Regulation of memory B-cell survival by the BH3-only protein Puma

Apoptosis is crucial for immune system homeostasis, including selection and survival of long-lived antibody-forming cells and memory cells. The interactions between proapoptotic and pro-survival proteins of the Bcl-2 family are critical for this process. In this report, we show that expression of the proapoptotic BH3-only Bcl-2 family member Puma was selectively up-regulated on in vitro activation with antigens or mitogens of both human and mouse B cells. Puma expression coincided in vivo, with the prosurvival Bcl-2 family member Mcl-1 within the germinal centers and its expression correlates with the germinal center like phenotype of Burkitt lymphoma. Experiments performed in Puma-deficient mice revealed that Puma is essential for apoptosis of mitogen-activated B cells in vitro and for the control of memory B-cell survival. In conclusion, using both human and murine models, our data show that Puma has a major role in the T cell- dependent B-cell immune response. These data demonstrate that Puma is a major regulator of memory B lymphocyte survival and therefore a key molecule in the control of the immune response.     

Evolving abundance and clonal pattern of human germinal center B cells during childhood

Childhood is a critical period for the development of the memory B-lymphocyte repertoire necessary in protective humoral immunity. This study addressed the natural history of memory B cells based on the previous identification of germinal center and mantle zone cells as the probable precursor and mature memory cell populations, respectively. Using flow cytometric quantitation of these B-cell subpopulations in human tonsil, we found that germinal center cells were abundant (70% of tonsil B cells) during early childhood (2 to 3 years), but decline by early adolescence (8 to 14 years) to a low level (33%, P = .0003). To study the clonal evolution of these B-cell subpopulations, germinal center and mantle zone B cells were isolated using a preparative magnetic immunobead method, and analyzed using a novel polymerase chain reaction-based quantitative assay to measure the abundance of B-cell clones bearing certain rearranged VH subfamilies. Two VH subfamilies were informative: VH1N clones were uniquely deficient in germinal center B cells at the early age period, but became abundant in later childhood; and VH3L clones were absent among germinal center cells regardless of age. In contrast, B-cell clones bearing each VH subfamily were abundant in the mantle zone subpopulation throughout childhood. These findings suggest that the abundance and clonal pattern of germinal center B cells evolves during childhood, presumably due to changing antigenic or ontogenic processes. Moreover, the distinct clonal pattern of germinal center versus mantle zone B cells suggests that a major phase of clonal selection occurs after germinal center emigration.     

Memory B cells contribute to rapid Bcl6 expression by memory follicular helper T cells

In primary humoral responses, B-cell lymphoma 6 (Bcl6) is a master regulator of follicular helper T (T_FH) cell differentiation; however, its activation mechanisms and role in memory responses remain unclear. Here we demonstrate that survival of CXCR5⁺ T_FH memory cells, and thus subsequent recall antibody response, require Bcl6 expression. Furthermore, we show that, upon rechallenge with soluble antigen Bcl6 in memory T_FH cells is rapidly induced in a dendritic cell-independent manner and that peptide:class II complexes (pMHC) on cognate memory B cells significantly contribute to this induction. Given the previous evidence that antigen-specific B cells residing in the follicles acquire antigens within minutes of injection, our results suggest that memory B cells present antigens to the cognate T_FH memory cells, thereby contributing to rapid Bcl6 reexpression and differentiation of the T_FH memory cells during humoral memory responses.The development of high-affinity B-cell memory is essential in most effective vaccines that are in use today. Because most protein antigens require T-cell help to induce B-cell responses, understanding the mechanisms by which memory T and B cells are generated and maintained, as well as how their swift activation is executed, is of fundamental importance for vaccine development.In primary immune responses, it is widely accepted that among several differentiated helper T-cell subsets follicular helper CD4 T cells (T_FH cells) are the major subset to deliver help to B cells (1). T_FH cells express CXC-chemokine receptor 5 (CXCR5), the chemokine receptor for the B-cell homing chemokine CXCL13. Surface expression of CXCR5 enables T_FH cells to migrate into B-cell follicles, where they provide help to B cells to form germinal centers. In addition, T_FH cells are needed for the crucial affinity-maturation process of B cells in germinal centers, whereby Ag-specific B cells undergo repeated rounds of somatic hypermutation and positive selection by T_FH cells to rapidly evolve high-affinity somatically mutated B-cell receptors. B-cell lymphoma 6 (Bcl6) has recently been identified as a T_FH lineage regulator (2–4); it is highly expressed by T_FH cells and is required for their development. According to the current view, during a primary response Bcl6 expression by T cells is induced by priming with dendritic cells (5–7) and ICOS is a key coreceptor molecule for induction of Bcl6 (5, 8). The initial Bcl6 induction and subsequent CXCR5 expression allow CD4 T cells to migrate toward the T–B border, where T_FH cells interact with antigen-specific B cells. According to this model, cognate B cells are not required for the induction of Bcl6 but support the expansion of T_FH cells (9).Although the importance of Bcl6 and its expression kinetics in naïve T-cell differentiation have been well elucidated, its role and activation mechanisms in T_FH memory cells still remain obscure. Hence, in this paper we first focus upon the roles of Bcl6, demonstrating its importance for maintenance of T_FH memory cells. Then, we show that Bcl6 in memory T_FH cells was rapidly induced upon rechallenge with soluble antigen and that this response was mainly mediated through antigen presentation by the cognate memory B cells. Given the good association between Bcl6 with IL-21 expression in differentiated memory T_FH cells, our results suggest that memory B cells are the primary antigen-presenting cells (APCs) to induce the rapid differentiation of memory T_FH cells toward effector cells, further accelerating memory B-cell responses during recall.     

Proapoptotic BH3-only protein Bim is essential for developmentally programmed death of germinal center-derived memory B cells and antibody-forming cells

T cell-dependent B-cell immune responses induce germinal centers that are sites for expansion, diversification, and selection of antigen-specific B cells. During the immune response, antigen-specific B cells are removed in a process that favors the retention of cells with improved affinity for antigen, a cell death process inhibited by excess Bcl-2. In this study, we examined the role of the BH3-only protein Bim, an initiator of apoptosis in the Bcl-2-regulated pathway, in the programmed cell death accompanying an immune response. After immunization, Bim-deficient mice showed persistence of both memory B cells lacking affinity-enhancing mutations in their immunoglobulin genes and antibody-forming cells secreting low-affinity antibodies. This was accompanied by enhanced survival of both cell types in culture. We have identified for the first time the physiologic mechanisms for killing low-affinity antibody-expressing B cells in an immune response and have shown this to be dependent on the BH3-only protein Bim.     

Isolated follicular lymphoma cells are resistant to apoptosis and can be grown in vitro in the CD40/stromal cell system

Low-grade follicular non-Hodgkin's lymphomas are characterized by the presence of a t(14;18) chromosomal translocation that results in deregulation of the B-cell lymphoma (Bcl-2) gene. Studies in cell lines and transgenic animal models have suggested that this results in the suppression of apoptotic cell death in germinal centers. B lymphocytes from normal germinal centers and lymph nodes infiltrated by follicular lymphoma were isolated by immunomagnetic depletion of cells bearing CD4, CD8, or slgD for study in vitro. Follicular lymphoma cells expressing Bcl-2 protein were shown to resist apoptosis after isolation, and could be induced to proliferate in a culture system previously described for the growth of normal B lymphocytes. By the use of a mouse fibroblast monolayer transfected with the CDw32 Fc receptor to present CD40 monoclonal antibody in the presence of interleukin-4, prolonged culture was possible. Karyotypic analysis of cultured lymphoma cells showed the t(14;18) translocation, with clonal identity confirmed by polymerase chain reaction amplification of the breakpoints and direct sequence analysis. These findings support the hypothesis that resistance to apoptosis is an influence on the initiation of follicular lymphoma, and provide a novel means of studying in vitro the intercellular reactions that may be important in progression of the disease.     

VavP-Bcl2 transgenic mice develop follicular lymphoma preceded by germinal center hyperplasia

In human follicular lymphoma the t(14; 18) chromosome translocation activates the antiapoptotic oncogene Bcl2 by linking it to the immunoglobulin heavy chain (IGH) locus. Transgenic mice expressing Bcl2 controlled by an Igh enhancer (E mu) do not develop follicular lymphoma, although they do have an increased incidence of other B-lymphoid neoplasms. We have now analyzed tumorigenesis in mice bearing a Bcl2 transgene controlled by Vav gene regulatory sequences (VavP), which confer expression in multiple hematopoietic lineages. Unlike E mu-Bcl2 mice, many VavP-Bcl2 mice older than 10 months developed follicular lymphoma. Young VavP-Bcl2 mice had an overabundance of enlarged germinal centers and greatly elevated numbers of cycling B cells that had undergone IgH class switching and V-gene hypermutation. The peripheral T-cell compartment was larger in the VavP-Bcl2 mice than in E mu-Bcl2 strains and, notably, CD4 T cells were 5-fold increased over normal. The germinal center hyperplasia required CD4 T cells, because it could be abolished by anti-CD4 antibody in vivo. VavP-Bcl2 mice also had a propensity to develop kidney disease of the autoimmune type. We suggest that the increased survival capacity of B and T cells fosters prolonged germinal center reactions, and that autoreactivity and hypermutation conspire to generate follicular lymphoma.     

Germinal center-associated nuclear protein (GANP) has a phosphorylation-dependent DNA-primase activity that is up-regulated in germinal center regions

Antigen stimulation induces a rapid proliferation of B cells for expansion of specific B cell clones and their further differentiation into antibody-producing cells in germinal centers of T-dependent antigen-immunized mice. Previously, we identified a 210-kDa germinal center-associated nuclear protein (GANP) that is up-regulated selectively in germinal centers and carries an MCM-binding domain in the carboxyl-terminal side. In addition, here, we found a region (from 414 to 550 aa) in GANP molecule that is slightly similar to the known DNA-primase component p49. The recombinant GANP fragment covering this region synthesizes RNA primers for extension by DNA polymerase I with single-stranded DNA templates in vitro. GANP DNA-primase activity is controlled by phosphorylation at Ser(502) that is induced by CD40-mediated signaling in vitro and in the germinal center B cells stimulated with antigen in vivo. Overexpression of ganp cDNA in Daudi B cells caused the increased DNA synthesis more than the levels of the mock-transfectants. These evidences suggested that the novel DNA-primase GANP is involved in regulation of cell proliferation of antigen-driven B cells in germinal centers.     

滤泡辅助T细胞相关分子与支气管哮喘

滤泡辅助性T细胞(T follicular helper cells,Tfh)是近来发现的CD4+T细胞亚群,它能够迁移至次级淋巴中心的B细胞滤泡,促进生发中心反应,包括调节生发中心的形成、发展和成熟以及辅助B细胞产生具有免疫功能的球蛋白及影响类别转换.Tfh能连续产生趋化因子受体5,在其配体CXCL13的作用下,外周的Tfh被募集至生发中心的B淋巴滤泡,参加生发中心的形成.诱导性共刺激分子和CO40L可以与B细胞表面的配体结合可以调节Tfh分泌IL-21参与B细胞的免疫应答,参与体液免疫.支气管哮喘(简称哮喘)发病机制复杂,T淋巴细胞在哮喘的气道炎症反应中起重要作用.本文回顾了Tfh细胞相关分子在哮喘气道炎症中的作用.     

BCL-6 gene product, a 92- to 98-kD nuclear phosphoprotein, is highly expressed in germinal center B cells and their neoplastic counterparts

The BCL-6 gene is known to be located on chromosome 3q27, at the breakpoint of the 3q27-associated translocations that occur frequently in human non-Hodgkin's lymphomas (NHLs). To identify the BCL-6 protein, two antibodies that recognized distinct domains of this protein were raised in rabbits. Immunoprecipitation and immunoblotting of lysates of BCL-6-expressing cells using both antibodies showed a broad 92- to 98- kD band. Dephosphorylation of BCL-6 protein reduced the size of this band to 87 kD, suggesting that BCL-6 may be expressed in a phosphorylated form. Immunostaining with both antibodies showed that BCL-6 protein was localized in the nuclei of most of the germinal center B cells and a small number of marginal zone B cells. Furthermore, BCL-6 protein was expressed in follicular, Burkitt's, and diffuse large B-cell lymphomas. These results suggest that the BCL-6 protein, expressed in B cells of the germinal centers which are important in the maturation of immune responses, may play some physiological role(s) in the germinal center B cells.     

Bcl-x rather than Bcl-2 mediates CD40-dependent centrocyte survival in the germinal center

Both rapid B-cell proliferation and programmed cell death (PCD) occur during the differentiation and selection of B cells within the germinal center. To help elucidate the role of Bcl-x in B-cell antigen selection and PCD within the germinal center, we examined its expression in defined B-cell populations and by immunochemistry of tonsil tissue. Purified B-cell fractions enriched for centrocytes express high amounts of Bcl-x and relatively low amounts of Bcl-2, whereas fractions enriched for centroblasts lack significant levels of both proteins. Consistent with this observation, immunocytochemistry localized Bcl-x within cells scattered throughout the germinal center. Stimulation of tonsil B cells with either CD40 or Staphylococcus aureus Cowan increase bcl-x mRNA and protein levels. Treatment of a cell line with a germinal center phenotype (RAMOS) or the tonsillar B-cell centroblast fraction with CD40 rapidly increased Bcl-x levels and partially rescued B cells from PCD. These data suggest that Bcl-x rather than Bcl-2 may rescue centrocytes during selection in the germinal center.     

B cells from the bench to the clinical practice

B cells develop in bone marrow and undergo antigen-induced activation and terminal differentiation in germinal centres of secondary lymphoid organs. Each B cell is a clone which means that an individual B cell has a unique genetic code and produces only one type of antibody when stimulated by antigen being able to multiply itself and originate several B cells with the same antigen specificity (clonal selection theory). However their important role in adaptive immune responses is supported by the remarkable capacity of recognizing an unlimited array of antigens due to mechanisms of antibody diversity such as V(D)J recombination class switching and somatic hypermutation. B cells can also function as antigen presenting cells that can activate T cells improving the effectiveness of the immune response. Immune B cell tolerance surveillance through clonal deletion anergy and receptor editing is also necessary to avoid pathological conditions like autoimmune diseases. B cells can contribute to autoimmunity by autoantibody production cytokine synthesis antigen presentation T cell activation and ectopic lymphogenesis.     

Epstein-Barr virus infection in vitro can rescue germinal center B cells with inactivated immunoglobulin genes

Immunoglobulin genotyping of Epstein-Barr virus (EBV)-positive posttransplantation lymphoproliferative disease has suggested that such lesions often arise from atypical post-germinal center B cells, in some cases carrying functionally inactivated immunoglobulin genes. To investigate whether EBV can rescue cells that are failed products of the somatic hypermutation process occurring in germinal centers (GCs), we isolated GC cells from tonsillar cell suspensions and exposed them to EBV in vitro. Screening more than 100 EBV-transformed cell lines of GC origin identified 6 lines lacking surface immunoglobulin, a phenotype never seen among lines derived from circulating naive or memory B cells. Furthermore, 3 of the 6 surface immunoglobulin-negative GC lines carried inactivating mutations in the immunoglobulin H (IgH) variable gene sequence. The ability of EBV to rescue aberrant products of the germinal center reaction in vitro strengthens the probability that a parallel activity contributes to EBV's lymphomagenic potential in vivo.     

B-cell development in progressively transformed germinal centers: similarities and differences compared with classical germinal centers and lymphocyte-predominant Hodgkin disease

Progressively transformed germinal centers (PTGCs) are histologic structures mainly composed of small resting B cells and intermingled proliferating centroblast-like cells. The B-cell differentiation processes within PTGCs and their relation to classical germinal centers (GC) and to lymphocyte-predominant Hodgkin disease (LPHD), with which PTGCs are often associated, are largely unknown. To address these issues, single small resting (Ki67-) and proliferating (Ki67+) centroblast-like cells were isolated from 7 PTGCs of 5 lymph nodes, and rearranged immunoglobulin genes were amplified and sequenced. Most small resting B cells were clonally unrelated, and most carried unmutated immunoglobulin gene rearrangements resembling mantle zone B cells. Small resting B cells with mutated immunoglobulin gene rearrangements may represent centrocytes, memory B cells, or both. Among the centroblast-like Ki67+ cells, expanded B-cell clones were observed in 6 of 7 PTGCs analyzed. Clonally related V region genes showed extensive intraclonal diversity, and the mutation pattern indicated stringent selection of the cells for the expression of functional antigen receptors. Thus, somatic hypermutation, clonal expansion, and selection occur also in the disorganized PTGC microenvironment, as in classical GCs. In lymph nodes affected by PTGCs, no clonal expansion across the borders of individual PTGCs was observed, distinguishing PTGCs from LPHD.     

Telomere lengthening and telomerase activation during human B cell differentiation

The function of the immune system is highly dependent on cellular differentiation and clonal expansion of antigen-specific lymphocytes. However, little is known about mechanisms that may have evolved to protect replicative potential in actively dividing lymphocytes during immune differentiation and response. Here we report an analysis of telomere length and telomerase expression, factors implicated in the regulation of cellular replicative lifespan, in human B cell subsets. In contrast to previous observations, in which telomere shortening and concomitant loss of replicative potential occur in the process of somatic cell differentiation and cell division, it was found that germinal center (GC) B cells, a compartment characterized by extensive clonal expansion and selection, had significantly longer telomeric restriction fragments than those of precursor naive B cells. Furthermore, it was found that telomerase, a telomere-synthesizing enzyme, is expressed at high levels in GC B cells (at least 128-fold higher than those of naive and memory B cells), correlating with the long telomeres in this subset of B cells. Finally, both naive and memory B cells were capable of up-regulating telomerase activity in vitro in response to activation signals through the B cell antigen receptor in the presence of CD40 engagement and/or interleukin 4. These observations suggest that a novel process of telomere lengthening, possibly mediated by telomerase, functions in actively dividing GC B lymphocytes and may play a critical role in humoral immune response by maintaining the replicative potential of GC and descendant memory B cells.     

Proapoptotic protein Bim is differentially required during thymic clonal deletion to ubiquitous versus tissue-restricted antigens

Positive and negative selection of thymocytes in the thymus are critical for the development of a mature and self-tolerant T-cell repertoire. The proapoptotic Bcl-2 family member Bim is important for negative selection by inducing apoptosis in thymocytes receiving a strong signal through their antigen receptor. However, in the case of ubiquitous self-antigens (UbA), Bim is not required for the clonal deletion of self-reactive thymocytes, suggesting the existence of nonapoptotic clonal deletion mechanisms. Unlike UbA, clonal deletion to tissue-restricted antigens (TRAs) requires positive selection and CCR7-mediated migration to the medulla. This led us to hypothesize that Bim is required for the latter. To study the role of Bim in clonal deletion to TRA, we constructed bone marrow (BM) chimeras using OT-I Bim-deficient or -sufficient donor bone marrow and recipients that express membrane bound chicken ovalbumin under control of the rat insulin promoter (Rip-mOVA). We found that clonal deletion to TRA was completely abrogated in the absence of Bim and large numbers of mature OT-I CD8 T cells survived in the periphery. Despite the large numbers of autoreactive T cells, the chimeras did not develop diabetes and OT-I Bim-deficient T cells from these chimeras were functionally impaired. Collectively, these data provide unique evidence of a differential, thymocyte-intrinsic, molecular requirement downstream of the T-cell receptor (TCR) for clonal deletion to UbA versus TRA and highlight the profound ability of other tolerance mechanisms to control T-cell autoreactivity in the absence of thymic clonal deletion.     

Patterns of Epstein-Barr virus infection in non-neoplastic lymphoid tissue.

Taking advantage of the abundant expression of the small Epstein-Barr virus (EBV)-encoded RNAs (EBERs) in latently infected cells, we have analyzed 72 normal and hyperplastic lymph nodes and three tonsils of acute infectious mononucleosis (IM) for the presence and distribution of EBV+ cells using EBER-specific in situ hybridization, in some cases combined with immunohistologic demonstration of cell type-characteristic antigens. In IM, large numbers of EBV+ lymphoid B blasts were detectable in extrafollicular areas, whereas germinal centers were generally free of EBV+ cells. In reactive lymph nodes, the frequency of EBV+ cells varied with the degree of lymphoid hyperplasia and underlying immune status. The lowest numbers of EBV+ cells were detected in nonactivated lymph nodes and highest in human immunodeficiency virus-associated lymphadenopathy. If present in these lymph nodes, EBV+ cells were almost exclusively localized to extrafollicular areas, as also observed in IM. However, in contrast to IM, these cells were mainly small lymphocytes. Furthermore, in some instances, occasional scattered EBV+ cells were seen within germinal centers, and in two cases diffuse expansions of EBV+ cells occurred within a single germinal center each, indicating that under certain circumstances EBV+ B lymphocytes may participate in physiologic germinal center reactions. These findings reflect the interference of EBV with physiologic lymphoid differentiation pathways and provide a link to EBV-associated malignant lymphomas with a postulated origin from germinal center cells.     

Modulación del cambio de isotipo de las inmunoglobulinas por señales del sistema inmunitario innato

Mature B cells emerge from the bone marrow and continue to diversify their immunoglobulin genes through 2 antigen-dependent processes known as somatic hypermutation and class switch recombination. These processes require AID, a DNA-editing enzyme. Although both processes predominantly occur in germinal center B cells engaged in a T cell-dependent (TD) antibody response against protein antigens recent, evidence shows that B cells receive additional help from invariant natural killer T cells, dendritic cells, and various granulocytes, including neutrophils, eosinophils, and basophils. These innate immune cells enhance TD antibody responses by delivering B-cell helper signals whether in germinal centers, postgerminal lymphoid centers, or the bone marrow. In addition to enhancing and complementing the B-cell helper activity of canonical T cells, invariant natural killer T cells, dendritic cells, and granulocytes can deliver T cell-independent B-cell helper signals at the mucosal interface and in the marginal zone of the spleen to initiate rapid innate-like antibody responses. In this review, we discuss recent advances in the role of innate cells in B-cell helper signals and in antibody diversification and production.     

Role of T cell death in maintaining immune tolerance during persistent viral hepatitis

Virus-specific T cells play an important role in the resolution of hepatic infection. However, during chronic hepatitis infection these cells lack their effector functions and fail to control the virus. Hepatitis B virus and hepatitis C virus have developed several mechanisms to generate immune tolerance. One of these strategies is the depletion of virus-specific T cells by apoptosis. The immunotolerogenic liver has unique property to retain and activate na ve T cell to avoid the over reactivation of immune response against antigens which is exploited by hepatotropic viruses to persist. The deletion of the virus-specific T cells occurs by intrinsic (passive) apoptotic mechanism. The pro-apoptotic molecule Bcl-2 interacting mediator (Bim) has attracted increasing attention as a pivotal involvement in apoptosis, as a regulator of tissue homeostasis and an enhancer for the viral persistence. Here, we reviewed our current knowledge on the evidence showing critical role of Bim in viral-specific T cell death by apoptotic pathways and helps in the immune tolerance.