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.     

B lymphoblastoid cell lines with rearranged T cell receptor beta-chain genes retain conventional B cell surface antigen and Ig expression

Transformation of peripheral blood lymphocytes by co-incubation with EBV produces B lymphoblastoid cell lines, but rearrangement of TCR beta-chain genes was observed in three different cell lines derived from two individuals. Because rearrangement of TCR genes in B lymphocytes is considered a rare event, these B lymphoblastoid cell lines with rearranged TCR beta-genes were examined in detail to determine whether there were any additional characteristics to distinguish them from B lymphoblastoid cell lines with germ-line TCR beta-genes. All B lymphoblastoid cell lines contained rearranged Ig H and kappa L chain genes, secreted Ig, and expressed B and not T cell surface markers. Cell lines with rearranged TCR beta-genes had rearranged both IgH genes and had rearranged and subsequently deleted both kappa C region genes. Furthermore all three B lymphoblastoid cell lines with rearranged TCR beta-genes produced small amounts of Ig with lambda-L chains. Although the cellular mechanisms maintaining lineage-specific rearrangement events remain unknown, extensive Ig gene rearrangement and inefficient Ig production by B cells may be indicators of a cellular status where normally stringent lineage-specific control elements fail to function efficiently.     

Experience dictates stem cell fate in the adult hippocampus

Adult hippocampal neurogenesis has been implicated in cognitive and emotional processes, as well as in response to antidepressant treatment. However, little is known about how the adult stem cell lineage contributes to hippocampal structure and function and how this process is modulated by the animal's experience. Here we perform an indelible lineage analysis and report that neural stem cells can produce expanding and persisting populations of not only neurons, but also stem cells in the adult hippocampus. Furthermore, the ratio of stem cells to neurons depends on experiences of the animal or the location of the stem cell. Surprisingly, social isolation facilitated accumulation of stem cells, but not neurons. These results show that neural stem cells accumulate in the adult hippocampus and that the stem cell-lineage relationship is under control of anatomic and experiential niches. Our findings suggest that, in the hippocampus, fate specification may act as a form of cellular plasticity for adapting to environmental changes.     

Role of the polymeric Ig receptor in mucosal B cell homeostasis

Secretory IgA (SIgA) is the most characteristic component of the mucosal immune system and has long been considered the major protective factor that prevents pathogens from invading hosts through the mucosae. Recent studies, however, have suggested that complete immunity against a range of mucosal bacterial and viral pathogens can be achieved in the absence of IgA. Therefore, to further dissect the role of SIgA, we generated mice deficient in the polymeric Ig receptor (pIgR(-/-) mice). As a result of an inability to transport dimeric IgA to the secretions, pIgR(-/-) mice are deficient in SIgA and accumulate circulating dimeric IgA, with serum levels 100-fold greater than those observed in normal mice. Examination of lamina propria mononuclear cells showed that pIgR(-/-) mice had approximately 3 times as many IgA-secreting cells as C57BL/6 mice. Further analysis showed that these cells displayed the differentiated IgA(+) B220(-) phenotype and accounted for a 2-fold increase in the number of lamina propria blast cells in the pIgR(-/-) mice. Subsequent experiments showed that OVA-specific CD4(+) T cell expansion following OVA feeding was not elevated in pIgR(-/-) mice. Furthermore, no differences in CD8(+) T cell tolerance or induction of influenza virus-specific CD8(+) T cells were detected in pIgR(-/-) mice compared with controls. Therefore, while SIgA is clearly involved in maintaining some parameters of mucosal homeostasis in the intestine, the mechanisms associated with its barrier function and the clinical consequences of its deficiency are yet to be identified.     

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.     

B cell receptor basal signaling regulates antigen-induced Ig light chain rearrangements

BCR editing in the bone marrow contributes to B cell tolerance by orchestrating secondary Ig rearrangements in self-reactive B cells. We have recently shown that loss of the BCR or a pharmacologic blockade of BCR proximal signaling pathways results in a global "back-differentiation" response in which immature B cells down-regulate genes important for the mature B cell program and up-regulate genes characteristic of earlier stages of B cell development. These observations led us to test the hypothesis that self-Ag-induced down-regulation of the BCR, and not self-Ag-induced positive signals, lead to Rag induction and hence receptor editing. Supporting this hypothesis, we found that immature B cells from xid (x-linked immunodeficiency) mice induce re-expression of a Rag2-GFP bacterial artificial chromosome reporter as well as wild-type immature B cells following Ag incubation. Incubation of immature B cells with self-Ag leads to a striking reversal in differentiation to the pro-/pre-B stage of development, consistent with the idea that back-differentiation results in the reinduction of genes required for L chain rearrangement and receptor editing. Importantly, Rag induction, the back-differentiation response to Ag, and editing in immature and pre-B cells are inhibited by a combination of phorbol ester and calcium ionophore, agents that bypass proximal signaling pathways and mimic BCR signaling. Thus, mimicking positive BCR signals actually inhibits receptor editing. These findings support a model whereby Ag-induced receptor editing is inhibited by BCR basal signaling on developing B cells; BCR down-regulation removes this basal signal, thereby initiating receptor editing.     

Imprinting the fate of antigen-reactive B cells through the affinity of the B cell receptor

Long-lived plasma cells (PCs) and memory B cells (B(mem)) constitute the cellular components of enduring humoral immunity, whereas short-lived PCs that rapidly produce Ig correspond to the host's need for immediate protection against pathogens. In this study we show that the innate affinity of the BCR for Ag imprints upon naive B cells their differentiation fate to become short- or long-lived PCs and B(mem). Using BCR transgenic mice with varying affinities for Ag, naive B cells with high affinity lose their capacity to form germinal centers (GCs), develop neither B(mem) nor long-lived PCs, and are destined to a short-lived PC fate. Moderate affinity interactions result in hastened GC responses, and differentiation to long-lived PCs, but B(mem) remain extinct. In contrast, lower affinity interactions show tempered GCs, producing B(mem) and affinity-matured, long-lived PCs. Thus, a continuum of elementary to comprehensive humoral immune responses exists that is controlled by inherent BCR affinity.     

Ia antigen: a murine B lymphocyte receptor for transformed cell induction of interferon-alpha/beta

A receptor on the surface of nonsensitized mouse spleen cells that recognizes a glycoprotein from transformed mouse L-929 cells is described. The interaction of the receptor and glycoprotein inducer results in the production of MoIFN alpha/beta. An assay was developed to assess certain biologic and physicochemical characteristics of the receptor. The receptor and glycoprotein inducer bound in a concentration-dependent manner, which tends to indicate a direct interaction between the two. The receptor was not ubiquitous; spleen cells but not normal mouse embryo cells appeared to be the source. It was specific for MoIFN alpha/beta inducers from transformed cells, but not from other MoIFN alpha/beta or gamma inducers such as NDV, LPS, PWM, or SEA. The receptor appeared to be a cell surface protein in that its activity was abolished by trypsinization of whole spleen cells. Previous studies indicated that the receptor was probably located on B cells. Gel filtration indicated that the receptor had a m.w. of 30,000 to 60,000. Because the receptor appeared to be: 1) B lymphocyte associated, 2) a surface protein, and 3) 30,000 to 60,000 daltons, a similarity to Ia antigen was suggested. This possibility was confirmed by showing binding of the receptor to an anti-IaK antibody-Sepharose affinity column. PAGE analysis of the affinity-purified receptor revealed a single protein band with a m.w. of approximately 60,000. ELISA of the above gel slices with anti-Ia antibody further confirmed the specificity of the column. A physical association of the receptor and inducer was demonstrated by showing binding of the glycoprotein inducer to a receptor (Ia antigen)-Sepharose affinity column. Furthermore, the receptor (Ia antigen) was highly purified by a glycoprotein inducer-Sepharose affinity column.     

The C-terminus of the kinase-defective neuregulin receptor ErbB-3 confers mitogenic superiority and dictates endocytic routing.

Signaling by the epidermal growth factor (EGF) family and the neuregulin group of ligands is mediated by four ErbB receptor tyrosine kinases, that form homo- and heterodimeric complexes. Paradoxically, the neuregulin receptor ErbB-3 is devoid of catalytic activity, but its heterodimerization with other ErbBs, particularly the ligand-less ErbB-2 oncoprotein of carcinomas, reconstitutes superior mitogenic and transforming activities. To understand the underlying mechanism we constructed a chimeric EGF-receptor (ErbB-1) whose autophosphorylation C-terminal domain was replaced by the corresponding portion of ErbB-3. Consistent with the possibility that this domain recruits a relatively potent signaling pathway(s), the mitogenic signals generated by the recombinant fusion protein were superior to those generated by ErbB-1 homodimers and comparable to the proliferative activity of ErbB-2/ErbB-3 heterodimers. Upon ligand binding, the chimeric receptor recruited an ErbB-3-specific repertoire of signaling proteins, including Shc and the phosphatidylinositol 3-kinase, but excluding the ErbB-1-specific substrate, phospholipase Cgamma1. Unlike ErbB-1, which is destined to lysosomal degradation through a mechanism that includes recruitment of c-Cbl and receptor poly-ubiquitination, the C-terminal tail of ErbB-3 shunted the chimeric protein to the ErbB-3-characteristic recycling pathway. These observations attribute the mitogenic superiority of ErbB-3 to its C-terminal tail and imply that the flanking kinase domain has lost catalytic activity in order to restrain the relatively potent signaling capability of the C-terminus.     

Ig alpha and Ig beta are functionally homologous to the signaling proteins of the T-cell receptor.

Signal transduction by antigen receptors and some Fc receptors requires the activation of a family of receptor-associated transmembrane accessory proteins. One common feature of the cytoplasmic domains of these accessory molecules is the presence is at least two YXXA repeats that are potential sites for interaction with Src homology 2 domain-containing proteins. However, the degree of similarity between the different receptor-associated proteins varies from that of T-cell receptor (TCR) zeta and Fc receptor RIIIA gamma chains, which are homologous, to the distantly related Ig alpha and Ig beta proteins of the B-cell antigen receptor. To determine whether T- and B-cell antigen receptors are in fact functionally homologous, we have studied signal transduction by chimeric immunoglobulins bearing the Ig alpha or Ig beta cytoplasmic domain. We found that Ig alpha and Ig beta cytoplasmic domains were able to activate Ca2+ flux, interleukin-2 secretion, and phosphorylation of the same group of cellular substrates as the TCR in transfected T cells. Chimeric proteins were then used to examine the minimal requirements for activation of the Fyn, Lck, and ZAP kinases in T cells. Both Ig alpha and Ig beta were able to trigger Fyn, Lck, and ZAP directly without involvement of TCR components. Cytoplasmic tyrosine residues in Ig beta were required for recruitment and activation of ZAP-70, but these amino acids were not essential for the activation of Fyn and Lck. We conclude that Fyn and Lck are able to recognize a clustered nonphosphorylated immune recognition receptor, but activation of these kinases is not sufficient to induce cellular responses such as Ca2+ flux and interleukin-2 secretion. In addition, the molecular structures involved in antigen receptor signaling pathways are conserved between T and B cells.     

The pathway of antigen uptake and processing dictates MHC class II-mediated B cell survival and activation

The influence of the pathway of Ag uptake and processing on MHC class II (CII)-mediated B cell function is unknown. In this study, we investigate in resting and activated (via the BCR or CD40) B cells the biological properties of CII-peptide complexes (CII-peptide) generated by either the BCR-mediated Ag processing (type I complex) or fluid phase Ag processing (type II complex). Compared with type I complex, ligation of type II complex by either specific Ab or the TCR in Ag-presenting assay results in significant decreases in B cell survival rate (50-100%) and expression levels of CII, CD86, and CD54. Loss of B cells following ligation of type II complex occurs in the presence of a comparatively good level of specific CD4(+) T cell division, indicating that B cell loss is a late event following T cell stimulation. Comparative analysis of T and B cell conjugates after Ab ligation of type I or II complex reveals decreased efficiency of the latter in forming conjugates. Neither initial differential levels of CII and other studied surface markers, B cell type inherent differences, BCR signaling, T cell proliferation, nor initial density of CII-peptide complexes could explain the T cell-induced B cell loss. We propose that the context in which CII-peptide complexes are present in the membrane following BCR uptake and processing leads to B cell survival. Thus, appropriate targeting of Ag ensures generation of relevant immune responses.     

Aberrant trafficking of the B cell receptor Ig-alpha beta subunit in a B lymphoma cell line

The B cell Ag receptor (BCR) has two important functions: first, it binds and takes up Ag for presentation to T lymphocytes; and second, it transmits signals that regulate B cell development. Normal expression of the BCR requires the association of the Ag binding subunit, membrane IgM (mIgM), with the signaling component, the Ig-alpha beta heterodimer. After assembly in the endoplasmic reticulum, the intact BCR travels through the secretory pathway to the cell surface. In this paper, we report two variants of the B lymphoma cell lines, WEHI 279 and WEHI 231, that have both lost the ability to express mu heavy chain and consequently do not express mIgM. However, these variants do express the Ig-alpha beta heterodimer. In one variant, WEHI 279*, the Ig-alpha beta remained trapped intracellularly in the absence of mIgM. The other variant, 303.1.5.LM, expressed an aberrantly glycosylated Ig-alpha beta on the cell surface that was capable of signaling after cross-linking with anti-Ig-beta Abs. Further characterization uncovered a point mutation in the 303.1.5.LM mb1 gene that would change a proline for a leucine in the extracellular domain of Ig-alpha. The 303.1.5.LM Ig-alpha beta could not associate with a wild-type mIgM after mu heavy chain was reconstituted by DNA transfection. Thus, this mutation could define a region of the Ig-alpha polypeptide that is important for recognition by the endoplasmic reticulum quality control system, for association with glycosylating enzymes, and for the association of Ig-alpha beta subunits with mIgM subunits to create a complete BCR complex.     

Antigen receptor signaling competence and the determination of B cell fate in B-lymphopoiesis

Recent studies suggest that developmental check-points in B-lymphopoiesis are set in order to test the B cell receptor signaling competence. In these check-points ligand-independent and ligand-dependent receptor signals confer B-lymphopoiesis with positive and negative selection events. As a consequence, B-lymphocytes are forced to make crucial fate decisions to determine developmental progression, survival or apoptosis. In here we review recent progress in unraveling molecular and cellular mechanisms for the role of B cell receptor signaling competence in determination of the B cell fate.     

Mathematical models of the fate of lymphoma B cells after antigen receptor ligation with specific antibodies

We formulate models of the mechanism(s) by which B cell lymphoma cells stimulated with an antibody specific to the B cell receptor (IgM) become quiescent or apoptotic. In particular, we aim to reproduce experimental results by Marches et al. according to which the fate of the targeted cells (Daudi) depends on the levels of expression of p21(Waf1) (p21) cell-cycle inhibitor. A simple model is formulated in which the basic ingredients are p21 and caspase activity, and their mutual inhibition. We show that this model does not reproduce the experimental results and that further refinement is needed. A second model successfully reproduces the experimental observations, for a given set of parameter values, indicating a critical role for Myc in the fate decision process. We use bifurcation analysis and objective sensitivity analysis to assess the robustness of our results. Importantly, this analysis yields experimentally testable predictions on the role of Myc, which could have therapeutic implications.     

Role of antigen in the B cell response. Specific antigen and the lymphokine IL-5 synergize to drive B cell lymphoma proliferation and differentiation to Ig secretion

CH12 tumor B cells specific for SRBC require SRBC as Ag and the lymphokine IL-5 (formerly known as BCGFII) for optimal proliferation and differentiation to Ig-secreting cells. Lysed SRBC and IL-5 purified to homogeneity synergize markedly, especially at low B cell densities. A sizable proportion of CH12 cells differentiate into Ig-secreting plaque-forming cells when low numbers of the B lymphoma cells (100 to 3000) are cultured with Ag and IL-5. IL-2 or IL-4 have no effects. Intact SRBC or lysed SRBC are equally effective as sources of Ag. Even in the presence of the mitogens LPS and dextran sulfate, there is a striking requirement for Ag for both proliferation and differentiation at low B cell density. Because of the low cell numbers used, the results strongly suggest that the effects of Ag and lymphokine are directly on the B cell. The cell surface phenotype of the CH12 lymphoma and the kinetics of their response suggest that CH12 B cells have the characteristics of activated B cells. Thus, it appears that Ag binding to surface Ig gives a direct signal to at least some B cells that is critical in the later phases of the B cell response after initial activation during which proliferation and differentiation to Ig secretion occur and that the lymphokine IL-5 costimulates with Ag to mediate this phase of the response.     

The dissociation activation model of B cell antigen receptor triggering

To detect its cognate antigen, each B lymphocyte contains up to 120 000 B cell antigen receptor (BCR) complexes on its cell surface. How these abundant receptors remain silent on resting B cells and how they can be activated by a molecularly diverse set of ligands is poorly understood. The antigen-specific activation of the BCR is currently explained by the cross-linking model (CLM). This model predicts that the many BCR complexes on the surface of a B cell are dispersed signalling-inert monomers and that it is BCR dimerization that initiates signalling from the receptor. The finding that the BCR forms auto-inhibited oligomers on the surface of resting B cells falsifies these predictions of the CLM. We propose the dissociation activation model (DAM), which fits better with the existing body of experimental data.     

Association of protein kinase C-delta with the B cell antigen receptor complex

Protein kinase C (PKC)-delta is a diacylglycerol-dependent, calcium-independent novel PKC isoform and has been demonstrated to exert negative regulatory functions in B lymphocytes as well as in mast cells. Whereas in mast cells PKC-delta functionally interacts with the high-affinity receptor for IgE, FcepsilonR1, no such association has been described for the B cell antigen receptor (BCR). In this report, for the first time, we demonstrate the interaction of PKC-delta with different classes of BCR by means of affinity purification and native protein complex analysis. Using a C-terminally truncated Ig-alpha as well as non-phosphorylated and phosphorylated peptides representing C-terminal regions of Ig-alpha, the dependence of this BCR/PKC-delta interaction on tyrosine-phosphorylated Ig-alpha is shown. Finally, splenocytes from PKC-delta-deficient mice are found to exert reduced phosphorylation of PKD (a.k.a. PKC-mu) in response to BCR engagement, suggesting the early, membrane-proximal activation of an attenuating kinase complex including PKC-delta and PKD.