosis in the absence of EBV, these findings suggest that transformation by EBV might bypass negative selection of B cells within GCs.     

Chemotaxis in densely populated tissue determines germinal center anatomy and cell motility: a new paradigm for the development of complex tissues

Germinal centers (GCs) are complex dynamic structures that form within lymph nodes as an essential process in the humoral immune response. They represent a paradigm for studying the regulation of cell movement in the development of complex anatomical structures. We have developed a simulation of a modified cyclic re-entry model of GC dynamics which successfully employs chemotaxis to recapitulate the anatomy of the primary follicle and the development of a mature GC, including correctly structured mantle, dark and light zones. We then show that correct single cell movement dynamics (including persistent random walk and inter-zonal crossing) arise from this simulation as purely emergent properties. The major insight of our study is that chemotaxis can only achieve this when constrained by the known biological properties that cells are incompressible, exist in a densely packed environment, and must therefore compete for space. It is this interplay of chemotaxis and competition for limited space that generates all the complex and biologically accurate behaviors described here. Thus, from a single simple mechanism that is well documented in the biological literature, we can explain both higher level structure and single cell movement behaviors. To our knowledge this is the first GC model that is able to recapitulate both correctly detailed anatomy and single cell movement. This mechanism may have wide application for modeling other biological systems where cells undergo complex patterns of movement to produce defined anatomical structures with sharp tissue boundaries.     

From affinity selection to kinetic selection in Germinal Centre modelling

Affinity maturation is an evolutionary process by which the affinity of antibodies (Abs) against specific antigens (Ags) increases through rounds of B-cell proliferation, somatic hypermutation, and positive selection in germinal centres (GC). The positive selection of B cells depends on affinity, but the underlying mechanisms of affinity discrimination and affinity-based selection are not well understood. It has been suggested that selection in GC depends on both rapid binding of B-cell receptors (BcRs) to Ags which is kinetically favourable and tight binding of BcRs to Ags, which is thermodynamically favourable; however, it has not been shown whether a selection bias for kinetic properties is present in the GC. To investigate the GC selection bias towards rapid and tight binding, we developed an agent-based model of GC and compared the evolution of founder B cells with initially identical low affinities but with different association/dissociation rates for Ag presented by follicular dendritic cells in three Ag collection mechanisms. We compared an Ag collection mechanism based on association/dissociation rates of B-cell interaction with presented Ag, which includes a probabilistic rupture of bonds between the B-cell and Ag (Scenario-1) with a reference scenario based on an affinity-based Ag collection mechanism (Scenario-0). Simulations showed that the mechanism of Ag collection affects the GC dynamics and the GC outputs concerning fast/slow (un)binding of B cells to FDC-presented Ags. In particular, clones with lower dissociation rates outcompete clones with higher association rates in Scenario-1, while remaining B cells from clones with higher association rates reach higher affinities. Accordingly, plasma cell and memory B cell populations were biased towards B-cell clones with lower dissociation rates. Without such probabilistic ruptures during the Ag extraction process (Scenario-2), the selective advantage for clones with very low dissociation rates diminished, and the affinity maturation level of all clones decreased to the reference level.     

Positional Shift Between the Vegetative Nucleus and the Generative Cell in Amaryllis Pollen Tube

A modified technique, FITC-tubulin immunofluorescence and DAPI localization to demonstrate simultaneously both the generative cell (GC) and the vegetative nucleus (VN) in the pollen tube under ultra-violet excitation, was developed sucessfully. During the germination of the pollen tube of Amaryllis vittata Ait. the GC and the VN, either being the first one, entered the tube within the first 1—2 h from the pollen grain. However, before the time of GC division, the VN was always positioned distally near the tip of the tube. In case when the GC entered the tube first, then the VN must have a positional shift in order to pass over the GC. The detail processes of positional shift between the GC and the VN were observed. Three basic processes were demonstrated: 1. The anterior end of the VN first reached the vicinity of the posterior attenuated extension of the GC about 2 h following germination forming a temporal physical association. Sometimes their both ends could be inserted into one another for certain extent. 2. The whole VN moved forward and contacted in parallel with the GC until they became twisted together and 3. The VN passed over the GC and greatly elongted lengthwise. Its posterior part became inserted into the anterior end of the GC. The behavior of positional shift between the VN and the GC in the pollen tube seems to be an adjustment of their diameters to fit the narrow tube. A conclusion may be drawn that the rate of movement between the VN and the GC was apparently different during the passage through the tube. Such difference may presumably be accompanied by the independent motive mechanism and structural difference between the VN and GC themselves, which provide their motive force for movement in the tube.     

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.     

Common germinal-center B-cell origin of the malignant cells in two composite lymphomas, involving classical Hodgkin's disease and either follicular lymphoma or B-CLL

BACKGROUND: Classical Hodgkin's disease (HD) and B-cell non-Hodgkin lymphoma (NHL) occasionally occur in the same patient. Such composite lymphomas represent interesting models to study the pathogenesis of B-cell lymphomas and the relationship between HD and B-cell NHL. MATERIALS AND METHODS: We analyzed two composite lymphomas (a combination of classical HD with follicular lymphoma [FL] and a combination of classical HD with B-cell chronic lymphocytic leukemia [B-CLL]) by micromanipulation of single cells from tissue sections and amplification of immunoglobulin V region genes for the clonal relationship of the tumor cells. RESULTS: In both cases, clonally related variable (V) genes with both shared as well as distinct somatic mutations were obtained from the two lymphomas, showing that in each of the cases the distinct tumor cells were members of a common germinal center (GC) B-cell clone. FL cells from two different lymph nodes of patient 1 showed a similar mutation pattern, suggesting that infiltration of these lymph nodes by tumor cells was not restricted to a particular FL cell or subclone. In the FL, a single cell was identified with a mutation signature indicating that premalignant cells can persist in the tissue. CONCLUSIONS: The cases presented here further underline the close relationship between HD and B-cell NHL and the role of the GC in lymphomagenesis. Whereas the latter was already suggested for FL and HD, the present study indicates that also in the B-CLL subset characterized by mutated Ig genes, important steps in malignant transformation happen in the GC, and that HRS cells can derive from CD5-positive B cells.     

CCL2 regulation of MST1-mTOR-STAT1 signaling axis controls BCR signaling and B-cell differentiation

Chemokines are important regulators of the immune system, inducing specific cellular responses by binding to receptors on immune cells. In SLE patients, decreased expression of CCL2 on mesenchymal stem cells (MSC) prevents inhibition of B-cell proliferation, causing the characteristic autoimmune phenotype. Nevertheless, the intrinsic role of CCL2 on B-cell autoimmunity is unknown. In this study using Ccl2 KO mice, we found that CCL2 deficiency enhanced BCR signaling by upregulating the phosphorylation of the MST1-mTORC1-STAT1 axis, which led to reduced marginal zone (MZ) B cells and increased germinal center (GC) B cells. The abnormal differentiation of MZ and GC B cells were rescued by in vivo inhibition of mTORC1. Additionally, the inhibition of MST1-mTORC1-STAT1 with specific inhibitors in vitro also rescued the BCR signaling upon antigenic stimulation. The deficiency of CCL2 also enhanced the early activation of B cells including B-cell spreading, clustering and signalosome recruitment by upregulating the DOCK8-WASP-actin axis. Our study has revealed the intrinsic role and underlying molecular mechanism of CCL2 in BCR signaling, B-cell differentiation, and humoral response.Subject terms: B cells, Signal transduction     

Involvement of DNase gamma in the resected double-strand DNA breaks in immunoglobulin genes

Somatic hypermutation (SHM) of immunoglobulin variable (V) region genes occurs in the germinal center (GC) B cells during immune responses, depending on activation-induced cytidine deaminase (AID). SHM is associated with resected double-strand DNA breaks (DSBs) which were shown to occur specifically in rearranged V regions in the GC B cells and CD40-stimulated B cells expressing AID. So far, endonucleases responsible for the DSBs have not been identified. Here we show that DNase gamma, a member of DNase I family of endonucleases, is expressed in GC B cells and CD40-stimulated B cells. Overexpression of DNase gamma in the mutation-competent Ramos B-cell line resulted in a marked increase in the resected but not blunt DSBs in the V region. Conversely, a selective DNase gamma inhibitor, DR396, suppressed the generation of the resected DSBs. These results suggest that DNase gamma is involved in the generation of resected DSBs associated with SHM.     

Memory B cells in systemic and mucosal immune response: implications for successful vaccination

B-cell memory has been extensively analyzed in the systemic immune response elicited by hapten-carrier antigens, and the regulatory mechanisms underlying the process are beginning to be elucidated. Memory B cells can be generated through heterogeneous pathways within and outside germinal centers (GCs). Once developed, they appear to be maintained like stem cells for long periods by homeostatic proliferation. In response to reencountered antigens, memory B cells robustly secrete antibodies with help of the anti-apoptotic effect of Ras-mediated signals. We have recently found that following intranasal infection with an influenza virus, virus-specific memory B cells develop in the lungs and persist for a long time along with GC B cells and plasma cells; this appears to be unique feature of the mucosal memory response. Thus memory B cell responses in the systemic and mucosal sites are regulated by distinct processes and further understanding of them should provide a theoretical framework for the development of new vaccine strategies.     

Differential effects of tumor necrosis factor-alpha and CD40L on NF-kappa B inhibitory proteins I kappa B alpha, beta and epsilon and on the induction of the Jun amino-terminal kinase pathway in Ramos Burkitt lymphoma cells

Interaction between the CD40 ligand and its cognate receptor is known to affect various aspects of B-cell biology. Less is known about the biological consequences of B-cell signaling through tumor necrosis factor alpha (TNF-alpha) and its two receptors. We have used Ramos germinal center (GC)-derived Burkitt's lymphoma (BL) cells as a model system to compare some of the early signaling events of TNF-alpha and CD40L on the NF-kappaB and c-Jun amino-terminal kinase (JNK) pathways. We have previously found that both TNF-alpha and CD40L induced enhanced cell aggregation, adherence and modified cell surface morphology of Ramos cells. In the present report, it was found that treatment with either TNF-alpha or CD40L resulted in a rapid degradation (within 15 min) of IkappaBalpha, followed by a recovery period lasting up to a few hours. The level of IkappaBbeta, another inhibitory molecule of the NF-kappaB pathway, also decreased following treatment with CD40L or TNF-alpha. However, whereas CD40L induced a rapid drop without significant recovery within 2 h, TNF-alpha caused a slow and gradual decline of IkappaBbeta. In addition, treatment with CD40L resulted in a gradual and modest decline of up to 60% of the level of IkappaBepsilon within 2 h, whereas a much smaller decline was seen with TNF-alpha (approx. 20%) Our results thus show that in Ramos cells, TNF-alpha and CD40L have common, as well as differential, signaling effects on the IkappaBalpha, IkappaBbeta and IkappaBepsilon, which form inhibitory complex(es) with the NF-kappaB cytosolic proteins. We also found that CD40L, but not TNF-alpha activates the JNK pathway through transient phosphorylation of its threonine183/tyrosine185 residues. As expected, c-Jun, which is known to be a substrate of JNK, was also phosphorylated at serine residue 73 by treatment with CD40L, but not by TNF-alpha.     

Lysosomal destabilization contributes to apoptosis of germinal center B-lymphocytes.

During germinal center (GC) reactions, B-lymphocytes with high-affinity B-cell receptors are selected. Regulation of apoptosis is a key process in selecting such wanted B-cells and in eliminating B-cells with unwanted specificities. In this paper, we show that apoptosis in human GC B-cells involves lysosomal destabilization, which is strictly controlled by caspase-8 activity, but not by caspase-9 activity. Ligation of CD40 provides resistance to lysosomal destabilization. Experimental lysosomal rupture by the lysosomotropic drug O-methyl-l-serine dodecylamide hydrochloride (MSDH) induces apoptosis in GC B-cells, including phosphatidyl serine exposure, mitochondrial inactivation, and DNA fragmentation. These apoptotic features occur in the absence of caspase-3 activity. Follicular dendritic cells (FDCs) protect binding B-lymphocytes from lysosomal destabilization, in both the absence and the presence of MSDH. Our study demonstrates that lysosomal leakage induces apoptosis of GC B-cells in a caspase-3-independent manner and that high-affinity binding to FDCsprevents lysosomal leakage and apoptosis in GC B-cells.     

The BTB-kelch protein KLHL6 is involved in B-lymphocyte antigen receptor signaling and germinal center formation

BTB-kelch proteins can elicit diverse biological functions but very little is known about the physiological role of these proteins in vivo. Kelch-like protein 6 (KLHL6) is a BTB-kelch protein with a lymphoid tissue-restricted expression pattern. In the B-lymphocyte lineage, KLHL6 is expressed throughout ontogeny, and KLHL6 expression is strongly upregulated in germinal center (GC) B cells. To analyze the role of KLHL6 in vivo, we have generated mouse mutants of KLHL6. Development of pro- and pre-B cells was normal but numbers of subsequent stages, transitional 1 and 2, and mature B cells were reduced in KLHL6-deficient mice. The antigen-dependent GC reaction was blunted (smaller GCs, reduced B-cell expansion, and reduced memory antibody response) in the absence of KLHL6. Comparison of mutants with global loss of KLHL6 to mutants lacking KLHL6 specifically in B cells demonstrated a B-cell-intrinsic requirement for KLHL6 expression. Finally, B-cell antigen receptor (BCR) cross-linking was less sensitive in KLHL6-deficient B cells compared to wild-type B cells as measured by proliferation, Ca2+ response, and activation of phospholipase Cgamma2. Our results strongly point to a role for KLHL6 in BCR signal transduction and formation of the full germinal center response.     

GPR174-CCL21调控体液免疫应答的两性差异

体液免疫应答具有明显的两性差异。通常女性产生的针对外来病原体或者自身抗原的抗体水平相比男性更高,因此女性比男性更容易清除病原体的感染,但女性也比男性更容易患自身免疫疾病。长效且高亲和力的体液免疫应答依赖由B细胞形成的生发中心(germinal center,GC)反应,然而B细胞形成GC的能力是否存在两性差异从而直接导致体液免疫应答的两性差异尚不清楚。该研究提出了一套在分子和细胞水平上解释体液免疫应答两性差异的新机制,即雄激素可以增强B细胞表达的GPR174在接收到CCL21信号后与Gαi蛋白的结合水平,由此促进雄性B细胞更多地迁移在滤泡外周,而不能更多地迁移至滤泡中心形成GC及抗体应答,从而直接介导体液免疫应答的两性差异。该研究为解决在增强保护性疫苗抗体应答水平以及治疗自身免疫疾病时遇到的B细胞介导的两性差异问题提供了新的见解。     

Regulatory T cells and control of the germinal centre response

Germinal centres (GCs) are specialised lymphoid microenvironments that form in secondary B-cell follicles upon exposure to T-dependent antigens. In the GC, clonal expansion, selection and differentiation of GC B cells result in the production of high-affinity plasma cells and memory B cells that provide protection against subsequent infection. The GC is carefully regulated to fulfil its critical role in defence against infection and to ensure that immunological tolerance is not broken in the process. The GC response can be controlled by a number of mechanisms, one of which is by forkhead box p3 expressing regulatory T (Treg) cells, a suppressive population of CD4⁺ T cells. A specialised subset of Treg cells – follicular regulatory T (Tfr) cells – form after immunisation and are able to access the GC, where they control the size and output of the response. Our knowledge of Treg cell control of the GC is expanding. In this review we will discuss recent advances in the field, with a particular emphasis on the differentiation and function of Tfr cells in the GC.     

Growth cones contain a dynamic population of neurofilament subunits

Neurofilaments (NFs) are classically considered to transport in a primarily anterograde direction along axons, and to undergo bulk degradation within the synapse or growth cone (GC). We compared overall NF protein distribution with that of newly expressed NF subunits within NB2a/d1 cells by transfection with a construct encoding green fluorescent protein (GFP) conjugated NF-M subunits. GCs lacked phosphorylated NF epitopes, and steady-state levels of non-phosphosphorylated NF subunits within GC were markedly reduced compared to those of neurite shaft as indicated by conventional immunofluorescence. However, GCs contained significant levels of GFP-tagged subunits in the form of punctate or short filamentous structures that in some cases exceeded that visualized along the shaft itself, suggesting that GCs contained a relatively higher concentration of newly synthesized subunits. GFP-tagged NF subunits within GCs co-localized with non-phosphorylated NF immunoreactivity. GFP-tagged subunits were observed within GC filopodia in which steady-state levels of NF subunits were too low to be detected by conventional immunofluorescence. Selective localization of fluorescein versus rhodamine fluorescene was observed within GCs following expression of NF-M conjugated to DsRed1-E5, which shifts from fluorescein to rhodamine fluorescence within hours after expression; axonal shafts contained a more even distribution of fluorescein and rhodamine fluorescence, further indicating that GCs contained relatively higher levels of the most-recently expressed subunits. GFP-tagged structures were rapidly extracted from GCs under conditions that preserved axonal structures. These short filamentous and punctate structures underwent rapid bi-directional movement within GCs. Movement of GFP-tagged structures within GCs ceased following application of nocodazole, cytochalasin B, and the kinase inhibitor olomoucine, indicating that their motility was dependent upon microtubules and actin and, moreover, was due to active transport rather than simple diffusion. Treatment with the protease inhibitor calpeptin increased overall NF subunits, but increased those within the GC to a greater extent than those along the shaft, indicating that subunits in the GC undergo more rapid turnover than do those within the shaft. Some GCs contained coiled aggregates of GFP-tagged NFs that appeared to be contiguous with axonal NFs. NFs extended from these aggregates into the advancing GC as axonal neurites elongated. These data are consistent with the presence of a population of dynamic NF subunits within GCs that is apparently capable of participating in regional filament formation during axonal elongation, and support the notion that NF polymerization and transport need not necessarily occur in a uniform proximal-distal manner.     

Movement of generative cell and vegetative nucleus in tobacco pollen tubes is dependent on microtubule cytoskeleton but independent of the synthesis of callose plugs

The role of microtubules (MTs) in vegetative nucleus (VN) and generative cell (GC) transport was investigated by comparing VN and GC distribution with callose plug formation in tobacco pollen grains germinated and grown for 12 h with the plant-specific anti-MT drug oryzalin. The VN-GC complex or VN alone was located close to the tube tip in 100% of controls, but in only 5% of oryzalin-treated tubes. Instead, in 38% of oryzalin tubes, the complex or VN occurred close to the last-formed callose plug; in 40% between or in the middle of plugs; and in 17%, in or near the grain. An aberrant microfilament (MF) cytoskeleton was revealed by expression of a green fluorescent protein-talin fusion protein in living oryzalin-treated tubes. The abnormal MF structures probably resulted from the absence of MTs and impaired - or were a consequence of - VN and GC movement into the tube tip. In oryzalin tubes with several callose plugs, the VN and GC could be in or near the grain, indicating that callose plug synthesis is not dependent on the movement of VN and GC into the tube. VN and GC movement and callose plug formation are apparently independent events, in which the transport of the VN-GC complex must precede callose plug synthesis. Maintenance of the correct developmental program requires an intact MT cytoskeleton, otherwise no fertile pollen tubes are formed.