CD72 negatively regulates signaling through the antigen receptor of B cells

The immunoreceptor tyrosine-based inhibition motif (ITIM) is found in various membrane molecules such as CD22 and the low-affinity Fc receptor for IgG in B cells and the killer cell-inhibitory receptor and Ly-49 in NK cells. Upon tyrosine phosphorylation at the ITIMs, these molecules recruit SH2 domain-containing phosphatases such as SH2-containing tyrosine phosphatase-1 and negatively regulate cell activity. The B cell surface molecule CD72 carries an ITIM and an ITIM-like sequence. We have previously shown that CD72 is phosphorylated and recruits SH2-containing tyrosine phosphatase-1 upon cross-linking of the Ag receptor of B cells (BCR). However, whether CD72 modulates BCR signaling has not yet been elucidated. In this paper we demonstrate that expression of CD72 down-modulates both extracellular signal-related kinase (ERK) activation and Ca2+ mobilization induced by BCR ligation in the mouse B lymphoma line K46micromlambda, whereas BCR-mediated ERK activation was not reduced by the ITIM-mutated form of CD72. Moreover, coligation with CD72 with BCR reduces BCR-mediated ERK activation in spleen B cells of normal mice. These results indicate that CD72 negatively regulates BCR signaling. CD72 may play a regulatory role in B cell activation, probably by setting a threshold for BCR signaling.     

A novel interaction between protein kinase D and TNF receptor-associated factor molecules regulates B cell receptor-CD40 synergy

Signaling by Ag to the B cell Ag receptor (BCR) is enhanced by several cooperating signals, including several provided by B-T cell interactions. One of these, CD40, provides critical signals for B cell differentiation, isotype switching, and B cell memory. The molecular mechanisms by which BCR and CD40 signals synergize are not well understood. Although the BCR and CD40 share certain signaling pathways, we hypothesized that unique signals provided by each could provide mutual enhancement of their signaling pathways. The BCR, but not CD40, activates protein kinase D (PKD), while CD40, but not the BCR, employs the TNFR-associated factor (TRAF) adapter proteins in signaling. In this study, we show that genetic or pharmacologic inhibition of BCR-mediated PKD activation in B lymphocytes abrogated the synergy between the CD40 and the BCR, as measured by activation of Ig and cytokine secretion. Interestingly, the role of PKD was dependent upon the association of CD40 with TRAF2, and was inhibited by the binding of TRAF3, revealing a novel functional link between these two classes of signaling molecules.     

Down-regulation of B cell receptor signaling by hematopoietic progenitor kinase 1 (HPK1)-mediated Phosphorylation and Ubiquitination of Activated B cell linker protein (BLNK)

Hematopoietic progenitor kinase 1 (HPK1) is a Ste20-like serine/threonine kinase that suppresses immune responses and autoimmunity. B cell receptor (BCR) signaling activates HPK1 by inducing BLNK/HPK1 interaction. Whether HPK1 can reciprocally regulate BLNK during BCR signaling is unknown. Here, we show that HPK1-deficient B cells display hyper-proliferation and hyper-activation of IκB kinase and MAPKs (ERK, p38, and JNK) upon the ligation of BCR. HPK1 attenuates BCR-induced cell activation via inducing BLNK threonine 152 phosphorylation, which mediates BLNK/14-3-3 binding. Furthermore, threonine 152-phosphorylated BLNK is ubiquitinated at lysine residues 37, 38, and 42, leading to attenuation of MAPK and IκB kinase activation in B cells during BCR signaling. These results reveal a novel negative feedback regulation of BCR signaling by HPK1-mediated phosphorylation, ubiquitination, and subsequent degradation of the activated BLNK.     

Positive signaling through CD72 induces mitogen-activated protein kinase activation and synergizes with B cell receptor signals to induce X-linked immunodeficiency B cell proliferation

CD72 is a 45-kDa B cell transmembrane glycoprotein that has been shown to be important for B cell activation. However, whether CD72 ligation induces B cell activation by delivering positive signals or sequestering negative signals away from B cell receptor (BCR) signals remains unclear. Here, by comparing the late signaling events associated with the mitogen-activated protein kinase pathway, we identified many similarities and some differences between CD72 and BCR signaling. Thus, CD72 and BCR activated the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinase. Both CD72- and BCR-mediated ERK and JNK activation required protein kinase C activity, which was equally important for CD72- and BCR-induced B cell proliferation. However, CD72 induced stronger JNK activation compared with BCR. Surprisingly, the JNK activation induced by both BCR and CD72 is Btk independent. Although both CD72 and BCR induced Btk-dependent ERK activation, CD72-mediated proliferation is more resistant to blocking of ERK activity than that of BCR, as shown by the proliferation response of B cells treated with PD98059 and dibutyryl cAMP, agents that inhibit ERK activity. Most importantly, CD72 signaling compensated for defective BCR signaling in X-linked immunodeficiency B cells and partially restored the proliferation response of X-linked immunodeficiency B cells to anti-IgM ligation. These results suggest that CD72 signals B cells by inducing BCR-independent positive signaling pathways.     

Complementary roles for CD19 and Bruton's tyrosine kinase in B lymphocyte signal transduction

CD19 and Bruton's tyrosine kinase (Btk) may function along common signaling pathways in regulating intrinsic and B cell Ag receptor (BCR)-induced signals. To identify physical and functional interactions between CD19 and Btk, a CD19-negative variant of the A20 B cell line was isolated, and CD19-deficient (CD19(-/-)) and CD19-overexpressing mice with the X-linked immunodeficient (Xid; Btk) mutation were generated. In A20 cells, Btk physically associated with CD19 following BCR engagement. CD19 and Btk interactions were not required for initial Btk phosphorylation, but CD19 expression maintained Btk in an activated state following BCR engagement. In primary B cells, CD19 signaling also required downstream Btk function since CD19-induced intracellular Ca(2+) ([Ca(2+)](i)) responses were modest in Xid B cells. In addition, CD19 overexpression did not normalize the Xid phenotype and most phenotypic and functional hallmarks of CD19 overexpression were not evident in these mice. However, CD19 and Btk also regulate independent signaling pathways since their combined loss had additive inhibitory effects on BCR-induced [Ca(2+)](i) responses and CD19 deficiency induced a severe immunodeficiency in Xid mice. Thus, CD19 expression amplifies or prolongs Btk-mediated signaling, rather than serving as a required agent for Btk activation. Consistent with this, phosphatidylinositol 3-monophosphate kinase and Akt activation were normal in CD19(-/-) B cells following IgM engagement, although their kinetics of activation was altered. Thus, these biochemical and compound gene dosage studies indicate that Btk activation and [Ca(2+)](i) responses following BCR engagement are regulated through multiple pathways, including a CD19/Src family kinase-dependent pathway that promotes the longevity of Btk signaling.     

SHP-1 requires inhibitory co-receptors to down-modulate B cell antigen receptor-mediated phosphorylation of cellular substrates

Signaling through the B cell antigen receptor (BCR) is negatively regulated by the SH2 domain-containing protein-tyrosine phosphatase SHP-1, which requires association with tyrosine-phosphorylated proteins for activation. Upon BCR ligation, SHP-1 has been shown to associate with the BCR, the cytoplasmic protein-tyrosine kinases Lyn and Syk, and the inhibitory co-receptors CD22 and CD72. How SHP-1 is activated by BCR ligation and regulates BCR signaling is, however, not fully understood. Here we demonstrate that, in the BCR-expressing myeloma line J558L mu 3, CD72 expression reduces the BCR ligation-induced phosphorylation of the BCR component Ig alpha/Ig beta and its cytoplasmic effectors Syk and SLP-65. Substrate phosphorylation was restored by expression of dominant negative mutants of SHP-1, whereas the SHP-1 mutants failed to enhance phosphorylation of the cellular substrates in the absence of CD72. This indicates that SHP-1 is efficiently activated by CD72 but not by other pathways in J558L mu m3 cells and that inhibition of SHP-1 specifically activated by CD72 reverses CD72-induced dephosphorylation of cellular substrates in these cells. Taken together, BCR-induced SHP-1 activation is likely to require inhibitory co-receptors such as CD72, and SHP-1 appears to mediate the negative regulatory effect of CD72 on BCR signaling by dephosphorylating Ig alpha/Ig beta and its downstream signaling molecules Syk and SLP-65.     

Synergistic B cell activation by CD40 and the B cell antigen receptor: role of B lymphocyte antigen receptor-mediated kinase activation and tumor necrosis factor receptor-associated factor regulation

Optimal activation of B-lymphocytes depends both upon expression of various cell surface receptors and adequate integration of signaling pathways. This requires signals generated upon recognition of antigen by the B lymphocyte antigen receptor (BCR) as well as additional signals provided by cognate interaction with T helper cells, including the CD40-CD154 interaction. Engagement of both the BCR and CD40 results in synergistic activation of B cells. Previous studies identified tumor necrosis factor receptor-associated factor (TRAF)-2 and TRAF3 in the CD40-signaling pathway together with BCR-activated protein kinase D (PKD) as important cooperative factors in this synergy. To better understand the role of these factors in bridging the BCR and CD40 signaling pathways, BCR signal regulation of TRAF function was examined. Results show that phosphorylation of TRAF2 is increased upon BCR but not CD40 engagement and that of the potentially phosphorylated residues of TRAF2, tyrosine 484 is crucial for BCR-CD40 synergy. Additionally, wild type or constitutively active Bruton's tyrosine kinase (Btk) enhanced, whereas the xid mutant form of Btk prevented, BCR-CD40 synergy. These effects were dependent upon TRAF2 and PKD activity. These findings suggest a model in which Btk contributes to the enhancement of the CD40 response by TRAF2 in a PKD-dependent manner.     

CD19 amplifies B lymphocyte signal transduction by regulating Src-family protein tyrosine kinase activation.

Ligation of the B cell Ag receptor (BCR) induces cellular activation by stimulating Src-family protein tyrosine kinases (PTKs) to phosphorylate members of the BCR complex. Subsequently, Src-family PTKs, particularly Lyn, are proposed to phosphorylate and bind CD19, a cell-surface costimulatory molecule that regulates mature B cell activation. Herein, we show that B cells from CD19-deficient mice have diminished Lyn kinase activity and BCR phosphorylation following BCR ligation. Tyrosine phosphorylation of other Src-family PTKs was also decreased in CD19-deficient B cells. In wild-type B cells, CD19 was constitutively complexed with Vav, Lyn, and other Src-family PTKs, with CD19 phosphorylation and its associations with Lyn and Vav increased after BCR ligation. Constitutive CD19/Lyn/Vav complex signaling may therefore be responsible for the establishment of baseline signaling thresholds in B cells before Ag receptor ligation, in addition to accelerating signaling following BCR engagement or other transmembrane signals. In vitro kinase assays using purified CD19 and purified Lyn revealed that the kinase activity of Lyn was significantly increased when coincubated with CD19. Thus, constitutive and induced CD19/Lyn complexes are likely to regulate basal signaling thresholds and BCR signaling by amplifying the kinase activity of Lyn and other Src-family PTKs. These in vivo and in vitro findings demonstrate a novel mechanism by which CD19 regulates signal transduction in B lymphocytes. The absence of this CD19/Src-family kinase amplification loop may account for the hyporesponsive phenotype of CD19-deficient B cells.     

Galectin-1 binds different CD43 glycoforms to cluster CD43 and regulate T cell death

Galectin-1 kills immature thymocytes and activated peripheral T cells by binding to glycans on T cell glycoproteins including CD7, CD45, and CD43. Although roles for CD7 and CD45 in regulating galectin-1-induced death have been described, the requirement for CD43 remains unknown. We describe a novel role for CD43 in galectin-1-induced death, and the effects of O-glycan modification on galectin-1 binding to CD43. Loss of CD43 expression reduced galectin-1 death of murine thymocytes and human T lymphoblastoid cells, indicating that CD43 is required for maximal T cell susceptibility to galectin-1. CD43, which is heavily O-glycosylated, contributes a significant fraction of galectin-1 binding sites on T cells, as T cells lacking CD43 bound approximately 50% less galectin-1 than T cells expressing CD43. Although core 2 modification of O-glycans on other glycoprotein receptors is critical for galectin-1-induced cross-linking and T cell death, galectin-1 bound to CD43 fusion proteins modified with either unbranched core 1 or branched core 2 O-glycans and expression of core 2 O-glycans did not enhance galectin-1 binding to CD43 on T cells. Moreover, galectin-1 binding clustered CD43 modified with either core 1 or core 2 O-glycans on the T cell surface. Thus, CD43 bearing either core 1 or core 2 O-glycans can positively regulate T cell susceptibility to galectin-1, identifying a novel function for CD43 in controlling cell death. In addition, these studies demonstrate that different T cell glycoproteins on the same cell have distinct requirements for glycan modifications that allow recognition and cross-linking by galectin-1.     

B cell receptor (BCR) cross-talk: CD40 engagement creates an alternate pathway for BCR signaling that activates I kappa B kinase/I kappa B alpha/NF-kappa B without the need for PI3K and phospholipase C gamma

BCR signaling is propagated by a series of intermediaries and eventuates in NF-kappaB activation, among other outcomes. Interruption of several mediators that constitute the signalosome, such as PI3K and phospholipase Cgamma2, completely blocks BCR signaling for NF-kappaB. We show here that this accepted, conventional paradigm is, in fact, limited to naive B cells. CD40L treatment reprograms normal B cells such that a novel, alternate pathway for BCR signaling is created. Through this alternate pathway BCR triggering induces nuclear NF-kappaB without the need for PI3K or for phospholipase Cgamma2. Induction of NF-kappaB via the alternate pathway is accompanied by IkappaB kinase beta (IKKbeta) phosphorylation, IkappaBalpha phosphorylation, and IkappaBalpha degradation, and inhibition of IKKbeta blocked IkappaBalpha degradation. Several key events in the conventional pathway, including early protein tyrosine phosphorylation, were unimpeded by generation of the alternate pathway which appears to operate in parallel, rather than in competition, with classical BCR signaling. These results demonstrate cross-talk between CD40 and BCR, such that the requirements for BCR signaling are altered by prior B cell exposure to CD40L. The alternate BCR signaling pathway bypasses multiple signalosome elements and terminates in IKKbeta activation.     

Cutting edge: molecular mechanisms of synergy between CD40 and the B cell antigen receptor: role for TNF receptor-associated factor 2 in receptor interaction

Optimal Ag-specific B lymphocyte activation requires both recognition of Ag by the B cell Ag receptor (BCR) and contact-mediated interactions with Ag-specific Th lymphocytes. One of these interactions involves ligation of B cell CD40 by T cell-expressed CD154. CD40 signaling is crucial for Ab production, isotype switching, up-regulation of surface molecules, development of germinal centers, and the humoral memory response. The signaling pathways emanating from the BCR and CD40 are able to cooperate, but the molecular mechanisms responsible for this interaction are incompletely understood. The present study explored the roles of signaling motifs in the CD40 cytoplasmic tail in this synergy. We find that threonine in the PXQXT motif in the TNFR-associated factor-2 binding site is critical for synergistic effects of CD40 and BCR signals, independent of its phosphorylation. Furthermore, data suggest an indirect role for TNFR-associated factor-2 in the cooperative signaling.     

The c-Abl tyrosine kinase is regulated downstream of the B cell antigen receptor and interacts with CD19

c-Abl is a nonreceptor tyrosine kinase that we have recently linked to growth factor receptor signaling. The c-Abl kinase is ubiquitously expressed and localizes to the cytoplasm, plasma membrane, cytoskeleton, and nucleus. Thus, c-Abl may regulate signaling processes in multiple subcellular compartments. Targeted deletion or mutation of c-Abl in mice results in a variety of phenotypes, including splenic and thymic atrophy and lymphopenia. Additionally, lymphocytes isolated from specific compartments of c-Abl mutant mice have reduced responses to a variety of stimuli and an increased susceptibility to apoptosis following growth factor deprivation. Despite these observations, little is known regarding the signaling mechanisms responsible for these phenotypes. We report here that splenic B cells from c-Abl-deficient mice are hyporesponsive to the proliferative effects of B cell Ag receptor (BCR) stimulation. The c-Abl kinase activity and protein levels are elevated in the cytosol following activation of the BCR in B cell lines. We show that c-Abl associates with and phosphorylates the BCR coreceptor CD19, and that c-Abl and CD19 colocalize in lipid membrane rafts. These data suggest a role for c-Abl in the regulation of B cell proliferation downstream of the BCR, possibly through interactions with CD19.     

Cutting edge: B cell antigen receptor signaling occurs outside lipid rafts in immature B cells

B cell Ag receptor (BCR) signaling changes dramatically during B cell development, resulting in activation in mature B cells and apoptosis, receptor editing, or anergy in immature B cells. BCR signaling in mature B cells was shown to be initiated by the translocation of the BCR into cholesterol- and sphingolipid-enriched membrane microdomains that include the Src family kinase Lyn and exclude the phosphatase CD45. Subsequently the BCR is rapidly internalized into the cell. Here we show that the BCR in the immature B cell line, WEHI-231, does not translocate into lipid rafts following cross-linking nor is the BCR rapidly internalized. The immature BCR initiates signaling from outside lipid rafts as evidenced by the immediate induction of an array of phosphoproteins and subsequent apoptosis. The failure of the BCR in immature B cells to enter lipid rafts may contribute to the dramatic difference in the outcome of signaling in mature and immature B cells.     

Specific blockade by CD54 and MHC II of CD40-mediated signaling for B cell proliferation and survival

Regulation of B lymphocyte proliferation is critical to maintenance of self-tolerance, and intercellular interactions are likely to signal such regulation. Here, we show that coligation of either the adhesion molecule ICAM-1/CD54 or MHC II with CD40 inhibited cell cycle progression and promoted apoptosis of mouse splenic B cells. This resulted from specific blockade of NF-kappa B induction, which normally inhibits apoptosis. LPS- or B cell receptor (BCR)-induced proliferation was not inhibited by these treatments, and mAb-induced association of CD40 with other B cell surface molecules did not have these effects. Addition of BCR or IL-4 signals did not overcome the effect of ICAM-1 or MHC II on CD40-induced proliferation. FasL expression was not detected in B cell populations. These results show that MHC II and ICAM-1 specifically modulate CD40-mediated signaling, so inhibiting proliferation and preventing inhibition of apoptosis.     

CD20 homo-oligomers physically associate with the B cell antigen receptor. Dissociation upon receptor engagement and recruitment of phosphoproteins and calmodulin-binding proteins

B cell antigen receptor (BCR) signaling initiates sustained cellular calcium influx necessary for the development, differentiation, and activation of B lymphocytes. CD20 is a B cell-restricted tetraspanning protein organized in the plasma membrane as multimeric molecular complexes involved in BCR-activated calcium entry. Using coprecipitation of native CD20 with tagged or truncated forms of the molecule, we provide here direct evidence of CD20 homo-oligomerization into tetramers. Additionally, the function of CD20 was explored by examining its association with surface-labeled and intracellular proteins before and after BCR signaling. Two major surface-labeled proteins that coprecipitated with CD20 were identified as the heavy and light chains of cell surface IgM, the antigen-binding components of the BCR. After activation, BCR-CD20 complexes dissociated, and phosphoproteins and calmodulin-binding proteins were transiently recruited to CD20. These data provide new evidence of the involvement of CD20 in signaling downstream of the BCR and, together with the previously described involvement of CD20 in calcium influx, the first evidence of physical coupling of the BCR to a calcium entry pathway.     

Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development

Signaling through the Ag receptor is required for peripheral B lymphocyte maturation and maintenance. Defects in components of the B cell receptor (BCR) signalosome result in developmental blocks at the transition from immature (heat-stable Ag (HSA)(high)) to mature (HSA(low)) B cells. Recent studies have subdivided the immature, or transitional, splenic B cells into two subsets, transitional 1 (T1) and transitional 2 (T2) cells. T1 and T2 cells express distinct surface markers and are located in distinct anatomic locations. In this report, we evaluated the BCR signaling capacity of T1 and T2 B cell subsets. In response to BCR engagement, T2 cells rapidly entered cell cycle and resisted cell death. In contrast, T1 cells did not proliferate and instead died after BCR stimulation. Correlating with these results, T2 cells robustly induced expression of the cell cycle regulator cyclin D2 and the antiapoptotic factors A1/Bfl-1 and Bcl-x(L) and exhibited activation of Akt. In contrast, T1 cells failed to up-regulate these markers. BCR stimulation of T2 cells also led to down-regulation of CD21 and CD24 (HSA) expression, resulting in a mature B cell phenotype. In addition, T2 cells from Bruton's tyrosine kinase-deficient Xid mice failed to generate these proliferative and survival responses, suggesting a requirement for the BCR signalosome specifically at the T2 stage. Taken together, these data clearly demonstrate that T2 immature B cells comprise a discrete developmental subset that mediates BCR-dependent proliferative, prosurvival, and differentiation signals. Their distinct BCR-dependent responses suggest unique roles for T1 vs T2 cells in peripheral B cell selection.     

CD1d and CD1c expression in human B cells is regulated by activation and retinoic acid receptor signaling

B cell activation and Ab production in response to protein Ags requires presentation of peptides for recruitment of T cell help. We and others have recently demonstrated that B cells can also acquire innate help by presenting lipid Ags via CD1d to NKT cells. Given the newfound contribution of NKT cells to humoral immunity, we sought to identify the pathways that regulate CD1 molecule expression in human B cells. We show that ex vivo, activated and memory B cells expressed lower levels of CD1d compared with resting, naive, and marginal zone-like B cells. In vitro, CD1d was downregulated by all forms of B cell activation, leaving a narrow temporal window in which B cells could activate NKT cells. CD1c expression and function also decreased following activation by CD40L alone, whereas activation via the BCR significantly upregulated CD1c, particularly on marginal zone-like B cells. We found that the CD40L-induced downregulation of CD1d and CD1c correlated with diminished expression of retinoic acid receptor α (RARα) response genes, an effect that was reversed by RARα agonists. However, BCR-induced upregulation of CD1c was independent of the RAR pathway. Our findings that both CD1d and CD1c are upregulated by RARα signaling in human B cells is distinct from effects reported in dendritic cells, in which CD1c is inversely downregulated. One functional consequence of CD1d upregulation by retinoic acid was NKT cell cytotoxicity toward B cells. These results are central to our understanding of how CD1-restricted T cells may control humoral immunity.     

Continuous signaling of CD79b and CD19 is required for the fitness of Burkitt lymphoma B cells

Expression of the B‐cell antigen receptor (BCR) is essential not only for the development but also for the maintenance of mature B cells. Similarly, many B‐cell lymphomas, including Burkitt lymphoma (BL), require continuous BCR signaling for their tumor growth. This growth is driven by immunoreceptor tyrosine‐based activation motif (ITAM) and PI3 kinase (PI3K) signaling. Here, we employ CRISPR/Cas9 to delete BCR and B‐cell co‐receptor genes in the human BL cell line Ramos. We find that Ramos B cells require the expression of the BCR signaling component Igβ (CD79b), and the co‐receptor CD19, for their fitness and competitive growth in culture. Furthermore, we show that in the absence of any other BCR component, Igβ can be expressed on the B‐cell surface, where it is found in close proximity to CD19 and signals in an ITAM‐dependent manner. These data suggest that Igβ and CD19 are part of an alternative B‐cell signaling module that use continuous ITAM/PI3K signaling to promote the survival of B lymphoma and normal B cells.