Analysis of the individual contributions of Igalpha (CD79a)- and Igbeta (CD79b)-mediated tonic signaling for bone marrow B cell development and peripheral B cell maturation

The individual contribution of Igalpha and Igbeta for BCR-triggered fates is unclear. Prior evidence supports conflicting ideas concerning unique as well as redundant functions for these proteins in the context of BCR/pre-BCR signaling. Part of this ambiguity may reflect the recent appreciation that Igalpha and Igbeta participate in both Ag-independent (tonic) and Ag-dependent signaling. The present study undertook defining the individual requirement for Igalpha and Igbeta under conditions where only ligand-independent tonic signaling was operative. In this regard, we have constructed chimeric proteins containing one or two copies of the cytoplasmic domains of either Igalpha or Igbeta and Igalpha/Igbeta heterodimers with targeted Tyr-->Phe modifications. The ability of these proteins to act as surrogate receptors and trigger early bone marrow and peripheral B cell maturation was tested in RAG2(-/-) primary pro-B cell lines and in gene transfer experiments in the muMT mouse model. We considered that the threshold for a functional activity mediated by the pre-BCR/BCR might only be reached when two functional copies of the Igalpha/Igbeta ITAM domain are expressed together, and therefore the specificity conferred by these proteins can only be observed in these conditions. We found that the ligand-independent tonic signal is sufficient to drive development into mature follicular B cells and both Igalpha and Igbeta chains supported formation of this population. In contrast, neither marginal zone nor B1 mature B cell subsets develop from bone marrow precursors under conditions where only tonic signals are generated.     

Ig-independent Ig beta expression on the surface of B lymphocytes after B cell receptor aggregation

In order for humoral immune responses to develop, B cells must be able to recognize, bind, and internalize Ags. These functions are performed by the BCR, which is also responsible for initiating and transducing activation signals necessary for B cell proliferation and differentiation. We have examined surface expression patterns of individual components of the BCR following anti-Ig- and Ag-induced aggregation. Specifically, the localization and expression levels of the Ag-binding component, surface Ig (sIg), and the Igbeta component of the Igalpha/Igbeta signaling unit were investigated to determine their individual participation in the internalization and signal transduction. Using primary murine B cells, we found that while >95% of the sIg is internalized following anti-Ig-induced aggregation, 20-30% of Igbeta remains on the surface. These results suggest that sIg and Igbeta may function independently following the initial stages of signal transduction.     

Ig alpha/Ig beta complexes generate signals for B cell development independent of selective plasma membrane compartmentalization

Ligand-induced BCR association with detergent-resistant plasma membrane compartments (lipid rafts) has been argued to be essential for initiating and/or sustaining Igalpha/Igbeta-dependent BCR signaling. Because a fraction of the BCR and an even larger fraction of the preBCR associates with lipid rafts in the apparent absence of ligand stimulation, it has been proposed that raft-associated receptor complexes mediate the ligand-independent basal signaling events observed in resting B lineage cells. However, there is no direct evidence that localization of Igalpha/Igbeta-containing complexes to detergent-resistant membrane compartments is absolutely required for the signaling events that drive B cell development. To address these issues we have designed surrogate preBCR/Igalpha/Igbeta complexes that are incapable of ligand-induced aggregation and that are preferentially targeted to either raft or nonraft compartments. An analysis of their ability to promote the preBCR-dependent proB-->preB cell transition of murine B cell progenitors revealed that expression of these surrogate receptor complexes at levels that approximate that of the conventional preBCR can drive B cell development in a manner independent of both aggregation and lipid raft localization.     

Phosphoinositide 3-kinase activation by Igbeta controls de novo formation of an antigen-processing compartment

Antigens that bind B cell antigen receptor (BCR) are preferentially and rapidly processed for antigen presentation. The BCR is a multimeric complex containing a signaling module composed of Igalpha and Igbeta. Signaling pathways implicated in antigen presentation through the BCR are ill defined. Here we demonstrate that phosphoinositide 3-kinase (PI3K) inhibitors preclude antigen presentation induced by BCR or Igbeta but not Igalpha. Unraveling the mechanisms responsible for this inhibition, we show that PI3K inhibitors block neither antigen internalization nor degradation. Rather PI3K inhibitors block de novo formation of a multivesicular antigen processing compartment, which is induced by triggering of the BCR or Igbeta. Strikingly, we found using fluorescent probes binding specifically to PI3K products that BCR and Igbeta but not Igalpha induce PI3K activation in endocytic compartments wherein antigen is transported. Altogether, these results strongly suggest that Igbeta couples the BCR to PI3K activation that is instrumental for de novo formation of the antigen processing compartment and efficient antigen presentation.     

Dual requirement for the Ig alpha immunoreceptor tyrosine-based activation motif (ITAM) and a conserved non-Ig alpha ITAM tyrosine in supporting Ig alpha beta-mediated B cell development

Surface Ig (sIg) expression is a critical checkpoint during avian B cell development. Only cells that express sIg colonize bursal follicles, clonally expand, and undergo Ig diversification by gene conversion. Expression of a heterodimer, in which the extracellular and transmembrane domains of murine CD8alpha or CD8beta are fused to the cytoplasmic domains of chicken Igalpha (chIgalpha) or Igbeta, respectively (murine CD8alpha (mCD8alpha):chIgalpha + mCD8beta:chIgbeta), or an mCD8alpha:chIgalpha homodimer supported bursal B cell development as efficiently as endogenous sIg. In this study we demonstrate that B cell development, in the absence of chIgbeta, requires both the Igalpha ITAM and a conserved non-ITAM Igalpha tyrosine (Y3) that has been associated with binding to B cell linker protein (BLNK). When associated with the cytoplasmic domain of Igbeta, the Igalpha ITAM is not required for the induction of strong calcium mobilization or BLNK phosphorylation, but is still necessary to support B cell development. In contrast, mutation of the Igalpha Y3 severely compromised calcium mobilization when expressed as either a homodimer or a heterodimer with the cytoplasmic domain of Igbeta. However, coexpression of the cytoplasmic domain of Igbeta partially complemented the Igalpha Y3 mutation, rescuing higher levels of BLNK phosphorylation and, more strikingly, supporting B cell development.     

The cytoplasmic domain of Ig alpha is necessary and sufficient to support efficient early B cell development

The B cell receptor complex (BcR) is essential for normal B lymphocyte function, and surface BcR expression is a crucial checkpoint in B cell development. However, functional requirements for chains of the BcR during development remain controversial. We have used retroviral gene transfer to introduce components of the BcR into chicken B cell precursors during embryonic development. A chimeric heterodimer, in which the cytoplasmic domains of chicken Igalpha and Igbeta are expressed by fusion with the extracellular and transmembrane domains of murine CD8alpha and CD8beta, respectively, targeted the cytoplasmic domains of the BcR to the cell surface in the absence of extracellular BcR domains. Expression of this chimeric heterodimer supported all early stages of embryo B cell development: bursal colonization, clonal expansion, and induction of repertoire diversification by gene conversion. Expression of the cytoplasmic domain of Igalpha, in the absence of the cytoplasmic domain of Igbeta, was not only necessary, but sufficient to support B cell development as efficiently as the endogenous BcR. In contrast, expression of the cytoplasmic domain of Igbeta in the absence of the cytoplasmic domain of Igalpha failed to support B cell development. The ability of the cytoplasmic domain of Igalpha to support early B cell development required a functional Igalpha immunoreceptor tyrosine-based activation motif. These results support a model in which expression of surface IgM following productive V(D)J recombination in developing B cell precursors serves to chaperone the cytoplasmic domain of Igalpha to the B cell surface, thereby initiating subsequent stages of development.     

Membrane-targeted peptides derived from Igalpha attenuate B-cell antigen receptor function

Within the B-cell antigen receptor (BCR), heterodimers of Igalpha/Igbeta couple the receptor to intracellular signaling pathways. In the resting state, Igalpha associates with Src-family tyrosine kinases (SFTKs) which contain some basal activity. Upon engagement of the receptor, the SFTKs phosphorylate tyrosine residues in the BCR that recruit and activate the tyrosine kinase Syk, initiating signaling pathways. To test the hypothesis that disrupting the association between the resting receptor and the SFTKs would attenuate both basal and induced receptor activities, we expressed non-phosphorylatable membrane-targeted analogs of Igalpha (Igalpha/M) or Igbeta (Igbeta/M) in B lymphocytes. Both Igalpha/M and Igbeta/M inhibited BCR-induced calcium mobilization, but only Igalpha/M was able to diminish tyrosine phosphorylation. In an immature B-cell line, Igalpha/M attenuated both receptor-induced and basal apoptosis. Taken together, these data demonstrate the importance of the resting receptor complex and suggest therapeutic strategies for regulating receptor-mediated functions.     

B cells

B cells are an important component of adaptive immunity. They produce and secrete millions of different antibody molecules, each of which recognizes a different (foreign) antigen. The fact that humans express a very large repertoire of antibodies is due to the complex mechanism of V(D)J recombination of immunoglobulin (Ig) genes as well as other processes including somatic hypermutation, gene conversion and class switching. The B cell receptor (BCR) is an integral membrane protein complex that is composed of two Ig heavy chains, two Ig light chains and two heterodimers of Igalpha and Igbeta. To eliminate foreign antigens, B cells cooperate with other cells of the immune system including macrophages, dendritic cells and T cells. B cell development is a tightly controlled process in which over 75% of the developing cells become apoptotic because of inappropriate immunoglobulin gene rearrangements or recognition of self antigens by Igs. Hence, the majority of B cell-associated disorders are caused by the incorrect function of genes/proteins involved in B cell development.     

MHC class II antigen processing in B cells: accelerated intracellular targeting of antigens.

Processing and presentation by Ag-specific B cells is initiated by Ag binding to the B cell Ag receptor (BCR). Cross-linking of the BCR by Ag results in a rapid targeting of the BCR and bound Ag to the MHC class II peptide loading compartment (IIPLC). This accelerated delivery of Ag may be essential in vivo during periods of rapid Ag-driven B cell expansion and T cell-dependent selection. Here, we use both immunoelectron microscopy and a nondisruptive protein chemical polymerization method to define the intracellular pathway of the targeting of Ags by the BCR. We show that following cross-linking, the BCR is rapidly transported through transferrin receptor-containing early endosomes to a LAMP-1+, beta-hexosaminadase+, multivesicular compartment that is an active site of peptide-class II complex assembly, containing both class II-invariant chain complexes in the process of invariant chain proteolytic removal as well as mature peptide-class II complexes. The BCR enters the class II-containing compartment as an intact mIg/Igalpha/Igbeta complex bound to Ag. The pathway by which the BCR targets Ag to the IIPLC appears not to be identical to that by which Ags taken up by fluid phase pinocytosis traffick, suggesting that the accelerated BCR pathway may be specialized and potentially independently regulated.     

Basal Igalpha/Igbeta signals trigger the coordinated initiation of pre-B cell antigen receptor-dependent processes

The pro-B to pre-B transition during B cell development is dependent upon surface expression of a signaling competent pre-B cell Ag receptor (pre-BCR). Although the mature form of the BCR requires ligand-induced aggregation to trigger responses, the requirement for ligand-induced pre-BCR aggregation in promoting B cell development remains a matter of significant debate. In this study, we used transmission electron microscopy on murine primary pro-B cells and pre-B cells to analyze the aggregation state of the pre-BCR. Although aggregation can be induced and visualized following cross-linking by Abs to the pre-BCR complex, our analyses indicate that the pre-BCR is expressed on the surface of resting cells primarily in a nonaggregated state. To evaluate the degree to which basal signals mediated through nonaggregated pre-BCR complexes can promote pre-BCR-dependent processes, we used a surrogate pre-BCR consisting of the cytoplasmic regions of Igalpha/Igbeta that is targeted to the inner leaflet of the plasma membrane of primary pro-B cells. We observed enhanced proliferation in the presence of low IL-7, suppression of V(H)(D)J(H) recombination, and induced kappa light (L) chain recombination and cytoplasmic kappa L chain protein expression. Interestingly, Igalpha/Igbeta-mediated allelic exclusion was restricted to the B cell lineage as we observed normal TCRalphabeta expression on CD8-expressing splenocytes. This study directly demonstrates that basal signaling initiated through Igalpha/Igbeta-containing complexes facilitates the coordinated control of differentiation events that are associated with the pre-BCR-dependent transition through the pro-B to pre-B checkpoint. Furthermore, these results argue that pre-BCR aggregation is not a requirement for pre-BCR function.     

Receptor-facilitated antigen presentation requires the recruitment of B cell linker protein to Igalpha

Ags that cross-link the B cell Ag receptor are preferentially and rapidly delivered to the MHC class II-enriched compartment for processing into peptides and subsequent loading onto MHC class II. Proper sorting of Ag/receptor complexes requires the recruitment of Syk to the phosphorylated immunoreceptor tyrosine-based activation motif tyrosines of the B cell Ag receptor constituent Igalpha. We postulated that the Igalpha nonimmunoreceptor tyrosine-based activation motif tyrosines, Y(176) and Y(204), contributed to receptor trafficking. Igalpha(YDeltaF(176,204))/Igbeta receptors were targeted to late endosomes, but were excluded from the vesicle lumen and could not facilitate the presentation of Ag to T cells. Subsequent analysis demonstrated that phosphorylation of Y(176)/Y(204) recruited the B cell linker protein, Vav, and Grb2. Reconstitution of Igalpha(YDeltaF(176,204))/Igbeta with the B cell linker protein rescued both receptor-facilitated Ag presentation and entry into the MHC class II-enriched compartment. Thus, aggregation accelerates receptor trafficking by recruiting two separate signaling modules required for transit through sequential checkpoints.     

Membrane IgE binds and activates Fc epsilon RI in an antigen-independent manner

Interaction of secretory IgE with FcepsilonRI is the prerequisite for allergen-driven cellular responses, fundamental events in immediate and chronic allergic manifestations. Previous studies reported the binding of soluble FcepsilonRIalpha to membrane IgE exposed on B cells. In this study, the functional interaction between human membrane IgE and human FcepsilonRI is presented. Four different IgE versions were expressed in mouse B cell lines, namely: a truncation at the Cepsilon2-Cepsilon3 junction of membrane IgE isoform long, membrane IgE isoform long (without Igalpha/Igbeta BCR accessory proteins), and both epsilonBCRs (containing membrane IgE isoforms short and long). All membrane IgE versions activated a rat basophilic leukemia cell line transfected with human FcepsilonRI, as detected by measuring the release of both preformed and newly synthesized mediators. The interaction led also to Ca(2+) responses in the basophil cell line, while membrane IgE-FcepsilonRI complexes were detected by immunoprecipitation. FcepsilonRI activation by membrane IgE occurs in an Ag-independent manner. Noteworthily, human peripheral blood basophils and monocytes also were activated upon contact with cells bearing membrane IgE. In humans, the presence of FcepsilonRI in several cellular entities suggests a possible membrane IgE-FcepsilonRI-driven cell-cell dialogue, with likely implications for IgE homeostasis in physiology and pathology.     

Surrogate light chain-mediated interaction of a soluble pre-B cell receptor with adherent cell lines.

Signals initiated by the precursor B cell receptor (pre-BCR) are critical for B cell progenitors to mature into precursor B cells. The pre-BCR consists of a homodimer of microH chains, the covalently associated surrogate L (SL) chain composed of VpreB and lambda5, and the transmembrane signal molecules Ig(alpha) and Igbeta. One way to explain how maturation signals are initiated in late progenitor B cells is that the pre-BCR is transported to the cell surface and interacts from there with a ligand on stroma cells. To address this hypothesis, we first produced soluble Fab-like pre-BCR and BCR fragments, as well as SL chain, in baculovirus-infected insect cells. Flow cytometry revealed that, in contrast to Fab-like BCR fragments, the soluble pre-BCR binds to the surface of stroma and several other adherent cell lines, but not to B and T lymphoid suspension cells. The specific binding of the soluble pre-BCR to stroma cells is saturable, sensitive to trypsin digestion, and not dependent on bivalent cations. The binding of pre-BCR seems to be independent of the H chain of IgM (microH chain), because SL chain alone was able to interact with stroma cells. Finally, soluble pre-BCR specifically precipitated a 135-kDa protein from ST2 cells. These findings not only demonstrate for the first time the capacity of a pre-BCR to specifically bind to a structure on the surface of adherent cells, but also suggest that the pre-BCR interacts via its SL chain with a putative ligand on stroma cells.     

Homooligomerization of the cytoplasmic domain of the T cell receptor zeta chain and of other proteins containing the immunoreceptor tyrosine-based activation motif

Antigen receptors on T cells, B cells, mast cells, and basophils all have cytoplasmic domains containing one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor engagement in an early and obligatory event in the signaling cascade. How clustering of receptor extracellular domains leads to phosphorylation of cytoplasmic domain ITAMs is not known, and little structural or biochemical information is available for the ITAM-containing cytoplasmic domains. Here we investigate the conformation and oligomeric state of several immune receptor cytoplasmic domains, using purified recombinant proteins and a variety of biophysical and biochemical techniques. We show that all of the cytoplasmic domains of ITAM-containing signaling subunits studied are oligomeric in solution, namely, T cell antigen receptor zeta, CD3epsilon, CD3delta, and CD3gamma, B cell antigen receptor Igalpha and Igbeta, and Fc receptor FcepsilonRIgamma. For zeta(cyt), the oligomerization behavior is best described by a two-step monomer-dimer-tetramer fast dynamic equilibrium with dissociation constants in the order of approximately 10 microM (monomer-dimer) and approximately 1 mM (dimer-tetramer). In contrast to the other ITAM-containing proteins, Igalpha(cyt) forms stable dimers and tetramers even below 10 microM. Circular dichroic analysis reveals the lack of stable ordered structure of the cytoplasmic domains studied, and oligomerization does not change the random-coil-like conformation observed. The random-coil nature of zeta(cyt) was also confirmed by heteronuclear NMR. Phosphorylation of zeta(cyt) and FcepsilonRIgamma(cyt) does not significantly alter their oligomerization behavior. The implications of these results for transmembrane signaling and cellular activation by immune receptors are discussed.     

Spi-1 and Spi-B control the expression of the Grap2 gene in B cells

The Ets family members Spi-1 and Spi-B have been implicated in the regulation of genes important for B cell antigen receptor (BCR) signaling. Mice deficient in Spi-B exhibit reduced B cell proliferation in response to BCR cross-linking and impaired T cell-dependent immune responses. This defect is exacerbated in the presence of Spi-1 haplo-insufficiency (Spi1+/- SpiB-/-). Tyrosine phosphorylation and calcium mobilization induced by BCR engagement is diminished in Spi1+/- SpiB-/- B lymphocytes, although many key BCR signaling proteins are expressed, suggesting that Spi-1 and Spi-B regulate expression of additional, unidentified signaling molecules. We now demonstrate that expression of the adaptor protein Grap2 is impaired in Spi1+/- SpiB+/- and Spi1+/- SpiB-/- B lymphocytes. Analysis of two alternate murine Grap2 promoters revealed a functionally important Spi-1 and Spi-B DNA binding element located in the downstream promoter. Ectopic expression of Grap2 in Grap2-deficient B cells reduced the recruitment of BLNK to Igalpha and the phosphorylation of specific substrates. Regulation of BLNK recruitment was dependent upon the Grap2 proline-rich domain, while modulation of phosphorylation was dependent upon both the proline-rich and SH2 domains. These data indicate that Spi-1 and Spi-B directly regulate the expression of Grap2 and that Grap2 functions to modulate BCR signaling, but that reduced Grap2 expression is unlikely to account for the BCR signaling defects observed in Spi1+/- SpiB-/- B cells.     

Antinuclear antigen B cells that down-regulate surface B cell receptor during development to mature, follicular phenotype do not display features of anergy in vitro

We previously demonstrated that B cells expressing a transgenic BCR with "dual reactivity" for the hapten arsonate and nuclear autoantigens efficiently complete development to follicular phenotype and stably reside in follicles in vivo. These B cells express very low levels of surface IgM and IgD, suggesting that they avoid central deletion and peripheral anergy by reducing their avidity for autoantigen via surface BCR (sBCR) down-regulation. Since a variety of states of B cell anergy have been previously described, a thorough examination of the functional capabilities of these B cells was required to test this hypothesis. In this study, we show that surface Ig cross-linking induces amounts of proximal BCR signaling in these B cells commensurate with their reduced sBCR levels. Functionally, however, they are comparable to nonautoreactive B cells in cell cycle progression, up-regulation of activation and costimulatory molecules, and Ab-forming cell differentiation when treated with a variety of stimuli in vitro. In addition, these B cells can efficiently process and present Ag and are capable of undergoing cognate interaction with naive TCR-transgenic T cells, resulting in robust IL-2 production. Together, these data reveal a lack of intrinsic anergy involving any known mechanism, supporting the idea that this type of antinuclear Ag B cell becomes indifferent to cognate autoantigen by down-regulating sBCR.     

Cutting edge: constitutive B cell receptor signaling is critical for basal growth of B lymphoma

B lymphomas account for the majority of the lymphoma cases. BCR expression appears to be important for B lymphoma because most oncogenes are translocated to nonrearranged Ig loci and because all of the variants that arise in anti-idiotypic Ab-treated lymphoma patients remain BCR positive. Based on this and the fact that BCR is required for mature B cell survival, we tested the requirement for continued expression of BCR for the growth and survival of B lymphoma cells. Using Igalpha or Igbeta-specific small interfering RNA (siRNA) to inhibit BCR expression, we demonstrate for the first time that constitutive signaling by BCR is critical for survival and proliferation of both murine and human B lymphoma cells. The BCR signals in lymphoma appear to be mediated by Syk, as it is constitutively active in a variety of B lymphoma cells. Blocking Syk activity by selective inhibitors suppresses growth of several murine and human B lymphomas.     

Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development

Signaling through the Ag receptor is required for peripheral B lymphocyte maturation and maintenance. Defects in components of the B cell receptor (BCR) signalosome result in developmental blocks at the transition from immature (heat-stable Ag (HSA)(high)) to mature (HSA(low)) B cells. Recent studies have subdivided the immature, or transitional, splenic B cells into two subsets, transitional 1 (T1) and transitional 2 (T2) cells. T1 and T2 cells express distinct surface markers and are located in distinct anatomic locations. In this report, we evaluated the BCR signaling capacity of T1 and T2 B cell subsets. In response to BCR engagement, T2 cells rapidly entered cell cycle and resisted cell death. In contrast, T1 cells did not proliferate and instead died after BCR stimulation. Correlating with these results, T2 cells robustly induced expression of the cell cycle regulator cyclin D2 and the antiapoptotic factors A1/Bfl-1 and Bcl-x(L) and exhibited activation of Akt. In contrast, T1 cells failed to up-regulate these markers. BCR stimulation of T2 cells also led to down-regulation of CD21 and CD24 (HSA) expression, resulting in a mature B cell phenotype. In addition, T2 cells from Bruton's tyrosine kinase-deficient Xid mice failed to generate these proliferative and survival responses, suggesting a requirement for the BCR signalosome specifically at the T2 stage. Taken together, these data clearly demonstrate that T2 immature B cells comprise a discrete developmental subset that mediates BCR-dependent proliferative, prosurvival, and differentiation signals. Their distinct BCR-dependent responses suggest unique roles for T1 vs T2 cells in peripheral B cell selection.