RESUMEN
In contrast to other B-cell antigen receptor (BCR) classes, the function of IgD BCR on mature B cells remains largely elusive as mature B cells co-express IgM, which is sufficient for development, survival, and activation of B cells. Here, we show that IgD expression is regulated by the forkhead box transcription factor FoxO1, thereby shifting the responsiveness of mature B cells towards recognition of multivalent antigen. FoxO1 is repressed by phosphoinositide 3-kinase (PI3K) signaling and requires the lipid phosphatase Pten for its activation. Consequently, Pten-deficient B cells expressing knock-ins for BCR heavy and light chain genes are unable to upregulate IgD. Furthermore, in the presence of autoantigen, Pten-deficient B cells cannot eliminate the autoreactive BCR specificity by secondary light chain gene recombination. Instead, Pten-deficient B cells downregulate BCR expression and become unresponsive to further BCR-mediated stimulation. Notably, we observed a delayed germinal center (GC) reaction by IgD-deficient B cells after immunization with trinitrophenyl-ovalbumin (TNP-Ova), a commonly used antigen for T-cell-dependent antibody responses. Together, our data suggest that the activation of IgD expression by Pten/FoxO1 results in mature B cells that are selectively responsive to multivalent antigen and are capable of initiating rapid GC reactions and T-cell-dependent antibody responses.
Asunto(s)
Linfocitos B/fisiología , Centro Germinal/fisiología , Inmunoglobulina D/genética , Fosfohidrolasa PTEN/fisiología , Receptores de Antígenos de Linfocitos B/genética , Animales , Células Cultivadas , Proteína Forkhead Box O1/fisiología , Regulación de la Expresión Génica/inmunología , Centro Germinal/metabolismo , Inmunoglobulina D/inmunología , Inmunoglobulina D/metabolismo , Ratones , Ratones Transgénicos , Fosfohidrolasa PTEN/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify â¼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21-derived heavy chains (HCs) with IGLV3-21-derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110-expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR-BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01-expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110-expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors.
Asunto(s)
Cadenas lambda de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Linfocitos B/inmunología , Estudios de Cohortes , Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Predisposición Genética a la Enfermedad , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas lambda de Inmunoglobulina/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Mutación Puntual , Receptores de Antígenos de Linfocitos B/genéticaRESUMEN
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 Igß (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, Igß 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 Igß 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.
Asunto(s)
Antígenos CD19/genética , Linfoma de Burkitt/genética , Antígenos CD79/genética , Aptitud Genética/genética , Linfocitos B/patología , Linfoma de Burkitt/patología , Sistemas CRISPR-Cas , Regulación Leucémica de la Expresión Génica/genética , Humanos , Inmunoglobulinas/genética , Motivo de Activación del Inmunorreceptor Basado en Tirosina/genética , Fosfatidilinositol 3-Quinasas/genética , Transducción de SeñalRESUMEN
The random gene segment rearrangement during B cell development ensures Ab repertoire diversity. Because this process might generate autoreactive specificities, it has been proposed that stringent selection mechanisms prevent the development of autoreactive B cells. However, conventional assays to identify autoreactive B cells usually employ in vitro-generated Abs, which differ from membrane-bound BCRs. In this study, we used a cell-based assay to investigate the autoreactivity of membrane-bound BCRs derived from different B cell developmental stages of human peripheral blood. Contrasted to soluble Ab counterparts, only a few of the tested BCRs were autoreactive, although the cell-based assay sensitively detects feeble Ag recognition of a germline-reverted murine BCR that was selected after OVA immunization of mice, whereas conventional assays failed to do so. Together, these data suggest that proper identification of autoreactive B cells requires the membrane-bound BCR, as the soluble Ab may largely differ from its BCR counterpart in Ag binding.
Asunto(s)
Inmunoglobulina M/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Membrana Celular/inmunología , Células Cultivadas , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Mature B cells coexpress both IgM and IgD B-cell antigen receptor (BCR) classes, which are organized on the cell surface in distinct protein islands. The specific role of the IgD-BCR is still enigmatic, but it is colocalized with several other receptors on the B-cell surface, including the coreceptor CD19. Here, we report that the chemokine receptor CXCR4 is also found in proximity to the IgD-BCR. Furthermore, B cells from IgD-deficient mice show defects in CXCL12-mediated CXCR4 signaling and B-cell migration, whereas B cells from IgM-deficient mice are normal in this respect. CXCR4 activation results in actin cytoskeleton remodeling and PI3K/Akt and Erk signaling in an IgD-BCR-dependent manner. The defects in CXCR4 signaling in IgD-deficient B cells can be overcome by anti-CD19 antibody stimulation that also increases CXCL12-mediated B-cell migration of normal B cells. These results show that the IgD-BCR, CD19, and CXCR4 are not only colocalized at nanometer distances but are also functionally connected, thus providing a unique paradigm of receptor signaling cross talk and function.
Asunto(s)
Inmunoglobulina D/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores CXCR4/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Antígenos CD19/metabolismo , Linfocitos B/metabolismo , Citoesqueleto/metabolismo , Inmunoglobulina D/inmunología , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptor Cross-Talk/inmunología , Receptores CXCR4/inmunología , Transducción de SeñalRESUMEN
A high proportion of human B cells carry B-cell receptors (BCRs) that are autoreactive. Inhibitory receptors such as CD22 can downmodulate autoreactive BCR responses. With its extracellular domain, CD22 binds to sialic acids in α2,6 linkages in cis, on the surface of the same B cell or in trans, on other cells. Sialic acids are self ligands, as they are abundant in vertebrates, but are usually not expressed by pathogens. We show that cis-ligand binding of CD22 is crucial for the regulation of B-cell Ca(2+) signaling by controlling the CD22 association to the BCR. Mice with a mutated CD22 ligand-binding domain of CD22 showed strongly reduced Ca(2+) signaling. In contrast, mice with mutated CD22 immunoreceptor tyrosine-based inhibition motifs have increased B-cell Ca(2+) responses, increased B-cell turnover, and impaired survival of the B cells. Thus, the CD22 ligand-binding domain has a crucial function in regulating BCR signaling, which is relevant for controlling autoimmunity.
Asunto(s)
Linfocitos B/metabolismo , Señalización del Calcio , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismo , Animales , Linfocitos B/inmunología , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Unión ProteicaRESUMEN
In chronic lymphocytic leukemia (CLL), survival of neoplastic cells depends on microenvironmental signals at lymphoid sites where the crosstalk between the integrin VLA-4 (CD49d/CD29), expressed in ~40% of CLL, and the B-cell receptor (BCR) occurs. Here, BCR engagement inside-out activates VLA-4, thus enhancing VLA-4-mediated adhesion of CLL cells, which in turn obtain pro-survival signals from the surrounding microenvironment. We report that the BCR is also able to effectively inside-out activate the VLA-4 integrin in circulating CD49d-expressing CLL cells through an autonomous antigen-independent BCR signaling. As a consequence, circulating CLL cells exhibiting activated VLA-4 express markers of BCR pathway activation (phospho-BTK and phospho-PLC-γ2) along with higher levels of phospho-ERK and phospho-AKT indicating parallel activation of downstream pathways. Moreover, circulating CLL cells expressing activated VLA-4 bind soluble blood-borne VCAM-1 leading to increased VLA-4-dependent actin polymerization/re-organization and ERK phosphorylation. Finally, evidence is provided that ibrutinib treatment, by affecting autonomous BCR signaling, impairs the constitutive VLA-4 activation eventually decreasing soluble VCAM-1 binding and reducing downstream ERK phosphorylation by circulating CLL cells. This study describes a novel anchor-independent mechanism occurring in circulating CLL cells involving the BCR and the VLA-4 integrin, which help to unravel the peculiar biological and clinical features of CD49d+ CLL.
RESUMEN
Integrin-mediated interactions between hematopoietic cells and their microenvironment are important for the development and function of immune cells. Here, the role of the integrin adaptor Kindlin-3 in B cell homeostasis is studied. Comparing the individual steps of B cell development in B cell-specific Kindlin-3 or alpha4 integrin knockout mice, we found in both conditions a phenotype of reduced late immature, mature, and recirculating B cells in the bone marrow. In the spleen, constitutive B cell-specific Kindlin-3 knockout caused a loss of marginal zone B cells and an unexpected expansion of follicular B cells. Alpha4 integrin deficiency did not induce this phenotype. In Kindlin-3 knockout B cells VLA-4 as well as LFA-1-mediated adhesion was abrogated, and short-term homing of these cells in vivo was redirected to the spleen. Upon inducible Kindlin-3 knockout, marginal zone B cells were lost due to defective retention within 2 weeks, while follicular B cell numbers were unaltered. Kindlin-3 deficient follicular B cells displayed higher IgD, CD40, CD44, CXCR5, and EBI2 levels, and elevated PI3K signaling upon CXCR5 stimulation. They also showed transcriptional signatures of spontaneous follicular B cell activation. This activation manifested in scattered germinal centers in situ, early plasmablasts differentiation, and signs of IgG class switch.
Asunto(s)
Linfocitos B , Proteínas del Citoesqueleto , Animales , Linfocitos B/metabolismo , Adhesión Celular/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Integrina alfa4/metabolismo , Antígeno-1 Asociado a Función de Linfocito , Ratones , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
In chronic lymphocytic leukemia (CLL), the B cell receptor (BCR) plays a critical role in disease development and progression, as indicated by the therapeutic efficacy of drugs blocking BCR signaling. However, the mechanism(s) underlying BCR responsiveness are not completely defined. Selective engagement of membrane IgM or IgD on CLL cells, each coexpressed by more than 90% of cases, leads to distinct signaling events. Since both IgM and IgD carry the same antigen-binding domains, the divergent actions of the receptors are attributed to differences in immunoglobulin (Ig) structure or the outcome of signal transduction. We showed that IgM, not IgD, level and organization associated with CLL-cell birth rate and the type and consequences of BCR signaling in humans and mice. The latter IgM-driven effects were abrogated when BCR signaling was inhibited. Collectively, these studies demonstrated a critical, selective role for IgM in BCR signaling and B cell fate decisions, possibly opening new avenues for CLL therapy.
Asunto(s)
Linfocitos B/inmunología , Inmunoglobulina D/inmunología , Inmunoglobulina M/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Transducción de Señal/inmunología , Animales , Femenino , Humanos , Inmunoglobulina D/genética , Inmunoglobulina M/genética , Leucemia Linfocítica Crónica de Células B/genética , Masculino , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos B/genética , Transducción de Señal/genéticaRESUMEN
Expression of the membrane-bound form of the immunoglobulin (Ig) as part of the antigen receptor is indispensable for both the development and the effector function of B cells. Among five known isotypes, IgM and IgD are the common B cell antigen receptors (BCRs) that are co-expressed in naïve B cells. Despite having identical antigen specificity and being associated with the same signaling heterodimer Igα/Igß (CD79a/CD79b), IgM and IgD-BCR isotypes functionally differ from each other in the manner of antigen binding, the formation of isolated nanoclusters and in their interaction with co-receptors such as CD19 and CXCR4 on the plasma membrane. With recent developments in experimental techniques, it is now possible to investigate the nanoscale organization of the BCR and better understand early events of BCR engagement. Interestingly, the cytoskeleton network beneath the membrane controls the BCR isotype-specific organization and its interaction with co-receptors. BCR triggering results in reorganization of the cytoskeleton network, which is further modulated by isotype-specific signals from co-receptors. For instance, IgD-BCR is closely associated with CXCR4 on mature B cells and this close proximity allows CXCR4 to employ the BCR machinery as signaling hub. In this review, we discuss the functional specificity and nanocluster assembly of BCR isotypes and the consequences of cross-talk between CXCR4 and IgD-BCR. Furthermore, given the role of BCR and CXCR4 signaling in the development and survival of leukemic B cells, we discuss the consequences of the cross-talk between CXCR4 and the BCR for controlling the growth of transformed B cells.
Asunto(s)
Linfocitos B/inmunología , Citoesqueleto/inmunología , Receptor Cross-Talk/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores CXCR4/metabolismo , Linfocitos B/metabolismo , Citoesqueleto/metabolismo , Humanos , Isotipos de Inmunoglobulinas/inmunología , Isotipos de Inmunoglobulinas/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Receptores CXCR4/inmunología , Transducción de Señal/inmunologíaRESUMEN
Binding of antigen to the B cell antigen receptor (BCR) initiates a multitude of events resulting in B cell activation. How the BCR becomes signaling-competent upon antigen binding is still a matter of controversy. Using a high-resolution proximity ligation assay (PLA) to monitor the conformation of the BCR and its interactions with co-receptors at a 10-20 nm resolution, we provide direct evidence for the opening of BCR dimers during B cell activation. We also show that upon binding Syk opens the receptor by an inside-out signaling mechanism that amplifies BCR signaling. Furthermore, we found that on resting B cells, the coreceptor CD19 is in close proximity with the IgD-BCR and on activated B cells with the IgM-BCR, indicating nanoscale reorganization of receptor clusters during B cell activation.DOI: http://dx.doi.org/10.7554/eLife.02069.001.
Asunto(s)
Linfocitos B/metabolismo , Activación de Linfocitos , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Animales , Línea Celular , Dimerización , Humanos , RatonesRESUMEN
Cigarette smoking causes cellular oxidative stress resulting in inflammatory diseases of lung wherein transcription factor NF-κB plays an important role. It is possible that vitamin C, an antioxidant, may prevent cigarette smoke (CS)-induced NF-κB activation that involves degradation of I-κBε and nuclear translocation of c-Rel/p50 in alveolar epithelial cells. Therefore, to examine the hypothesis, we verified the effect of vitamin C on CS-induced expression of NF-κB driven luciferase reporter and NF-κB binding at its target DNA by EMSA in alveolar epithelial A549 cells. We also examined the level of I-κBε and sub-cellular distribution of c-Rel by western blotting and immunofluorescence respectively in CSE-treated A549 cells with or without vitamin C pretreatment. We observed a significant reduction in CSE induced luciferase expression, NF-κB DNA binding, I-κBε degradation and c-Rel nuclear translocation in cells pretreated with vitamin C. To further validate the result, we examined sub-cellular distribution of c-Rel in lungs of CS-exposed guinea pigs treated or untreated with vitamin C. Result showed that vitamin C treatment resulted in markedly reduced c-Rel nuclear translocation. All these results demonstrate that vitamin C prevents CS(E)-induced NF-κB activation and thus it could be used for the prevention of CS-induced inflammatory diseases.