B Cell Antigen Receptor Ligation Research Articles

Quantitative description of the phase-separation behavior of the multivalent SLP65-CIN85 complex.

Biomolecular condensates play a major role in cell compartmentalization, besides membrane-enclosed organelles. The multivalent SLP65 and CIN85 proteins are proximal B-cell antigen receptor (BCR) signal effectors and critical for proper immune responses. In association with intracellular vesicles, the two effector proteins form phase separated condensates prior to antigen stimulation, thereby preparing B lymphocytes for rapid and effective activation upon BCR ligation. Within this tripartite system, 6 proline-rich motifs (PRMs) of SLP65 interact promiscuously with 3 SH3 domains of the CIN85 monomer, establishing 18 individual SH3-PRM interactions whose individual dissociation constants we determined. Based on these 18 dissociation constants, we measured the phase-separation properties of the natural SLP65/CIN85 system as well as designer constructs that emphasize the strongest SH3/PRM interactions. By modeling these various SLP65/CIN85 constructs with the program LASSI (LAttice simulation engine for Sticker and Spacer Interactions), we reproduced the observed phase-separation properties. In addition, LASSI revealed a deviation in the experimental measurement, which was independently identified as a previously unknown intramolecular interaction. Thus, thermodynamic properties of the individual PRM/SH3 interactions allow us to model the phase-separation behavior of the SLP65/CIN85 system faithfully.

The inhibitory coreceptor CD22 restores B cell signaling by developmentally regulating Cd45-/- immunodeficient B cells.

The protein tyrosine phosphatase CD45 plays a crucial role in B cell antigen receptor (BCR) signaling by activating Src family kinases. Cd45-/- mice show altered B cell development and a phenotype likely due to reduced steady-state signaling; however, Cd45-/- B cells show relatively normal BCR ligation-induced signaling. In our investigation of how BCR signaling was restored in Cd45-/- cells, we found that the coreceptor CD22 switched from an inhibitory to a stimulatory function in these cells. We disrupted the ability of CD22 to interact with its ligands in Cd45-/- B cells by generating Cd45-/-St6galI-/- mice, which cannot synthesize the glycan ligand of CD22, or by treating Cd45-/- B cells in vitro with the sialoside GSC718, which inhibits ligand binding to CD22. BCR ligation-induced signaling was reduced by ST6GalI deficiency, but not by GSC718 treatment, suggesting that CD22 restored BCR ligation-induced signaling in Cd45-/- mature B cells by altering cellular phenotypes during development. CD22 was required for the increase in the surface amount of IgM-BCR on Cd45-/- B cells, which augmented signaling. Because B cell survival depends on steady-state BCR signaling, IgM-BCR abundance was likely increased by the selective survival of IgM-BCRhi Cd45-/- B cells because of CD22-mediated signaling under conditions of substantially reduced steady-state signaling. Because the amount of surface IgM-BCR is increased on B cells from patients with other BCR signaling deficiencies, including X-linked agammaglobulinemia, our findings suggest that CD22 may contribute to the partial restoration of B cell function in these patients.

CD22-Binding Synthetic Sialosides Regulate B Lymphocyte Proliferation Through CD22 Ligand-Dependent and Independent Pathways, and Enhance Antibody Production in Mice

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are expressed in various immune cells and most of them carry signaling functions. High-affinity synthetic sialoside ligands have been developed for various Siglecs. Therapeutic potentials of the nanoparticles and compounds that contain multiple numbers of these sialosides and other reagents such as toxins and antigens have been demonstrated. However, whether immune responses can be regulated by monomeric sialoside ligands has not yet been known. CD22 (also known as Siglec-2) is an inhibitory molecule preferentially expressed in B lymphocytes (B cells) and is constitutively bound and functionally regulated by α2,6 sialic acids expressed on the same cell (cis-ligands). Here, we developed synthetic sialosides GSC718 and GSC839 that bind to CD22 with high affinity (IC50 ~100 nM), and inhibit ligand binding of CD22. When B cells are activated by B cell antigen receptor (BCR) ligation, both GSC718 and GSC839 downregulate proliferation of B cells, and this regulation requires both CD22 and α2,6 sialic acids. This result suggests that these sialosides regulate BCR ligation-induced B cell activation by reversing endogenous ligand-mediated regulation of CD22. By contrast, GSC718 and GSC839 augment B cell proliferation induced by TLR ligands or CD40 ligation, and this augmentation requires CD22 but not α2,6 sialic acids. Thus, these sialosides appear to enhance B cell activation by directly suppressing the inhibitory function of CD22 independently of endogenous ligand-mediated regulation. Moreover, GSC839 augments B cell proliferation that depends on both BCR ligation and CD40 ligation as is the case for in vivo B cell responses to antigens, and enhanced antibody production to the extent comparable to CpG oligonuleotides or a small amount of alum. Although these known adjuvants induce production of the inflammatory cytokines or accumulation of inflammatory cells, CD22-binding sialosides do not. Thus, synthetic sialosides that bind to CD22 with high-affinity modulate B cell activation through endogenous ligand-dependent and independent pathways, and carry an adjuvant activity without inducing inflammation.

Transitional B cells commit to marginal zone B cell fate by Taok3-mediated surface expression of ADAM10.

Notch2 and B cell antigen receptor (BCR) signaling determine whether transitional B cells become marginal zone B (MZB) or follicular B (FoB) cells in the spleen, but it is unknown how these pathways are related. We generated Taok3-/- mice, lacking the serine/threonine kinase Taok3, and found cell-intrinsic defects in the development of MZB but not FoB cells. Type 1 transitional (T1) B cells required Taok3 to rapidly respond to ligation by the Notch ligand Delta-like 1. BCR ligation by endogenous or exogenous ligands induced the surface expression of the metalloproteinase ADAM10 on T1 B cells in a Taok3-dependent manner. T1 B cells expressing surface ADAM10 were committed to becoming MZB cells in vivo, whereas T1 B cells lacking expression of ADAM10 were not. Thus, during positive selection in the spleen, BCR signaling causes immature T1 B cells to become receptive to Notch ligands via Taok3-mediated surface expression of ADAM10.

The regulation and role of L-glutamine in B-lymphocyte activation (LYM7P.618)

Abstract B lymphocyte activation is a highly energetic event that promotes cell survival and growth. Aberrant activation of B lymphocytes can lead to disease states such as lymphomas or autoimmune diseases. To meet the metabolic demands of activation, B cells increase nutrient uptake to provide energy and intermediates for macromolecule synthesis. We have investigated the role of L-glutamine, which is emerging as a critical immunomodulatory nutrient; however, the precise role of L-glutamine in B cell growth responses is poorly understood. Depletion of extracellular L-glutamine results in impaired cell growth and proliferation in response to B-cell antigen receptor (BCR) ligation, although, early activation events such as induction of CD86 are unaffected. We show that L-glutamine uptake is induced in response to BCR ligation. This corresponds to an increase in the surface expression of the high affinity L-glutamine transporter, ASCT2, indicating its involvement in L-glutamine uptake. Further, inhibition of ASCT2 by the small molecule N-glutaryl-L-phenylalanine p-nitroanilide (GPNA) caused impaired B lymphocyte activation similar to that observed in response to L-glutamine depletion. The addition of L-leucine abated some of the effects of GPNA treatment on B lymphocyte growth responses, indicating mTORC involvement. These data provide new insight on the requirement of L-glutamine in B lymphocyte activation and suggest a possible underlying mechanism of growth regulation by mTORC.

Methotrexate and a spleen tyrosine kinase inhibitor cooperate to inhibit responses to peripheral blood B cells in rheumatoid arthritis.

BackgroundSelective disruption of the spleen tyrosine kinase (Syk) represents a novel strategy to control B-cell functional responses by inhibition of B-cell antigen receptor (BCR) signaling. PRT062607 (P505-15) is a highly selective small molecule Syk inhibitor that potently suppresses B-cell function in human and rodent blood, and reduces inflammation in rodent models of rheumatoid arthritis (RA).AimsIn this study, we sought to determine the potency of Syk inhibition by PRT062607 in whole blood from RA patients, and elucidate covariates that affect the potency of immune-regulation by this compound.Materials and MethodsWhole blood was collected from 30 patients diagnosed with RA as part of a single-center outpatient study. Disease severity, serum protein markers of inflammation, and co-medications were related to each other, and to PRT062607 activity in ex vivo Syk-mediated immune function assays.ResultsWe report here that PRT062607 exhibited greater potency in suppressing BCR mediated B-cell functional responses in whole blood from RA patients who received stable methotrexate (MTX) therapy. We demonstrate that the B-cell functional response to BCR ligation is influenced by cytokines and JAK/STAT signaling.DiscussionMTX is a known cytokine modulating agent, and this mechanism may act in concert with PRT062607 to control B-cell function.ConclusionThese data have important implications for the co-administration of Syk inhibitors and MTX for the treatment of RA.

The ERM family protein Ezrin associates with the JNK signaling complex to regulate its activation and antigen-dependent B cell survival. (109.7)

Abstract Antigen binding to the B cell antigen receptor (BCR) results in initiation of a highly orchestrated series of enzymatic reactions and protein-protein interactions that transmit the signals for B cell survival and proliferation. The highly conserved Ezrin-Radixin-Moesin (ERM) family members are thought to regulate lymphocyte activation via their membrane-actin crosslinking property, which is in turn regulated by phosphorylation of a conserved threonine residue at their C-terminus. Here, we report a novel signal transduction axis that links antigen-dependent tyrosine phosphorylation of ezrin with activation of the c-Jun N-terminal kinase (JNK). BCR ligation induces phosphorylation of Y353 in ezrin, a residue that is not conserved in other ERM proteins. Phosphorylation of Y353 is dependent on Src and Syk family kinase activity, occurs preferentially on the open conformer of ezrin, and correlates with the strength of BCR signaling in mature B cell subsets. Tyrosine phosphorylated ezrin associates with components of the JNK signaling complex in response to BCR stimulation. A non-phosphorylatable (Y353F) mutant of ezrin acts in a dominant negative manner to specifically impair antigen-induced JNK activation, and reduces B cell survival. Our results demonstrate a unique and novel signaling role for ezrin in regulating antigen-dependent JNK activation and B cell survival.

BCR-induced cell death of B cells from CD22 deficient mice is mediated by a novel ssRNA-directed endonuclease (136.33)

Abstract B cells from CD22 deficient mice backcrossed onto a C57BL/6 genetic background (B6 CD22-/-) undergo B cell antigen receptor (BCR)-induced apoptosis. By contrast, B cells from parental inbred CD22-/- mice on a mixed B6x129 genetic background (B6/129 CD22-/-) survive and proliferate normally following BCR ligation. Through an extensive reverse genetic screen, a single locus was identified as being responsible for the proliferation defect of B6 CD22-/- B cells. In B6 CD22-/- mice this locus was homozygous for B6 germline DNA (locusB6), and in B6/129 CD22-/- mice this region was homozygous for 129 germline DNA (locus129). In locusB6 CD22-/- B cells, a novel single-stranded RNA (ssRNA)-directed endonuclease was specifically overexpressed compared to locus129 CD22-/- B cells, suggesting a direct role in BCR-induced cell death. Targeted disruption of the endonuclease gene in locusB6 CD22-/- mice restored BCR-mediated B cell survival and proliferation to normal levels. These results identify a novel ssRNA-directed endonuclease as a potent inhibitor of B cell survival when expressed following antigen receptor stimulation. This research was supported by NIH grant R01CA096547.

Induction of autophagy by B cell antigen receptor stimulation and its inhibition by costimulation

Autophagy is a major pathway for degradation of cytoplasmic components, and is induced by some apoptotic stimuli mostly in cancer cells under the condition in which apoptosis is blocked. Ligation of the B cell antigen receptor (BCR) induces apoptosis and plays a crucial role in self-tolerance. However, whether BCR ligation induces autophagy is not clear. Here, we demonstrate that autophagosomes are extensively formed in normal mouse B cells as well as the WEHI-231 B cell line upon induction of BCR ligation-induced apoptosis regardless of whether apoptosis is blocked by overexpression of Bcl-2. In contrast, autophagosomes were not formed during apoptosis of spleen B cells cultured with medium alone or in BCR-ligated BAL17 cells which do not undergo apoptosis. Moreover, autophagy is not induced when apoptotic BCR signaling is abrogated by CD40 signaling. These results indicate that autophagy is induced specifically by apoptotic BCR signaling even in unmanipulated normal B cells.

Induction of Cytosolic Calcium Flux by CD20 Is Dependent upon B Cell Antigen Receptor Signaling

The anti-CD20 monoclonal antibody (mAb) rituximab is now routinely used for the treatment of non-Hodgkins lymphoma and is being examined in a wide range of other B-cell disorders, such as rheumatoid arthritis. Despite intensive study, the mechanism of action still remains uncertain. In the current study, anti-CD20 mAb-induced calcium signaling was investigated. Previously, we grouped anti-CD20 mAbs into Type I (rituximab-like) and Type II (B1-like) based upon various characteristics such as their ability to induce complement activation and redistribute CD20 into detergent-insoluble membrane domains. Here we show that only Type I mAbs are capable of inducing a calcium flux in B cells and that this is tightly correlated with the expression of the B-cell antigen receptor (BCR). Inhibitor analysis revealed that the signaling cascade employed by CD20 was strikingly similar to that utilized by the BCR, with inhibitors of Syk, Src, and PI3K, but not EGTA, p38, or ERK1/2, completely ablating calcium flux. Furthermore, binding of Type I but not Type II mAbs caused direct association of CD20 with the BCR as measured by FRET and resulted in the phosphorylation of BCR-specific adaptor proteins BLNK and SLP-76. Crucially, variant Ramos cells lacking BCR expression but with unchanged CD20 expression were completely unable to induce calcium flux following ligation of CD20. Collectively, these data indicate that CD20 induces cytosolic calcium flux through its ability to associate with and "hijack" the signaling potential of the BCR.

Distinctive tyrosine phosphorylation pattern of CD19 during BCR and CD40 signaling

CD19 regulates constitutive and B cell antigen receptor (BCR)‐induced signaling through its cytoplasmic domain that contains nine conserved tyrosine residues. Of nine tyrosine residues, studies have clarified that three tyrosines, CD19‐Y513, CD19‐Y482, and CD19‐Y391, have central roles. Herein, we demonstrate distinctive patterns of tyrosine phophorylation of CD19 following various stimulations using antibodies reactive with each phosphorylated tyrosine residue. BCR ligation caused tyrosine phophorylation of Y513, followed by Y391 phosphorylation that preceded Y482 phosphorylation. IgG‐BCR induced more rapid phosphorylation of Y513 and more intense phosphorylation of Y482 and Y391 when compared with IgM‐BCR. Furthermore, simultaneous CD40 stimulation with BCR ligation resulted in much enhanced and prolonged phosphorylation of the three tyrosine residues. Phosphorylated tyrosine residues of CD19 also exhibited unique distribution: majority of phosphorylated Y513 located within the lipid rafts, while Y482 and Y391 were phosphorylated mainly out of the lipid rafts following the BCR ligation. Collectively, these observations contribute to better understanding of CD19 functions.

FRET-based Ca2+ measurement in B lymphocyte by flow cytometry and confocal microscopy

Upon the B cell antigen receptor (BCR) ligation Ca2+ mobilization is induced, which is essential for activation of downstream signaling molecules such as MAP kinase. Although synthetic fluorescent chelators such as Fluo-4 and Indo-1 are widely used for Ca2+ measurement upon BCR ligation, they are leaked or unfavorably localized into some organelles with time post loading. To solve these problems, we introduce a genetically encoded fluorescent indicator cameleon which is a fluorescence resonance energy transfer (FRET)-based indicator comprising two fluorescent proteins (CFP and YFP) and two Ca2+-responsive elements (a variant of calmodulin (CaM) and a CaM-binding peptide). Here, we demonstrate that cameleon as well as a conventional synthetic Ca2+ indicator enables Ca2+ measurement by flow cytometry clearly upon BCR ligation. In addition, confocal microscopy analysis allows us to detect cameleon-based Ca2+ mobilization in a single cell upon BCR ligation.

ER stress is involved in B cell antigen receptor ligation-induced apoptosis

Apoptosis of B cells upon ligation of the B cell antigen receptor (BCR) plays a role in elimination of self-reactive B cells. Previously, BCR ligation was shown to induce expression of the molecules involved in the unfolded protein response (UPR). However, the role of the UPR in BCR-mediated apoptosis is poorly understood. Here, we demonstrate that activation of various UPR molecules are induced when BCR ligation induces apoptosis in the B cell line WEHI-231 and mouse spleen B cells. BCR ligation-induced UPR is attenuated by survival signaling through CD40 in these cells. When overexpression of BiP suppresses the UPR in WEHI-231 cells, activation of p38 MAPK is blocked and apoptosis is reduced. Moreover, the p38 MAPK inhibitor SB203580 reduces BCR ligation-induced apoptosis. These results suggest that the UPR is involved in BCR ligation-induced apoptosis and that p38 MAPK is crucial for apoptosis during the UPR in B cells.

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.

Expression and function of the adaptor protein Gads in murine B cells

Nearly all hematopoietic receptors are dependent on adaptor proteins for the activation of downstream signaling pathways. The Gads adaptor protein is expressed in many hematopoietic tissues, including bone marrow, lymph node, and spleen. Using intracellular staining, we detected Gads protein in a number cells, including B cells, T cells, NK cells, monocytes, and plasmacytoid DC, but not in macrophages, neutrophils, or monocyte-derived DC. In the B cell compartment, Gads was first expressed after immature B cells leave the bone marrow and was down-regulated after B cell antigen receptor (BCR) ligation. Female Gads(-/-) mice had increased numbers of splenic B cells, as compared to female Gads(+/+) mice, suggesting a role for Gads in B cell homeostasis. Although B cell production and turnover of splenic B cell subsets appeared normal in Gads(-/-) mice, homeostatic proliferation was significantly impaired in Gads(-/-) B cells. Whereas BCR ligation can induce apoptosis in wild-type transitional stage 1 (T1) B cells, Gads(-/-) T1 B cells were resistant to BCR-induced apoptosis. Gads(-/-) B cells also showed increased BCR-mediated calcium mobilization. We conclude that Gads may have a negative regulatory role in signaling through survival pathways, and is necessary for normal homeostatic proliferation in B cells.

The Adaptor Protein HSH2 Attenuates Apoptosis in Response to Ligation of the B Cell Antigen Receptor Complex on the B Lymphoma Cell Line, WEHI-231

Signals transduced by the B cell antigen receptor (BCR) play a central role in regulating the functional response of the cell to antigen. Depending on the nature of the antigenic signal and the developmental or differentiation state of the B cell, antigen receptor signaling can promote either apoptosis or survival and activation. Understanding the molecular mechanisms underlying BCR-mediated apoptosis constitutes an important area of research because aberrations in programmed cell death can result in the development of autoimmunity or cancer. Expression of the adaptor protein hematopoietic Src homology 2 (HSH2) was found to significantly decrease BCR-mediated apoptosis in the murine WEHI-231 cell line. Analysis of signal transduction pathways activated in response to BCR ligation revealed that HSH2 does not significantly alter total protein tyrosine phosphorylation or Ca2+ mobilization. HSH2 does not potentiate the activation-dependent phosphorylation of AKT either. With respect to MAPK activation, HSH2 was not observed to alter the activation of ERK or p38 in response to BCR ligation, but it does significantly potentiate JNK activation. Analysis of processes directly associated with apoptosis revealed that HSH2 inhibits mitochondrial depolarization to a significant degree, whereas it has only a slight effect on caspase activation and poly ADP-ribose polymerase cleavage. BCR-induced apoptosis of WEHI-231 cells is associated with the loss of endogenous HSH2 expression within 12 h, whereas inhibition of apoptosis in response to CD40-mediated signaling leads to stabilization of HSH2 expression. Thus, endogenous HSH2 expression correlates directly with survival of WEHI-231 cells, which supports the hypothesis that HSH2 modulates the apoptotic response through its ability to directly or indirectly promote mitochondrial stability.

The tyrosine kinase Syk is required for light chain isotype exclusion but dispensable for the negative selection of B cells.

In this study we set out to test whether Syk was required for negative selection of immature B cells. B cells expressing a B cell antigen receptor (BCR) transgene (3-83, anti-H-2K(k)) underwent negative selection independently of Syk in both fetal liver organ culture and radiation chimera models. Furthermore, Syk-independent negative selection was not reversed by transgenic overexpression of Bcl-2. Receptor editing was not apparent in Syk-deficient B cells, presumably as a consequence of the failure of mature edited B cells to develop in the absence of Syk. Interestingly, light chain isotype exclusion by the BCR transgene failed in the absence of Syk. We observed a dramatic reduction in the overall BCR-mediated tyrosine phosphorylation of cellular proteins in Syk-deficient immature B cells. However, the tyrosine phosphorylation of a number of substrates including phospholipase C gamma 2, although reduced, was not completely abrogated. BCR ligation triggered an increase in calcium flux in the absence of Syk. Thus signaling events that mediate negative selection can still occur in the absence of Syk. This may be due to redundancy with zeta-associated protein 70 (ZAP-70), which we demonstrate to be expressed in immature B cells.

Loss of the pro-apoptotic BH3-only Bcl-2 family member Bim inhibits BCR stimulation-induced apoptosis and deletion of autoreactive B cells.

During development, the stochastic process assembling the genes encoding antigen receptors invariably generates B and T lymphocytes that can recognize self-antigens. Several mechanisms have evolved to prevent the activation of these cells and the concomitant development of autoimmune disease. One such mechanism is the induction of apoptosis in developing or mature B cells by engagement of the B cell antigen receptor (BCR) in the absence of T cell help. Here we report that B lymphocytes lacking the pro-apoptotic Bcl-2 family member Bim are refractory to apoptosis induced by BCR ligation in vitro. The loss of Bim also inhibited deletion of autoreactive B cells in vivo in two transgenic systems of B cell tolerance. Bim loss prevented deletion of autoreactive B cells induced by soluble self-antigen and promoted accumulation of self-reactive B cells developing in the presence of membrane-bound self-antigen, although their numbers were considerably lower compared with antigen-free mice. Mechanistically, we determined that BCR ligation promoted interaction of Bim with Bcl-2, inhibiting its survival function. These findings demonstrate that Bim is a critical player in BCR-mediated apoptosis and in B lymphocyte deletion.

Slow and persistent increase of [Ca2+]c in response to ligation of surface IgM in WEHI-231 cells

WEHI-231 and Bal 17 B cell lines are representative models for immature and mature B cells, respectively. Their regulation of cytosolic Ca2+ concentration ([Ca2+]c) was compared using fura-2 fluorescence ratiometry. The ligation of B cell antigen receptor (BCR) by anti-IgM antibody induced a slow but large increase of [Ca2+]c in WEHI-231 cells while not in Bal 17 cells. The thapsigargin-induced store-operated Ca2+ entry (SOCE) of Bal 17 cells reached a steady state which was blocked by 2-aminoethoxydiphenyl borate (2-APB). On the contrary, the thapsigargin-induced SOCE of WEHI-231 cells increased continuously, which was accelerated by 2-APB. The increase of [Ca2+]c by BCR ligation was also enhanced by 2-APB in WEHI-231 cells while blocked in Bal 17 cells. The Mn2+ quenching study showed that the thapsigargin-, or the BCR ligation-induced Ca2+ influx pathway of WEHI-231 was hardly permeable to Mn2+. The intractable increase of [Ca2+]c may explain the mechanism of BCR-driven apoptosis of WEHI-231 cells, a well-known model of clonal deletion of autoreactive immature B cells.

Bruton’s tyrosine kinase targets NF-κB to the bcl-x promoter via a mechanism involving phospholipase C-γ2 following B cell antigen receptor engagement

Disruption of Bruton’s tyrosine kinase (BTK) function leads to x-linked immunodeficiency ( xid) in mice. BTK-deficient ( btk −/−) B cells are defective for survival. Prior studies show that BTK is required for the induction of Bcl-x L following B cell antigen receptor (BCR) engagement. However, the mechanism underlying Bcl-x L induction in response to BCR ligation remains unresolved. We now demonstrate that BTK regulates bcl-x expression by transcriptional control in response to BCR engagement. BTK targets nuclear factor-κB (NF-κB) to activate the bcl-x promoter via a phospholipase C-γ2 (PLC-γ2)-dependent mechanism. Perturbation of the BTK/PLC-γ2/NF-κB signaling axis likely contributes to the defective expression of bcl-x and compromised survival of xid B cells.