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Of the thousands of long noncoding RNAs (lncRNA) identified in lymphocytes, very few have defined functions. In this study, we report the discovery and functional elucidation of a human B cell-specific lncRNA with high levels of expression in three types of B cell cancer and normal B cells. The AC099524.1 gene is upstream of the gene encoding the B cell-specific phospholipase C γ 2 ( PLCG2 ), a B cell-specific enzyme that stimulates intracellular Ca 2+ signaling in response to BCR activation. AC099524.1 (B cell-associated lncRNA modulator of BCR-mediated Ca + signaling [BCALM]) transcripts are localized in the cytoplasm and, as expected, CRISPR/Cas9 knockout of AC099524.1 did not affect PLCG2 mRNA or protein expression. lncRNA interactome, RNA immunoprecipitation, and coimmunoprecipitation studies identified BCALM-interacting proteins in B cells, including phospholipase D 1 (PLD1), and kinase adaptor proteins AKAP9 (AKAP450) and AKAP13 (AKAP-Lbc). These two AKAP proteins form signaling complexes containing protein kinases A and C, which phosphorylate and activate PLD1 to produce phosphatidic acid (PA). BCR stimulation of BCALM-deficient B cells resulted in decreased PLD1 phosphorylation and increased intracellular Ca + flux relative to wild-type cells. These results suggest that BCALM promotes negative feedback that downmodulates BCR-mediated Ca + signaling by promoting phosphorylation of PLD1 by AKAP-associated kinases, enhancing production of PA. PA activates SHP-1, which negatively regulates BCR signaling. We propose the name BCALM for B-Cell Associated LncRNA Modulator of BCR-mediated Ca + signaling. Our findings suggest a new, to our knowledge, paradigm for lncRNA-mediated modulation of lymphocyte activation and signaling, with implications for B cell immune response and BCR-dependent cancers., (Copyright © 2020 by The American Association of Immunologists, Inc.)

While B cells play a significant role in the onset of type-1 diabetes (T1D), little is know about their role in those early stages. Thus, to gain new insights into the role of B cells in T1D, we converted a physiological early pancreas-infiltrating B cell into a novel BCR mouse model using Somatic Cell Nuclear Transfer (SCNT). Strikingly, SCNT-derived B1411 model displayed neither developmental block nor anergy. Instead, B1411 underwent spontaneous germinal center reactions. Without T cell help, B1411-Rag1 -/- was capable of forming peri-/intra-pancreatic lymph nodes, and undergoing class-switching. RNA-Seq analysis identified 93 differentially expressed genes in B1411 compared to WT B cells, including Irf7, Usp18 , and Mda5 that had been linked to a potential viral etiology of T1D. We also found various members of the oligoadenylate synthase (OAS) family to be enriched in B1411, such as Oas1 , which had recently also been linked to T1D. Strikingly, when challenged with glucose B1411-Rag1 -/- mice displayed impaired glucose tolerance., (Copyright © 2020 Phillips, Ke, Nham, Seidl, Freeman, Abadejos, Xiao, Nemazee, Ku and Kirak.)

Background: Dysregulation in calcium (Ca 2+ ) signaling is a hallmark of chronic lymphocytic leukemia (CLL). While the role of the B cell receptor (BCR) Ca 2+ pathway has been associated with disease progression, the importance of the newly described constitutive Ca 2+ entry (CE) pathway is less clear. In addition, we hypothesized that these differences reflect modifications of the CE pathway and Ca 2+ actors such as Orai1, transient receptor potential canonical (TRPC) 1, and stromal interaction molecule 1 (STIM1), the latter being the focus of this study., Methods: An extensive analysis of the Ca 2+ entry (CE) pathway in CLL B cells was performed including constitutive Ca 2+ entry, basal Ca 2+ levels, and store operated Ca 2+ entry (SOCE) activated following B cell receptor engagement or using Thapsigargin. The molecular characterization of the calcium channels Orai1 and TRPC1 and to their partner STIM1 was performed by flow cytometry and/or Western blotting. Specific siRNAs for Orai1, TRPC1 and STIM1 plus the Orai1 channel blocker Synta66 were used. CLL B cell viability was tested in the presence of an anti-STIM1 monoclonal antibody (mAb, clone GOK) coupled or not with an anti-CD20 mAb, rituximab. The Cox regression model was used to determine the optimal threshold and to stratify patients., Results: Seeking to explore the CE pathway, we found in untreated CLL patients that an abnormal CE pathway was (i) highly associated with the disease outcome; (ii) positively correlated with basal Ca 2+ concentrations; (iii) independent from the BCR-PLCγ2-InsP 3 R (SOCE) Ca 2+ signaling pathway; (iv) supported by Orai1 and TRPC1 channels; (v) regulated by the pool of STIM1 located in the plasma membrane (STIM1 PM ); and (vi) blocked when using a mAb targeting STIM1 PM . Next, we further established an association between an elevated expression of STIM1 PM and clinical outcome. In addition, combining an anti-STIM1 mAb with rituximab significantly reduced in vitro CLL B cell viability within the high STIM1 PM CLL subgroup., Conclusions: These data establish the critical role of a newly discovered BCR independent Ca 2+ entry in CLL evolution, provide new insights into CLL pathophysiology, and support innovative therapeutic perspectives such as targeting STIM1 located at the plasma membrane.

In addition to functioning as a ligand to engage TCRs and drive TCR signaling, MHC class II molecules are signaling molecules that generate a number of signals within APCs, such as B lymphocytes. Moreover, MHC class II signaling is critical for B cell activation and development of a robust humoral immune response. Murine class II molecules exist in two distinct conformational states, based primarily on the differential pairing of transmembrane domain GxxxG dimerization motifs (i.e., M1- and M2-paired class II). This laboratory has previously reported that the binding of a multimerized form of an anti-class II mAb that selectively recognizes M1-paired I-A k class II drives intracellular calcium signaling in resting murine B cells and that this signaling is dependent on both src and Syk protein tyrosine kinase activity. In contrast, multimerized forms of two different anti-I-A k mAbs that bind both M1- and M2-paired class II fail to elicit a response. In this report, a flow cytometry-based calcium flux assay is used to demonstrate that coligation of M1- and M2-paired I-A k class II results in the active and selective inhibition of M1-paired I-A k class II B cell calcium signaling by M2-paired class II molecules. Because M1- and M2-paired class II can be loaded with different sets of peptides derived from Ags acquired by distinct pathways of endocytosis, these findings suggest an MHC class II signaling-based mechanism by which CD4 T cells of differing specificities can either enhance or suppress B cell activation., (Copyright © 2019 The Authors.)

Self-tolerance by clonal anergy of B cells is marked by an increase in IgD and decrease in IgM antigen receptor surface expression, yet the function of IgD on anergic cells is obscure. Here we define the RNA landscape of the in vivo anergy response, comprising 220 induced sequences including a core set of 97. Failure to co-express IgD with IgM decreases overall expression of receptors for self-antigen, but paradoxically increases the core anergy response, exemplified by increased Sdc1 encoding the cell surface marker syndecan-1. IgD expressed on its own is nevertheless competent to induce calcium signalling and the core anergy mRNA response. Syndecan-1 induction correlates with reduction of surface IgM and is exaggerated without surface IgD in many transitional and mature B cells. These results show that IgD attenuates the response to self-antigen in anergic cells and promotes their accumulation. In this way, IgD minimizes tolerance-induced holes in the pre-immune antibody repertoire.

Calcium signals are crucial for diverse cellular functions including adhesion, differentiation, proliferation, effector functions and gene expression. After engagement of the B cell receptor, the intracellular calcium ion (Ca(2+)) concentration is increased promoting the activation of various signaling cascades. While elevated Ca(2+) in the cytosol initially comes from the endoplasmic reticulum (ER), a continuous influx of extracellular Ca(2+) is required to maintain the increased level of cytosolic Ca(2+). Store-operated Ca(2+) entry manages this process, which is regulated by an ER calcium sensor, stromal interaction molecule (STIM). STIM proteins sense changes in the levels of Ca(2+) stored within the ER lumen and regulates the Ca(2+)-release activated Ca(2+) channel in the plasma membrane. This review focuses on the signaling pathways leading to Ca(2+) influx and the role of Ca(2+) signals in B cell functions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

T follicular helper (T(FH)) cells select high-affinity, antibody-producing B cells for clonal expansion in germinal centers (GCs), but the nature of their interaction is not well defined. Using intravital imaging, we found that selection is mediated by large but transient contacts between T(FH) and GC B cells presenting the highest levels of cognate peptide bound to major histocompatibility complex II. These interactions elicited transient and sustained increases in T(FH) intracellular free calcium (Ca(2+)) that were associated with T(FH) cell coexpression of the cytokines interleukin-4 and -21. However, increased intracellular Ca(2+) did not arrest TFH cell migration. Instead, T(FH) cells remained motile and continually scanned the surface of many GC B cells, forming short-lived contacts that induced selection through further repeated transient elevations in intracellular Ca(2+)., (Copyright © 2014, American Association for the Advancement of Science.)

The Lyn tyrosine kinase regulates inhibitory signaling in B and myeloid cells: loss of Lyn results in a lupus-like autoimmune disease with hyperactive B cells and myeloproliferation. We have characterized the relative contribution of Lyn-regulated signaling pathways in B cells specifically to the development of autoimmunity by crossing the novel lyn(flox/flox) animals with mice carrying the Cre recombinase under the control of the Cd79a promoter, resulting in deletion of Lyn in B cells. The specific deletion of Lyn in B cells is sufficient for the development of immune complex-mediated glomerulonephritis. The B cell-specific Lyn-deficient mice have no defects in early bone marrow B cell development but have reduced numbers of mature B cells with poor germinal centers, as well as increased numbers of plasma and B1a cells, similar to the lyn(-/-) animals. Within 8 mo of life, B cell-specific Lyn mutant mice develop high titers of IgG anti-Smith Ag ribonucleoprotein and anti-dsDNA autoantibodies, which deposit in their kidneys, resulting in glomerulonephritis. B cell-specific Lyn mutant mice also develop myeloproliferation, similar to the lyn(-/-) animals. The additional deletion of MyD88 in B cells, achieved by crossing lyn(flox/flox)Cd79a-cre mice with myd88(flox/flox) animals, reversed the autoimmune phenotype observed in B cell-specific Lyn-deficient mice by blocking production of class-switched pathogenic IgG autoantibodies. Our results demonstrate that B cell-intrinsic Lyn-dependent signaling pathways regulate B cell homeostasis and activation, which in concert with B cell-specific MyD88 signaling pathways can drive the development of autoimmune disease.

Background: Chronic inflammatory and autoimmune diseases are largely due to inappropriate response of hyperactive or autoreactive B cells. These autoreactive B cells can evade central tolerance checkpoints and migrate to the periphery, where they would be silenced by anergy. Such anergic cells are characterized by B-cell receptor (BCR) desensitization and altered downstream signaling., Objective: We sought to determine whether intravenous immunoglobulin (IVIg) induces a nonresponsive state of B cells and to address the similarities of this mechanism to those described in anergy., Methods: Human B cells were stimulated with anti-IgM antibody, and effects of IVIg on several parameters, such as calcium release, tyrosine phosphorylation, BCR aggregation, BCR internalization, or transcriptional activity, were studied by using flow cytometry, confocal microscopy, Western blotting, and a quantitative PCR array., Results: IVIg-treated B cells show defects in activating coreceptor expression, calcium signaling, and BCR aggregation on engagement by antigen. IVIg also induces suppression of phosphoinositide 3-kinase signaling, which plays a central role in determining B-cell fate. All these events ultimately lead to profound modifications in gene expression, resulting in long-term functional but reversible silencing of IVIg-treated B cells., Conclusion: Our findings provide insights into the effectiveness of IVIg in treating autoimmune or inflammatory pathologies associated with the loss of B-cell tolerance. Furthermore, these data provide a model to explore the complexity of positive versus negative selection in B cells., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Light chain receptor editing is an important mechanism that prevents B cell self-reactivity. We have previously shown that tonic signaling through the BCR represses RAG expression at the immature B cell stage, and that initiation of light chain rearrangements occurs in the absence of these tonic signals in an in vitro model of B cell development. To further test our hypothesis we studied the effect of itpkb deficiency (itpkb(-/-) mice) or Raf hyper-activation (Raf-CAAX transgenic mice), two mutations that enhance BCR signaling, on receptor editing in an in vivo model. This model relies on transferring bone marrow from wild-type or mutant mice into mice expressing an anti-kappa light chain transgene. The anti-kappa transgene induces receptor editing of all kappa light chain expressing B cells, leading to a high frequency of lambda light chain expressing B cells. Anti-κ transgenic recipients of bone marrow from itpkb(-/-) or Raf-CAAX mice showed lower levels of editing to λ light chain than did non-transgenic control recipients. These results provide evidence in an in vivo model that enhanced BCR signaling at the immature B cell stage of development suppresses light chain receptor editing., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A subset of patients with common variable immunodeficiency (CVID), group 1a of the Freiburg classification, is characterized by increased B cells expressing low levels of CD21 (CD21(low) ), lymphoproliferation and autoimmunity. The CD21(low) B cells have been shown to be profoundly anergic, and defects of BCR-mediated calcium signaling and of T cells have been described in CVID 1a. We found that also the classical naïve B cells from CVID 1a patients, but not from CVID non-1a patients, proliferated poorly. The B cells of CVID 1a patients had a reduced capacity to divide reminiscent of the proliferative arrest associated with replicative senescence. Thus, we investigated whether lymphocyte dysfunction in CVID 1a was related to telomere-dependent replicative senescence, and found that both the B and the T cells from CVID 1a patients had significantly shorter telomeres compared with B and T cells from CVID non-1a patients. Telomere lengths in B and T cells were significantly correlated, indicating that the rate of telomere attrition in lymphocytes is an individual characteristic of CVID patients. Our findings suggest that telomere-dependent replicative senescence contributes to the immune dysfunction of CVID 1a patients, and may provide an important clue for a better understanding of the pathogenesis of CVID., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ikaros family transcription factors have a key role in lymphoid development, and their aberrant function contributes to a multitude of lymphoid malignancies. Ikaros and Helios bind to similar DNA sequences, and Helios associates with Ikaros-containing chromatin remodeling complexes. Previously, we have shown that loss of Ikaros leads to diminished BCR-signaling strength. In this study, we describe a Helios-deficient chicken DT40 B-cell line with a BCR signaling phenotype that is the opposite to that of Ikaros-deficient cells. In contrast to Ikaros-deficient cells, Helios(-/-) B cells exhibit increased calcium release to the cytoplasm after BCR crosslinking, but diminished BCR-induced phosphorylation of signaling molecules. The inositol 5-phosphatase SHIP, an important regulator in several signaling pathways, is differentially expressed in Ikaros- and Helios-deficient cells. In the absence of Ikaros, SHIP is upregulated, whereas Helios deficiency leads to the downregulation of SHIP expression. We also show with ChIP that Ikaros binds to the promoter of the INPP5D gene-encoding SHIP. Considering the critical role of SHIP in the BCR signaling pathway, our findings provide insight into the mechanism of how both Helios and Ikaros are involved in the regulation of BCR signaling.

Several lines of evidence have demonstrated B cell intrinsic activation defects in patients with common variable immunodeficiency (CVID). The rapid increase of intracellular free calcium concentrations after engagement of the BCR represents one crucial element in this activation process. The analysis of 53 patients with CVID for BCR-induced calcium flux identified a subgroup of patients with significantly reduced Ca2+ signals in primary B cells. This subgroup strongly corresponded to the class Ia of the Freiburg classification. Comparison at the level of defined B cell subpopulations revealed reduced Ca2+ signals in all mature B cell populations of patients with CVID class Ia when compared with healthy individuals and other groups of patients with CVID but not in circulating transitional B cells. BCR-induced Ca2+ responses were the lowest in CD21low B cells in patients as well as healthy donors, indicating an additional cell-specific mechanism inhibiting the Ca2+ flux. Although proximal BCR signaling events are unperturbed in patients' B cells, including normal phospholipase Cgamma2 phosphorylation and Ca2+ release from intracellular stores, Ca2+ influx from the extracellular space is significantly impaired. CD22, a negative regulator of calcium signals in B cells, is highly expressed on CD21low B cells from patients with CVID Ia and might be involved in the attenuated Ca2+ response of this B cell subpopulation. These data from patients with CVID suggest that a defect leading to impaired BCR-induced calcium signaling is associated with the expansion of CD21low B cells, hypogammaglobulinemia, autoimmune dysregulation, and lymphadenopathy.

Signals processed through the B cell antigen receptor (BCR) control both the proliferation and differentiation of B lymphocytes. How these different signaling modes are established at the BCR is poorly understood. We show that a conserved arginine in the tail sequence of the Igalpha subunit of the BCR is methylated by the protein arginine methyltransferase 1. This modification negatively regulates the calcium and PI-3 kinase pathways of the BCR while promoting signals leading to B cell differentiation. Thus, Igalpha arginine methylation can play an important role in specifying the outcome of BCR signaling.

CD22 and Siglec-G are inhibitory coreceptors for BCR-mediated signaling. Although CD22-deficient mice show increased calcium signaling in their conventional B2 cells and a quite normal B cell maturation, Siglec-G-deficient mice have increased calcium mobilization just in B1 cells and show a large expansion of the B1 cell population. Neither CD22-deficient, nor Siglec-G-deficient mice on a pure C57BL/6 or BALB/c background, respectively, develop autoimmunity. Using Siglec-G x CD22 double-deficient mice, we addressed whether Siglec-G and CD22 have redundant functions. Siglec-G x CD22 double-deficient mice show elevated calcium responses in both B1 cells and B2 cells, increased serum IgM levels and an enlarged population of B1 cells. The enlargement of B1 cell numbers is even higher than in Siglecg(-/-) mice. This expansion seems to happen at the expense of B2 cells, which are reduced in absolute cell numbers, but show an activated phenotype. Furthermore, Siglec-G x CD22 double-deficient mice show a diminished immune response to both thymus-dependent and thymus-independent type II Ags. In contrast, B cells from Siglec-G x CD22 double-deficient mice exhibit a hyperproliferative response to stimulation with several TLR ligands. Aged Siglec-G x CD22 double-deficient mice spontaneously develop anti-DNA and antinuclear autoantibodies. These resulted in a moderate form of immune complex glomerulonephritis. These results show that Siglec-G and CD22 have partly compensatory functions and together are crucial in maintaining the B cell tolerance.

Lysophospholipids (LPLs) such as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are chemotactic for lymphocytes, and increases of in cytosolic [Ca(2+)] signal the regulation of lymphocyte activation and migration. Here, the authors investigated the effects of LPA and S1P on [Ca(2+)](c) in mouse B cell lines (WEHI-231 and Bal-17) and primary B cells isolated from mouse spleen and bone marrow, and focused on the modulation of store-operated Ca(2+) entry (SOCE) by LPLs. In Bal-17 (a mature B cell line) both LPA and S1P induced a transient [Ca(2+)](c) increase via a phospholipase C pathway. In addition, pretreatment with LPLs was found to augment thapsigargin-induced SOCE in Bal-17 cells. However, in WEHI-231 (an immature B cell line) LPLs had no significant effect on [Ca(2+)](c) or SOCE. Furthermore, in freshly isolated splenic B cells (SBCs) and bone marrow B cells (BMBCs), LPLs induced only a small increase in [Ca(2+)](c). Interestingly, however, pretreatment with LPLs markedly increased SOCE in primary B cells, and this augmentation was more prominent in BMBCs than SBCs. The unidirectional influx of Ca(2+) was measured using Ba(2+) as a surrogate ion. Similarly, Ba(2+) influx was also found to be markedly increased by LPLs in SBCs and BMBCs. Summarizing, LPLs were found to strongly augment SOCE-mediated Ca(2+)-signaling in mouse B cells. However, unlike the mature Bal-17 cell line, PLC-dependent Ca(2+) release was insignificant in primary B cells and inWEHI-231.

The calcitonin receptor (CTR) is a seven-transmembrane-domain G-protein-coupled receptor that regulates calcium metabolism and bone resorption by osteoclasts. Here we demonstrate that high levels are expressed by normal human T and B lymphocytes from tonsils and peripheral blood in relation to their activation status, as CTR(+) T cells are prone to produce IFN-gamma after TCR stimulation. The receptor is also highly expressed on B cells from chronic lymphocytic leukemia patients, thus suggesting a correlation between its expression, their proliferative extent as well as their memory, antigen-experienced phenotype. Moreover, we found that binding of the receptor with salmon calcitonin induces an increase of intracellular calcium(2+) in peripheral lymphocytes. This effect is involved in several lymphocyte immune functions, as cytosolic calcium(2+) levels regulate both cell proliferation and cytokine production. In our hands, the increase of calcium(2+) levels by CTR binding with sCT induced a dose-dependent cell proliferation. We therefore suppose that expression of this functional receptor may contribute to the modulation of cytoplasmic calcium(2+) levels needed to regulate T and B cell activation and perhaps other immune functions.

Coronin 1 is a leukocyte specific regulator of Ca(2+)-dependent signaling and is essential for the survival of peripheral T lymphocytes, but its role in B cells is unknown. In this study, we show that coronin 1 is essential for intracellular Ca(2+) mobilization and proliferation upon triggering of the BCR. However, the presence of costimulatory signals rendered coronin 1 dispensable for B cell signaling, consistent with the generation of normal immune responses against a variety of Ags in coronin 1-deficient mice. We conclude that coronin 1, while being essential for T cell function and survival, is dispensable for B cell function in vivo.

Chemokines orchestrate the organization of leucocyte recruitment during inflammation and homeostasis. Despite growing knowledge of chemokine receptors, some orphan chemokine receptors are still not characterized. The gene CCRL2 encodes such a receptor that exists in two splice variants, CRAM-A and CRAM-B. Here, we report that CRAM is expressed by human peripheral blood and bone marrow B cells, and by different B-cell lines dependent on the B-cell maturation stage. Intriguingly, CRAM surface expression on the pre-B-cell lines Nalm6 and G2 is specifically upregulated in response to the inflammatory chemokine CCL5 (RANTES), a chemokine that is well known to play an important role in modulating immune responses. Although Nalm6 cells do not express any of the known CCL5 binding receptors, extracellular signal-regulated kinases 1 and 2 (ERK1/2) are phosphorylated upon CCL5 stimulation, suggesting a direct effect of CCL5 through the CRAM receptor. However, no calcium mobilization or migratory responses upon CCL5 stimulation are induced in B-cell lines or in transfected cells. Also, ERK1/2 phosphorylation cannot be inhibited by pertussis toxin, suggesting that CRAM does not couple to Gi proteins. Our results describe the expression of a novel, non-classical chemokine receptor on B cells that is potentially involved in immunomodulatory functions together with CCL5.

Calcium is a second messenger for many signaling pathways in B cells, but its role as a receptor ligand has not been well characterized. However, pulses of free calcium were found to cause the rapid release of internal calcium stores in normal human B cells. This response appeared to be mediated by a cell surface protein with receptor properties as it could be blocked by pretreatment with trypsin and with kinase and phospholipase Cgamma inhibitors. The calcium receptor on B cells was not the conventional calcium-sensing receptor (CaSR) since B cells did not express CaSR and calcium-induced responses could not be blocked by specific CaSR inhibitors. B-cell responses to extracellular calcium activated phosphoinositide-3 kinase/AKT, calcineurin, extracellular signal regulated kinase, p38 mitogen-activated protein kinase, protein kinase C, Ca(2+)/calmodulin kinase II, and nuclear factor-kappaB signaling pathways, and resulted in transcription of the early response gene, CD83. This extracellular calcium sensor enhanced B-cell responses to Toll-like receptor, B-cell receptor, and cytokine receptor agonists. These findings suggest a means by which B cells prepare to engage in immune responses by responding to calcium fluctuations in their environment.

The murine transmembrane glycoprotein CD83 is an important regulator for both thymic T cell maturation and peripheral T cell responses. Recently, we reported that CD83 also has a function on B cells: Ubiquitous transgenic (Tg) expression of CD83 interfered with the immunoglobulin (Ig) response to infectious agents and to T cell dependent as well as T cell independent model antigen immunization. Here we compare the function of CD83Tg B cells that overexpress CD83 and CD83 mutant (CD83mu) B cells that display a drastically reduced CD83 expression. Correlating with CD83 expression, the basic as well as the lipopolysaccharide (LPS) induced expression of the activation markers CD86 and MHC-II are significantly increased in CD83Tg B cells and reciprocally decreased in CD83mu B cells. Wild-type B cells rapidly upregulate CD83 within three hours post BCR or TLR engagement by de novo protein synthesis. The forced premature overexpression of CD83 on the CD83Tg B cells results in reduced calcium signaling, reduced Ig secretion and a reciprocally increased IL-10 production upon in vitro activation. This altered phenotype is mediated by CD83 expressed on the B cells themselves, since it is observed in the absence of accessory cells. In line with this finding, purified CD83mu B cells displayed a reduced IL-10 production and slightly increased Ig secretion upon LPS stimulation in vitro. Taken together, our data strongly suggest that CD83 is expressed by B cells upon activation and contributes to the regulation of B cell function.

B cells respond to antigen stimulation with mobilization of the Ca(2+) second messenger in two phases operated by two distinct sets of effector proteins. First, an antigen receptor-specific Ca(2+) initiation complex is assembled, activated, and targeted to the plasma membrane to trigger the transient release of Ca(2+) from intracellular stores of the endoplasmic reticulum. Second, more ubiquitously expressed Ca(2+) channels of the plasma membrane are opened to allow for sustained Ca(2+) influx from the extracellular medium. Depending on the developmental stage of the B cell, the kinetics and profile of the two phases are adjusted at multiple levels of positive and negative regulation. A molecular basis for the Ca(2+) signaling plasticity is provided by cytosolic and transmembrane adapter proteins. They act as signal organizers, which control enzyme/substrate interactions by directing the different signaling modules into specific subcellular compartments. These arrangements orchestrate a graduated activation of Ca(2+)-sensitive downstream pathways, which ultimately determine appropriate cellular responses, namely elimination of autoreactive B cells or proliferation and differentiation of immunocompetent B cells into antibody-secreting plasma cells.

TACI (transmembrane activator and calcium modulator and cyclophilin ligand [CAML] interactor) is a part of a novel network of ligands and receptors involved in B-cell survival and isotype switching. The TACI protein mediates its effects through CAML, an endoplasmic reticulum (ER)-localized protein that controls Ca(2+) efflux. The adenovirus E3-6.7K protein prevents inflammatory responses and also confers resistance from a variety of apoptotic stimuli and maintains ER Ca(2+) homeostasis; however, the mechanism of action is unknown. Here, we provide evidence that E3-6.7K shares sequence homology with TACI and inhibits apoptosis and ER Ca(2+) efflux through an interaction with CAML, a Ca(2+)-modulating protein. We demonstrate a direct interaction between E3-6.7K and CAML and reveal that the two proteins colocalize in an ER-like compartment. Furthermore, the interaction between the two proteins is localized to the N-terminal domain of CAML and to a 22-amino-acid region near the C terminus of E3-6.7K termed the CAML-binding domain (CBD). Mutational analysis of the CBD showed that an interaction with CAML is required for E3-6.7K to inhibit thapsigargin-induced apoptosis and ER Ca(2+) efflux. E3-6.7K appears to be the first virologue of TACI to be identified. It targets CAML in a novel immunosubversive mechanism to alter ER Ca(2+) homeostasis, which consequently inhibits inflammation and protects infected cells from apoptosis.

Bruton tyrosine kinase (Btk) and phospholipase Cgamma2 (PLCgamma2) are 2 key molecules involved in B-cell receptor (BCR) signaling. Biochemical studies have placed them in a linear signaling pathway, with Btk acting upstream of PLCgamma2. Consistent with this, mice lacking either molecule display a leaky but similar block in B-cell development. Here, we generated Btk(-/-) PLCgamma2(-/-) mice and showed that combined deficiencies in Btk and PLCgamma2 severely arrested B lymphopoiesis at the large pre-B-cell stage. In contrast to either single mutant, Btk(-/-) PLCgamma2(-/-) pre-B cells expressed high levels of pre-BCR on their cell surfaces and exhibited reduced immunoglobulin light chain gene rearrangements. Pre-BCR-induced calcium signaling was also drastically compromised in Btk(-/-) PLCgamma2(-/-) pre-B cells compared with wild-type and single-mutant cells. Interestingly, immunoglobulin heavy chain allelic exclusion remained intact in the absence of Btk and PLCgamma2. Overall, our results suggest that Btk and PLCgamma2 have combinatorial roles in regulating pre-B cell differentiation.

An encounter of B cells with cognate self Ags in the periphery can lead to anergy, a condition characterized by altered anatomical localization, shortened life span, and refractility to Ag stimulation. We recently reported that an immature B cell encounter with cognate self-Ag in the bone marrow can also lead to anergy. In this study we show that anergic as well as acutely Ag-stimulated immature B cells are defective in stromal cell-derived factor-1 (SDF-1)-induced calcium mobilization and migration and do not localize to bone marrow following adoptive transfer. This hyporesponsiveness does not involve CXCR4 modulation. However, BCR signal-mediated hyporesponsiveness to SDF-1 is associated with phosphorylation of the 5-inositol phosphatase SHIP1 and requires SHIP1 expression. Therefore, an encounter with cognate Ag may, by preventing SDF-1-induced phosphatidylinositol 3,4,5-triphosphate accumulation, trigger premature emigration of immature B cells from bone marrow.

SLP-65 and the linker for activation of T cells (LAT) are central adaptor proteins that link the activated pre-BCR to downstream events in pre-B cells. Recently, a new transmembrane adaptor called NTAL/LAB/LAT2 (hereafter called NTAL for non-T cell activation linker) with striking functional and structural similarity to LAT has been identified in B cells. In this study, we compare the function of NTAL and LAT in pre-BCR signaling and show that, in contrast to LAT, NTAL does not induce pre-BCR down-regulation, calcium flux, or pre-B cell differentiation. To test whether differences between NTAL-mediated and LAT-mediated signaling are caused by the missing phospholipase C (PLC)-gamma binding motif in NTAL, we inserted the PLC-gamma1/2 binding motif of LAT into NTAL. This insertion rendered NTAL capable of activating pre-BCR down-regulation and calcium flux. Unexpectedly however, the ability of NTAL to induce calcium flux was not sufficient to promote pre-B cell differentiation, suggesting that the PLC-gamma binding motif has only partial effects on NTAL-mediated pre-BCR signaling. By generating chimeric swap mutants, we identified the N terminus of NTAL as an inhibitory domain that prevents pre-B cell differentiation while allowing pre-BCR down-regulation and receptor-mediated calcium flux. Our data suggest that, in addition to the missing PLC-gamma1/2 binding motif, the N terminus is responsible for the functional differences between NTAL and LAT in pre-B cells.

Cross-linking of CD38 on hematopoietic cells induces activation, proliferation and differentiation of mature T and B cells and mediates apoptosis of myeloid and lymphoid progenitor cells. In addition to acting as a signaling receptor, CD38 is also an enzyme capable of producing several calcium-mobilizing metabolites, including cyclic adenosine diphosphate ribose (cADPR). It has been previously postulated that the calcium-mobilizing metabolites produced by CD38 may regulate its receptor-based activities. To test this hypothesis, we examined whether the enzyme activity of CD38 controls the apoptosis of an anti-CD38-stimulated leukemic B cell. We show that anti-CD38-induced apoptosis of Ba/F3 cells, a murine pro-B cell line, is not affected by blocking the calcium-mobilizing activity of cADPR or by inhibiting intracellular or extracellular calcium mobilization. In addition, we demonstrate that blocking CD38 enzyme activity with 2'-deoxy-2'-fluoro-nicotinamide arabinoside adenine dinucleotide has no effect on apoptosis and that Ba/F3 cells expressing catalytically inactive mutant forms of CD38 still undergo apoptosis upon CD38 cross-linking. Instead, we find that anti-CD38-induced apoptosis is dependent on tyrosine kinase and caspase activation, and that this process appears to be potentiated by the presence of membrane microdomains. Thus, the receptor-mediated functions of CD38 can be separated from its enzyme activity in a murine leukemic cell line, suggesting that CD38 plays multiple, but independent, biologic roles.

We have generated transgenic mice expressing the amino-terminal fragment of the phosphatidylinositol 3-kinase (PI3K) catalytic subunit (p110ABD) in thymocytes. Expression of p110ABD results in constitutive activation of PI3K and in significant increases in the numbers of mature, single-positive thymocytes. We previously reported that the increase in mature cells was in part due to a defect in thymic emigration. In this study we identify another component to this phenotype. Expression of p110ABD results in an enhancement of positive selection, without alterations in thymocyte lifespan or negative selection. Since PI3K can affect activation of Btk, which in turn potentiates calcium fluxes, during B cell development, our results suggest that PI3K could play a role in the regulation of Itk kinases in T cells, and that both cell types share a common signaling network to modulate calcium responses downstream of their antigen receptor.

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 phosphatidylinositol 3-kinase (PI3K) pathway plays a critical role in B cell activation and differentiation. Recruitment of pleckstrin homology (PH) domain-containing signal transduction proteins to the plasma membrane through binding to 3-phosphoinositide second messengers represents a major effector mechanism for PI3Ks. We have found that the PH domains of Bam32 and tandem PH domain-containing protein 2 (TAPP2) specify a temporally distinct wave of membrane recruitment compared with that of Bruton's tyrosine kinase (Btk), with recruitment of these two adaptors representing a later stage of the response. In this study we provide direct evidence that PH domain-dependent recruitment of Btk to the membrane is blocked by coligation of the inhibitory receptor FcgammaRII in human B lymphoma cells. In contrast, recruitment specified by the Bam32 or TAPP2 PH domains is completely insensitive to FcgammaRII inhibition. This differential regulation can be accounted for by Src homology 2-containing inositol polyphosphate 5-phosphatase (SHIP) activity alone, as expression of membrane-targeted SHIP completely abrogated Btk recruitment, but had no inhibitory effect on Bam32 or TAPP2 recruitment. Strikingly, kinetic analysis revealed that membrane recruitment of Bam32 and TAPP2 is actually more rapid under "inhibitory" signaling conditions. Analysis of 3-phosphoinositide generation under activating and inhibitory signaling conditions indicated that recruitment of Bam32 and TAPP2 is inversely correlated with the SHIP substrate/product ratio (phosphatidylinositol 3,4,5-trisphosphate/phosphatidylinositol 3,4-bisphosphate). Overexpression of TAPP2 in B cells led to an increase in the sustained phase of the calcium response and increased NF-AT-dependent transcriptional activation after B cell Ag receptor ligation. Together, these results suggest that Bam32 and TAPP2 adaptors define a novel group of SHIP-activated targets of PI3K that regulate B cell Ag receptor signaling.

Signal transduction by the B-cell antigen receptor (BCR) regulates development, survival, and clonal expansion of B cells. The BCR complex comprises the membrane-bound immunoglobulin molecule and the Ig-alpha/Ig-beta heterodimer, and was shown to form oligomeric structures. Antigen-mediated engagement of the BCR results in the tyrosine phosphorylation of multiple signaling proteins leading to calcium mobilization and the activation of downstream serine/threonine kinases as well as transcription factors. In pervanadate (PV)-treated B cells, comparable pathways are activated on expression of the BCR, indicating that the BCR can signal in an antigen-independent fashion as well. In this chapter, we describe the analysis of antigen-dependent and -independent tyrosine phosphorylation events as well as a method to study calcium mobilization from differentially stimulated B cells. Furthermore, we emphasize the use of phospho-specific antibodies (Abs) and low-molecular-weight enzyme inhibitors in the process of mapping BCR-activated signaling pathways as well as determining activation states of signaling proteins.

Objective: To provide a comprehensive understanding of the humoral immune response that takes place at the site of inflammation in rheumatoid arthritis (RA), we studied the functional properties of synovial B cells. In particular, the response to various modes of mitogen stimulation was investigated., Methods: Purified synovial fluid (SF) B cells were cultured in the presence of CD40 ligand (CD40L)-expressing fibroblasts and cytokines, activated T cells, or phorbol myristate acetate (PMA)/ionomycin. Proliferation was determined by 3H-thymidine incorporation. Release of intracellular calcium was studied by flow cytometry., Results: The inflamed joints of RA patients contained a population of CD20+,CD38- B cells with dramatically impaired mitogen responsiveness. Although the Ig-producing capacity was intact, these cells failed to proliferate in response to (a) CD40 in the presence of interleukin-2 (IL-2) and IL-10, (b) activated T cells, or (c) stimulation via the B cell receptor. Moreover, SF CD20+,CD38- B cells revealed a defective B cell receptor-induced Ca2+ influx, reminiscent of anergic B cells. Release of intracellular Ca2+ by ionomycin in the presence of the protein kinase C activator PMA did not restore the proliferative capacity. These findings indicate blockades in the proximal and distal intermediates involved in mitogen signaling., Conclusion: SF CD20+,CD38- B cells have functionally impaired proliferative responsiveness. The capacity of these cells to respond to activation by the production of Ig supports the notion that these cells might serve as Ig-producing effector cells and, as such, play a role in the pathophysiology of RA.

FcgammaRIIB functions as an inhibitory receptor to dampen B cell Ag receptor signals and immune responses. Accumulating evidence indicates that ex vivo B cells require the inositol 5-phosphatase, Src homology domain 2-containing inositol 5-phosphatase (SHIP), for FcgammaRIIB-mediated inhibitory signaling. However, we report here that LPS-activated primary B cells do not require SHIP and thus differ from resting B cells. SHIP-deficient B cell blasts display efficient FcgammaRIIB-dependent inhibition of calcium mobilization as well as Akt and extracellular signal-related protein kinase phosphorylation. Surprisingly, FcgammaRIIB-dependent degradation of phosphatidylinositol 3,4,5-trisphosphate and conversion into phosphatidylinositol 3,4-bisphosphate occur in SHIP-deficient B cell blasts, demonstrating the function of an additional inositol 5-phosphatase. Further analysis reveals that while resting cells express only SHIP, B cell blasts also express the recently described inositol 5-phosphatase, SHIP-2. Finally, data suggest that both SHIP-2 and SHIP can mediate downstream biologic consequences of FcgammaRIIB signaling, including inhibition of the proliferative response.

An increase in the intracellular Ca(2+) concentration by B cell receptor (BCR) cross-linking plays important roles in the regulation of B cell functions. [Ca(2+)](i) is regulated by Ca(2+) release from the Ca(2+) store as well as store-operated Ca(2+) influx (SOC). Protein tyrosine kinases downstream of BCR cross-linking were shown to regulate the mechanism for Ca(2+) release. However, it remains elusive whether BCR cross-linking regulates SOC or not. In this study, we examined the effect of BCR cross-linking on thapsigargin-induced SOC in the DT40 B cells. We found that the SOC-mediated increase in intracellular Ca(2+) concentration was inhibited by BCR cross-linking. Using a membrane-potential-sensitive dye, we found that BCR cross-linking induced depolarization, which is expected to decrease the driving force of Ca(2+) influx and SOC channel conductance. When membrane potential was held constant by the transmembrane K(+) concentration gradient in the presence of valinomycin, the BCR-mediated inhibition of SOC was still observed. Thus, the BCR-mediated inhibition of SOC involves both depolarization-dependent and depolarization-independent mechanisms of SOC inhibition. The depolarization-independent inhibition of the SOC was abolished in Lyn-deficient, but not in Bruton's tyrosine kinase-, Syk- or SHIP (Src homology 2 domain containing phosphatidylinositol 5'-phosphatase)-deficient cells, indicating that Lyn is involved in the inhibition. These results show novel pathways of BCR-mediated SOC regulations.

The B cell receptor (BCR) regulates B cell development and function through immunoglobulin (Ig)alpha and Ig beta, a pair of membrane-bound Ig superfamily proteins, each of which contains a single cytoplasmic immunoreceptor tyrosine activation motif (ITAM). To determine the function of Ig beta, we produced mice that carry a deletion of the cytoplasmic domain of Ig beta (Ig beta Delta C mice) and compared them to mice that carry a similar mutation in Ig alpha (MB1 Delta C, herein referred to as Ig alpha Delta C mice). Ig beta Delta C mice differ from Ig alpha Delta C mice in that they show little impairment in early B cell development and they produce immature B cells that respond normally to BCR cross-linking as determined by Ca(2+) flux. However, Ig beta Delta C B cells are arrested at the immature stage of B cell development in the bone marrow and die by apoptosis. We conclude that the cytoplasmic domain Ig beta is required for B cell development beyond the immature B cell stage and that Ig alpha and Ig beta have distinct biologic activities in vivo.

Membrane microdomains (lipid rafts) are enriched in selected signaling molecules and may compartmentalize receptor-mediated signals. Here, we report that in primary human B lymphocytes and in Ramos B cells B cell receptor (BCR) stimulation induces rapid and transient redistribution of a subset of engaged BCRs to lipid rafts and phosphorylation of raft-associated tyrosine kinase substrates. Cholesterol sequestration disrupted the lipid rafts, preventing BCR redistribution, but did not inhibit tyrosine kinase activation or phosphorylation of mitogen-activated protein kinase/extracellular regulated kinase. However, raft disruption enhanced the release of calcium from intracellular stores, suggesting that rafts may sequester early signaling events that down-regulate calcium flux. Consistent with this, BCR stimulation induced rapid and transient translocation of the Src homology 2 domain-containing inositol phosphatase, SHIP, into lipid rafts.

CD19 is a coreceptor on B cells that enhances the increase in cytoplasmic calcium and ERK2 activation when coligated with the B cell Ag receptor. Constructs containing point mutations and truncations were expressed in Daudi human B lymphoblastoid cells to systematically determine the requirement for individual CD19 cytoplasmic tyrosines in these responses. Evidence for activity was found for Y330, Y360, and Y421 as well as that previously published for Y391. Precipitates formed with phosphopeptides consisting of CD19 sequences flanking these residues were used to screen for cytoplasmic proteins that mediate signaling. Phosphopeptide Y330 precipitated Grb2 and Sos, whereas phosphopeptides Y391 and Y421 both precipitated Vav and phospholipase C-gamma2. These molecules also were found associated with native CD19. In mapping studies with altered constructs, CD19 Y330 and/or Y360 were necessary for binding Grb2 and Sos. Vav associated with CD19 constitutively in unstimulated cells by a tyrosine-independent mechanism requiring the portion of CD19 encoded by exons 9-12. After B cell Ag receptor stimulation, Vav association was tyrosine-dependent, but binding was influenced by multiple residues. However, when maximally phosphorylated by pervanadate, Y391 and, to a lesser extent, Y421 were sufficient. CD19 Y391 was also both necessary and sufficient for binding phospholipase C-gamma2. Thus, different tyrosines along the CD19 cytoplasmic domain provide scaffolding for the formation of complexes of different signaling molecules.