BAFF receptor signaling aids the differentiation of immature B cells into transitional B cells following tonic BCR signaling.
ABSTRACT: BAFF is an important prosurvival cytokine for mature B cells. However, previous studies have shown that BAFFR is already expressed at the immature B cell stage, and that the prosurvival protein Bcl-2 does not completely complement the B cell defects resulting from the absence of BAFFR or BAFF. Thus, we hypothesized that BAFF also functions to aid the differentiation of nonautoreactive immature B cells into transitional B cells and to promote their positive selection. We found that BAFFR is expressed at higher levels on nonautoreactive than on autoreactive immature B cells and that its expression correlates with that of surface IgM and with tonic BCR signaling. Our data indicate that BAFFR signaling enhances the generation of transitional CD23(-) B cells in vitro by increasing cell survival. In vivo, however, BAFFR signaling is dispensable for the generation of CD23(-) transitional B cells in the bone marrow, but it is important for the development of transitional CD23(-) T1 B cells in the spleen. Additionally, we show that BAFF is essential for the differentiation of CD23(-) into CD23(+) transitional B cells both in vitro and in vivo through a mechanism distinct from that mediating cell survival, but requiring tonic BCR signaling. In summary, our data indicate that BAFFR and tonic BCR signals cooperate to enable nonautoreactive immature B cells to differentiate into transitional B cells and to be positively selected into the naive B cell repertoire.
Project description:B cell receptors (BCRs) generate tonic signals critical for B cell survival and early B cell development. To determine whether these signals also mediate the development of transitional and mature B cells, we examined B cell development using a mouse strain in which nonautoreactive immunoglobulin heavy and light chain-targeted B cells express low surface BCR levels. We found that reduced BCR expression translated into diminished tonic BCR signals that strongly impaired the development of transitional and mature B cells. Constitutive expression of Bcl-2 did not rescue the differentiation of BCR-low B cells, suggesting that this defect was not related to decreased cell survival. In contrast, activation of the Ras pathway rescued the differentiation of BCR-low immature B cells both in vitro and in vivo, whereas extracellular signal-regulated kinase (Erk) inhibition impaired the differentiation of normal immature B cells. These results strongly suggest that tonic BCR signaling mediates the differentiation of immature into transitional and mature B cells via activation of Erk, likely through a pathway requiring Ras.
Project description:The BAFF-receptor (BAFFR) is encoded by the TNFRSF13C gene and is one of the main pro-survival receptors in B cells. Its function is impressively documented in humans by a homozygous deletion within exon 2, which leads to an almost complete block of B cell development at the stage of immature/transitional B cells. The resulting immunodeficiency is characterized by B-lymphopenia, agammaglobulinemia, and impaired humoral immune responses. However, different from mutations affecting pathway components coupled to B cell antigen receptor (BCR) signaling, BAFFR-deficient B cells can still develop into IgA-secreting plasma cells. Therefore, BAFFR deficiency in humans is characterized by very few circulating B cells, very low IgM and IgG serum concentrations but normal or high IgA levels.
Project description:Follicular B cell survival requires signaling from BAFFR, a receptor for BAFF and the B cell antigen receptor (BCR). This "tonic" BCR survival signal is distinct from that induced by antigen binding and may be ligand-independent. We show that inducible inactivation of the Syk tyrosine kinase, a key signal transducer from the BCR following antigen binding, resulted in the death of most follicular B cells because Syk-deficient cells were unable to survive in response to BAFF. Genetic rescue studies demonstrated that Syk transduces BAFFR survival signals via ERK and PI3 kinase. Surprisingly, BAFFR signaling directly induced phosphorylation of both Syk and the BCR-associated Igα signaling subunit, and this Syk phosphorylation required the BCR. We conclude that the BCR and Igα may be required for B cell survival because they function as adaptor proteins in a BAFFR signaling pathway leading to activation of Syk, demonstrating previously unrecognized crosstalk between the two receptors.
Project description:Newly generated bone marrow B cells are positively selected into the peripheral lymphoid tissue only when they express a B cell receptor (BCR) that is nonautoreactive or one that binds self-antigen with only minimal avidity. This positive selection process, moreover, is critically contingent on the ligand-independent tonic signals transduced by the BCR. We have previously shown that when autoreactive B cells express an active form of the rat sarcoma (RAS) oncogene, they upregulate the receptor for the B cell activating factor (BAFFR) and undergo differentiation in vitro and positive selection into the spleen in vivo, overcoming central tolerance. Based on the in vitro use of pharmacologic inhibitors, we further showed that this cell differentiation process is critically dependent on the activation of the mitogen-activated protein kinase kinase pathway MEK (MAPKK)-extracellular signal-regulated kinase (ERK), which is downstream of RAS. Here, we next investigated if activation of ERK is not only necessary but also sufficient to break central B cell tolerance and induce differentiation of autoreactive B cells in vitro and in vivo. Our results demonstrate that activation of ERK is critical for upregulating BAFFR and overcoming suboptimal levels of tonic BCR signals or low amounts of antigen-induced BCR signals during in vitro B cell differentiation. However, direct activation of ERK does not lead high avidity autoreactive B cells to increase BAFFR levels and undergo positive selection and differentiation in vivo. B cell-specific MEK-ERK activation in mice is also unable to lead to autoantibody secretion, and this in spite of a general increase of serum immunoglobulin levels. These findings indicate that additional pathways downstream of RAS are required for high avidity autoreactive B cells to break central and/or peripheral tolerance.
Project description:Memory B cells (MBCs) are long-lived cells that form a critical part of immunological memory, providing rapid antibody responses to recurring infections. However, very little is known about signals controlling MBC survival. Previous work has shown that antigen is not required for MBC survival, but a requirement for the B cell antigen receptor (BCR) has not been tested. Other studies have shown that, unlike naive B cells, MBCs do not express BAFFR and their survival is independent of BAFF, the ligand for BAFFR. Here, using inducible genetic ablation, we show that survival of MBCs is critically dependent on the BCR and on signaling through the associated CD79A protein. Unexpectedly, we found that MBCs express BAFFR and that their survival requires BAFF and BAFFR; hence, loss of BAFF or BAFFR impairs recall responses. Finally, we show that MBC survival requires IKK2, a kinase that transduces BAFFR signals. Thus, MBC survival is critically dependent on signaling from BCR and BAFFR.
Project description:Newly generated immature B cells are selected to enter the peripheral mature B-cell pool only if they do not bind (or bind limited amount of) self-antigen. We previously suggested that this selection relies on basal extracellular signal-regulated kinase (Erk) activation mediated by tonic B-cell antigen receptor (BCR) signaling and that this signal can be replaced by an active rat sarcoma (Ras), which are small GTPase proteins. In this study we compared the activity of Ras and Erk in nonautoreactive and autoreactive immature B cells and investigated whether activation of Ras can break tolerance. Our results demonstrate lower levels of active Erk and Ras in autoreactive immature B cells, although this is evident only when these cells display medium/high avidity for self-antigen. Basal activation of Erk in immature B cells is proportional to surface IgM and dependent on sarcoma family kinases, whereas it is independent of B-cell activating factor, IFN, and Toll-like receptor signaling. Ectopic expression of the constitutively active mutant Ras form N-RasD12 in autoreactive cells raises active Erk, halts receptor editing via PI3 kinase, and promotes differentiation via Erk, breaking central tolerance. Moreover, when B cells coexpress autoreactive and nonautoreactive BCRs, N-RasD12 leads also to a break in peripheral tolerance with the production of autoantibodies. Our findings indicate that in immature B cells, basal activation of Ras and Erk are controlled by tonic BCR signaling, and that positive changes in Ras activity can lead to a break in both central and peripheral B-cell tolerance.
Project description:The capacity of immature B cells of the spleen and bone marrow to differentiate in vitro into cells representing mature end stage cells was investigated using B-cell activating factor belonging to the TNF family (BAFF) and Notch pathway activators. Immature splenic and bone marrow B cells were found, in the presence of both of these activators, to mature into cells with follicular mature (FM) and marginal zone (MZ) cell phenotypes. Such cells were functionally responsive to B-cell-specific activation. The derivation in vitro of cells with an MZ phenotype was more robust from CD23(-) populations than CD23(+) immature/transitional B cells, suggesting a direct immature/T1 B cell to MZ cell differentiation pathway. Transcript analysis of the in vitro-derived B-cell populations demonstrated expression profiles similar to maturing B cells in vivo. FACS-purified populations of B220(+)CD19(+)CD21(-)CD23(-) cells from bone marrow of 2-wk-old mice gave rise to populations of CD21(+)CD23(-) cells with MZ cell phenotypes as well as CD21(+)CD23(+) cells with FM cell phenotypes in percentages similar to those found in vivo. These data suggest that the commitment to an MZ and FM B cell phenotype is set prior to immature B-cell release from the marrow.
Project description:BAFF, APRIL and their receptors regulate the survival, maturation and homeostasis of mature B-cells. Despite the lack of a functional role of BAFF/APRIL system during normal early B-cell development, previous studies indicated a contribution of these molecules in the pathogenesis of B-lineage acute lymphoblastic leukemia (B-ALL). Here, we evaluated the expression of this system in B-ALL and its involvement in spontaneous and drug-induced apoptosis of B-lymphoblasts, taking into consideration the distinct disease subtypes. We found that BAFFR is the most predominant aberrantly expressed receptor in B-ALL and that its expression, along with BCMA and APRIL, positively correlates with the maturation stage of B-lymphoblasts. Moreover, the binding of the E2A-PBX1 chimeric protein to the <i>BAFFR</i> promoter suggests that the transcriptional activator promotes the increase in <i>BAFFR</i> expression observed in about 50% of pre-B-ALL patients carrying the <i>t</i> <sub>(1, 19)</sub> translocation. BAFF binding to BAFFR led to the processing of NF-?B2 p100 in pre-B ALL cells suggesting that BAFFR can activate the NF-?B2 pathway in pre-B ALL cells. Surprisingly, we found that BAFF treatment promotes the cell death of primary BCR-ABL<sup>+</sup> BAFFR<sup>+</sup> pre-B-lymphoblasts in adult B-ALL. It also enhances glucocorticoid-induced apoptosis in the E2A-PBX1<sup>+</sup> pre-B-ALL cell line 697. These data suggest that BAFF/BAFFR signaling in B-ALL cells differs from normal B cells and that it may affect the pathogenesis of the disease.
Project description:The B cell survival cytokine BAFF has been linked with the pathogenesis of systemic lupus erythematosus (SLE). BAFF binds distinct BAFF-family surface receptors, including the BAFF-R and transmembrane activator and CAML interactor (TACI). Although originally characterized as a negative regulator of B cell activation, TACI signals are critical for class-switched autoantibody (autoAb) production in BAFF transgenic mice. Consistent with this finding, a subset of transitional splenic B cells upregulate surface TACI expression and contribute to BAFF-driven autoAb. In the current study, we interrogated the B cell signals required for transitional B cell TACI expression and Ab production. Surprisingly, despite established roles for dual BCR and TLR signals in autoAb production in SLE, signals downstream of these receptors exerted distinct impacts on transitional B cell TACI expression and autoAb titers. Whereas loss of BCR signals prevented transitional B cell TACI expression and resulted in loss of serum autoAb across all Ig isotypes, lack of TLR signals exerted a more limited impact restricted to autoAb class-switch recombination without altering transitional B cell TACI expression. Finally, in parallel with the protective effect of TACI deletion, loss of BAFF-R signaling also protected against BAFF-driven autoimmunity. Together, these findings highlight how multiple signaling pathways integrate to promote class-switched autoAb production by transitional B cells, events that likely impact the pathogenesis of SLE and other BAFF-dependent autoimmune diseases.
Project description:<h4>Background</h4>B cells, key cells in allergic inflammation, differentiate in the bone marrow and their precursors include pro-B, pre-B and immature B cells. Eosinophil progenitor cells increase in the lung after allergen exposure. However, the existence and possible role of B cell precursors in the lung during allergic inflammation remains elusive.<h4>Methods</h4>A BALB/c mouse model of allergic airway inflammation was utilized to perform phenotypic and quantification analyses of pro-B and pre-B cells in the lung by flow cytometry. B cell maturation factors IL-7 and B cell-activating factor (BAFF) and their receptors (CD127 and BAFFR, BCMA, TACI, respectively) were also evaluated in the lung and serum. The effect of anti-BAFF treatment was investigated both in vivo (i.p. administration of BAFF-R-Ig fusion protein) and in vitro (colony forming cell assay). Finally, BAFF levels were examined in the bronchoalveolar lavage (BAL) of asthmatic patients and healthy controls.<h4>Results</h4>Precursor pro and pre-B cells increase in the lung after allergen exposure, proliferate in the lung tissue in vivo, express markers of chemotaxis (CCR10 and CXCR4) and co-stimulation (CD40, CD86) and are resistant to apoptosis (Bax). Precursor B cells express receptors for BAFF at baseline, while after allergen challenge both their ligand BAFF and the BCMA receptor expression increases in B cell precursors. Blocking BAFFR in the lung in vivo decreases eosinophils and proliferating precursor B cells. Blocking BAFFR in bone marrow cultures in vitro reduces pre-B colony formation units. BAFF is increased in the BAL of severe asthmatics.<h4>Conclusion</h4>Our data support the concept of a BAFF-mediated role for B cell precursors in allergic airway inflammation.