The Amount of BCL6 in B Cells Shortly after Antigen Engagement Determines Their Representation in Subsequent Germinal Centers

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Optimality of Mutation and Selection in Germinal Centers

The population dynamics theory of B cells in a typical germinal center could play an important role in revealing how affinity maturation is achieved. However, the existing models encountered some conflicts with experiments. To resolve these conflicts, we present a coarse-grained model to calculate the B cell population development in affinity maturation, which allows a comprehensive analysis of its parameter space to look for optimal values of mutation rate, selection strength, and initial antibody-antigen binding level that maximize the affinity improvement. With these optimized parameters, the model is compatible with the experimental observations such as the ∼100-fold affinity improvements, the number of mutations, the hypermutation rate, and the “all or none” phenomenon. Moreover, we study the reasons behind the optimal parameters. The optimal mutation rate, in agreement with the hypermutation rate in vivo, results from a tradeoff between accumulating enough beneficial mutations and avoiding too many deleterious or lethal mutations. The optimal selection strength evolves as a balance between the need for affinity improvement and the requirement to pass the population bottleneck. These findings point to the conclusion that germinal centers have been optimized by evolution to generate strong affinity antibodies effectively and rapidly. In addition, we study the enhancement of affinity improvement due to B cell migration between germinal centers. These results could enhance our understanding of the functions of germinal centers.     

Artesunate Abolishes Germinal Center B Cells and Inhibits Autoimmune Arthritis

The antimalarial drug artemisinin and its derivatives exhibit potent immunosuppressive activity in several autoimmune disease models, however the mechanisms are not well-understood. This study was designed to investigate the therapeutic effects and the underlying mechanisms of the artemisinin analog artesunate using the K/BxN mouse model of rheumatoid arthritis. The well-studied disease mechanisms of K/BxN model allowed us to pinpoint the effect of artesunate on disease. Artesunate treatment prevented arthritis development in young K/BxN mice by inhibiting germinal center (GC) formation and production of autoantibodies. In adult K/BxN mice with established arthritis, artesunate diminished GC B cells in a few days. However, artesunate did not affect the follicular helper T cells (Tfh). In contrast to the spontaneous K/BxN model, artesunate treatment exerted minor influence on K/BxN serum transfer induced arthritis suggesting that artesunate has minimal effect on inflammatory responses downstream of antibody production. Finally, we showed that artesunate preferentially inhibits proliferating GC B cells. These results identify GC B cells as a target of artesunate and provide a new rationale for using artemisinin analogues to treat autoimmune diseases mediated by autoantibodies.     

Speckled-like Pattern in the Germinal Center (SLIP-GC), a Nuclear GTPase Expressed in Activation-induced Deaminase-expressing Lymphomas and Germinal Center B Cells

We identified a novel GTPase, SLIP-GC, with expression limited to a few tissues, in particular germinal center B cells. It lacks homology to any known proteins, indicating that it may belong to a novel family of GTPases. SLIP-GC is expressed in germinal center B cells and in lymphomas derived from germinal center B cells such as large diffuse B cell lymphomas. In cell lines, SLIP-GC is expressed in lymphomas that express activation-induced deaminase (AID) and that likely undergo somatic hypermutation. SLIP-GC is a nuclear protein, and it localizes to replication factories. Reduction of SLIP-GC levels in the Burkitt lymphoma cell line Raji and in non-Hodgkin lymphoma cell lines resulted in an increase in DNA breaks and apoptosis that was AID-dependent, as simultaneous reduction of AID abrogated the deleterious effects of SLIP-GC reduction. These results strongly suggest that SLIP-GC is a replication-related protein in germinal center B cells whose reduction is toxic to cells through an AID-dependent mechanism.The germinal center (GC)³ is a transient structure formed during T-dependent B cell responses wherein B cell affinity maturation to a specific antigen occurs, leading to the formation of high affinity memory B cells (1–3). Many features of this reaction are unique in biology such as the somatic hypermutation (SHM) of immunoglobulin (Ig) genes, the genetic rearrangement of the constant domains in class switch recombination to generate B cells bearing receptors of downstream isotypes such as IgG, IgE, and IgA, and the cellular selection process that recruits high affinity variants generated via SHM. In SHM the variable (V) regions of the heavy and light chain loci of Ig genes undergo a directed process of hypermutation where base substitutions accumulate, particularly in regions encoding the antigen binding pockets of the B cell receptor. The molecular basis for SHM is not fully understood, but it is known to be triggered by a cytosine deaminase, AID (4, 5). However, it is clear that novel factors are yet to be discovered in SHM. For example, AID alone is not sufficient for proper targeting to the Ig locus, and it is likely that a novel factor targets AID to the Ig locus (6). In addition, AID-mediated deamination of cytosines explains only mutations at G:C base pairs, yet mutations at A:T base pairs occur at approximately the same rate as G:C mutations. Although A:T mutations have been linked to the activities of the mismatch repair (MMR) proteins MSH/MSH6 and the error-prone DNA polymerase η, hypermutating Burkitt lymphoma cell lines have intact MMR and polymerase η, yet mutations at A:T base pairs are markedly reduced (7). The class switch recombination reaction is also only partly understood. Targeting of AID, the DNA substrate subjected to AID deamination, and the subsequent DNA breaks and their repair also remain only partially defined for class switch recombination. Finally, it remains unclear how these reactions are coordinated in the GC environment with both cellular selection for increased affinity to foreign antigen and tolerance mechanisms to prevent or minimize autoreactivity acquired during hypermutation that can lead to high affinity pathogenic IgG antibodies (8, 9). Clearly, efforts to understand these mechanisms and to identify novel proteins that contribute to this unique environment are needed.To identify proteins that may contribute to SHM or other aspects of the GC reaction, we mined expression libraries generated by the I.M.A.G.E. Consortium (10) through informatics tools in the Cancer Genome Anatomy Group website (11). Given that BCL6 is a critical protein for the GC reaction (12, 13), we pooled libraries derived from GC B cells with BCL6 expression and compared them to all other libraries (see Fig. 1A for the scheme). This strategy led us to the discovery of a novel protein, SLIP-GC (speckled-like pattern in the germinal center), expressed in GC B cells, and its expression profile was similar to that of AID. Subsequent experiments showed that this protein is expressed in GC B cells and localizes to replication factories in the nucleus and when reduced in AID⁺ lymphoma cell lines results in an increase in DNA breaks and in cell death. These studies reveal SLIP-GC to be a novel factor that likely contributes to the unique reactions in GCs. The data also suggest that SLIP-GC reduction is toxic to B cells through an AID-mediated mechanism.Open in a separate windowFIGURE 1.Identification of a novel GTPase expressed in germinal center B cells. A, Shown is a schematic representation of the method used to identify novel proteins primarily expressed in germinal center B cells by mining EST libraries. B, shown is the amino acid sequence of SLIP-GC. Italic boldface motifs are potential nuclear localization signals. The underlined boldfaced motif is a GTPase motif (P-loop), whereas the underlined italic sequence near the C terminus is the coiled-coil region. C, shown is a representative graph of GTPase assay of immunoprecipitated SLIP-GC from lipopolysaccharide-activated and unstimulated B cells. CRL-2289 is a cell line with endogenous SLIP-GC. As a positive control, RhoA, a ubiquitous GTPase, was immunoprecipitated with specific antibodies and tested the same way as SLIP-GC. We also performed GTPase assays on CRL-2631, which does not express SLIP-GC. Accordingly, a Coomassie Blue gel of immunoprecipitated SLIP-GC only shows a SLIP-GC band in the CRL-2289 extracts (data not shown), and SLIP-GC GTPase activity in lipopolysaccharide (LPS)-activated CRL-2631 was negligible, whereas RhoA GTPase activity was high (SLIP-GC, 0.36 nmol GDP/min; RhoA, 28.48 nmol GDP/min). The assay was done at least two times.     

Clusterin Is a Potential Lymphotoxin Beta Receptor Target That Is Upregulated and Accumulates in Germinal Centers of Mouse Spleen during Immune Response

Clusterin is a multifunctional protein that participates in tissue remodeling, apoptosis, lipid transport, complement-mediated cell lysis and serves as an extracellular chaperone. The role of clusterin in cancer and neurodegeneration has been extensively studied, however little is known about its functions in the immune system. Using expression profiling we found that clusterin mRNA is considerably down-regulated in mouse spleen stroma upon knock-out of lymphotoxin β receptor which plays pivotal role in secondary lymphoid organ development, maintenance and function. Using immunohistochemistry and western blot we studied clusterin protein level and distribution in mouse spleen and mesenteric lymph nodes in steady state and upon immunization with sheep red blood cells. We showed that clusterin protein, represented mainly by the secreted heterodimeric form, is present in all stromal compartments of secondary lymphoid organs except for marginal reticular cells. Clusterin protein level rose after immunization and accumulated in light zones of germinal centers in spleen - the effect that was not observed in lymph nodes. Regulation of clusterin expression by the lymphotoxin beta signaling pathway and its protein dynamics during immune response suggest a specific role of this enigmatic protein in the immune system that needs further study.     

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.     

B Cell Diversification Is Uncoupled from SAP-Mediated Selection Forces in Chronic Germinal Centers within Peyer’s Patches

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Affinity Inequality among Serum Antibodies That Originate in Lymphoid Germinal Centers

Upon natural infection with pathogens or vaccination, antibodies are produced by a process called affinity maturation. As affinity maturation ensues, average affinity values between an antibody and ligand increase with time. Purified antibodies isolated from serum are invariably heterogeneous with respect to their affinity for the ligands they bind, whether macromolecular antigens or haptens (low molecular weight approximations of epitopes on antigens). However, less is known about how the extent of this heterogeneity evolves with time during affinity maturation. To shed light on this issue, we have taken advantage of previously published data from Eisen and Siskind (1964). Using the ratio of the strongest to the weakest binding subsets as a metric of heterogeneity (or affinity inequality), we analyzed antibodies isolated from individual serum samples. The ratios were initially as high as 50-fold, and decreased over a few weeks after a single injection of small antigen doses to around unity. This decrease in the effective heterogeneity of antibody affinities with time is consistent with Darwinian evolution in the strong selection limit. By contrast, neither the average affinity nor the heterogeneity evolves much with time for high doses of antigen, as competition between clones of the same affinity is minimal.     

Polyclonal B Cell Differentiation and Loss of Gastrointestinal Tract Germinal Centers in the Earliest Stages of HIV-1 Infection

Background

The antibody response to HIV-1 does not appear in the plasma until approximately 2–5 weeks after transmission, and neutralizing antibodies to autologous HIV-1 generally do not become detectable until 12 weeks or more after transmission. Moreover, levels of HIV-1–specific antibodies decline on antiretroviral treatment. The mechanisms of this delay in the appearance of anti-HIV-1 antibodies and of their subsequent rapid decline are not known. While the effect of HIV-1 on depletion of gut CD4⁺ T cells in acute HIV-1 infection is well described, we studied blood and tissue B cells soon after infection to determine the effect of early HIV-1 on these cells.

Methods and Findings

In human participants, we analyzed B cells in blood as early as 17 days after HIV-1 infection, and in terminal ileum inductive and effector microenvironments beginning at 47 days after infection. We found that HIV-1 infection rapidly induced polyclonal activation and terminal differentiation of B cells in blood and in gut-associated lymphoid tissue (GALT) B cells. The specificities of antibodies produced by GALT memory B cells in acute HIV-1 infection (AHI) included not only HIV-1–specific antibodies, but also influenza-specific and autoreactive antibodies, indicating very early onset of HIV-1–induced polyclonal B cell activation. Follicular damage or germinal center loss in terminal ileum Peyer''s patches was seen with 88% of follicles exhibiting B or T cell apoptosis and follicular lysis.

Conclusions

Early induction of polyclonal B cell differentiation, coupled with follicular damage and germinal center loss soon after HIV-1 infection, may explain both the high rate of decline in HIV-1–induced antibody responses and the delay in plasma antibody responses to HIV-1. Please see later in the article for Editors'' Summary     

Cannabinoid Receptor 2 (CB2) Plays a Role in the Generation of Germinal Center and Memory B Cells,but Not in the Production of Antigen-Specific IgG and IgM,in Response to T-dependent Antigens

The cannabinoid receptor 2 (CB2) has been reported to modulate B cell functions including migration, proliferation and isotype class switching. Since these processes are required for the generation of the germinal center (GC) and antigen-specific plasma and memory cells following immunization with a T-dependent antigen, CB2 has the capacity to alter the quality and magnitude of T-dependent immune responses. To address this question, we immunized WT and CB2^−/− mice with the T-dependent antigen 4-hydroxy-3-nitrophenylacetyl (NP)-chicken-gamma-globulin (CGG) and measured GC B cell formation and the generation of antigen-specific B cells and serum immunoglobulin (Ig). While there was a significant reduction in the number of splenic GC B cells in CB2^−/− mice early in the response there was no detectable difference in the number of NP-specific IgM and IgG₁ plasma cells. There was also no difference in NP-specific IgM and class switched IgG₁ in the serum. In addition, we found no defect in the homing of plasma cells to the bone marrow (BM) and affinity maturation, although memory B cell cells in the spleen were reduced in CB2^−/− mice. CB2-deficient mice also generated similar levels of antigen-specific IgM and IgG in the serum as WT following immunization with sheep red blood cells (sRBC). This study demonstrates that although CB2 plays a role in promoting GC and memory B cell formation/maintenance in the spleen, it is dispensable on all immune cell types required for the generation of antigen-specific IgM and IgG in T-dependent immune responses.     

Germinal Center B Cell Depletion Diminishes CD4+ Follicular T Helper Cells in Autoimmune Mice

Background

Continuous support from follicular CD4⁺ T helper (Tfh) cells drives germinal center (GC) responses, which last for several weeks to produce high affinity memory B cells and plasma cells. In autoimmune Sle1 and NZB/W F1 mice, elevated numbers of Tfh cells persist, promoting the expansion of self-reactive B cells. Expansion of circulating Tfh like cells have also been described in several autoimmune diseases. Although, the signals required for Tfh differentiation have now been well described, the mechanisms that sustain the maintenance of fully differentiated Tfh are less understood. Recent data demonstrate a role for GC B cells for Tfh maintenance after protein immunization.

Methods and Finding

Given the pathogenic role Tfh play in autoimmune disease, we explored whether B cells are required for maintenance of autoreactive Tfh. Our data suggest that the number of mature autoreactive Tfh cells is controlled by GC B cells. Depletion of B cells in Sle1 autoimmune mice leads to a dramatic reduction in Tfh cells. In NZB/W F1 autoimmune mice, similar to the SRBC immunization model, GC B cells support the maintenance of mature Tfh, which is dependent mainly on ICOS. The CD28-associated pathway is dispensable for Tfh maintenance in SRBC immunized mice, but is required in the spontaneous NZB/W F1 model.

Conclusion

These data suggest that mature Tfh cells require signals from GC B cells to sustain their optimal numbers and function in both autoimmune and immunization models. Thus, immunotherapies targeting B cells in autoimmune disease may affect pathogenic Tfh cells.     

Mechanisms of Organelle Inheritance in Dividing Plant Cells

Organelles form essential compartments of all eukaryotic cells. Mechanisms that ensure the unbiased inheritance of organelles during cell division are therefore necessary to maintain the viability of future cell generations. Although inheritance of organelles represents a fundamental component of the cell cycle, surprisingly little is known about the underlying mechanisms that facilitate unbiased organelle inheritance. Evidence from a select number of studies, however, indicates that ordered organelle inheritance strategies exist in dividing cells of higher plants. The basic requirement for unbiased organelle inheritance is the duplication of organelle volume and distribution of the resulting organelle populations in a manner that facilitates unbiased partitioning of the organelle population to each daughter cell. Often, partitioning strategies are specific to the organelle, being influenced by the functional requirements of the organelle and whether the cells are mitotically active or re-entering into the cell cycle. Organelle partitioning mechanisms frequently depend on interactions with either the actin or microtubule cytoskeleton. In this focused review, we attempt to summarize key findings regarding organelle partitioning strategies in dividing cells of higher plants. We particularly concentrate on the role of the cytoskeleton in mediating unbiased organelle partitioning.     

Nerve Growth Factor Receptor TrkA,a New Receptor in Insulin Signaling Pathway in PC12 Cells

TrkA is a cell surface transmembrane receptor tyrosine kinase for nerve growth factor (NGF). TrkA has an NPXY motif and kinase regulatory loop similar to insulin receptor (INSR) suggesting that NGF→TrkA signaling might overlap with insulin→INSR signaling. During insulin or NGF stimulation TrkA, insulin receptor substrate-1 (IRS-1), INSR (and presumably other proteins) forms a complex in PC12 cells. In PC12 cells, tyrosine phosphorylation of INSR and IRS-1 is dependent upon the functional TrkA kinase domain. Moreover, expression of TrkA kinase-inactive mutant blocked the activation of Akt and Erk5 in response to insulin or NGF. Based on these data, we propose that TrkA participates in insulin signaling pathway in PC12 cells.     

Receptor for Antibody—Antigen Complexes used to Separate T Cells from B Cells

LYMPHOCYTES with the capacity to bind antibody-antigen complexes to their surface^1–3 are probably bone marrow-derived, B, cells, not thymus-derived, T, cells³. We now have definite evidence that such lymphocytes are indeed B cells and will describe how this property can be utilized in a practical way for separating T cells from B cells.     

Toll样受体调节树突状细胞中腺苷受体A2B亚型的表达及功能

树突状细胞（dendritic cell,DC）表面所表达的腺苷受体A2B亚型（ADOR-A2B）可促进DC对辅助性T淋巴细胞（T helper cell,Th）的激活,导致自身免疫性疾病的发生或加重. 本文旨在研究作为免疫反应的诱导分子Toll样受体（Toll-like receptors, TLRs）是否可调节ADOR-A2B在DC中的表达并籍此影响其功能. 体外诱导小鼠骨髓细胞分化为树突状细胞（BM-DC）,以多种TLRs的配体,Pam3csk4、polyIC、LPS及CpG进行干预. 提取细胞总RNA,real-time PCR测定ador-a2a、ador-a2b的表达;放射性配体结合实验测定BM-DC对3H 腺苷结合能力的变化.以LPS及选择性ADOR-A2B激动剂BAY 60-6583协同干预BM-DC,ELISA测定培养基中IL-1、IL-6及IL-12的含量. 以干预后的BM DC刺激naive CD4细胞,ELISA测定培养基中IL-17A、IFNγ的含量,荧光抗体染色及流式细胞仪分析检测CD4细胞的分化. 结果显示, TLR 4的配体LPS可显著提高BM DC中ador-a2b的表达及对腺苷的结合能力. BAY 60-6583与LPS相协同可刺激BM DC分泌多种致炎因子,并增加其诱导CD4细胞向Th1及Th17分化的能力. 由此可见,Toll样受体可上调ador-a2b在DC中的表达,并可籍此增加DC分泌促炎因子的能力及对CD4细胞的刺激作用.     

Generation and Fates of Supernumerary Centrioles in Dividing Cells

The centrosome is a subcellular organelle from which a cilium assembles. Since centrosomes function as spindle poles during mitosis, they have to be present as a pair in a cell. How the correct number of centrosomes is maintained in a cell has been a major issue in the fields of cell cycle and cancer biology. Centrioles, the core of centrosomes, assemble and segregate in close connection to the cell cycle. Abnormalities in centriole numbers are attributed to decoupling from cell cycle regulation. Interestingly, supernumerary centrioles are commonly observed in cancer cells. In this review, we discuss how supernumerary centrioles are generated in diverse cellular conditions. We also discuss how the cells cope with supernumerary centrioles during the cell cycle.