Functional properties of human germinal center B cells.

Germinal centers (GCs) are histologically defined areas where B cells undergo extensive proliferation and maturation, or die of apoptosis. GC B cells isolated from human tonsils can be phenotypically identified by expression of peanut agglutinin (PNA)-binding sites and can be further divided into subpopulations based on their expression of CD77. To assess the functional potential of GC B cells, we studied CD77+ PNA+ B cells isolated from tonsils by examining their differentiation status and their ability to proliferate in vitro to various cytokines and costimulants. We found that CD77+ GC B cells are less differentiated than CD77- GC B cells; GC B cells less frequently express cytoplasmic IgG and IgM, and spontaneously secrete less Ig compared to CD77- GC B cells. To identify conditions capable of inducing GC B cell proliferation, we examined IL-4, IL-2, IFN-gamma, low molecular weight BCGF (LMW-BCGF), and an MLR supernatant along with costimulants such as anti-IgM antibody, Staphylococcus aureus Cowan I (SAC), PMA, and pokeweed mitogen (PWM). While non-GC B cells proliferate strongly in response to these stimuli, GC B cells did not proliferate. However, CD77+ as well as CD77- GC B cells mounted a rapid and strong proliferative response upon stimulation with IL-4, but only in the presence of anti-CD40 antibody. Moreover, although nine additional cytokines were examined, only IL-4 was capable of supporting CD77+ GC B cell proliferation in the presence of anti-CD40 antibody. When cells were stimulated with IL-4 and anti-CD40 antibody, we also found that IFN-gamma consistently decreased the proliferative response of CD77+ GC B cells without affecting the response of non-GC B cells. Taken together, these data indicate that GC B cells have characteristic growth requirements and that IL-4 may be important for GC B cell growth in vivo.     

AID-targeting and hypermutation of non-immunoglobulin genes does not correlate with proximity to immunoglobulin genes in germinal center B cells

Upon activation, B cells divide, form a germinal center, and express the activation induced deaminase (AID), an enzyme that triggers somatic hypermutation of the variable regions of immunoglobulin (Ig) loci. Recent evidence indicates that at least 25% of expressed genes in germinal center B cells are mutated or deaminated by AID. One of the most deaminated genes, c-Myc, frequently appears as a translocation partner with the Ig heavy chain gene (Igh) in mouse plasmacytomas and human Burkitt's lymphomas. This indicates that the two genes or their double-strand break ends come into close proximity at a biologically relevant frequency. However, the proximity of c-Myc and Igh has never been measured in germinal center B cells, where many such translocations are thought to occur. We hypothesized that in germinal center B cells, not only is c-Myc near Igh, but other mutating non-Ig genes are deaminated by AID because they are near Ig genes, the primary targets of AID. We tested this "collateral damage" model using 3D-fluorescence in situ hybridization (3D-FISH) to measure the distance from non-Ig genes to Ig genes in germinal center B cells. We also made mice transgenic for human MYC and measured expression and mutation of the transgenes. We found that there is no correlation between proximity to Ig genes and levels of AID targeting or gene mutation, and that c-Myc was not closer to Igh than were other non-Ig genes. In addition, the human MYC transgenes did not accumulate mutations and were not deaminated by AID. We conclude that proximity to Ig loci is unlikely to be a major determinant of AID targeting or mutation of non-Ig genes, and that the MYC transgenes are either missing important regulatory elements that allow mutation or are unable to mutate because their new nuclear position is not conducive to AID deamination.     

Shining a light on germinal center B cells

The mechanisms of B cell selection in lymphoid tissues are poorly understood. In this issue, Victora et?al. (2010) use imaging of photoactivatable green fluorescent protein to define the movements of B?cells in germinal centers and provide evidence that antibody affinity maturation is driven by competition for T?cell help.     

Abundance of early functional HIV-specific CD8+ T cells does not predict AIDS-free survival time

Background

T-cell immunity is thought to play an important role in controlling HIV infection, and is a main target for HIV vaccine development. HIV-specific central memory CD8⁺ and CD4⁺ T cells producing IFNγ and IL-2 have been associated with control of viremia and are therefore hypothesized to be truly protective and determine subsequent clinical outcome. However, the cause-effect relationship between HIV-specific cellular immunity and disease progression is unknown. We investigated in a large prospective cohort study involving 96 individuals of the Amsterdam Cohort Studies with a known date of seroconversion whether the presence of cytokine-producing HIV-specific CD8⁺ T cells early in infection was associated with AIDS-free survival time.

Methods and Findings

The number and percentage of IFNγ and IL-2 producing CD8⁺ T cells was measured after in vitro stimulation with an overlapping Gag-peptide pool in T cells sampled approximately one year after seroconversion. Kaplan-Meier survival analysis and Cox proportional hazard models showed that frequencies of cytokine-producing Gag-specific CD8⁺ T cells (IFNγ, IL-2 or both) shortly after seroconversion were neither associated with time to AIDS nor with the rate of CD4⁺ T-cell decline.

Conclusions

These data show that high numbers of functional HIV-specific CD8⁺ T cells can be found early in HIV infection, irrespective of subsequent clinical outcome. The fact that both progressors and long-term non-progressors have abundant T cell immunity of the specificity associated with low viral load shortly after seroconversion suggests that the more rapid loss of T cell immunity observed in progressors may be a consequence rather than a cause of disease progression.     

Novel diversity in IL-4-mediated responses in resting human naive B cells versus germinal center/memory B cells

Recent studies have defined several phenotypic and molecular changes associated with the maturation of naive human B cells within the milieu of germinal centers. Although naive B cells serve as natural precursors to germinal center (GC)/memory (M) subpopulations, little is known about the physiological requirements for the survival of the naive B cell pool in the absence of cell-cell contact or Ag-mediated activation. Because IL-4 induces expression of several membrane receptors such as CD23 which are uniquely present on resting human naive B lymphocytes, we hypothesized that these cells might be intrinsically programmed to respond to IL-4 in the absence of cell division. Using buoyant density-dependent isolation and further enrichment by negative/positive selection of human naive and GC/M subpopulations, we characterized cytokine receptor moieties on these cells and analyzed their survival and growth in the presence of IL-4 or IL-10. Resting naive B cells expressed significantly higher IL-4 receptor alpha-chain on their cell surface than the combined GC/M subpopulation. The IL-10 receptor and the IL-2 receptor gammac chain were almost equally expressed on both subpopulations. When cultured in vitro, the addition of IL-4, but not IL-10, protected naive B cells from apoptosis in the absence of activation and growth. However, IL-4 exerted no such effect on resting GC/M B cells. These data support the hypothesis that IL-4 plays a pivotal role in the survival and maintenance of resting human naive B cells.     

Immunobiology of chicken germinal center: II. Accumulation of apoptotic cells within the germinal center

The germinal center (GC) develops after antigen stimulation and is thought to occur at the site of various immune responses. We observed apoptotic cells within the GC using in situ end labeling (TUNEL), small amount DNA ladder assay, and RT-PCR analysis of Bcl-2 mRNA expression. Apoptosis was detected within GCs at all phases of the GC reaction by both TUNEL and DNA ladder assays. The number of TUNEL⁺ nuclei within the GC did not increase over the course of the GC reaction. However, the density of DNA in the ladder assay was higher in later-phase GCs. Bcl-2 mRNA expression was detected within GCs during the early phases of the GC reaction. These results indicate that accumulation of apoptotic cells and rescue from apoptosis occur within chicken GCs. In the present paper, the reasons for the accumulation of apoptotic cells will be discussed.This work was supported by Grants-in-Aid for Scientific Research (Nos. 11670322 and 10306017) from the Ministry of Education, Science, Sport and Culture, and the Ministry of Agriculture, Forestry and Fisheries of Japan (Special Scientific Research and Pioneering Research Project in Biotechnology), as well as from the Bio-oriented Technology Research Advancement Institution (BRAIN)     

Tracking murine gammaherpesvirus 68 infection of germinal center B cells in vivo

Infection of mice with murine gammaherpesvirus 68 (MHV68) provides a tractable small animal model to study various aspects of persistent gammaherpesvirus infection. We have previously utilized a transgenic MHV68 that expresses enhanced yellow fluorescent protein (EYFP) to identify infected cells. While this recombinant MHV68 has been useful for identifying infected cell populations by flow cytometry, it has been suboptimal for identification of infected cells in tissue sections due to the high solubility of EYFP. Efficient detection of EYFP expressed from the MHV68 genome in tissue sections requires fixation of whole organs prior to sectioning, which frequently leads to over-fixation of some cellular antigens precluding their detection. To circumvent this issue, we describe the generation and characterization of a transgenic MHV68 harboring a fusion gene composed of the EYFP coding sequence fused to the histone H2B open reading frame. Because the H2bYFP fusion protein is tightly bound in nucleosomes in the nucleus it does not freely diffuse out of unfixed tissue sections, and thus eliminates the need for tissue fixation. We have used the MHV68-H2bYFP recombinant virus to assess the location and distribution of virus infected B cells in germinal centers during the peak of MHV68 latency in vivo. These analyses show that the physical location of distinct populations of infected germinal center B cells correlates well with their surface phenotype. Furthermore, analysis of the distribution of virus infection within germinal center B cell populations revealed that ca. 70% of MHV68 infected GC B cells are rapidly dividing centroblasts, while ca. 20% have a clear centrocyte phenotype. Finally, we have shown that marking of infected cells with MHV68-H2bYFP is extended long after the onset of latency - which should facilitate studies to track MHV68 latently infected cells at late times post-infection.     

Standard percent DNA sequence difference for insects does not predict species boundaries

Diagnosis and assessment of species boundaries of economically important insects are often problematic because of limited morphological and/or biological characters. DNA data can help to identify and revise species. Nonoverlapping intra- and interspecific sequence divergences are often used as evidence for species. Thus, the establishment of a standardized percent nucleotide divergence to predict species boundaries would aid in cases where species status is suspect. However, given variation in nucleotide mutation rates and species concepts, association between a standard percent sequence divergence and species is questionable. This review surveys the percent DNA sequence difference found between sister-species of economically important insects, to assess whether a standard divergence associates with all taxa. Sixty-two comparisons of intra- and interspecific pairwise DNA differences were made for mitochondrial and nuclear loci spanning families of Isoptera, Phthiraptera, Hemiptera, Coleoptera, Lepidoptera, Diptera, and Hymenoptera. Intra- and interspecific sequence divergences varied widely among insects, 0.04-26.0 and 1.0-30.7%, respectively. The ranges of intra- and interspecific sequence divergences overlapped in 28 of 62 comparisons. This implies that a standardized percent sequence divergence would fail to correctly diagnose species for 45% of the cases. Common occurrence of nonmonophyly among closely related species probably explains this observation. Nonmonophyly and overlap of intra- and interspecific divergences were significantly associated. The reviewed studies suggest that a standard percent sequence divergence does not predict species boundaries among economically important insects. DNA data can help best to predict species boundaries via its inclusion in nonphenetic phylogenetic analysis and subsequent systematic expert scrutiny.     

Influence of IgH V-region genes on the growth kinetics of a murine B cell leukemia (BCL1)

The growth kinetics of an IgM-bearing B cell leukemia of BALB/c (Ig-1a) origin, designated BCL1, has been investigated in 2 allotype immunoglobulin (Ig) heavy (H) chain congenic strains, C.B-20 (Ig-1b) and C.AL-20 (Ig-1d), and an (H) chain recombinant strain, BAB-14 (Ig-1a/1b), that carries Ig-1a genes in the variable (V)-region and Ig-1b genes in the constant (C)-region. When large numbers (10(6) to 10(7)) of BCL1 cells were injected into these mice, leukemia, as measured by the appearance of leukemic cells in peripheral blood with subsequent mortality, did not occur in C.B-20, was delayed in C.AL-20, and progressed at the same rate in BAB-14 relative to BALB/c control mice. These results indicate that an immune response directed against an antigen encoded for by an H chain V region gene (idiotype or variable-region allotype) or linked gene (minor histocompatibility antigen) prevents the growth of the BCL1 leukemia in the C.B-20 mice. Tumor resistance appears to be due to T cell activity since adoptive transfer of such cells from C.B-20 tumor rejectors protected sublethally irradiation recipients from subsequent tumor challenge. Although H-2 restricted, anti-BCL1 cytotoxic cells were detected in C.B-20 mice challenged in vivo and restimulated in vitro with BCL1 cells, evidence is discussed that suggests that the resistance observed is not due to these effector cells. The resistance of allotype congenic mice to BCL1 was not absolute; a small inoculum (10(2)) was as lethal in C.B-20 and C.AL-20 as BALB/c mice. Thus, Ig-encoded cell surface antigens, although immunogenic, in no way ensure ultimate host survival.     

CD80 expression on B cells regulates murine T follicular helper development, germinal center B cell survival, and plasma cell generation

Germinal center (GC) B cells and T follicular helper (T(FH)) cells interact in the production of high-affinity long-lived plasma cells (PCs) and memory B cells, although the mechanisms regulating the formation of these long-lived populations remain unclear. Because CD80 is one of the few markers shared by human and murine memory B cells, we investigated its role in the development of GCs, memory cells, and PCs. In CD80-deficient mice, fewer long-lived PCs were generated upon immunization compared with that in B6 controls. In concert, the absence of CD80 resulted in an increase in apoptotic GC B cells during the contraction phase of the GC. CD80(-/-) mice had fewer T(FH) cells compared with that of B6, and residual T(FH) cells failed to mature, with decreased ICOS and PD-1 expression and decreased synthesis of IL-21 mRNA. Mixed bone marrow chimeras demonstrated a B cell-intrinsic requirement for CD80 expression for normal T(FH) cell and PC development. Therefore, B cell expression of CD80 plays a critical role in regulating B-T interactions in both early and late GC responses. This, in turn, results in impaired ability to produce long-lived PCs. These data provide new insights into the development of GCs and Ab-forming cells and the functions of CD80 in humoral immunity.     

SOCS3 deletion in B cells alters cytokine responses and germinal center output

B cell behavior is fine-tuned by internal regulatory mechanisms and external cues such as cytokines and chemokines. Suppressor of cytokine signaling 3 (SOCS3) is a key regulator of STAT3-dependent cytokine responses in many cell types and has been reported to inhibit CXCL12-induced retention of immature B cells in the bone marrow. Using mice with SOCS3 exclusively deleted in the B cell lineage (Socs3(Δ/Δ)mb1cre(+)), we analyzed the role of SOCS3 in the response of these cells to CXCL12 and the STAT3-inducing cytokines IL-6 and IL-21. Our findings refute a B cell-intrinsic role for SOCS3 in B cell development, because SOCS3 deletion in the B lineage did not affect B cell populations in naive mice. SOCS3 was strongly induced in B cells stimulated with IL-21 and in plasma cells exposed to IL-6. Its deletion permitted excessive and prolonged STAT3 signaling following IL-6 stimulation of plasma cells and, in a T cell-dependent immunization model, reduced the number of germinal center B cells formed and altered the production of Ag-specific IgM and IgE. These data demonstrate a novel regulatory signal transduction circuit in plasma cells, providing, to our knowledge, the first evidence of how these long-lived, sessile cells respond to the external signals that mediate their longevity.     

STAT3 signaling in B cells controls germinal center zone organization and recycling

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In vivo obtained antigen presented by germinal center B cells to T cells in vitro

Shortly after secondary immunization germinal center (GC) B cells obtain antigen from follicular dendritic cells (FDC) in the form of immune complexes. This antigen appears to be degraded by the GC B cells and may be processed for presentation to T cells. The present study was undertaken to determine whether GC B cells can process and present antigen obtained from FDC in vivo to appropriate T cells in vitro. GC B cells were isolated from immune mice with the use of Percoll density separation followed by a panning procedure which utilizes the ability of the plant lectin, peanut agglutinin (PNA), to selectively bind to GC B cells. The enriched GC B cells were approximately 80% highly positive for PNA, 97% positive for Ia and surface IgM, but less than 0.01% positive for Thy-1.2 or esterase. In some experiments, this population was further purified to near 100% highly PNA-positive cells with the use of fluoresceinated PNA and a fluorescence-activated cell sorter. Cell sorting analysis indicated that the antigen (125I-labeled ovalbumin (OVA)) was restricted to the highly PNA-positive cell fraction. The capacity of these highly PNA-positive B cells to present antigen was assessed by monitoring interleukin 2 (IL-2) production by the OVA-specific T cell hybridoma, 3DO-54.8. GC B cells obtained from mice 3 wk or more after secondary immunization did not elicit IL-2 production in the absence of added OVA. However, GC B cells isolated as early as 1 day and for over 1 wk after a challenge with OVA, were able to stimulate high levels of IL-2 production, in the absence of adding OVA to the cell cultures. This response was maximal on day 5 and corresponded precisely with the kinetics of the ultrastructural studies which document the uptake of antigen by GC B cells in vivo. The FDC-derived antigen was remarkably immunogenic when compared with exogenous antigen. These findings demonstrated that antigen obtained in vivo by GC B cells could be processed and presented to T cells. In vivo, GC B cells may induce the T cell help needed for the germinal center reaction, generate B memory cells, and help induce the high titers of antibody associated with the secondary antibody response.     

Cutting edge: cellular Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein protects germinal center B cells from apoptosis during germinal center reactions

During germinal center (GC) reactions, follicular dendritic cells are believed to select memory B lymphocytes by switching off apoptosis in the successfully binding B cells. The cellular signals involved in this process are largely unknown. Here, we show that GC B lymphocytes have a long isoform of the cellular homologue of the viral Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein (cFLIP(L)), which is capable of inhibiting death receptor-induced caspase activation. In isolated GC B cells, cFLIP(L) decays rapidly even without Fas ligation, and this results in activation of caspase activity and apoptosis. Contact with follicular dendritic cells prevents cFLIP(L) degradation and blocks all signs of apoptosis, even in the presence of anti-Fas ABS: cFLIP(L) expression is sustained by CD40 ligation as well, suggesting that at least at some stage of the GC reaction activated T cells may help selected B cells to leave the follicular dendritic cell network without becoming apoptotic.     

FcgammaRIIB regulates autoreactive primary antibody-forming cell, but not germinal center B cell, activity

The low-affinity FcR for IgG FcgammaRIIB suppresses the development of IgG autoantibodies and autoimmune disease in normal individuals, but how this effect is mediated is incompletely understood. To investigate this issue, we created FcgammaRIIB-deficient versions of two previously described targeted BCR-transgenic lines of mice that contain follicular B cells with specificity for the hapten arsonate, but with different levels of antinuclear autoantigen reactivity. The primary development and tolerance of both types of B cells were unaltered by the absence of FcgammaRIIB. Moreover, the reduced p-azophenylarsonate-driven germinal center and memory responses characteristic of the highly autoreactive clonotype were not reversed by an intrinsic FcgammaRIIB deficiency. In contrast, the p-azophenylarsonate-driven primary Ab-forming cell responses of both clonotypes were equivalently increased by such a deficiency. In total, our data do not support the idea that FcgammaRIIB directly participates in the action of primary or germinal center tolerance checkpoints. In contrast, this receptor apparently contributes to the prevention of autoimmunity by suppressing the production of autoreactive IgGs from B cells that have breached tolerance checkpoints and entered the Ab-forming cell pathway due to spontaneous, or cross-reactive, Ag-mediated activation.     

Fas-mediated apoptosis eliminates B cells that acquire self-reactivity during the germinal center response to NP

C57Bl/6 mice with the lpr mutation of Fas (CD95) were tested for deviation from the genetically restricted antibody response to the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). lambda1+ germinal centers (GC) with the canonical v186.2 V(H) gene element develop in lpr/lpr mice with the same time course as in wild-type (+/+) mice. In contrast to +/+ mice, however, lambda1+ GC persist in the spleens of lpr/lpr mice 25 days after immunization. Virtually all of the lambda1+ GC are reactive with NP 10 days after immunization. Sixteen days after immunization, however, many of the lambda1+ GC are not reactive with NP, and few of the lambda1+ GC are reactive with NP 25 days after immunization. The V(H) gene elements of three lambda1+NP- GC 25 days after immunization are derived by somatic mutation of v186.2, but have lost reactivity with NP. The mutated VDJs from these GC react with cells in spleen sections from +/+ and lpr/lpr mice, indicating that they represented secondary antibody responses induced by self antigens that are available as presented antigen. These data indicate that Fas-mediated apoptosis serves to eliminate a (limited) population of B cells that acquire reactivity to "self antigens" by somatic mutation of VDJs in the GC.     

Cyclin D3 is selectively required for proliferative expansion of germinal center B cells

The generation of robust T-cell-dependent humoral immune responses requires the formation and expansion of germinal center structures within the follicular regions of the secondary lymphoid tissues. B-cell proliferation in the germinal center drives ongoing antigen-dependent selection and the generation of high-affinity class-switched plasma and memory B cells. However, the mechanisms regulating B-cell proliferation within this microenvironment are largely unknown. Here, we report that cyclin D3 is uniquely required for germinal center progression. Ccnd3(-/-) mice exhibit a B-cell-intrinsic defect in germinal center maturation and fail to generate an affinity-matured IgG response. We determined that the defect resulted from failed proliferative expansion of GL7(+) IgD(-) PNA(+) B cells. Mechanistically, sustained expression of cyclin D3 was found to be regulated at the level of protein stability and controlled by glycogen synthase kinase 3 in a cyclic AMP-protein kinase A-dependent manner. The specific defect in proliferative expansion of GL7(+) IgD(-) PNA(+) B cells in Ccnd3(-/-) mice defines an underappreciated step in germinal center progression and solidifies a role for cyclin D3 in the immune response, and as a potential therapeutic target for germinal center-derived B-cell malignancies.     

生发中心起源的B细胞淋巴瘤的表观遗传致病机制

生发中心(germinal center, GC)反应过程中,B细胞(B cell)经历了体细胞高频突变,类别转换,亲和力成熟等环节。在此过程中需要对GC B细胞特殊调控,从而维持GC B细胞的增殖和分化。但是一旦关键转录因子调控失调,可能导致B细胞淋巴瘤的形成。过去对淋巴瘤的研究大多停留在遗传学角度,随着新的基因组学技术的出现,大量研究成果表明,表观遗传修饰(如DNA甲基化,组蛋白修饰和微RNA)也在B细胞淋巴瘤(B cell lymphoma)中扮演着极其重要的角色。表观遗传修饰是可逆的,使其为靶点治疗相关癌症药物取得了良好的临床疗效。但表观遗传修饰是一个极其复杂的转录调控信号通路,仍需进一步的研究来揭示B细胞淋巴瘤的发生机制,以期寻找更好的治疗靶点以及更好的药物来防治癌症。