RESUMEN
Although the essential role of the adaptor protein SLP-65 in pre-B cell differentiation is established, the molecular mechanism underlying its function is poorly understood. In this study, we uncover a link between SLP-65-dependent signaling and the phosphoinositide-3-OH kinase (PI(3)K)-protein kinase B (PKB)-Foxo pathway. We show that the forkhead box transcription factor Foxo3a promotes light chain rearrangement in pre-B cells. Our data suggest that PKB suppresses light chain recombination by phosphorylating Foxo proteins, whereas reconstitution of SLP-65 function counteracts PKB activation and promotes Foxo3a and Foxo1 activity in pre-B cells. Together, these data illuminate a molecular function of SLP-65 and identify a key role for Foxo proteins in the regulation of light chain recombination, receptor editing and B cell selection.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Linfocitos B/inmunología , Factores de Transcripción Forkhead/metabolismo , Genes de las Cadenas Ligeras de las Inmunoglobulinas/genética , Proteínas Tirosina Quinasas/metabolismo , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/genética , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Recombinación GenéticaRESUMEN
The majority of early immature B cells express autoreactive B cell receptors (BCRs) that are, according to the current view, negatively selected to avoid the production of self-reactive antibodies. Here, we show that polyreactive BCRs, which recognize multiple self-antigens, induced autonomous signaling and selective expansion of B cell precursors in a manner comparable to the pre-BCR. We found that the pre-BCR was capable of recognizing multiple self-antigens and that a signaling-deficient pre-BCR lacking the non-Ig region of the surrogate-light-chain component lambda5 was rescued by the complementarity-determining region 3 derived from heavy chains of polyreactive receptors. Importantly, bone marrow B cells from mice carrying Ig transgenes for an autoreactive BCR showed increased cell-cycle activity, which could not be detected in cells lacking the transgenic BCR. Together, the pre-BCR has evolved to ensure self-recognition because autoreactivity is required for positive selection of B cell precursors.
Asunto(s)
Autoantígenos/inmunología , Linfocitos B/inmunología , Imitación Molecular , Receptores de Células Precursoras de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Linfocitos B/metabolismo , Línea Celular , Proteínas de Unión al ADN/genética , Ratones , Ratones Transgénicos , Imitación Molecular/genética , Receptores de Células Precursoras de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Transducción de SeñalRESUMEN
DEC-205 is a type I transmembrane multilectin receptor that is predominantly expressed on dendritic cells (DCs). Therefore, previous studies primarily focused on processing of DEC-205targeted antigens by this potent antigen presenting cell type. Here we show that Epstein-Barr virus (EBV) transformed lymphoblastoid B-cell lines (LCLs) not only express DEC-205 at similar levels to DCs, but also efficiently present targeted EBV nuclear antigen 1 (EBNA1) and EBV-latent membrane protein 1 (LMP1) to EBNA1- and LMP1-specific CD4+ and CD8+ T-cell clones in vitro. Targeting of antigens to DEC-205 on B cells led to more efficient MHC class II than I loading, and stimulated T cells more efficiently than targeting to DEC-205 on DCs. Although LCLs internalized DEC-205targeted antigens less efficiently than DCs, they retained them for longer time periods and delivered them to endosomal compartments that receive also B-cell receptor targeted proteins. This could facilitate prolonged T-cell stimulation and efficient MHC class II loading, and, indeed, CD4+ T-cell expansion by DEC-205targeted vaccination was significantly compromised in B-cell deficient mice. These studies suggest that B cells, activated by virus transformation or other means, can contribute to T-cell stimulation after DEC-205 targeting of antigens during vaccination.
Asunto(s)
Antígenos CD/metabolismo , Linfocitos B/inmunología , Linfocitos B/virología , Células Dendríticas/inmunología , Herpesvirus Humano 4/fisiología , Lectinas Tipo C/metabolismo , Receptores de Superficie Celular/metabolismo , Linfocitos T/inmunología , Animales , Presentación de Antígeno/fisiología , Antígenos CD/inmunología , Linfocitos B/metabolismo , Línea Celular Transformada , Transformación Celular Viral/inmunología , Células Cultivadas , Células Dendríticas/fisiología , Herpesvirus Humano 4/inmunología , Lectinas Tipo C/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor , Terapia Molecular Dirigida , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Superficie Celular/inmunología , Linfocitos T/metabolismo , Linfocitos T/fisiología , Vacunación/métodosRESUMEN
Functional differences between human dendritic cell (DC) subsets and the potential benefits of targeting them with vaccines remain poorly defined. Here we describe that mice with reconstituted human immune system components (huNSG mice) develop all human conventional and plasmacytoid DC compartments in lymphoid organs. Testing different Toll-like receptor agonists for DC maturation in vivo, we found that IL-12p70 and interferon (IFN)-α production correlated with the maturation of CD141+ (BDCA3+) conventional DCs in huNSG mice. Furthermore, depletion of CD141+ DCs before stimulation significantly reduced IFN-α levels in vivo. This DC subset produced similar total amounts but different subtypes of IFN-α in response to synthetic double-stranded RNA compared with plasmacytoid DCs in response to a single-stranded RNA equivalent. Moreover, synthetic double-stranded RNA as adjuvant and antigen targeting to the endocytic receptor DEC-205, a combination that focuses antigen presentation for T-cell priming on CD141+ DCs, stimulated antigen-specific human CD4+ T-cell responses. Thus, the human CD141+ DC subset is a prominent source of IFN-α and interleukin-12 production and should be further evaluated for vaccine development.
Asunto(s)
Antígenos CD/inmunología , Células Dendríticas/inmunología , Interferón-alfa/biosíntesis , Lectinas Tipo C/inmunología , Activación de Linfocitos/inmunología , ARN Bicatenario/inmunología , Receptores de Superficie Celular/inmunología , Animales , Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/citología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Interferón-alfa/inmunología , Ratones , Antígenos de Histocompatibilidad MenorRESUMEN
The nonreceptor protein spleen tyrosine kinase (Syk) is a key mediator of signal transduction in a variety of cell types, including B lymphocytes. We show that deregulated Syk activity allows growth factor-independent proliferation and transforms bone marrow-derived pre-B cells that are then able to induce leukemia in mice. Syk-transformed pre-B cells show a characteristic pattern of tyrosine phosphorylation, increased c-Myc expression, and defective differentiation. Treatment of Syk-transformed pre-B cells with a novel Syk-specific inhibitor (R406) reduces tyrosine phosphorylation and c-Myc expression. In addition, R406 treatment removes the developmental block and allows the differentiation of the Syk-transformed pre-B cells into immature B cells. Because R406 treatment also prevents the proliferation of c-Myc-transformed pre-B cells, our data indicate that endogenous Syk kinase activity may be required for the survival of pre-B cells transformed by other oncogenes. Collectively, our data suggest that Syk is a protooncogene involved in the transformation of lymphocytes, thus making Syk a potential target for the treatment of leukemia.
Asunto(s)
Linfocitos B/metabolismo , Diferenciación Celular/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Traslado Adoptivo , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/trasplante , Benzamidas , Diferenciación Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Péptidos y Proteínas de Señalización Intercelular/farmacología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Leucemia/genética , Leucemia/patología , Leucemia/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Oxazinas/farmacología , Fosfolipasa C gamma/genética , Fosfolipasa C gamma/metabolismo , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Piridinas/farmacología , Pirimidinas/farmacología , Receptores de Antígenos de Linfocitos B/genética , Bazo/efectos de los fármacos , Bazo/metabolismo , Bazo/patología , Quinasa Syk , TransfecciónRESUMEN
To investigate human natural killer (NK)-cell reactivity in vivo we have reconstituted human immune system components by transplantation of human hematopoietic progenitor cells into NOD-scid IL2Rγ(null) mice. We demonstrate here that this model allows the development of all NK-cell subsets that are also found in human adult peripheral and cord blood, including NKp46(+)CD56(-) NK cells. Similar to human cord blood, NK cells from these reconstituted mice require preactivation by interleukin-15 to reach the functional competence of human adult NK cells. Mainly the terminally differentiated CD16(+) NK cells demonstrate lower reactivity without this stimulation. After preactivation, both CD16(+) and CD16(-) NK cells efficiently produce interferon-γ and degranulate in response to stimulation with NK cell-susceptible targets, including K562 erythroleukemia cells. NK-cell lines, established from reconstituted mice, demonstrate cytotoxicity against this tumor cell line. Importantly, preactivation can as well be achieved by bystander cell maturation via poly I:C stimulation in vitro and injection of this maturation stimulus in vivo. Preactivation in vivo enhances killing of human leukocyte antigen class I negative tumor cells after their adoptive transfer. These data suggest that a functional, but resting, NK-cell compartment can be established in immune-compromised mice after human hematopoietic progenitor cell transfer.
Asunto(s)
Sistema Inmunológico/inmunología , Inmunocompetencia/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Adulto , Animales , Antígenos CD34/metabolismo , Antígeno CD56/metabolismo , Degranulación de la Célula/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Citotoxicidad Inmunológica/efectos de los fármacos , Sangre Fetal/citología , Granzimas/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/inmunología , Humanos , Sistema Inmunológico/efectos de los fármacos , Inmunocompetencia/efectos de los fármacos , Interferón gamma/biosíntesis , Interleucina-15/farmacología , Células Asesinas Naturales/citología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/fisiología , Activación de Linfocitos/efectos de los fármacos , Subgrupos Linfocitarios/citología , Subgrupos Linfocitarios/efectos de los fármacos , Subgrupos Linfocitarios/inmunología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptor 1 Gatillante de la Citotoxidad Natural/metabolismo , Perforina/metabolismo , Poli I-C/administración & dosificación , Poli I-C/farmacología , Receptores de Interleucina-2/metabolismoRESUMEN
Many pathogens relevant to human disease do not infect other animal species. Therefore, animal models that reconstitute or harbor human tissues are explored as hosts for these. In this review, we will summarize recent advances to utilize mice with human immune system components, reconstituted from hematopoietic progenitor cells in vivo. Such mice can be used to study human pathogens that replicate in leukocytes. In addition to studying the replication of these pathogens, the reconstituted human immune system components can also be analyzed for initiating immune responses and control against these infections. Moreover, these new animal models of human infectious disease should replicate the reactivity of the human immune system to vaccine candidates and, especially, the adjuvants contained in them, more faithfully.
Asunto(s)
Sistema Inmunológico/inmunología , Inmunidad Adaptativa/inmunología , Animales , Humanos , Inmunidad Innata/inmunología , Ratones , Modelos Animales , VacunaciónRESUMEN
Mutations in genes involved in macroautophagy have been found to be associated with Morbus Crohn, also called Crohn's disease (CD), an inflammatory bowel disease. Taking this disease as an example for pathogenesis due to altered macroautophagy, we discuss here how macroautophagy supports innate and adaptive immunity. This support ranges from maintenance of components of the immune system, antigen processing for presentation to the immune system, to education of the immune system in order to distinguish self from dangerous non-self. A better understanding of these mechanisms should allow us not only to develop therapeutical strategies for CD but also to utilize macroautophagy for enhanced immunity against pathogens and tumors.
Asunto(s)
Autofagia/inmunología , Proteínas Portadoras/inmunología , Enfermedad de Crohn/inmunología , Citocinas/inmunología , Proteínas Asociadas a Microtúbulos/inmunología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/inmunología , Linfocitos T/inmunología , Inmunidad Adaptativa , Animales , Presentación de Antígeno/inmunología , Proteínas Portadoras/metabolismo , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/genética , Citocinas/metabolismo , Humanos , Inmunidad Innata , Inmunosupresores/uso terapéutico , Proteínas Asociadas a Microtúbulos/metabolismo , Sirolimus/uso terapéutico , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Linfocitos T/metabolismoRESUMEN
Epstein-Barr virus (EBV) persistently infects more than 90% of the human population and is etiologically linked to several B cell malignancies, including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B cell lymphoma (DLBCL). Despite its growth transforming properties, most immune-competent individuals control EBV infection throughout their lives. EBV encodes various oncogenes, and of the 6 latency-associated EBV-encoded nuclear antigens, only EBNA3B is completely dispensable for B cell transformation in vitro. Here, we report that infection with EBV lacking EBNA3B leads to aggressive, immune-evading monomorphic DLBCL-like tumors in NOD/SCID/γc-/- mice with reconstituted human immune system components. Infection with EBNA3B-knockout EBV (EBNA3BKO) induced expansion of EBV-specific T cells that failed to infiltrate the tumors. EBNA3BKO-infected B cells expanded more rapidly and secreted less T cell-chemoattractant CXCL10, reducing T cell recruitment in vitro and T cell-mediated killing in vivo. B cell lines from 2 EBV-positive human lymphomas encoding truncated EBNA3B exhibited gene expression profiles and phenotypic characteristics similar to those of tumor-derived lines from the humanized mice, including reduced CXCL10 secretion. Screening EBV-positive DLBCL, HL, and BL human samples identified additional EBNA3B mutations. Thus, EBNA3B is a virus-encoded tumor suppressor whose inactivation promotes immune evasion and virus-driven lymphomagenesis.
Asunto(s)
Transformación Celular Viral/genética , Infecciones por Virus de Epstein-Barr/virología , Antígenos Nucleares del Virus de Epstein-Barr/fisiología , Genes Supresores de Tumor , Genes Virales , Herpesvirus Humano 4/fisiología , Linfoma de Células B/virología , Trastornos Linfoproliferativos/virología , Complicaciones Posoperatorias/virología , Proteínas Supresoras de Tumor/fisiología , Infecciones Tumorales por Virus/virología , Animales , Línea Celular Transformada/trasplante , Línea Celular Transformada/virología , Quimiocina CXCL10/biosíntesis , Quimiocina CXCL10/deficiencia , Quimiocina CXCL10/genética , Quimera , Análisis Mutacional de ADN , Infecciones por Virus de Epstein-Barr/genética , Antígenos Nucleares del Virus de Epstein-Barr/genética , Eliminación de Gen , Trasplante de Células Madre Hematopoyéticas , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidad , Humanos , Interferón gamma/deficiencia , Interferón gamma/genética , Linfoma de Células B/genética , Trastornos Linfoproliferativos/genética , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Mutación , Complicaciones Posoperatorias/genética , Trasplante Heterólogo , Proteínas Supresoras de Tumor/genética , Infecciones Tumorales por Virus/genéticaRESUMEN
Signals from the pre-B cell receptor (pre-BCR) mediated by the cytoplasmic tails of Ig-alpha/Ig-beta are essential for developing B cells. To analyze the role of Ig-alpha ITAM and non-ITAM tyrosines in pre-BCR signaling, we reconstituted individual tyrosine mutants of Ig-alpha in src homology 2 domain-containing leukocyte protein of 65 kDa (SLP-65)/Ig-alpha double-deficient pre-B cells. We show that the Ig-alpha mutants led to comparable pre-BCR expression on the cell surface, while the pre-BCR-induced tyrosine phosphorylation was different. We further show that the reconstitution of Ig-alpha and the resulting pre-BCR expression led to enrichment of the pre-BCR-expressing cells in vitro irrespective of the introduced Ig-alpha mutation. We show that, even though the enrichment rate increased by lowering the IL-7 concentration, residual amounts of IL-7 were required for optimal enrichment. Our results indicate that surface IL-7 receptor expression is modulated by the pre-BCR, thereby increasing the IL-7 sensitivity of the respective cells. In contrast to the comparable pre-B cell proliferation, however, the Ig-alpha mutants differed in their capacity to induce calcium flux and activate efficient pre-B cell differentiation. Together, our data suggest that ITAM tyrosines and Y204 are required for efficient pre-B cell differentiation but not proliferation.
Asunto(s)
Subgrupos de Linfocitos B/citología , Antígenos CD79/fisiología , Diferenciación Celular/inmunología , Proliferación Celular , Células Madre/citología , Células Madre/inmunología , Tirosina , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencias de Aminoácidos/genética , Animales , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Antígenos CD79/deficiencia , Antígenos CD79/genética , Diferenciación Celular/genética , Células Cultivadas , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Mutagénesis Sitio-Dirigida , Fosforilación , Receptores de Células Precursoras de Linfocitos B , Receptores de Antígenos de Linfocitos B/biosíntesis , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/fisiología , Transducción de Señal/genética , Transducción de Señal/inmunología , Células Madre/metabolismo , Tirosina/genéticaRESUMEN
Signals from the B cell antigen receptor (BCR), consisting of mu heavy chain (muHC) and conventional light chain (LC), and its precursor the pre-BCR, consisting of muHC and surrogate light chain (SLC), via the adaptor protein SLP-65 regulate the development and function of B cells. Here, we compare the effect of SLC and conventional LC expression on receptor-induced Ca(2+) flux in B cells expressing an inducible form of SLP-65. We found that SLC expression strongly enhanced an autonomous ability of muHC to induce Ca(2+) flux irrespective of additional receptor crosslinking. In contrast, LC expression reduced this autonomous muHC ability and resulted in antigen-dependent Ca(2+) flux. These data indicate that autonomous ligand-independent signaling can be induced by receptor forms other than the pre-BCR. In addition, our data suggest that conventional LCs play an important role in the inhibition of autonomous receptor signaling, thereby allowing further B cell differentiation.
Asunto(s)
Linfocitos B/inmunología , Cadenas Ligeras de Inmunoglobulina/metabolismo , Receptores de Antígenos de Linfocitos B/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Calcio/metabolismo , Línea Celular Tumoral , Humanos , Inmunoglobulina de Cadenas Ligeras Subrogadas , Ligandos , Glicoproteínas de Membrana/metabolismo , Ratones , Fosforilación , Transducción de SeñalRESUMEN
Ikaros DNA-binding proteins are critical for the development of lymphocytes and other hematopoietic lineages, but it remains unclear how they cooperate with other regulators of signaling and transcription to achieve ordered gene expression during development. Here, we show that Ikaros proteins regulate the pre-BCR component lambda5 in a stage-specific manner. In pre-BI cells, Ikaros modulated lambda5 expression in competition with the transcriptional activator EBF. This required Ikaros binding to the Igll1 (lambda5) promoter and was abolished either by mutation of the Ikaros DNA-binding domain or by deletion of a single Ikaros site from the Igll1 promoter. At the transition from the pre-BI to pre-BII stage, the expression of the Ikaros family member Aiolos was upregulated and required for the efficient silencing of Igll1. Aiolos expression was controlled by pre-BCR signals via the adaptor protein SLP-65. Thus, pre-BCR signaling regulates Aiolos and the silencing of Igll1 via a developmental-stage-specific feedback loop.