Ig Beta Research Articles

Proteomics Comparison of Breast Cancer with Adjacent Normal Tissues in Women with Breast Cancer

Introduction: The molecular mechanisms involved in the development and progression of breast cancer have yet to be determined. In the present study, the proteome of cancerous beast and adjacent normal tissues were compared. Materials & Methods: In a cohort study, the cancer tissues and adjacent normal tissues were obtained from 5 female patients with ductal carcinoma in stage 3. The total protein contents of cancer and adjacent normal tissues were extracted. The protein expression levels were examined by Image Master 2D Platinum software following two-dimensional gel electrophoresis. MALDI-TOF MS/MS mass spectrometry was used for proteins identification. Findings: The constant region of Ig gamma-1 chain and beta subunit of hemoglobin were exclusively detected in the cancer and adjacent normal tissues, respectively. The expression of serum albumin and collagen VI alpha chain in the cancer tissue was significantly lower than the normal tissue (P less-than 0.05). In contrast, the expression of a single peptide matching to cytoskeletal type I and II keratin significantly increased in the cancer tissue compared to the normal tissues (P less-than 0.05). Discussion & Conclusion: As the output of our investigation, it seems that proteome of cancerous tissue is extensively different from the adjacent one. Therefore, proteomic approach might be a promising tool for monitoring breast tumorigenesis. However, this needs to be confirmed in future.

Igβ deficiency in humans

To describe novel immunological and molecular findings regarding early B cell development arrest resulting in autosomal recessive agammaglobulinemia. Recently two different groups identified mutations in Ig beta, a component of the pre-B cell receptor, responsible for agammaglobulinemia in humans. These are the first two patients ever described with mutations in Ig beta. These novel findings broaden the spectrum of genetic defects underlying this rare condition. This novel cause of agammaglobulinemia not only sheds light into early B cell development in humans but also sets the basis for potential alternative therapeutic approaches such as gene therapy.

High CD79b Expression Predicts a Poor Outcome in B-Cell Chronic Lymphocytic Leukemia (B-CLL).

[Display omitted] The immunoglobulin (Ig) beta protein (CD79b) dimerizes with Ig alpha (CD79a) to form the signaling component of the B-cell antigen receptor complex (BCR). Given the critical role of Ig beta in BCR signaling, it has been suggested that its variable expression in CLL may induce either cell proliferation or apoptosis. High CD79b expression has been associated with atypical morphology, strong expression of surface Ig, advanced clinical stage and short overall survival in B-CLL (Garcia Vela, 1999; Zucchetto, 2006). Moreover, high CD38 expression was correlated with high CD40, CD69 and CD79b which are consistent with an activation phenotype (Damle, 2002). Furthermore, we have demonstrated that the ZAP-70+ CLL subgroup shows a rapid disease progression and an inferior overall survival (Del Principe, 2006). Since it has been described that BCR engagement has significant effects on B-CLL cell survival, activation and cell cycle progression (Deglesne, 2006), we decided to evaluate the real impact of CD79b expression on B-CLL prognosis. The primary aims of our research were: 1) to determine progression-free survival (PFS) and overall survival (OS) upon CD79b expression; 2) whether CD79b could predict varied outcome within ZAP-70+ and ZAP-70 negative subgroups; and finally 3) whether CD79b was an independent prognostic factor. Therefore, we investigated 401 patients (pts), median age 65 years (range 33–89), 213 males and 188 females. With regard to modified Rai stages, 123 pts had a low stage, 261 an intermediate stage and 17 a high stage. CD79b was determined by multicolor flow cytometry, using the monoclonal antibody anti-CD79 beta-FITC (clone SN8, Dako) and fixing a cut-off value of 30%. CD79b+ B-CLL pts were 207/401 (52%). CD79b>30% was significantly associated with the intermediate/high Rai stage (p=0.00001), beta-2 microglobulin >2.2 mg/dl (p<0.0001) and with multiple intrathoracic/abdominal lymphadenopathies and/or splenomegaly (p<0.0001). Low CD79b was significantly correlated either with IgVH mutated status (>2%) (86/105; p<0.0001) or lower soluble CD23 levels (125/161; p<0.00001). Significant associations were found either between CD38<30% and lower CD79b (172/194; p<0.0001) or ZAP- 70<20% and CD79b<30% (145/193; p<0.0001). With regard to cytogenetics, there were strict correlations either between high CD79b and trisomy 12 (25/33; p=0.001) or high CD79b and del17p (16/20; p=0.001). Median follow up duration from diagnosis was 68 months (range 6–230). Concerning clinical outcome, both a shorter PFS (Figure) and OS were observed in CD79b+ pts (12% vs 58% at 16 years; p<0.0001 and 49% vs 85% at 16 years; p<0.0001) as well as in ZAP-70+ pts (5% vs 52% at 11 years; p<0.0001 and 33% vs 87% at 18 years; p<0.0001). To further explore the prognostic value of CD79b, we investigated its expression within ZAP-70+ (159 pts) and ZAP-70 negative (242 pts) subsets. As a matter of fact, CD79b+ pts showed a shorter PFS and OS both within the ZAP-70+ subset (7% vs 19% at 10 years; p=0.00006 and 25% vs 65% at 16 years; p=0.006) and within the ZAP-70 negative subset (29% vs 67% at 14 years, p<0.00001 and 86% vs 100% at 14 years; p=0.01). In multivariate analysis of PFS and OS, in which age, Rai stages, CD38, CD69, CD79b and ZAP-70 entered, ZAP-70 (p=0.001 and p=0.001), CD69 (p=0.001 and p=0.01) and CD79b (p=0.0007 and p=0.03) resulted to be independent prognostic factors. Therefore CD79b, easily determined by flow cytometry, should be considered another important prognostic parameter and could be used to early identify and stratify B-CLL progressive pts.

Inhibition of Apoptosis Reduces Immunogeneic Potential of Adenoviral-Treated Syngeneic Liver Grafts

Effects of adenoviral therapy and reduced apoptosis on immune response were investigated in a rat liver transplantation model after prolonged ischemia-reperfusion. Liver donors were treated i.v. either with an adenoviral construct, expressing bcl-2, green-fluorescent-protein, or doxycyclin. Intrahepatic apoptosis was assessed by terminal transferase dUTP nick end labeling assay. The intrahepatic presence of CD4, CD8a, CD163, immunoglobulin (Ig)beta, tumor necrosis factor (TNF)-alpha and myeloperoxidase (MPO) was quantified by realtime polymerase chain reaction at 24 hours and seven days after transplantation. Bcl-2 expression abrogated the TNF-alpha elevation and reduced apoptosis of hepatocytes and sinusoidal endothelial cells as compared to advCMV green fluorescent protein. No effects on CD4, CD8a, CD163 and MPO expression were noticed in bcl-2 pretreated livers, whereas Igbeta was slightly enhanced compared to controls. Adenoviral infected liver grafts trigger an immune response but reduced apoptosis resulted in down-regulation of TNF-alpha. Thus, bcl-2 transfer might simultaneously reduce graft ischemia reperfusion injury and immunogenicity.

Ig beta tyrosine residues contribute to the control of B cell receptor signaling by regulating receptor internalization.

Immunoglobulin (Ig)α and Igβ initiate B cell receptor (BCR) signaling through immune receptor tyrosine activation motifs (ITAMs) that are targets of SH2 domain–containing kinases. To examine the function of Igβ ITAM tyrosine resides in mature B cells in vivo, we exchanged these residues for alanine by gene targeting (IgβAA). Mutant mice showed normal development of all B cell subtypes with the exception of B1 cells that were reduced by fivefold. However, primary B cells purified from IgβAA mice showed significantly decreased steady-state and ligand-mediated BCR internalization and higher levels of cell surface IgM and IgD. BCR cross-linking resulted in decreased Src and Syk activation but paradoxically enhanced and prolonged BCR signaling, as measured by cellular tyrosine phosphorylation, Ca++ flux, AKT, and ERK activation. In addition, B cells with the ITAM mutant receptor showed an enhanced response to a T-independent antigen. Thus, Igβ ITAM tyrosines help set BCR signaling threshold by regulating receptor internalization.

IL-6 Induced CD45RO, RB Translocation to Lipid Rafts in Myeloma Cells.

We have previously reported that CD45 functions as a positive regulator for myeloma cell proliferation in the downstream of interleukin-6 (IL-6) by regulating activation of Lyn (Blood 99: 2172, 2002). It remains to be clarified how CD45 regulates IL-6-induced proliferation in myeloma cells. In this study, we aim to explore the regulation and isoform-specific difference on the mechanism of CD45 translocation induced by IL-6 in human myeloma cells. We used myeloma cell lines, U266 and ILKM2, which express CD45RO and CD45RB but not CD45RA in this study. We showed that methyl-beta-cyclodextrin, a lipid rafts inhibitor, significantly inhibited IL-6 induced proliferation, diminished phosphorylation of Lyn and signal transducer and activator of transcription 3 (STAT3), and abolished nuclear localization of STAT3, suggesting the significant role of lipid rafts on IL-6 signaling. Using sucrose density gradient fractionation method, we observed the dramatic change of CD45 distribution by IL-6 stimulation. Without IL-6 stimulation, CD45RO and RB molecules were excluded from lipid rafts. But, IL-6 stimulation results in significant translocation of both isoforms to lipid rafts. On the other hand, IL-6R alpha, gp130, Lyn and a raft integral protein, flotillin-2, were always localized inside lipid rafts. Furthermore, it was also confirmed that IL-6 induced CD45RO and CD45RB translocation to lipid rafts by confocal laser scanning microscopy, while IL-6R alpha always remained inside rafts. These observations have also been confirmed by the exogenous expression of CD45RO, RB tagged with green fluorescent protein in CD45 negative U266 cells. As negative control, Epstein-Barr Virus transformed B cells, C066 or KUS, which express CD45RA and CD45RB, but not CD45RO, were used. BCR cross-linking using anti-IgM stimulation induced IgM and Ig beta translocation to lipid rafts, but both CD45RB and RA showed no marked translocation. And we couldn't observe marked translocation of CD45RB nor CD45RA to rafts after IL-6 stimulation. As the level of IL-6R alpha was very low in these cells, we introduced IL-6R alpha gene into KUS or C066 cells. Then, translocation of CD45RB to lipid rafts was observed while CD45RA did not in response to IL-6 stimulation. In addition, we confirmed IL-6 induced complex formation of CD45 with IL-6R alpha, Lyn on lipid rafts. We also checked the phosphorylation state of two tyrosine residues (Tyr396 and Tyr507) of Lyn. In CD45 positive cells, IL-6 induced dephosphorylation of the negative regulatory tyrosine, Tyr507, after that, Tyr396, the positive regulatory site of kinase activity, was phosphorylated. While, in CD45 negative cells, Tyr507 dephosphorylation and significant phosphorylation of Tyr396 were not observed. And after IL-6 stimulation, we also observed PAG dephosphorylation and Csk release from lipid rafts, which might finally lead to the dephosphorylation of Tyr507 of Lyn. Considering these results, we conclude that CD45RO and RB translocation to lipid rafts is significant for myeloma cells to proliferate in response to IL-6.

A conserved sequence upstream of the B29 (Ig beta, CD79b) gene interacts with YY1.

The human, murine, and rat B29 (Ig beta, CD79b) genes are highly conserved in sequence and organization and exhibit strict B cell-specific expression. In the human and rat genomes, the B29 gene is located between the skeletal muscle-specific Na-channel alpha subunit (SCN4A) gene and the pituitary-specific growth hormone (GH-N) gene. The human pituitary-specific GH-N gene is controlled by a tissue-specific locus control region (LCR) located just upstream of the B29 promoter that mediates tissue-specific enhancement, histone acetylation, and an open chromatin conformation across the B29 gene in growth hormone (GH)-expressing pituitary cells. Here we show that B29 mRNA is not detected in a GH-expressing pituitary cell line and that GH-N mRNA is not detected in B cells. This differential expression suggests that the B29 gene is insulated or otherwise protected from the regulatory influences of the closely proximal GH LCR. We searched available sequences upstream of the human, mouse, and rat B29 genes and found a highly conserved sequence that fulfills the criteria recently established for non-coding DNA elements potentially involved in gene control. This B29 conserved sequence (BCS) bound ubiquitously expressed nuclear protein complexes. DNase I protection analysis of the BCS revealed a central 'footprinted' core which was confirmed to bind the multifunctional transcription factor, YY1. However, neither the BCS nor the YY1-binding core motif exhibited silencer or enhancer activity in transient transfections or position-independent insulator activity in enhancer-blocking assays. Thus, the BCS may function as a tissue-specific LCR or position-dependent insulator specifically countering the influences of the 5' GH LCR and controlling B29 gene expression.

The variable, C(H)1, C(H)2 and C(H)3 domains of Ig heavy chain are dispensable for pre-BCR function in transgenic mice.

The pre-BCR consists of Ig micro protein, the product of a heavy chain gene assembled by V(D)J recombination in pro-B cells, the surrogate light chains V(pre-B) and lambda 5, and the signaling chains Ig alpha and Ig beta. Signaling by the pre-BCR is a checkpoint required for further maturation of pro-B cells in the adult bone marrow. However, it is currently not known whether an extracellular ligand is required to initiate pre-BCR signaling. We reasoned that if the ectodomain of the pre-BCR is required to interact with a ligand, then a truncated heavy chain protein would not support B cell development. To test this notion, we produced transgenic mice expressing a heavy chain protein whose extracellular domains except for C(H)4 were replaced by an irrelevant Ig superfamily ectodomain from the human CD8 alpha protein. This transgene resulted in pre-BCR-like signaling since it rescued development of pre-B cells in recombinase-activating gene (RAG)1-deficient mice and resulted in allelic exclusion of the endogenous Ig heavy chain gene in RAG-proficient mice. These findings lead us to suggest that the majority of the extracellular region of the pre-BCR is not required for pre-BCR function and, thus, ligand binding is unlikely to be required for pre-BCR function.

Bob1 (OCA-B/OBF-1) differential transactivation of the B cell-specific B29 (Ig beta) and mb-1 (Ig alpha) promoters.

The B29 (Igbeta) and mb-1 (Igalpha) gene products are B cell-specific essential components of the B cell receptor that are coexpressed at all stages of B cell differentiation, with the exception of plasma cells, which lack mb-1 expression. Transcription of both genes is governed by a similar cassette of interactive transcription factor-binding elements, including octamer motifs, in TATA-less promoters. In this study, we show the B cell-specific B29 gene promoter is transactivated in B and non-B cells by cotransfection with the B cell-specific octamer cofactor gene, Bob1 (OCA-B/OBF-1). The expression of Bob1 is also sufficient to override the silencing effects of the B29 silencer. This indicates that Bob1 plays a critical role in B cell-specific B29 promoter expression. In contrast, coexpression of Bob1 had no effect on mb-1 promoter activity. Bob1 transactivation only occurs with select octamer sequences that have an adenosine at position 5 (ATGCAAAT). The B29 promoter conforms to this consensus octamer motif, while the mb-1 promoter octamer motif does not. Octamer motif swapping between B29 and mb-1 promoters renders B29 unresponsive to Bob1 transactivation and makes mb-1 competent for Bob1 transactivation, thereby indicating that the B29 octamer motif is solely responsible for Bob1 interaction. Additionally, the mb-1 construct containing the B29 octamer motif is expressed in a plasmacytoma cell line, while the wild-type mb-1 promoter is not. Bob1 transactivation of B29 and the lack of this transactivation of mb-1 account for the differential expression of B29 and mb-1 in terminally differentiated plasma cells.

Structure of the β-chain (B29) gene of the chicken B-cell receptor and conserved collinearity with genes for potential skeletal muscle sodium channel and growth hormone

Abstract. Reported here is the cloning of the gene for the beta-chain (Ig beta) of the chicken B-cell receptor for antigen. A cDNA library made from B lymphocyte-derived DT40 cells was first screened with a beta-chain cDNA clone from rat. A full-length clone (1556 bp) was obtained encoding a 226-amino-acid beta-chain having 40% sequence identity with the highly conserved beta-chains present in mammals and producing mRNA of 1.7 kb. Clones containing the full B29 gene encoding chicken Ig beta were isolated and fully sequenced. As in mammals, the chicken B29 gene consists of six exons. The chicken B29 gene is somewhat larger (4336 bp) than that of rat (3.1 kb) and human (3.6 kb). Genome collinearity was found in the region of the chicken B29 gene. Genes for potential skeletal muscle sodium channel (2.2 kb upstream) and for growth hormone (2.3 kb downstream) were found in the same order and transcriptional orientation as in mammals, though the genes are more closely positioned in the chicken.

PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting src homology 2-domain-containing tyrosine phosphatase 2 to phosphotyrosine.

PD-1 is an immunoreceptor that belongs to the immunoglobulin (Ig) superfamily and contains two tyrosine residues in the cytoplasmic region. Studies on PD-1-deficient mice have shown that PD-1 plays critical roles in establishment and/or maintenance of peripheral tolerance, but the mode of action is totally unknown. To study the molecular mechanism for negative regulation of lymphocytes through the PD-1 receptor, we generated chimeric molecules composed of the IgG Fc receptor type IIB (Fc gamma RIIB) extracellular region and the PD-1 cytoplasmic region and expressed them in a B lymphoma cell line, IIA1.6. Coligation of the cytoplasmic region of PD-1 with the B cell receptor (BCR) in IIA1.6 transformants inhibited BCR-mediated growth retardation, Ca(2+) mobilization, and tyrosine phosphorylation of effector molecules, including Ig beta, Syk, phospholipase C-gamma 2 (PLC gamma 2), and ERK1/2, whereas phosphorylation of Lyn and Dok was not affected. Mutagenesis studies indicated that these inhibitory effects do not require the N-terminal tyrosine in the immunoreceptor tyrosine-based inhibitory motif-like sequence, but do require the other tyrosine residue in the C-terminal tail. This tyrosine was phosphorylated and recruited src homology 2-domain-containing tyrosine phosphatase 2 (SHP-2) on coligation of PD-1 with BCR. These results show that PD-1 can inhibit BCR signaling by recruiting SHP-2 to its phosphotyrosine and dephosphorylating key signal transducers of BCR signaling.

Antibody regulation of B cell development.

Antibodies on the surface of B lymphocytes trigger adaptive immune responses and control a series of antigen-independent checkpoints during B cell development. These physiologic processes are regulated by a complex of membrane immunoglobulin and two signal transducing proteins known as Ig alpha and Ig beta. Here we focus on the role of antibodies in governing the maturation of B cells from early antigen-independent through the final antigen-dependent stages.

An upstream Oct-1- and Oct-2-binding silencer governs B29 (Ig beta) gene expression.

The B cell-specific B29 (Igbeta) gene is activated in the earliest B cell precursors and is expressed throughout B cell development. Tissue-specific expression of the murine B29 gene is controlled by a B cell-specific promoter whose activity is governed by a cassette of upstream transcriptional silencers. This study describes a potent new silencer that is located 5' of the previously identified B29 silencer elements, FROG and TOAD. Like these known elements, the new B29 silencer is not restricted to the B29 promoter. Nuclear proteins from all cell lines tested interacted with this A+T-rich sequence, which closely resembled a noncanonical octamer binding motif and also conformed to the consensus sequence for nuclear matrix attachment regions. Interaction of Oct-1 and Oct-2 with the B29 A+T-rich sequence was confirmed using octamer-specific Abs. Oct-1/Oct-2 binding was required for the inhibitory activity of this sequence because mutations that blocked Oct-1/Oct-2 binding also eliminated inhibition of the B29 promoter. This B29 A+T-rich sequence specifically interacted with isolated nuclear matrix proteins in vitro, suggesting that it may also function as a matrix attachment region element. Maintenance of the level of B29 gene expression through the interaction of the minimal promoter and the upstream silencer elements FROG, TOAD, and the A+T-rich Oct-1/Oct-2 binding motif may be essential for normal B cell development and/or function.

Tyrosine kinase activation in the decision between growth, differentiation, and death responses initiated from the B cell antigen receptor

Immunoglobulin-containing receptors expressed on B lineage lymphocytes play critical roles in the development and function of the humoral arm of the immune system. The preB cell antigen receptor (preBCR) contains the immunoglobulin mu heavy chain (Ig mu) and signals to the preB cell that heavy chain rearrangement has been successful, a process termed heavy chain selection. The B cell antigen receptor (BCR) contains both Ig heavy and light chains and is expressed on immature and mature B cells before and after antigen encounter. Both receptor types from a complex with the Ig alpha and Ig beta proteins that link the predominantly extracellular Ig with intracellular signal transduction pathways. Signaling through the BCR induces different cellular responses depending on the nature of the signaling agent and the development stage of the target cell. These responses include clonal anergy and apoptotic deletion in immature B cells and survival, proliferation, and differentiation in mature B and preB cells. Several protein tyrosine kinases are activated rapidly following engagement of the BCR/preBCR complexes, including members of the Src family (Lyn and Blk), the Syk/ZAP70 family (Syk), and the Tec family (Btk). In this review, we discuss possible mechanisms by which engagement of these similar receptor complexes can give rise to different cellular responses and the role that these kinases play in this process.

CD38 ligation in human B cell progenitors triggers tyrosine phosphorylation of CD19 and association of CD19 with lyn and phosphatidylinositol 3-kinase.

CD38 is a 45-kDa transmembrane glycoprotein highly expressed in lymphoid progenitors. Ligation of CD38 with specific Abs inhibits growth and induces apoptosis in human immature B cells. CD38 ligation also triggers tyrosine phosphorylation of syk, c-cbl, and phospholipase C-gamma and activates phosphatidylinositol 3-kinase (PI3-K). In the present study, we investigated whether the cell surface membrane molecules used in B cell receptor-mediated signaling, such as Ig alpha, Ig beta, and CD19, could be involved in the CD38-mediated signaling cascade. In the B cell receptor-negative immature B cell lines RS4;11, 380, and REH, Ig alpha and Ig beta were expressed exclusively in the cytoplasm and were not tyrosine phosphorylated after CD38 ligation. By contrast, CD19 was markedly tyrosine phosphorylated and was associated with lyn and PI3-K. PI3-K activation appears to be directly linked to the growth-arresting effects of CD38 ligation, which are reduced by PI3-K inhibitors. Ligation of either CD38 or CD19 resulted in a similar pattern of protein tyrosine phosphorylation; both signaling pathways caused tyrosine phosphorylation of c-cbl. Levels of CD38 surface expression were not affected by prolonged incubation with anti-CD19 Ab, while CD19 expression markedly decreased. These results indicate that CD19 is a major component of the CD38 signaling cascade in B cell precursors, serving as a cell surface membrane docking site for cytoplasmic kinases. CD38 and CD19 are not physically linked, but activate an overlapping set of kinases in human immature B cells.

B cell antigen receptor desensitization: disruption of receptor coupling to tyrosine kinase activation.

Antigen binding to the B cell receptor (BCR) induces receptor desensitization, a condition characterized by cellular unresponsiveness to subsequent Ag stimulation despite the continued ability to bind Ag. To better understand the molecular mechanism of this unresponsiveness, we have used complementary lymphoma (K46 mu) and Ig transgenic (3-83 mu delta) mouse models to study regulation of BCR signaling. Our findings in the lymphoma model show that an initial Ag encounter renders receptors unresponsive to subsequent Ag challenge, as measured by their inability to mobilize Ca2+ and to mediate phosphorylation of receptor-proximal kinases, including Lyn, Blk, and Syk. Most importantly, the Ig alpha and Ig beta components of desensitized receptors are not phosphorylated, and receptor-associated kinases are not activated upon Ag challenge. The molecular defect does not appear to result from Lyn inactivation, sequestration, or repression, since Lyn from desensitized cell lysates is activated in vitro by synthetic doubly phosphorylated immunoreceptor tyrosine-based activation motif peptides. A similar deficit in Ag-induced receptor phosphorylation was observed in desensitized B cells from 3-83 mu delta transgenic mice. These studies indicate that Ag receptor desensitization reflects an inability to initiate activation of receptor-associated kinases that normally phosphorylate receptor Ig alphabeta subunits, leading to signal propagation.

The roles of preB cell receptor in early B cell development and its signal transduction.

The preB cell receptor is expressed for a short period after mu heavy chain is produced, that is, at the large preB cell stage in B cell development. The severe impairment of B cell differentiation observed in mice deficient for the preB cell receptor clearly demonstrated the importance of the preB cell receptor in B cell development. Analyses of bone marrow precursor B cells in normal and B cell-deficient mutant mice indicated the preB cell receptor transduced signals to drive cell cycle and to induce allelic exclusion. The proliferation of the preB cell receptor-expressing cells leads to the selective expansion of cells which have succeeded in the productive rearrangement of mu heavy chain gene. This process builds up a preB cell pool large enough to generate sufficient numbers of mature B cells. The preB cell receptor appears to induce allelic exclusion by shutting off the expression of recombinase activation gene (RAG). In order to analyse the signal transduction pathway downstream of the preB cell receptor, we have developed a new system in which cross-linking of Ig beta expressed on bone marrow proB cells mimics the signalling through the preB cell receptor to induce differentiation from proB to small preB cells.

Counterselection against D mu is mediated through immunoglobulin (Ig)alpha-Igbeta.

The pre-B cell receptor is a key checkpoint regulator in developing B cells. Early events that are controlled by the pre-B cell receptor include positive selection for cells express membrane immunoglobulin heavy chains and negative selection against cells expressing truncated immunoglobulins that lack a complete variable region (D mu). Positive selection is known to be mediated by membrane immunoglobulin heavy chains through Ig alpha-Ig beta, whereas the mechanism for counterselection against D mu has not been determined. We have examined the role of the Ig alpha-Ig beta signal transducers in counterselection against D mu using mice that lack Ig beta. We found that D mu expression is not selected against in developing B cells in Ig beta mutant mice. Thus, the molecular mechanism for counterselection against D mu in pre-B cells resembles positive selection in that it requires interaction between mD mu and Ig alpha-Ig beta.

Signaling through CD38 augments B cell antigen receptor (BCR) responses and is dependent on BCR expression.

mAbs directed against the ectoenzyme CD38 will induce B cell proliferation in normal resting B lymphocytes, but cannot induce proliferation in B cells that are unresponsive to B cell Ag receptor (BCR) cross-linking. Using the CD38- murine B cell line A20 we have examined the relationship between CD38- and BCR-mediated signaling after transfection of wild-type or mutant CD38 molecules. Although association between CD38 and the BCR was not detectable, co-cross-linking of CD38 and the BCR gave rise to a synergistic response, and expression of CD38 lowered the threshold for BCR-induced responses. Generation of Ig loss variant clones established that coexpression of the BCR was required for CD38-mediated signal transduction. The cytoplasmic tail of Ig alpha or Ig beta rescued CD 38 responsiveness in the CD38+Ig- cells provided that the chimeric molecules were coligated with CD38. Separate experiments indicated that the cytoplasmic tail of CD38 is not required for CD38 signaling. The anti-CD38-induced response was dependent on the influx of extracellular calcium but was not accompanied by detectable tyrosine phosphorylation of any cellular proteins. Together, these data demonstrate that the CD38 molecule can influence BCR-induced responses and that CD38 signaling is dependent on the BCR complex, perhaps to utilize a functional cytoplasmic tail(s) for intracellular signaling.

Regulation of an Early Developmental Checkpoint in the B Cell Pathway by Igβ

Many of the cell fate decisions in precursor B cells and more mature B cells are controlled by membrane immunoglobulin (Ig)M heavy chain (mu) and the Ig alpha-Ig beta signal transducers. The role of Ig beta in regulating early B cell development was examined in mice that lack Ig beta (Ig beta-/-). These mice had a complete block in B cell development at the immature CD43+B220+ stage. Immunoglobulin heavy chain diversity (DH) and joining (JH) segments rearranged, but variable (VH) to DJH recombination and immunoglobulin messenger RNA expression were compromised. These experiments define an unexpected, early requirement for Ig(beta) to produce B cells that can complete VDJH recombination.