RESUMEN
The generation, differentiation, survival and activation of B cells are coordinated by signals emerging from the B cell antigen receptor (BCR) or its precursor, the pre-BCR. The adaptor protein SLP65 (also known as BLNK) is an important signaling factor that controls pre-B cell differentiation by down-regulation of PI3K signaling. Here, we investigated the mechanism by which SLP65 interferes with PI3K signaling. We found that SLP65 induces the activity of the small GTPase RHOA, which activates PTEN, a negative regulator of PI3K signaling, by enabling its translocation to the plasma membrane. The essential role of RHOA is confirmed by the complete block in early B cell development in conditional RhoA-deficient mice. The RhoA-deficient progenitor B cells showed defects in activation of immunoglobulin gene rearrangement and fail to survive both in vitro and in vivo. Reconstituting the RhoA-deficient cells with RhoA or Foxo1, a transcription factor repressed by PI3K signaling and activated by PTEN, completely restores the survival defect. However, the defect in differentiation can only be restored by RhoA suggesting a unique role for RHOA in B cell generation and selection. In full agreement, conditional RhoA-deficient mice develop increased amounts of autoreactive antibodies with age. RHOA function is also required at later stage, as inactivation of RhoA in peripheral B cells or in a transformed mature B cell line resulted in cell loss. Together, these data show that RHOA is the key signaling factor for B cell development and function by providing a crucial SLP65-activated link between BCR signaling and activation of PTEN. Moreover, the identified essential role of RHOA for the survival of transformed B cells offers the opportunity for targeting B cell malignancies by blocking RHOA function.
Asunto(s)
Proteínas de Unión al GTP Monoméricas , Células Precursoras de Linfocitos B , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Ratones , Proteínas de Unión al GTP Monoméricas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/genética , Proteína de Unión al GTP rhoARESUMEN
Homeostasis of metabolism by hormone production is crucial for maintaining physiological integrity, as disbalance can cause severe metabolic disorders such as diabetes mellitus. Here, we show that antibody-deficient mice and immunodeficiency patients have subphysiological blood glucose concentrations. Restoring blood glucose physiology required total IgG injections and insulin-specific IgG antibodies detected in total IgG preparations and in the serum of healthy individuals. In addition to the insulin-neutralizing anti-insulin IgG, we identified two fractions of anti-insulin IgM in the serum of healthy individuals. These autoreactive IgM fractions differ in their affinity to insulin. Interestingly, the low-affinity IgM fraction (anti-insulin IgMlow) neutralizes insulin and leads to increased blood glucose, whereas the high-affinity IgM fraction (anti-insulin IgMhigh) protects insulin from neutralization by anti-insulin IgG, thereby preventing blood glucose dysregulation. To demonstrate that anti-insulin IgMhigh acts as a protector of insulin and counteracts insulin neutralization by anti-insulin IgG, we expressed the variable regions of a high-affinity anti-insulin antibody as IgG and IgM. Remarkably, the recombinant anti-insulin IgMhigh normalized insulin function and prevented IgG-mediated insulin neutralization. These results suggest that autoreactive antibodies recognizing insulin are key regulators of blood glucose and metabolism, as they control the concentration of insulin in the blood. Moreover, our data suggest that preventing autoimmune damage and maintaining physiological homeostasis requires adaptive tolerance mechanisms generating high-affinity autoreactive IgM antibodies during memory responses.
Asunto(s)
Autoanticuerpos/inmunología , Glucemia/inmunología , Homeostasis/inmunología , Insulina/inmunología , Animales , Afinidad de Anticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Femenino , Humanos , Tolerancia Inmunológica/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Ratones , Ratones Endogámicos C57BLRESUMEN
Mature B cells co-express IgM and IgD B cell antigen receptors (BCR) on their surface. While IgM BCR expression is already essential at early stages of development, the role of the IgD-class BCR remains unclear as most B cell functions appeared unchanged in IgD-deficient mice. Here, we show that IgD-deficient mice have an accelerated rate of B cell responsiveness as they activate antibody production within 24h after immunization, whereas wildtype (WT) animals required 3 days to activate primary antibody responses. Strikingly, soluble monovalent antigen suppresses IgG antibody production induced by multivalent antigen in WT mice. In contrast, IgD-deficient mice were not able to modulate IgG responses suggesting that IgD controls the activation rate of B cells and subsequent antibody production by sensing and distinguishing antigen-valences. Using an insulin-derived peptide we tested the role of IgD in autoimmunity. We show that primary autoreactive antibody responses are generated in WT and in IgD-deficient mice. However, insulin-specific autoantibodies were detected earlier and caused more severe symptoms of autoimmune diabetes in IgD-deficient mice as compared to WT mice. The rapid control of autoimmune diabetes in WT animals was associated with the generation of high-affinity IgM that protects insulin from autoimmune degradation. In IgD-deficient mice, however, the generation of high-affinity protective IgM is delayed resulting in prolonged autoimmune diabetes. Our data suggest that IgD is required for the transition from primary, highly autoreactive, to secondary antigen-specific antibody responses generated by affinity maturation.
Asunto(s)
Afinidad de Anticuerpos , Formación de Anticuerpos , Inmunoglobulina D/fisiología , Animales , Autoantígenos/inmunología , Autoinmunidad , Linfocitos B/inmunología , Femenino , Inmunoglobulina G/biosíntesis , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos B/inmunologíaRESUMEN
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
Activation of phosphoinositide 3-kinase (PI3K) signaling plays a central role in regulating proliferation and survival of B cells. Here, we tested the hypothesis that B cell receptor (BCR)-mediated activation of PI3K induces the terminal differentiation factor Blimp-1 that interferes with proliferation and survival, thereby controlling the expansion of activated B cells. In fact, B-cell-specific inactivation of Pten, the negative regulator of PI3K signaling, leads to deregulated PI3K activity and elevated Blimp-1 expression. Combined deficiency for Pten and Blimp-1 results in abnormal expansion of B-1 B cells and splenomegaly. Interestingly, Blimp-1 also acts at early stages of B cell development to regulate B cell selection, as Blimp-1 deficiency results in an increased proportion of autoreactive B cells. Together, our data suggest that the combined requirement of deregulated PI3K signaling in addition to defective terminal differentiation represents the basis for proper selection and expansion of developing B cells.
Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Homeostasis , Fosfatidilinositol 3-Quinasas/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Animales , Compartimento Celular , Muerte Celular , Diferenciación Celular , Proliferación Celular , Citoprotección , Femenino , Masculino , Ratones Transgénicos , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva/deficiencia , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismoRESUMEN
Human cytomegalovirus (HCMV) persistently infects 40-90% of the human population but in the face of a normal immune system, viral spread and dissemination are efficiently controlled thus preventing clinically signs and disease. HCMV-infected hosts produce a remarkably large amount of HCMV-specific CD4+ and CD8+ T cells that can even reach 20-50% of total T memory cells in the elderly. How HCMV may elicit such large and long-lasting T-cell responses in the absence of detectable viremia has not been elucidated yet. Additionally, HCMV is known to encode several gene products that potently inhibit T-cell recognition of infected cells. The best characterized are the four immune evasive US2, US3, US6, and US11 genes that by different mechanisms account for major histocompatibility complex (MHC) class I and class II degradation and intracellular retention in infected cells. By infecting M1 and M2 human macrophages (Mφ) with the wild-type HCMV strain TB40E or a mutant virus deleted of the four immune evasive genes US2, US3, US6, and US11, we demonstrated that human Mφ counteract the inhibitory potential of the US2-11 genes and remain capable to present peptides via MHC class I and class II molecules. Moreover, by sorting the infected and bystander cells, we provide evidence that both infected and bystander Mφ contribute to antigen presentation to CD4+ and CD8+ T cells. The T cells responding to TB40E-infected Mφ show markers of the T effector memory compartment, produce interferon-γ, and express the lytic granule marker CD107a on the cell surface, thus mirroring the HCMV-specific T cells present in healthy seropositive individuals. All together, our findings reveal that human Mφ escape inhibition of MHC-dependent antigen presentation by HCMV and continue to support T cell proliferation and activation after HCMV infection. Taking into account that Mφ are natural targets of HCMV infection and a site of viral reactivation from latency, our findings support the hypothesis that Mφ play crucial roles for the lifelong maintenance and expansion of HCMV-committed T cells in the human host.
Asunto(s)
Presentación de Antígeno/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Macrófagos/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Antígenos Virales/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Línea Celular , Citomegalovirus/genética , Infecciones por Citomegalovirus/metabolismo , Infecciones por Citomegalovirus/virología , Regulación Viral de la Expresión Génica , Humanos , Memoria Inmunológica , Activación de Linfocitos/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Proteínas Virales/genética , Proteínas Virales/inmunologíaRESUMEN
Phosphoinositide-3 kinase (PI3K) signaling is important for the survival of numerous cell types and class IA of PI3K is specifically required for the development of B cells but not for T cell development. Here, we show that class IA PI3K-mediated signals induce the expression of the transcription factor Pax5, which plays a central role in B cell commitment and differentiation by activating the expression of central B cell-specific signaling proteins such as SLP-65 and CD19. Defective class IA PI3K function leads to reduction in Pax5 expression and prevents B cell development beyond the stage expressing the precursor B cell receptor (pre-BCR). Investigating the mechanism of PI3K-induced Pax5 expression revealed that it involves a network of transcription factors including FoxO1 and Irf4 that directly binds to the Pax5 gene. Together, our results suggest that PI3K signaling links survival and differentiation of developing B cells with B cell identity and that decreased PI3K activity in pre-B cells results in reduced Pax5 expression and lineage plasticity.
Asunto(s)
Linfocitos B/metabolismo , Linfopoyesis , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antígenos CD19/metabolismo , Linfocitos B/citología , Línea Celular , Linaje de la Célula , Células Cultivadas , Proteína Forkhead Box O1/metabolismo , Factores Reguladores del Interferón/metabolismo , Ratones , Factor de Transcripción PAX5/metabolismoRESUMEN
B-cell antigen receptor (BCR) expression is a key feature of most B-cell lymphomas, but the mechanisms of BCR signal induction and the involvement of autoantigen recognition remain unclear. In follicular lymphoma (FL) B cells, BCR expression is retained despite a chromosomal translocation that links the antiapoptotic gene BCL2 to the regulatory elements of immunoglobulin genes, thereby disrupting 1 heavy-chain allele. A remarkable feature of FL-BCRs is the acquisition of potential N-glycosylation sites during somatic hypermutation. The introduced glycans carry mannose termini, which create potential novel binding sites for mannose-specific lectins. Here, we investigated the effect of N-linked variable-region glycosylation for BCR interaction with cognate antigen and with lectins of different origins. N-glycans were found to severely impair BCR specificity and affinity to the initial cognate antigen. In addition, we found that lectins from Pseudomonas aeruginosa and Burkholderia cenocepacia bind and stimulate FL cells. Human exposure to these bacteria can occur by contact with soil and water. In addition, they represent opportunistic pathogens in susceptible hosts. Understanding the role of bacterial lectins might elucidate the pathogenesis of FL and establish novel therapeutic approaches.