Regulated expression of BAFF-binding receptors during human B cell differentiation

BAFF plays a central role in B-lineage cell biology; however, the regulation of BAFF-binding receptor (BBR) expression during B cell activation and differentiation is not completely understood. In this study, we provide a comprehensive ex vivo analysis of BBRs in human B-lineage cells at various stages of maturation, as well as describe the events that drive and regulate receptor expression. Our data reveal that B-lineage cells ranging from naive to plasma cells (PCs), excluding bone marrow PCs, express BAFF-R uniformly. In contrast, only tonsillar memory B cells (MB) and PCs, from both tonsil and bone marrow tissues, express BCMA. Furthermore, we show that TACI is expressed by MB cells and PCs, as well as a subpopulation of activated CD27(neg) B cells. In this regard, we demonstrate that TACI is inducible early upon B cell activation and this is independent of B cell turnover. In addition, we found that TACI expression requires activation of the ERK1/2 pathway, since its expression was blocked by ERK1/2-specific inhibitors. Expression of BAFF-R and B cell maturation Ag (BCMA) is also highly regulated and we demonstrate that BCMA expression is only acquired in MB cells and in a manner accompanied by loss of BAFF-R expression. This inverse expression coincides with MB cell differentiation into Ig-secreting cells (ISC), since blocking differentiation inhibited both induction of BCMA expression and loss of BAFF-R. Collectively, our data suggest that the BBR profile may serve as a footprint of the activation history and stage of differentiation of normal human B cells.     

T cell costimulation by the TNF ligand BAFF.

The TNF ligand family member BAFF (B cell activating factor belonging to the TNF family, also called Blys, TALL-1, zTNF-4, or THANK) is an important survival factor for B cells [corrected]. In this study, we show that BAFF is able to regulate T cell activation. rBAFF induced responses (thymidine incorporation and cytokine secretion) of T cells, suboptimally stimulated through their TCR. BAFF activity was observed on naive, as well as on effector/memory T cells (both CD4+ and CD8+ subsets), indicating that BAFF has a wide function on T cell responses. Analysis of the signal transduced by BAFF into T cells shows that BAFF has no obvious effect on T cell survival upon activation, but is able to deliver a complete costimulation signal into T cells. Indeed, BAFF is sufficient to induce IL-2 secretion and T cell division, when added to an anti-TCR stimulation. This highlights some differences in the BAFF signaling pathway in T and B cells. In conclusion, our results indicate that BAFF may play a role in the development of T cell responses, in addition to its role in B cell homeostasis.     

A division-linked mechanism for the rapid generation of Ig-secreting cells from human memory B cells

Memory B cells, when re-exposed to Ag and T cell help, differentiate into Ig-secreting cells (ISC) at the same time as maintaining a residual pool of non-Ig-secreting cells with memory capabilities. To investigate the mechanism underlying this dual process, we followed the fate of human B cells activated in vitro with the T cell-derived signals CD40 ligand (CD40L), IL-2, and IL-10 using CFSE to monitor cell division. A substantial number of ISCs detected by ELISPOT, intracellular Ig staining, and Ig secretion could be generated from memory but not naive B cells. The proportion of ISCs increased with successive cell divisions and was markedly enhanced by IL-10 at each division. Within ISCs, two distinct populations were detected after withdrawal of CD40L. The first had acquired the plasma cell marker CD38 and continued to proliferate despite the absence of CD40L. In contrast, the second population remained CD38(-), ceased dividing, and underwent rapid apoptosis. The former most likely represent the immediate precursors of long-lived plasma cells, which preferentially home to the bone marrow in vivo, whereas the latter contain short-lived ISCs responsible for the initial Ab response to stimulation with Ag and T cell help. Taken together, the results point to a division-based mechanism responsible not only for regulating differentiation of short- and long-lived ISCs from memory B cells, but for preserving the memory B cell pool for reactivation upon subsequent Ag exposure.     

Cutting edge: germinal centers can be induced in the absence of T cells.

Immunization of mice containing mutations that inactivate the TCR Cbeta and Cdelta genes with the T cell-independent (TI) type 2 Ag (4-hydroxy-3-nitrophenyl)acetyl-Ficoll induces clusters of peanut agglutinin-binding B cells in the spleen. These clusters are histologically indistinguishable from germinal centers (GCs) typical of T cell-dependent immune responses. They are located in follicles, and contain mature follicular dendritic cells, immune complex deposits, and B cells that display the phenotypic qualities of conventional GC B cells. However, the kinetics of this TI GC response differ from T cell-dependent GC responses in being rapidly induced and of short duration. Moreover, the Ab V genes expressed in TI GCs have not undergone somatic hypermutation. Therefore, T cells may be required for B cell differentiation processes associated with the intermediate and latter stages of the GC reaction, but they are dispensable for the induction and initial development of this response.     

The roles of T cell factors in activation, cell cycle progression, and differentiation of human B cells

The relationship of the T cell influences involved in human B cell activation and differentiation into immunoglobulin-secreting cells (ISC) was investigated. T cell supernatants (T supt) generated by stimulating T cells with phytohemagglutinin and phorbol myristate acetate contained activities capable of augmenting DNA synthesis and the growth of mitogen-stimulated B cells and supporting the differentiation of ISC. To examine the role of T supt in B cell activation and the progression through the cell cycle, T cell- and monocyte-depleted B cells were stimulated with formalinized Cowan I strain Staphylococcus aureus (SA), and the percentages of cells in G1, S, and G2 + M were determined by acridine orange staining and analysis. In all experiments, a similar percentage of cells entered G1 during the first 24 to 36 hr of culture when stimulated with SA or SA + T supt. Similar results were seen when B cell activation was analyzed by acquisition of a number of other markers of cell activation. Analysis of cell cycle progression with mithramycin staining of cellular DNA in the presence or absence of vinblastine to arrest mitosis indicated that SA-activated B cells were able to complete S and divide in the absence of T supt. Although an effect of T supt on the progression of B cells through the S phase was evident during the first cell cycle, the major effect only became apparent after the first round of cell division. Although T supt was not necessary for initial B cell activation, T cell influences were absolutely necessary for the differentiation of ISC. T supt did not need to be present during the initial 24 to 36 hr of incubation to permit subsequent generation of ISC. However, when T supt was present initially, an increased number of ISC were produced. Hydroxyurea elimination of cells traversing the G1-S interphase indicated that reception of the differentiation signal occurred before the S phase, but that the generation of ISC required subsequent DNA synthesis and/or cell division. Although precursors of ISC were entirely contained within the population triggered to divide by SA alone, there was no preferential expansion of such precursors as a result of SA stimulation. These results indicate that T cell signals are not absolutely necessary for initial B cell activation and progression through the first cell cycle, although T cell factors promote DNA synthesis by some activated B cells. In contrast, differentiation into ISC is completely dependent on T cell influences.(ABSTRACT TRUNCATED AT 400 WORDS)     

BAFF/APRIL inhibition decreases selection of naive but not antigen-induced autoreactive B cells in murine systemic lupus erythematosus

BAFF inhibition is a new B cell-directed therapeutic strategy for autoimmune disease. Our purpose was to analyze the effect of BAFF/APRIL availability on the naive and Ag-activated B cell repertoires in systemic lupus erythematosus, using the autoreactive germline D42 H chain (glD42H) site-directed transgenic NZB/W mouse. In this article, we show that the naive Vκ repertoire in both young and diseased glD42H NZB/W mice is dominated by five L chains that confer no or low-affinity polyreactivity. In contrast, glD42H B cells expressing L chains that confer high-affinity autoreactivity are mostly deleted before the mature B cell stage, but are positively selected and expanded in the germinal centers (GCs) as the mice age. Of these, the most abundant is VκRF (Vκ16-104*01), which is expressed by almost all IgG anti-DNA hybridomas derived from the glD42H mouse. Competition with nonautoreactive B cells or BAFF/APRIL inhibition significantly inhibited selection of glD42H B cells at the late transitional stage, with only subtle effects on the glD42H-associated L chain repertoire. However, glD42H/VκRF-encoded B cells were still vastly overrepresented in the GC, and serum IgG anti-DNA Abs arose with only a slight delay. Thus, although BAFF/APRIL inhibition increases the stringency of negative selection of the naive autoreactive B cell repertoire in NZB/W mice, it does not correct the major breach in B cell tolerance that occurs at the GC checkpoint.     

Identification of murine germinal center B cell subsets defined by the expression of surface isotypes and differentiation antigens

Germinal centers (GCs) are inducible lymphoid microenvironments that support the generation of memory B cells, affinity maturation, and isotype switching. Previously, phenotypic transitions following in vivo B cell activation have been exploited to discriminate GC from non-GC B cells in the mouse and to delineate as many as seven distinct human peripheral B cell subsets. To better understand the differentiative processes occurring within murine GCs, we sought to identify subpopulations of GC B cells corresponding to discrete stages of GC B cell ontogeny. We performed multiparameter flow-cytometric analyses of GC B cells at consecutive time points following immunization of BALB/c mice with SRBC. We resolved the murine GC compartment into subsets based on the differential expression of activation markers, surface Ig isotypes, and differentiation Ags. Class-switched and nonswitched GC B cells emerged contemporaneously, and their relative frequencies remained nearly constant throughout the GC reaction, perhaps reflecting the establishment of a steady state. A significant percentage of the nonswitched B cells with a GC phenotype exhibited surface markers associated with naive B cells, including CD23, surface IgD, and high levels of CD38 consistent with either prolonged recruitment into the GC reaction or protracted expression of these markers during differentiation within the GC. Expression of the activation marker BLA-1 was dynamic over time, with all GC B cells being positive early after immunization, followed by progressive loss as the GC reaction matured into the second and third week. Implications of these results concerning GC evolution are discussed.     

The dendritic cell-specific CC-chemokine DC-CK1 is expressed by germinal center dendritic cells and attracts CD38-negative mantle zone B lymphocytes

DC-CK1 (CCL18) is a dendritic cell (DC)-specific chemokine expressed in both T and B cell areas of secondary lymphoid organs that preferentially attracts CD45RA(+) T cells. In this study, we further explored the nature of DC-CK1 expressing cells in germinal centers (GCs) of secondary lymphoid organs using a newly developed anti-DC-CK1 mAb. Immunohistochemical analysis demonstrated a remarkable difference in the number of DC-CK1 expressing cells in adjacent GCs within one tonsil, implicating that the expression of DC-CK1 in GCs depends on the activation and/or progression stage of the GC reaction. Using immunohistology and RNA analysis, we demonstrated that GCDC are the source of DC-CK1 production in the GCs. Considering the recently described function of GCDC in (naive) B cell proliferation, isotype switching and Ab production, we investigated the ability of DC-CK1 to attract B lymphocytes. Here we demonstrate that DC-CK1 is a pertussis toxin-dependent chemoattractant for B lymphocytes with a preference in attracting mantle zone (CD38(-)) B cells. The findings that GCDC produce DC-CK1 and attract mantle zone B cells support a key role for GCDC in the development of GCs and memory B cell formation.     

Cytokine-mediated regulation of human B cell differentiation into Ig-secreting cells: predominant role of IL-21 produced by CXCR5+ T follicular helper cells

Differentiation of B cells into Ig-secreting cells (ISC) is critical for the generation of protective humoral immune responses. Because of the important role played by secreted Ig in host protection against infection, it is necessary to identify molecules that control B cell differentiation. Recently, IL-21 was reported to generate ISC from activated human B cells. In this study, we examined the effects of IL-21 on the differentiation of all human mature B cell subsets--neonatal, transitional, naive, germinal center, IgM-memory, and isotype-switched memory cells--into ISC and compared its efficacy to that of IL-10, a well-known mediator of human B cell differentiation. IL-21 rapidly induced the generation of ISC and the secretion of vast quantities IgM, IgG and IgA from all of these B cell subsets. Its effect exceeded that of IL-10 by up to 100-fold, highlighting the potency of IL-21 as a B cell differentiation factor. Strikingly, IL-4 suppressed the stimulatory effects of IL-21 on naive B cells by reducing the expression of B-lymphocyte induced maturation protein-1 (Blimp-1). In contrast, memory B cells were resistant to the inhibitory effects of IL-4. Finally, the ability of human tonsillar CD4+CXCR5+CCR7- T follicular helper (TFH) cells, known to be a rich source of IL-21, to induce the differentiation of autologous B cells into ISC was mediated by the production of IL-21. These findings suggest that IL-21 produced by TFH cells during the primary as well as the subsequent responses to T cell-dependent Ag makes a major contribution to eliciting and maintaining long-lived humoral immunity.     

TACI, an enigmatic BAFF/APRIL receptor, with new unappreciated biochemical and biological properties

BAFF is a B cell survival factor that binds to three receptors BAFF-R, TACI and BCMA. BAFF-R is the receptor triggering na?ve B cell survival and maturation while BCMA supports the survival of plasma cells in the bone marrow. Excessive BAFF production leads to autoimmunity, presumably as the consequence of inappropriate survival of self-reactive B cells. The function of TACI has been more elusive with TACI(-/-) mice revealing two sides of this receptor, a positive one driving T cell-independent immune responses and a negative one down-regulating B cell activation and expansion. Recent work has revealed that the regulation of TACI expression is intimately linked to the activation of innate receptors on B cells and that TACI signalling in response to multimeric BAFF and APRIL provides positive signals to plasmablasts. How TACI negatively regulates B cells remains elusive but may involve an indirect control of BAFF levels. The discovery of TACI mutations associated with common variable immunodeficiency (CVID) in humans not only reinforces its important role for humoral responses but also suggests a more complex role than first anticipated from knockout animals. TACI is emerging as an unusual TNF receptor-like molecule with a sophisticated mode of action.     

LKB1 inhibition of NF‐κB in B cells prevents T follicular helper cell differentiation and germinal center formation

T-cell-dependent antigenic stimulation drives the differentiation of B cells into antibody-secreting plasma cells and memory B cells, but how B cells regulate this process is unclear. We show that LKB1 expression in B cells maintains B-cell quiescence and prevents the premature formation of germinal centers (GCs). Lkb1-deficient B cells (BKO) undergo spontaneous B-cell activation and secretion of multiple inflammatory cytokines, which leads to splenomegaly caused by an unexpected expansion of T cells. Within this cytokine response, increased IL-6 production results from heightened activation of NF-κB, which is suppressed by active LKB1. Secreted IL-6 drives T-cell activation and IL-21 production, promoting T follicular helper (T_FH) cell differentiation and expansion to support a ∽100-fold increase in steady-state GC B cells. Blockade of IL-6 secretion by BKO B cells inhibits IL-21 expression, a known inducer of T_FH-cell differentiation and expansion. Together, these data reveal cell intrinsic and surprising cell extrinsic roles for LKB1 in B cells that control T_FH-cell differentiation and GC formation, and place LKB1 as a central regulator of T-cell-dependent humoral immunity.     

Long-lived bone marrow plasma cells are induced early in response to T cell-independent or T cell-dependent antigens

The signals required to generate long-lived plasma cells remain unresolved. One widely cited model posits that long-lived plasma cells derive from germinal centers (GCs) in response to T cell-dependent (TD) Ags. Thus, T cell-independent (TI) Ags, which fail to sustain GCs, are considered ineffective at generating long-lived plasma cells. However, we show that long-lived hapten-specific plasma cells are readily induced without formation of GCs. Long-lived plasma cells developed in T cell-deficient mice after a single immunization with haptenated LPS, a widely used TI Ag. Long-lived plasma cells also formed in response to TD Ag when the GC response was experimentally prevented. These observations establish that long-lived plasma cells are induced in both TI and TD responses, and can arise independently of B cell maturation in GCs.     

The function of BAFF on T helper cells in autoimmunity

B cell-activating factor belonging to the TNF family (BAFF) exerts its pathogenic role in supporting the survival and proliferation of B cells, regulating class switch recombination as well as the selection of autoreactive B cells. Overexpression of BAFF induces a dramatic expansion of activated B cells, particularly marginal zone B cells, as well as hypergammaglobulinemia, autoantibody production and immune complex deposition. However, in addition to its effect on B cells, recent work has also demonstrated that BAFF can promote T cell activation, proliferation and differentiation. In this review, we have discussed the recent progress on the function and role of BAFF on T cells and T cell-mediated diseases.     

Dexamethasone counteracts the immunostimulatory effects of triiodothyronine (T3) on dendritic cells

Glucocorticoids (GCs) are widely used as anti-inflammatory and immunosuppressive agents. Several studies have indicated the important role of dendritic cells (DCs), highly specialized antigen-presenting and immunomodulatory cells, in GC-mediated suppression of adaptive immune responses. Recently, we demonstrated that triiodothyronine (T3) has potent immunostimulatory effects on bone marrow-derived mouse DCs through a mechanism involving T3 binding to cytosolic thyroid hormone receptor (TR) β1, rapid and sustained Akt activation and IL-12 production. Here we explored the impact of GCs on T3-mediated DC maturation and function and the intracellular events underlying these effects. Dexamethasone (Dex), a synthetic GC, potently inhibited T3-induced stimulation of DCs by preventing the augmented expression of maturation markers and the enhanced IL-12 secretion through mechanisms involving the GC receptor. These effects were accompanied by increased IL-10 levels following exposure of T3-conditioned DCs to Dex. Accordingly, Dex inhibited the immunostimulatory capacity of T3-matured DCs on naive T-cell proliferation and IFN-γ production while increased IL-10 synthesis by allogeneic T cell cultures. A mechanistic analysis revealed the ability of Dex to dampen T3 responses through modulation of Akt phosphorylation and cytoplasmic-nuclear shuttling of nuclear factor-κB (NF-κB). In addition, Dex decreased TRβ1 expression in both immature and T3-maturated DCs through mechanisms involving the GC receptor. Thus GCs, which are increased during the resolution of inflammatory responses, counteract the immunostimulatory effects of T3 on DCs and their ability to polarize adaptive immune responses toward a T helper (Th)-1-type through mechanisms involving, at least in part, NF-κB- and TRβ1-dependent pathways. Our data provide an alternative mechanism for the anti-inflammatory effects of GCs with critical implications in immunopathology at the cross-roads of the immune-endocrine circuits.     

Cutting edge: germinal centers formed in the absence of B cell-activating factor belonging to the TNF family exhibit impaired maturation and function

Germinal centers (GCs) form in B cell follicles and require specific signals for development and maintenance. B cell-activating factor belonging to the TNF family (BAFF) is a fundamental B cell survival factor and therefore may influence GC reactions and subsequent Ab responses. To test this possibility, the effect of BAFF neutralization in immunized mice was assessed. Using B cell maturation Ag-Fc, we demonstrate that BAFF blockade does not inhibit GC formation or somatic hypermutation. However, GCs in B cell maturation Ag-Fc-treated mice dissipated more rapidly than those of control mice and did not form a mature follicular dendritic cell reticulum. Examination of immunized BAFF-null mice validated the BAFF-independent nature of GC formation. Furthermore, Ab responses, including high-affinity responses, were attenuated. This is the first evidence that BAFF is required for maintenance, but not initiation, of the GC reaction, and it further hints that somatic hypermutation within the GC and selection of Ag-specific high-affinity Ab could be uncoupled.     

Germinal center helper T cells are dual functional regulatory cells with suppressive activity to conventional CD4+ T cells

Germinal center (GC) reaction is a T cell-dependent process in which activated B cells mature to produce high-affinity Abs and differentiate into memory B cells. The GC microenvironment is almost exclusively reserved for the optimal Ag-specific B cell clonal expansion, selection, and maturation, but lack significant conventional CD4(+) T cell responses. The mechanisms that ensure such a focused B cell response in the GC are not known. In this study, we report that human CD4(+)CD57(+) T cells, which are the major helper T cells in GCs, actively suppress the activation of conventional CD4(+) T cells, particularly Th1 cells, via a direct contact-dependent mechanism and soluble mediators. Our findings demonstrate that GC T cells are unique regulatory cells that provide critical help signals for B cell response but suppress conventional effector T cells in the same local environment.     

Human IgM+CD27+ B cells: memory B cells or "memory" B cells?

Memory B cells are generated in germinal centers (GC) and contribute to serological immunity by rapidly differentiating into plasma cells. Human memory B cells can be identified by the expression of CD27. These cells exhibit more rapid responses than naive (CD27-) B cells following stimulation in vitro, consistent with the heightened kinetics of secondary responses in vivo. CD27+ B cells express mutated Ig V region genes; however a significant proportion continue to express IgM, suggesting the existence of IgM+ memory B cells. The observation that mutated IgM+CD27+ B cells are generated in humans who cannot form GC led to the conclusions that these cells are generated independently of GC and thus are not memory cells and that they mediate responses to T cell-independent Ag. Although some studies support the idea that IgM+CD27+ B cells participate in T cell-independent responses, many others do not. In this review we will provide alternate interpretations of the biology of IgM+CD27+ B cells and propose that they are indeed memory cells.     

Notch-Mediated Suppression of TSC2 Expression Regulates Cell Differentiation in the Drosophila Intestinal Stem Cell Lineage

Epithelial homeostasis in the posterior midgut of Drosophila is maintained by multipotent intestinal stem cells (ISCs). ISCs self-renew and produce enteroblasts (EBs) that differentiate into either enterocytes (ECs) or enteroendocrine cells (EEs) in response to differential Notch (N) activation. Various environmental and growth signals dynamically regulate ISC activity, but their integration with differentiation cues in the ISC lineage remains unclear. Here we identify Notch-mediated repression of Tuberous Sclerosis Complex 2 (TSC2) in EBs as a required step in the commitment of EBs into the EC fate. The TSC1/2 complex inhibits TOR signaling, acting as a tumor suppressor in vertebrates and regulating cell growth. We find that TSC2 is expressed highly in ISCs, where it maintains stem cell identity, and that N-mediated repression of TSC2 in EBs is required and sufficient to promote EC differentiation. Regulation of TSC/TOR activity by N signaling thus emerges as critical for maintenance and differentiation in somatic stem cell lineages.