B Cell Antigen Receptor Cross-linking Research Articles

Prolactin Rescues Immature B-Cells from Apoptosis Induced by B-Cell Receptor Cross-Linking.

Prolactin has an immunomodulatory effect and has been associated with B-cell-triggered autoimmune diseases, such as systemic lupus erythematosus (SLE). In mice that develop SLE, the PRL receptor is expressed in early bone marrow B-cells, and increased levels of PRL hasten disease manifestations, which are correlated with a reduction in the absolute number of immature B-cells. The aim of this work was to determine the effect of PRL in an in vitro system of B-cell tolerance using WEHI-231 cells and immature B-cells from lupus prone MRL/lpr mice. WEHI-231 cells express the long isoform of the PRL receptor, and PRL rescued the cells from cell death by decreasing the apoptosis induced by the cross-linking of the B-cell antigen receptor (BCR) as measured by Annexin V and active caspase-3. This decrease in apoptosis may have been due to the PRL and receptor interaction, which increased the relative expression of antiapoptotic Bcl-xL and decreased the relative expression of proapoptotic Bad. In immature B-cells from MRL/lpr mice, PRL increased the viability and decreased the apoptosis induced by the cross-linking of BCR, which may favor the maturation of self-reactive B-cells and contribute to the onset of disease.

Protein kinase Cβ is critical for the metabolic switch to glycolysis following B-cell antigen receptor engagement

Signals derived from the BCR (B-cell antigen receptor) control survival, development and antigenic responses. One mechanism by which BCR signals may mediate these responses is by regulating cell metabolism. Indeed, the bioenergetic demands of naïve B-cells increase following BCR engagement and are characterized by a metabolic switch to aerobic glycolysis; however, the signalling pathways involved in this metabolic reprogramming are poorly defined. The PKC (protein kinase C) family plays an integral role in B-cell survival and antigenic responses. Using pharmacological inhibition and mice deficient in PKCβ, we demonstrate an essential role of PKCβ in BCR-induced glycolysis in B-cells. In contrast, mice deficient in PKCδ exhibit glycolytic rates comparable with those of wild-type B-cells following BCR cross-linking. The induction of several glycolytic genes following BCR engagement is impaired in PKCβ-deficient B-cells. Moreover, blocking glycolysis results in decreased survival of B-cells despite BCR engagement. The results establish a definitive role for PKCβ in the metabolic switch to glycolysis following BCR engagement of naïve B-cells.

Dasatinib inhibits B cell receptor signalling in chronic lymphocytic leukaemia but novel combination approaches are required to overcome additional pro‐survival microenvironmental signals

As antigenic stimulation of the B cell antigen receptor (BCR) is key to chronic lymphocytic leukaemia (CLL) pathogenesis, targeting dysregulated kinases involved in BCR signalling is an attractive therapeutic approach. We studied the effects of the Src/c-Abl tyrosine kinase inhibitor dasatinib on BCR signal transduction in CLL cells. Treatment of CLL cells with 100 nmol/l dasatinib induced apoptosis by an average reduction in viability of 33·7% at 48 h, with dasatinib sensitivity correlating with inhibition of Syk(Y348) phosphorylation. Dasatinib inhibited calcium flux, phosphatidylinositol-3-kinase and mitogen-activated protein kinase activation following BCR crosslinking, and blocked the Mcl-1-dependent increase in CLL cell survival on prolonged BCR stimulation. However, the pro-apoptotic effect of dasatinib was abrogated by stromal cell contact alone or in the presence of CD154 and interleukin (IL)-4 (CD154L/IL-4 system). Whilst dasatinib retained the ability to sensitize CLL cells in stromal co-culture to both fludarabine and chlorambucil, the addition of CD154 and IL-4 rendered cells resistant to these drug combinations. We demonstrate that the HSP90 inhibitor 17-DMAG exhibited synergy with dasatinib in vitro, and moreover, induced apoptosis of CLL cells in the CD154L/IL-4 system. Our data provide evidence that dasatinib would be most clinically effective in combination with agents able to target antigen-independent microenvironmental signals.

NF-κB Signaling In Response to CpG Stratifies Mantle Cell Lymphoma Patient Outcome

AbstractAbstract 144 Introduction:Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin's lymphoma. Although newer treatment regimens including intensive chemo-immunotherapy followed by stem cell transplant have improved survival to a median approaching 6 years, MCL is still incurable in many cases. The use of flow cytometry to investigate stimulation induced signaling at the single cell level represents an opportunity to obtain each patient's signaling profile as well as to discover tumor cell heterogeneity and identify signaling events potentially responsible for poor outcomes. We recently used this method to characterize signaling in subpopulations of tumor samples from patients with follicular lymphoma (FL). In FL, we identified a lymphoma negative prognostic (LNP) cell subset with impaired B cell antigen receptor (BCR) signaling, the prevalence of which correlated with adverse clinical outcome (Irish et al., PNAS 2010). In the present study we used the same approach to identify signaling responses with large variation within MCL patient samples, and tested whether these signaling events might predict patients' clinical outcomes. By understanding the biology based on lymphoma signaling profile, we might identify prognostic significant factors such as the LNP subset observed in FL. Method:Single cell flow cytometry measurements of signaling were acquired for samples of MCL (n=25). Of these, 21 MCL specimens were obtained prior to any treatment. Median age was 61 years, and follow up time ranged from ½ until 15 years. Treatment regimens varied. Samples from diffuse large B cell lymphoma (DLBCL, n=12), FL (n=14), chronic lymphocytic leukemia (CLL, n=14) and tonsils from healthy donors (n=4) were also included for comparison. Phosphorylation of 14 signaling proteins was measured under 12 different stimulation conditions in every cell within the patient specimens, including lymphoma B cells and in tumor-infiltrating T cells. Stimulation conditions included BCR crosslinking, CD40 ligand, CpG oligodeoxynucleotides (CpG ODN) and several cytokines (IL-4, IL-10, IL-21, IFN-γ). Result:The magnitude of most cytokine induced signaling responses in lymphoma cells from MCL patients was higher than in lymphoma cells from FL. This included IL-10 induced phosphorylation of STAT3 (p<0.0001) and IL-4 induced phosphorylation of STAT6 (p<0.0001). In contrast, IL-21 induced phosphorylation of STAT3 in lymphoma cells was similar in FL and MCL patients. Furthermore, MCL tumor cells responded better to BCR crosslinking at 9 measured downstream signaling proteins, including SYK, Src family kinases (SFK) and PLC-γ, than tumor cells from FL and CLL patients. Although signaling responses overall were higher in MCL, there was a large variation in the lymphoma cells' signaling capacity within each lymphoma histology. To identify possible signaling events that correlated with clinical outcome in MCL, we first identified the signaling events with the largest variance. This included phosphorylation of SYK and PLC-γ by BCR crosslinking, phosphorylation of NF-κB (p65) induced by CpG ODN, CD40L, or by PMA and ionomycin, and phosphorylation of STAT3 induced by IL-10 and IL-21. Of these seven signaling events, only CpG ODN induced p-p65 significantly stratified overall survival when MCL patients were split into two even groups based on higher and lower signaling response, compared to healthy B cells from tonsils. Hence, patients whose MCL cells displayed high CpG ODN induced NF-κB (p65) signaling had an adverse prognosis (p<0.008, HR=5.1). In contrast with our observations of FL, BCR signaling did not stratify the outcome of MCL patients. Conclusion:Potentiated NF-kB p65 signaling in response to CpG ODN was associated with poor prognosis in MCL. The tumor cells capacity to respond to CpG induced signaling likely represents a biological advantage for the tumor cells in this lymphoma entity. Disclosures:No relevant conflicts of interest to declare.

A Subpopulation of Follicular Lymphoma Tumor Infiltrating T Cells Shows Suppressed Common Gamma Chain Cytokine Signaling.

Abstract 759 Introduction:Tumor infiltrating T cells present within biopsy specimens of human B cell non-Hodgkin's lymphomas (NHL) provide a valuable opportunity to examine immune system function in the presence of cancer. We recently used flow cytometry to characterize signaling in subpopulations of tumor samples from patients with follicular lymphoma (FL). In FL, we identified a novel lymphoma cell subset with impaired B cell antigen receptor (BCR) signaling, the prevalence of which correlated with adverse clinical outcome. Here, we turned our attention to signaling differences in subsets of the tumor-infiltrating T cells from FL and two other NHLs, diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). Signaling differences that distinguish the tumor infiltrating T cells from each malignancy might be expected to be a reflection of the specific disease microenvironment, whereas T cell signaling differences distinguishing cases of the same malignancy might be related to the biology of each patient's tumor. Methods:Single cell flow cytometry measurements of signaling were acquired for samples of DLBCL (N=13), MCL (N=20), and FL (N=14). Phosphorylation of 14 signaling proteins was measured under 12 stimulation conditions in every cell, including lymphoma B cells and tumor-infiltrating T cells within the same specimen. Stimulation conditions included those that were B cell specific (BCR crosslinking, CD40 ligand), T cell specific (IL-7), and those that stimulated both B and T cells (IL-4, IL-10, IL-21, PMA + ionomycin, and IFN-γ). Results:Striking differences were observed in the signaling responses of tumor infiltrating T cells. T cells infiltrating FL patient samples showed significantly lower responses to cytokines where signal transduction is mediated by the common γ chain receptor. Specifically, we observed significant lower induction of p-STAT6 after IL-4 stimulation, p-STAT5 after IL-7 stimulation, and p-STAT3 after IL-21 stimulation (p < 0.001 for FL vs. MCL in all cases). In contrast, receptor-independent signaling was not significantly different as FL tumor infiltrating T cells responded at a level comparable to MCL and DLBCL tumor infiltrating T cells when stimulated with PMA and ionomycin. The lower response to common γ chain family cytokines could be the result of a partial suppression of all tumor infiltrating T cells or a complete suppression of a distinct subset. To distinguish between these possibilities, we analyzed signaling in tumor infiltrating T cell subsets. This single cell approach showed that tumor infiltrating T cells were a heterogeneous mixture of non-responsive cells and highly responsive T cells in response to cytokines. Specifically, the mean percentage of T cells that did not induce p-STAT3 after IL-21 stimulation was 50.3% in FL samples in contrast to only 26.2% in MCL samples. Phenotypic analysis showed that the vast majority of T cells infiltrating FL patient samples were CD4+CD45RO memory cells, and the single cell signaling approach revealed that the FL nonresponsive T cell subset had this phenotype. Furthermore, FL T cells were composed of a significantly larger fraction of T regulatory cells than MCL T cells, on average 17% FoxP3+CD25+ cells compared to only 9% in MCL (p<0.0002). Experiments are ongoing to test whether the prevalence of T regulatory cells influence the signaling capacity of the remaining CD4 conventional T cells. Conclusions:A subpopulation of tumor infiltrating T cells within FL patient samples has reduced responsiveness to the common gamma chain family members IL-4, IL-7 and IL-21, and distinguishes FL from DLBCL and MCL. These results may reflect a more suppressive microenvironment in FL. Disclosures:No relevant conflicts of interest to declare.

Dasatinib-Induced Apoptosis in Chronic Lymphocytic Leukaemia Is Abrogated by Selective Micro-Environmental Signals.

Abstract 1264Poster Board I-286 BackgroundRecent advances in chronic lymphocytic leukaemia (CLL) biology indicate that a plethora of micro-environmental factors are important in promoting the pathogenesis of the disease. As B cell antigen receptor (BCR) signalling plays a critical role in CLL progression, we chose to study the effect of dasatinib, a dual src/abl tyrosine kinase inhibitor, on CLL cells, thus targeting the key, BCR-proximal tyrosine kinases Lyn and c-Abl that are over-expressed in CLL. We were interested to determine the impact of BCR-signalling directed therapy on CLL cell viability in the context of the leukaemic micro-environment. ResultsClinically relevant concentrations of dasatinib (100 nM) both inhibited ERK-MAPK and Akt phosphorylation, and prevented calcium mobilisation following BCR crosslinking. Furthermore, dasatinib prevented up-regulation of Mcl-1 observed on prolonged BCR stimulation, abrogating the BCR-driven increase in CLL cell survival. Dasatinib induced apoptosis of CLL cells, with a mean reduction in viability of 35.2% ± 3.8% following 48 hr exposure (n=26). Neither ZAP-70 expression nor cytogenetic abnormalities were predictive of response. In addition, dasatinib exhibited synergy with both fludarabine and chlorambucil, with mean ED50 combination indices of 0.29 and 0.62 respectively. Additional micro-environmental elements responsible for the maintenance and progression of CLL include stromal or ‘nurse-like' cells and activated T lymphocytes, the latter of which express CD40 ligand and secrete IL-4. CLL cell co-culture with the murine bone marrow stromal cell line NT-L significantly inhibited both spontaneous and dasatinib induced apoptosis. As we observed that stromal co-culture significantly increased phosphorylation of both ERK-MAPK and the Akt/mTOR target p70 S6 kinase in CLL cells, we investigated whether pharmacological inhibition these signalling pathways may re-sensitise CLL cells to dasatinib in co-culture. Both the MEK inhibitor PD98059 and PI3K inhibitor LY294002 significantly increased the level of apoptosis observed on dasatinib treatment of CLL cells in stromal co-culture. Dasatinib also retained the ability to potentiate the effects of fludarabine and chlorambucil in stromal co-culture, however, CLL cells co-cultured with NT-L cells stably transfected with CD40 ligand (CD154L cells) were resistant to all drug combinations. Furthermore, on CD154L/IL-4 co-culture, dasatinib failed to prevent up-regulation of Bcl-xL and Mcl-1, or inhibit CLL cell proliferation. Conclusions:While dasatinib was capable of reversing BCR-mediated survival signals, it was unable to overcome stromal-derived survival signals, indicating that combination of dasatinib with agents targeting antigen-independent micro-environmental pathways are required to effectively target CLL cells in tissue niches. DisclosuresNo relevant conflicts of interest to declare.

Interdomain A is crucial for ITAM-dependent and -independent regulation of Syk

Non-receptor type protein tyrosine kinase (PTK) Syk is essential for the signaling via the B cell antigen receptor (BCR). Upon BCR crosslinking, Syk is recruited via its tandem SH2 domains to tyrosine-phosphorylated Ig-α/Ig-β constituting components of BCR, and is then activated. The interdomain A lying between the two SH2 domains is highly conserved among different species of Syk and between Syk and ZAP-70. The mutant Syk carrying a deletion in the interdomain A (Δ140–159) became phosphorylated regardless of BCR ligation and did not induce Ca 2+ mobilization upon crosslinking of BCR. Furthermore, in vitro binding assay revealed that deletion of a part of the interdomain A abolished its binding activity to phosphorylated Ig-α/Ig-β. These results indicate that the interdomain A of Syk is required for activation of Syk by binding to the phosphorylated Ig-α/Ig-β upon BCR ligation and inhibition of spontaneous activation at the resting state.

Signaling from the B cell antigen receptor (BCR) induces VH replacement in human immature B cells (B33)

Abstract VH replacement occurs through RAG-mediated secondary IgH recombination between a cryptic RSS (cRSS) from a previously rearranged VH gene and a 23-bp RSS from an upstream VH gene. Our previous studies have shown that VH replacement occurs in human bone marrow immature B cells and contributes to about 5% of the primary B cell repertoire. However, how VH replacement is regulated is not clear. The human EU12 cells, which undergo spontaneous VH replacement, are sensitive to BCR stimulation. BCR crosslinking strongly induces double stranded DNA breaks at the VH3 cRSS border in the μHC+ EU12 cells as measured by semi quantitative ligation-mediated PCR (LM-PCR) and real time LM-PCR, suggesting the induction of VH replacement. The VH3 to VH1 VH replacement excision circles are also induced upon BCR crosslinking. In addition, ongoing VH replacement was found in the newly emigrated immature B cells from peripheral blood of three healthy donors, which can be further induced by BCR crosslinking. These results provide the first evidence that VH replacement in human immature B cells can be regulated by BCR mediated signaling.

Differential regulation of NF-κB survival signaling in transitional and mature Fo B cells (86.13)

Abstract The earliest transitional (T1) B cells are significantly more sensitive to cell death than the late transitional (T2) and mature follicular (Fo) B cells and aberrant survival of T1 B cells can lead to autoimmune disease. While it is known that survival of splenic B cells is predominantly regulated by B cell antigen receptor (BCR) and B cell activation factor receptor (BAFF-R), the mechanisms by which these receptors mediate survival is less clear. We have investigated the developmental stage-specific regulation of anti-apoptotic genes and the transcription factor NF-κB, which is a major modulator of these genes. We found that in vivo, T1 B cells express the highest levels of Mcl-1 and Bcl-xL, whereas mature Fo B cells predominantly express Bcl-2. Consistent with T1 cell sensitivity to cell death following BCR crosslinking, T1 B cells only weakly induced the expression of anti-apoptotic genes (Mcl-1 and Bcl-xL) relative to robust induction in T2 and mature Fo B cells following BCR stimulation. BCR-induced expression of NF-κB (NF-κB1, RelA, cRel, NF-κB2 and RelB) coincides with that of anti-apoptotic genes; lowest in T1 and highest in mature Fo B cells. These findings suggest that T2 and mature Fo B cell survival may be linked to sustained expression of NF-κB and anti-apoptotic genes. This study is supported by NIH RO1 A1060729

B-cell receptor signaling in chronic lymphocytic leukemia cells is regulated by overexpressed active protein kinase CβII

AbstractSignals through the B-cell antigen receptor (BCR) are important for the survival of chronic lymphocytic leukemia (CLL) cells. Therefore, factors that influence these signals have important pathophysiological roles in this disease. One key mediator of BCR signaling is protein kinase C β (PKCβ), which regulates the activation of I-κB kinases and the deactivation of Bruton tyrosine kinase within the signaling pathways initiated by BCR engagement. The present study demonstrates that overexpression of the PKCβII isoform is a feature of CLL cells and that activity of this enzyme strongly correlates with CLL cell response to BCR engagement. Thus, intracellular Ca2+ release and increases in cell survival after BCR cross-linking were significantly greater in CLL patients with low levels than in CLL patients with high levels of active PKCβII. Furthermore, BCR-induced Ca2+ fluxes could be restored in CLL patients with high levels of active PKCβII by pretreating the cells with the PKCβ-specific inhibitor LY379196. Conversely, BCR-mediated intracellular Ca2+ release could be inhibited in CLL cells with low levels of active PKCβII by pretreatment with the PKC agonist bryostatin. Taken together, these results demonstrate that overexpressed active PKCβII plays a role in the regulation and outcome of BCR signals that can be important for the progression of CLL.

Btk regulates multiple stages in the development and survival of B-1 cells

B-1 cells are important players in the first line of defense against pathogens. According to current models for the origin of B-1 cells, they either represent a separate lineage from conventional B-2 cells or differentiate from conventional B-2 cells via an intermediate, B-1int, in response to positive selection by antigen. Here we show that Btk, a Tec family kinase that mediates B cell antigen receptor (BCR) signaling, is required at multiple stages of B-1 cell development. VH12 anti-phosphatidylcholine (PtC) IgH transgenic mice provide a model for the induced differentiation of B-1 cells. This transgene selects for PtC-reactive cells and induces them to adopt a B-1 phenotype. Both processes have been shown to depend on Btk. To determine whether this is secondary to a requirement for Btk in the development of mature B-2 cells, we crossed VH12 transgenic mice to mice expressing low levels of Btk. B-2 cell development occurs normally in Btklo mice despite reduced responsiveness to BCR crosslinking. Analysis of VH12.Btklo mice reveals that Btk regulates the B-1int to B-1 transition and/or the survival of splenic B-1 cells, in part via a mechanism independent of its role in BCR signaling. We also show that Btk mediates the survival of, and expression of IL-10 by, those B-1 cells that do develop and migrate to the peritoneum. Multiple roles for Btk in B-1 cell development and maintenance may explain the particular sensitivity of this population to mutations in components of Btk signaling pathways.

Tonic B-Cell Receptor Signaling Promotes the Survival of Diffuse Large B-Cell Lymphomas: Identification of SYK as a Rational Treatment Target.

The role of BCR-mediated survival signals in diffuse large B-cell lymphoma (DLBCL) remains undefined. B-cell antigen receptor (BCR) signaling induces receptor oligomerization and Ig alpha/beta ITAM phosphorylation. Thereafter, the protein tyrosine kinase (PTK) SYK is recruited and activated, initiating downstream events and amplifying the original BCR signal. Recent studies indicate that BCRs also transmit low-level survival signals in the absence of receptor engagement and highlight the critical role of SYK in this “tonic” BCR signaling. We recently found that SYK is a major substrate of the protein tyrosine phosphatase, PTPROt. PTPROt-mediated dephosphorylation and inactivation of SYK inhibited DLBCL proliferation and induced apoptosis in the absence of BCR cross-linking. These observations suggested that tonic BCR signaling may be an important survival mechanism in certain DLBCLs and identified SYK as a possible rational therapeutic target. For these reasons, we assessed the efficacy of a recently developed ATP-competitive inhibitor of SYK, R406, in a panel of 8 DLBCL cell lines. R406 inhibited the proliferation of 6 DLBCL cell lines at IC50s of 0.9–1.8 micromolar. The SYK inhibitor was cytotoxic (30–85% apoptotic cells) at low micromolar doses in 6 of these lines. To delineate the relationship between tonic BCR signaling, associated SYK activity and R406 responsiveness, we assessed SYK phosphorylation (pTYR 352 and 525/526) in the DLBCL cell line panel. Upon BCR signaling, SYK is phosphorylated at TYR 352; thereafter, the kinase undergoes autophosphorylation at TYR 525/526, which is required for SYK activity. In sensitive DLBCL cell lines, R406 specifically inhibited TYR 525/526 phosphorylation and additional downstream signals (p-ERK). In addition, all 6 R406-sensitive DLBCL cell lines exhibited basal SYK 352 phosphorylation whereas the 2 R406-resistant lines lacked pTyr 352 phosphorylation. Basal SYK pTYR 352 could be detected by immunoblotting total SYK immunoprecipitates with a phospho-specific SYK 352 antibody or using the same pTYR 352 antibody for intracellular phospho-specific flow cytometry. We conclude that :tonic BCR signaling is an important survival mechanism in many DLBCLs;SYK is a rational therapeutic target in these tumors;R406 is a promising SYK inhibitor; andSYK pTYR 352 is an excellent indicator of tonic BCR signaling and predictor of R406 sensitivity in DLBCL.

Depletion of Hsp90β Induces Multiple Defects in B Cell Receptor Signaling

Hsp90 participates in many distinct aspects of cellular functions and accomplishes these roles by interacting with multiple client proteins. To gain insight into the interactions between Hsp90 and its clients, here we have reduced the protein level of Hsp90 in avian cells by gene targeting in an attempt to elicit the otherwise undetectable (because of the vast amount of cellular Hsp90) Hsp90-interacting proteins. Hsp90beta-deficient cells can grow, albeit more slowly than wild-type cells. B cell antigen receptor signaling is multiply impaired in these mutant cells; in particular, the amount of immunoglobulin M heavy chain protein is markedly reduced. Furthermore, serum activation does not promote ERK phosphorylation in Hsp90beta-deficient cells. These multifaceted depressive effects seem to be provoked independently of each other and possibly recapitulate the proteome-wide in vivo functions of Hsp90. Reintroduction of the Hsp90beta gene efficiently restores all of the defects. Unexpectedly, however, introducing the Hsp90alpha gene is also effective in restoration; thus, these defects might be caused by a reduction in the total expression of Hsp90 rather than by loss of Hsp90beta-specific function.

Single and Combined Deletions of the NTAL/LAB and LAT Adaptors Minimally Affect B-Cell Development and Function

NTAL (non-T-cell activation linker, also called LAB) and LAT (linker for activation of T cells) are evolutionarily related transmembrane adaptor proteins that are phosphorylated upon immunoreceptor engagement. Using quantitative reverse transcription-PCR, both NTAL and LAT were found to be expressed in B cells. However, LAT expression was limited to early B cells, whereas NTAL expression typified mature B cells. To delineate their roles in B-cell development and function, Ntal-deficient mice were generated and crossed with Lat-deficient mice. B cells developed in Lat(-/-) Ntal(-/-) double-deficient mice and in mice lacking either of the two adaptors with the same efficiency as in wild-type mice. Upon B-cell antigen receptor cross-linking, Ntal(-/-) B cells exhibited slightly increased Ca(2+) mobilization and proliferation. In addition, Ntal-deficient mice had increased levels of natural antibodies and slightly increased humoral response to a T-dependent antigen. Normal titers of serum-specific immunoglobulins were produced in response to a T-cell-independent antigen. Although NTAL is also expressed in plasma cells, its absence did not affect the hypergammaglobulinemia E and G1 that developed in mice with a mutation in tyrosine 136 of LAT. Therefore, NTAL does not play a role in B cells symmetric to the role played by LAT in T cells.

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

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

B Cell Antigen Receptor Signaling Enhances IFN-γ-Induced Stat1 Target Gene Expression Through Calcium Mobilization and Activation of Multiple Serine Kinase Pathways

The signal transducers and activators of transcription 1 (Stat1) are essential for the majority of interferon-gamma (IFN-gamma)-regulated gene expression. Phosphorylation of serine 727 in the transcription activation domain of Stat1 is induced in response to IFN-gamma for maximal transcription activity. In this report, we show that crosslinking of B cell antigen receptor (BCR) or T cell antigen receptor (TCR) can enhance S727 phosphorylation in Stat1 and result in increased expression of Stat1 target genes. We further demonstrate that this enhancement by BCR cross-linking involves the widely used secondary messenger Ca2+ and simultaneous activation of multiple serine kinase pathways. When cells are exposed to both IFN-gamma and a Ca2+ fluxing reagent, the level of S727 phosphorylation is enhanced, resulting in increased transcription activation of Stat1 target genes. We directly demonstrate that the biochemical function of phospho-Ser-727 is to enhance the recruitment of transcription coactivator CBP/p300 to the promoters of Stat1 target genes. Furthermore, we show that both the p38 mitogen-activated protein kinase (MAPK) and the Ca(2+)/calmodulin-dependent kinase (CaMKII) are activated in response to BCR signaling to converge on Stat1 S727 for maximal gene expression. These studies demonstrate that a wide variety of noncytokine signaling pathways can modulate cytokine signaling through modulation of Stat1 serine phosphorylation.

Dissection of the signalling mechanisms underlying FcγRIIB-mediated apoptosis of mature B-cells

The low-affinity receptor for IgG, FcgammaRIIB, negatively regulates BCR (B-cell antigen receptor)-mediated proliferative signalling and thus plays an important role in feedback inhibition of the humoral immune response. Whereas crosslinking of BCR on mature B-cells results in proliferation, co-ligation of FcgammaRIIB results in growth arrest and apoptosis. We have now investigated the signals underlying FcgammaRIIB-driven apoptosis and found this to be dependent on disruption of mitochondrial potential (Deltapsi), involve the pro-apoptotic Bcl-2 family members, Bid and Bad, and be caspase-independent.

The Adaptor Protein Bam32 Regulates Rac1 Activation and Actin Remodeling through a Phosphorylation-dependent Mechanism

The B cell adaptor molecule of 32 kDa (Bam32) is an adaptor that links the B cell antigen receptor (BCR) to ERK and JNK activation and ultimately to mitogenesis. After BCR cross-linking, Bam32 is recruited to the plasma membrane and accumulates within F-actin-rich membrane ruffles. Bam32 contains one Src homology 2 and one pleckstrin homology domain and is phosphorylated at a single site, tyrosine 139. To define the function of Bam32 in membrane-proximal signaling events, we established human B cell lines overexpressing wild-type or mutant Bam32 proteins. The basal level of F-actin increased in cells expressing wild-type or myristoylated Bam32 but decreased in cells expressing either an Src homology-2 or Tyr-139 Bam32 mutant. Overexpression of wild-type Bam32 also affected BCR-induced actin remodeling, which was visualized as increases in F-actin-rich membrane ruffles. In contrast, Bam32 mutants largely blocked the BCR-induced increase in cellular F-actin. The positive and negative effects of Bam32 variants on F-actin levels were closely mirrored by their effects on the activation of the GTPase Rac1, which is known to regulate actin remodeling in lymphocytes. Bam32-deficient DT40 B cells showed decreased Rac1 activation and a failure of Rac1 to co-localize with the BCR, whereas cells overexpressing Bam32 had increased constitutive Rac1 activation. These results suggest that Bam32 regulates the cytoskeleton through Rac1. Bam32 variants also affected downstream signaling to JNK in a manner similar to that of Rac1, suggesting that the effect of Bam32 on JNK activation may be at least partially mediated through Rac1. Our results demonstrate a novel phosphorylation-dependent function of Bam32 in regulating Rac1 activation and actin remodeling.

Syk-mediated Tyrosine Phosphorylation Is Required for the Association of Hematopoietic Lineage Cell-specific Protein 1 with Lipid Rafts and B Cell Antigen Receptor Signalosome Complex

Hematopoietic lineage cell-specific protein 1 (HS1) is an F-actin- and actin-related proteins 2 and 3 (Arp2/3)-binding protein that undergoes a rapid tyrosine phosphorylation upon B cell antigen receptor (BCR) activation. Density gradient centrifugation of Triton X-100 lysates from B lymphocytes demonstrated that HS1 was translocated in response to BCR cross-linking into lipid raft microdomain along with Arp2/3 complex and Wiskott-Aldrich syndrome protein. HS1-green fluorescent protein was localized in membrane patches enriched with GM1 gangliosides and BCR in the cells treated with anti-IgM antibody. Colocalization of HS1-green fluorescent protein with BCR was also correlated with tyrosine phosphorylation of HS1. Interestingly a murine HS1 mutant at the tyrosine residues Tyr388 and Tyr405 targeted by Syk failed to respond to BCR cross-linking for either translocation into lipid rafts or colocalization with BCR within cells. Furthermore HS1 was unable to translocate into lipid rafts in a chicken B cell line deficient in Syk. Reintroducing a Syk construct into the Syk knock-out cells recovered effectively both tyrosine phosphorylation and translocation of HS1 into lipid rafts. In contrast, translocation of HS1 into rafts was normal in a Lyn knock-out B cell line, and an HS1 mutant at the tyrosine residue Tyr222 targeted by Lyn maintained the ability to partition into rafts upon BCR cross-linking. These data indicate that Syk plays an important role in the translocation of HS1 into lipid rafts and may be responsible for actin assembly recruitment to rafts and subsequent antigen presentations.

Engagement of the B-cell antigen receptor (BCR) allows efficient transduction of ZAP-70-positive primary B-CLL cells by recombinant adeno-associated virus (rAAV) vectors

Engagement of the B-cell antigen receptor (BCR) by crosslinking of the surface immunoglobulin (sIg) homodimer was studied for recombinant adeno-associated virus (rAAV)-mediated gene transfer into B-cell chronic lymphocytic leukaemia (B-CLL) cells. Leukemic cells obtained from 20 patients were stimulated with anti-sIg-directed antibodies and transduced with rAAV vectors coding for enhanced green fluorescent protein (EGFP) (AAV/EGFP) or CD40L (AAV/CD40L). Transduction of B-CLL cells was enhanced after BCR engagement compared to unstimulated controls (P=0.0356). BCR crosslinking induced a significant, dose- and time-dependent upregulation of heparan sulfate proteoglycan (HSPG), the primary receptor for AAV, on B-CLL cells (mean: 38.2 versus 1.7%; P=0.0006). A correlation of HSPG expression after BCR crosslinking with transduction efficiency by AAV/EGFP (P=0.0153) and AAV/CD40L (P=0.0347) was observed. High expression of zeta-associated protein 70 (ZAP-70) in B-CLL cells correlated with a better transduction efficiency by AAV/EGFP (P<0.0001) and AAV/CD40L (P=0.002), respectively: 48 h after transduction of ZAP-70-positive samples, transgene expression was seen in a mean of 33.8% (s.e.m. 3.7%) and 28.9% (s.e.m. 6.7%) of cells, respectively, and could be specifically blocked by heparin, a soluble competitor of HSPG (P<0.0001). In summary, engagement of the BCR on ZAP-70 positive B-CLL cells allows efficient rAAV-mediated gene delivery.