Molecular interactions of FDCs with B cells in aging

Follicular dendritic cells (FDCs), as accessory cells to B cells, promote germinal center (GC) development. Age-related defects in the role of FDCs are well documented in vivo. In old mice, FDCs bind fewer immune complexes (ICs) and produce few iccosomes for endocytosis by B cells, antigen processing, and presentation to T cells. We recently studied whether these defects are due to changes in the FDC microenvironment or to changes in FDCs and their surface molecules. In vitro evidence suggests that age-related defects in both B cell stimulation via the BCR and co-stimulation via CD21/CD21L are related to IC-trapping by FDCs in vivo-a defect which is repairable, at least, in vitro.     

T cells and follicular dendritic cells in germinal center B-cell formation and selection

Summary: Germinal centers (GCs) are specialized microenvironments formed after infection where activated B cells can mutate their B-cell receptors to undergo affinity maturation. A stringent process of selection allows high affinity, non-self-reactive B cells to become long-lived memory B cells and plasma cells. While the precise mechanism of selection is still poorly understood, the last decade has advanced our understanding of the role of T cells and follicular dendritic cells (FDCs) in GC B-cell formation and selection. T cells and non-T-cell-derived CD40 ligands on FDCs are essential for T-dependent (TD) and T-independent GC formation, respectively. TD-GC formation requires Bcl-6-expressing T cells capable of signaling through SAP, which promotes formation of stable T:B conjugates. By contrast, differentiation of B blasts along the extrafollicular pathway is less dependent on SAP. T-follicular helper (Tfh) cell-derived CD40L, interleukin-21, and interleukin-4 play important roles in GC B-cell proliferation, survival, and affinity maturation. A role for FDC-derived integrin signals has also emerged: GC B cells capable of forming an immune synapse with FDCs have a survival advantage. This emerges as a powerful mechanism to ensure death of B cells that bind self-reactive antigen, which would not normally be presented on FDCs.     

Follicular dendritic cells in aging, a "bottle-neck" in the humoral immune response

Senescence leads to the appearance of atrophic follicular dendritic cells (FDCs) that trap and retain little immune complexes (IC), generate few memory B cells, and induce a reduced number of germinal centers (GC). Deficiencies in antibody responses to T cell dependent exogenous antigens such as pneumonia and influenza vaccines may reflect intrinsic FDC defects or altered FDC-B cell interactions. We recently studied antigen handling capacity and co-stimulatory activity of old FDCs and determined age-related changes in the expression or function of FcgammaRII or CR1 and 2 on FDCs. Here, we present an overview of FDC function in recall responses with known deficiencies in FDCs and GC development. Then, we review our recent work on aged FDCs and discuss age-related changes in molecular interactions between FDCs and B cells. We also discuss the causes underlying the impaired humoral immune response with respect to age-related molecular changes in FDC and B cell interactions. In vitro evidence suggests that FcgammaRII on aged FDCs is regulated abnormally and this in turn might cause the development of a defective FDC-network (reticulum) that retains few ICs, promotes ITIM signaling, prevents B cell proliferation and GC formation, and antibody production.     

Isolation of functionally active murine follicular dendritic cells

Biochemical, genetic, and immunological studies of follicular dendritic cells (FDCs) have been hampered by difficulty in obtaining adequate numbers of purified cells in a functional state. To address this obstacle, we enriched FDCs by irradiating mice to destroy most lymphocytes, excised the lymph nodes, and gently digested the nodes with an enzyme cocktail to form single cell suspensions. The FDCs in suspension were selected using the specific mAb FDC-M1 with magnetic cell separation technology. We were able to get nearly a million viable lymph node FDCs per mouse at about 90% purity. When examined under light and transmission electron microscopy, the cytological features were characteristic of FDCs. Furthermore, the cells were able to trap and retain immune complexes and were positive for important phenotypic markers including FDC-M1, CD21/35, CD32, CD40, and CD54. Moreover, the purified FDCs exhibited classical FDC accessory activities including: the ability to co-stimulate B cell proliferation, augment antibody responses induced by mitogens or antigens, maintain B cell viability for weeks, and protect B lymphocytes from anti-FAS induced apoptosis. In short, this combination of methods made it possible to obtain a substantial number of highly enriched functional murine FDCs.     

Lymphotoxin α1β2 expression on B cells is required for follicular dendritic cell activation during the germinal center response

CD19‐deficient mice were used as a model to study follicular dendritic cell (FDC) activation because these mice have normal numbers of FDC‐containing primary follicles, but lack the ability to activate FDCs or form GCs. It was hypothesized that CD19 expression is necessary for B‐cell activation and upregulation of membrane lymphotoxin (mLT) expression, which promotes FDC activation. Using VCAM‐1 and FcγRII/III as FDC activation markers, it was determined that the adoptive transfer of CD19⁺ wild‐type B cells into CD19‐deficient hosts rescued GC formation and FDC activation, demonstrating that CD19 expression on B cells is required for FDC activation. In contrast, CD19⁺ donor B cells lacking mLT were unable to induce VCAM‐1 expression on FDCs, furthermore FcγRII/III upregulation was impaired in FDCs stimulated with mLT‐deficient B cells. VCAM‐1 expression on FDCs, but not FcγRII/III, was rescued when CD19‐deficient B cells expressing transgenic mLT were cotransferred into recipient mice with CD19⁺, mLT‐deficient B cells, suggesting that FDC activation requires the CD19‐dependent upregulation of mLT on activated B cells. Collectively, these data demonstrate that activated B cells are responsible for the initiation of FDC activation resulting in a microenvironment supportive of GC development and maintenance.     

Germinal center B cells rescued from apoptosis by CD40 ligation or attachment to follicular dendritic cells,but not by engagement of surface immunoglobulin or adhesion receptors,become resistant to CD95-induced apoptosis

Germinal centers (GC) constitute a specialized microenvironment essential for the formation of memory B cells, B cell affinity maturation and isotype switching. Within the GC, the B cells closely interact with follicular dendritic cells (FDC) and T cells, which both provide stimuli to the B cells that prevent their entry into apoptosis and promote their differentiation into memory cells or plasma cells. Cross-linking of B cell immunoglobulin (Ig) receptors by antigen, stimulation of the integrin adhesion molecules LFA-1 and VLA-4 on the B cell through interaction with their counter receptors ICAM-1 and VCAM-1 on the FDC and cross-linking of CD40 on the B cells through interaction with the CD40 ligand (CD40L) on T cells have been shown to prevent entry into apoptosis of GC B cells. Triggering of CD95, on the other hand, has been shown to induce apoptosis. We therefore investigated the interaction between adhesion-mediated signals, Ig, CD40, and CD95. The spontaneous apoptosis of GC B cells was not further increased by adding anti-CD95. However. CD95 stimulation did result in apoptosis of GC B cells in the presence of anti-Ig or adhesion-mediated rescue signals, which indicates that CD95 expressed on GC B cells is functionally active. In contrast, anti-CD95 was unable to induce apoptosis in cells rescued via CD40 stimulation, suggesting an important role for CD40L expressed on GC T cells in apoptosis regulation. We also studied apoptosis of B cells adhering to FDC, and found that B cells that interact with FDC were also rescued from CD95-induced apoptosis. A human CD40.Fcu fusion protein that blocks CD40 ligation failed to inhibit this effect. Our studies therefore indicate that neither CD40, Ig receptors, nor adhesion receptors mediate rescue from apoptosis by FDC.     

Fas receptor expression in germinal-center B cells is essential for T and B lymphocyte homeostasis

Fas is highly expressed in activated and germinal center (GC) B cells but can potentially be inactivated by misguided somatic hypermutation. We employed conditional Fas-deficient mice to investigate the physiological functions of Fas in various B cell subsets. B cell-specific Fas-deficient mice developed fatal lymphoproliferation due to activation of B cells and T cells. Ablation of Fas specifically in GC B cells reproduced the phenotype, indicating that the lymphoproliferation initiates in the GC environment. B cell-specific Fas-deficient mice also showed an accumulation of IgG1(+) memory B cells expressing high amounts of CD80 and the expansion of CD28-expressing CD4(+) Th cells. Blocking T cell-B cell interaction and GC formation completely prevented the fatal lymphoproliferation. Thus, Fas-mediated selection of GC B cells and the resulting memory B cell compartment is essential for maintaining the homeostasis of both T and B lymphocytes.     

Marginal zone B cells transport and deposit IgM-containing immune complexes onto follicular dendritic cells

Secreted IgM and complement are important mediators in the optimal initiation of primary T-dependent humoral immune responses. Secreted IgM serves as a natural adjuvant by enhancing the immunogenicity of protein antigens, perhaps as a result of IgM's ability to facilitate antigen deposition onto follicular dendritic cells (FDCs) and promote rapid germinal center (GC) formation. To understand how IgM enhances adaptive immune responses, we investigated the mechanism by which IgM-containing immune complexes (IgM-IC) are transported to FDCs as a first step in GC formation. We demonstrate that IgM-IC localize first to the splenic marginal zone (MZ) where the IgM-IC bind MZ B cells in a complement and complement receptor (CR1/2) dependent process. MZ B cells then transport the IgM-IC into the follicle for deposition onto FDCs. Mice with reduced numbers of MZ B cells trap IgM-IC on FDC less efficiently, whereas mice with reduced numbers of follicular B cells trap IgM-IC normally. The functional elimination of MZ B cells abrogates the ability of FDCs to trap IgM-IC. Transfer of B cells with associated IgM-IC into naive mice results in deposition of IgM-IC onto FDC by MZ B cells. The results demonstrate an IgM and complement-dependent role for MZ B cells in the fate of antigen early in the initial phases of T-dependent immune responses. The data also establish an important role for CR1/2 on MZ B cells in the efficient binding and transport of IgM-IC to FDCs, which we suggest is an important first step in initiating adaptive immune responses.     

CD4-deficient T helper cells are capable of supporting somatic hypermutation and affinity maturation of germinal center B cells

Collaborative interactions between B lymphocytes and CD4+ helper T cells are necessary for the induction of Ab responses to most protein Ag and for the generation of memory B cells in germinal centers. To study the role of the CD4 molecule in the germinal center response and in the development of B cell memory, we have investigated T helper function in the initiation and maturation of humoral immunity in CD4-deficient mice. In the absence of CD4+ T cells, immunization with thymus-dependent Ag was able to induce germinal center formation and Ig somatic hypermutation. In addition, Ag-driven affinity maturation and development of B cell memory were largely intact in CD4-deficient mice. Thus, CD4-deficient T helper cells are able to collaborate with Ag-activated B cells to elicit the germinal center reaction, switch on the mutational machinery, and deliver signals necessary for B cell memory development.     

滤泡树突状细胞与生发中心反应研究的新进展

生发中心是在T细胞依赖性抗体应答过程中于外周淋巴组织内形成的一个特殊的结构。在GC内,受抗原刺激而活化的B细胞进行克隆扩增、IgV区基因的体细胞高度突变、亲和力成熟以及同类型转换,最终形成记忆性B细胞或是产生Ig的浆细胞。在GC内B细胞增殖的同时,也启动了凋亡机制,以确保最终形成的记忆B细胞或浆细胞对抗原的高度特异性。FDCs是参与再次免疫应答的重要细胞,它主要是通过表面的FcR和CR将免疫复合物结合在细胞膜上,并选择性的将抗原递呈给表达高亲和力BCR的B细胞,使之激活并产生抗体或形成记忆B细胞。因此,FDCs在生发中心反应、免疫记忆的维持、B细胞的分化、成熟以及记忆B细胞的形成具有极其重要的作用。但最近的研究对FDCs及其结合的免疫复合物的重要性提出了质疑,认为FDCs在生发中心反应、B细胞的分化、成熟以及记忆B细胞的形成中的作用很可能是非特异性的,并对驻留在FDCs表面的免疫复合物的其它潜在功能进行了讨论。     

Follicular dendritic cells: beyond the necessity of T-cell help.

Follicular dendritic cells (FDCs) are potent accessory cells for B cells, but the molecular basis of their activity is not understood. Several important molecules involved in FDC-B-cell interactions are indicated by blocking the ligands and receptors on FDCs and/or B cells. The engagement of CD21 in the B-cell coreceptor complex by complement-derived CD21 ligand on FDCs delivers a crucial signal that dramatically augments the stimulation delivered by the binding of antigen to the B-cell receptor (BCR). The engagement of Fc gamma receptor IIB (FcgammaRIIB) by the Ig crystallizable fragment (Fc) in antigen-antibody complexes held on FDCs decreases the activation of immunoreceptor tyrosine-based inhibition motifs (ITIMs), mediated by the crosslinking of BCR and FcgammaRIIB. Thus, FDCs minimize a negative B-cell signal. In short, these ligand-receptor interactions help to signal to B cells and meet a requirement for B-cell stimulation that goes beyond the necessity of T-cell help.     

CD73 expression identifies a subset of IgM+ antigen‐experienced cells with memory attributes that is T cell and CD40 signalling dependent

B‐cell memory was long characterized as isotype‐switched, somatically mutated and germinal centre (GC)‐derived. However, it is now clear that the memory pool is a complex mixture that includes unswitched and unmutated cells. Further, expression of CD73, CD80 and CD273 has allowed the categorization of B‐cell memory into multiple subsets, with combinatorial expression of the markers increasing with GC progression, isotype‐switching and acquisition of somatic mutations. We have extended these findings to determine whether these markers can be used to identify IgM memory phenotypically as arising from T‐dependent versus T‐independent responses. We report that CD73 expression identifies a subset of antigen‐experienced IgM⁺ cells that share attributes of functional B‐cell memory. This subset is reduced in the spleens of T‐cell‐deficient and CD40‐deficient mice and in mixed marrow chimeras made with mutant and wild‐type marrow, the proportion of CD73⁺ IgM memory is restored in the T‐cell‐deficient donor compartment but not in the CD40‐deficient donor compartment, indicating that CD40 ligation is involved in its generation. We also report that CD40 signalling supports optimal expression of CD73 on splenic T cells and age‐associated B cells (ABCs), but not on other immune cells such as neutrophils, marginal zone B cells, peritoneal cavity B‐1 B cells and regulatory T and B cells. Our data indicate that in addition to promoting GC‐associated memory generation during B‐cell differentiation, CD40‐signalling can influence the composition of the unswitched memory B‐cell pool. They also raise the possibility that a fraction of ABCs may represent T‐cell‐dependent IgM memory.     

Regulation of humoral immunity by complement

The complement system of innate immunity is important in regulating humoral immunity largely through the complement receptor CR2, which forms a coreceptor on B cells during antigen-induced activation. However, CR2 also retains antigens on follicular dendritic cells (FDCs). Display of antigen on FDCs is critical for clonal selection and affinity maturation of activated B cells. This review will discuss the role of complement in adaptive immunity in general with a focus on the interplay between CR2-associated antigen on B cells with CR2 expressed on FDCs. This latter interaction provides an opportunity for memory B cells to sample antigen over prolonged periods. The cocrystal structure of CR2 with its ligand C3d provides insight into?how the complement system regulates access of antigen by B cells with implications for therapeutic manipulations to modulate aberrant B cell responses in the case of autoimmunity.     

CD137 is expressed by follicular dendritic cells and costimulates B lymphocyte activation in germinal centers

CD137, a member of the TNF receptor family, and its ligand are expressed on T lymphocytes and antigen-presenting cells (APC), respectively. During interaction with APC, T lymphocytes receive a potent, costimulatory signal through CD137. Reverse signaling has been demonstrated for the CD137 ligand, which causes activation in monocytes. Here we show that B lymphocytes also receive costimulatory signals through the CD137 ligand. Immobilized CD137 augmented proliferation of preactivated B lymphocytes up to fivefold and immunoglobulin synthesis, up to threefold. CD137 had no effect on resting cells. Further, we show that CD137 is expressed in vivo by follicular dendritic cells (FDC) in germinal centers. Germinal centers form during humoral immune responses and are essential for B lymphocyte affinity maturation. These data imply that, similar to the CD40 receptor/ligand system, which mediates T lymphocyte help to B lymphocytes after the first antigen encounter, the CD137 receptor/ligand system may mediate costimulation of B lymphocytes by FDC during affinity maturation.     

T cell activation by anti-CD3 antibodies: function of Fc receptors on B cell blasts, but not resting B cells, and CD18 on the responding T cells

Mouse anti-human CD3 (T3) antibodies can induce T cell proliferation in the presence of Fc receptor (FcR)-bearing accessory cells. Depending on whether the particular antibody can interact with the FcR, it can be mitogenic or otherwise. Previously, some of us (Smith, K. G. C. et al., Eur. J. Immunol. 1986. 16:478) examined human T cell responses to the murine anti-CD3 antibody switch variants UCHT1 (IgG1) and UCHT1B (IgG2b). Using a novel xenogeneic system with mouse macrophages (M phi) and an anti-FcR antibody, 2.4G2, we obtained direct evidence for accessory function of FcR in these responses. However, mouse B cells which also possess FcR were not accessory cells. Here we show that resting B cells do not inhibit anti-CD3 responses in the presence of other accessory cells, and they do not synergize with them. They appear to be inert in these responses but this is not simply because of their radiosensitivity. In contrast, B cell blasts proved to be potent stimulators of responses with UCHT1, UCHT1B and OKT3 (IgG2a). All three responses were inhibited by 2.4G2, whereas we have shown previously that the OKT3 response with M phi was not, in keeping with the known specificities of B cell and M phi FcR. These findings are discussed in relation to the molecular cloning of FcR, and we consider the possibility that distinct FcR could be expressed on resting and activated B cells. A report that anti-CD18 (LFA-1) antibodies blocked the UCHT1 response with human monocytes raised the possibility that this molecule might also be involved in accessory function. However, we show that this inhibition is in fact at the level of the T cell, since anti-human, but not anti-mouse CD18 antibodies, inhibited proliferative responses and clustering with both human and mouse accessory cells. Our results demonstrate that the principal contribution of accessory cells to anti-CD3 responses may be the provision of an FcR, and that CD18 is most probably required at the level of the T cell.     

Antigen targeting reveals splenic CD169+ macrophages as promoters of germinal center B‐cell responses

Ag delivery to specific APCs is an attractive approach in developing strategies for vaccination. CD169⁺ macrophages in the marginal zone of the spleen represent a suitable target for delivery of Ag because of their strategic location, which is optimal for the capture of blood‐borne Ag and their close proximity to B cells and T cells in the white pulp. Here we show that Ag targeting to CD169⁺ macrophages in mice resulted in strong, isotype‐switched, high‐affinity Ab production and the preferential induction and long‐term persistence of Ag‐specific GC B cells and follicular Th cells. In agreement with these observations, CD169⁺ macrophages retained intact Ag, induced cognate activation of B cells, and increased expression of costimulatory molecules upon activation. In addition, macrophages were required for the production of cytokines that promote B‐cell responses. Our results identify CD169⁺ macrophages as promoters of high‐affinity humoral immune responses and emphasize the value of CD169 as target for Ag delivery to improve vaccine responses.     

The role of germinal centers for antiviral B cell responses

Germinal centers (GCs) are crucially involved in T cell-dependent B cell responses. B cells rapidly proliferate within GCs and their Ig variable region genes undergo hypermutation. Cognate T helper cells and antigen presented in native form on follicular dendritic cells (FDCs) select B cells expressing high-affinity Igs, leading to affinity maturation and the generation of memory B cells. In addition to these well-established functions of GCs, this article presents evidence that they also play a crucial role for the maintenance of specific memory Ig titers and for the prevention of viral antibody escape mutants.