RESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Linfócitos B/imunologia , Fatores de Transcrição Forkhead/metabolismo , Genes de Cadeia Leve de Imunoglobulina/genética , Proteínas Tirosina Quinases/metabolismo , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/genética , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Recombinação GenéticaRESUMO
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.
Assuntos
Autoantígenos/imunologia , Linfócitos B/imunologia , Mimetismo Molecular , Receptores de Células Precursoras de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Linfócitos B/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , Camundongos , Camundongos Transgênicos , Mimetismo Molecular/genética , Receptores de Células Precursoras de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Antígenos CD/metabolismo , Linfócitos B/imunologia , Linfócitos B/virologia , Células Dendríticas/imunologia , Herpesvirus Humano 4/fisiologia , Lectinas Tipo C/metabolismo , Receptores de Superfície Celular/metabolismo , Linfócitos T/imunologia , Animais , Apresentação de Antígeno/fisiologia , Antígenos CD/imunologia , Linfócitos B/metabolismo , Linhagem Celular Transformada , Transformação Celular Viral/imunologia , Células Cultivadas , Células Dendríticas/fisiologia , Herpesvirus Humano 4/imunologia , Lectinas Tipo C/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Terapia de Alvo Molecular , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Superfície Celular/imunologia , Linfócitos T/metabolismo , Linfócitos T/fisiologia , Vacinação/métodosRESUMO
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.
Assuntos
Antígenos CD/imunologia , Células Dendríticas/imunologia , Interferon-alfa/biossíntese , Lectinas Tipo C/imunologia , Ativação Linfocitária/imunologia , RNA de Cadeia Dupla/imunologia , Receptores de Superfície Celular/imunologia , Animais , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/citologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Interferon-alfa/imunologia , Camundongos , Antígenos de Histocompatibilidade MenorRESUMO
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.
Assuntos
Linfócitos B/metabolismo , Diferenciação Celular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transferência Adotiva , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/transplante , Benzamidas , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia/genética , Leucemia/patologia , Leucemia/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Oxazinas/farmacologia , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Receptores de Antígenos de Linfócitos B/genética , Baço/efeitos dos fármacos , Baço/metabolismo , Baço/patologia , Quinase Syk , TransfecçãoRESUMO
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.
Assuntos
Sistema Imunitário/imunologia , Imunocompetência/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Adulto , Animais , Antígenos CD34/metabolismo , Antígeno CD56/metabolismo , Degranulação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Sangue Fetal/citologia , Granzimas/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/imunologia , Humanos , Sistema Imunitário/efeitos dos fármacos , Imunocompetência/efeitos dos fármacos , Interferon gama/biossíntese , Interleucina-15/farmacologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/fisiologia , Ativação Linfocitária/efeitos dos fármacos , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptor 1 Desencadeador da Citotoxicidade Natural/metabolismo , Perforina/metabolismo , Poli I-C/administração & dosagem , Poli I-C/farmacologia , Receptores de Interleucina-2/metabolismoRESUMO
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.
Assuntos
Sistema Imunitário/imunologia , Imunidade Adaptativa/imunologia , Animais , Humanos , Imunidade Inata/imunologia , Camundongos , Modelos Animais , VacinaçãoRESUMO
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.
Assuntos
Autofagia/imunologia , Proteínas de Transporte/imunologia , Doença de Crohn/imunologia , Citocinas/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/imunologia , Linfócitos T/imunologia , Imunidade Adaptativa , Animais , Apresentação de Antígeno/imunologia , Proteínas de Transporte/metabolismo , Doença de Crohn/tratamento farmacológico , Doença de Crohn/genética , Citocinas/metabolismo , Humanos , Imunidade Inata , Imunossupressores/uso terapêutico , Proteínas Associadas aos Microtúbulos/metabolismo , Sirolimo/uso terapêutico , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Linfócitos T/metabolismoRESUMO
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.
Assuntos
Transformação Celular Viral/genética , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/fisiologia , Genes Supressores de Tumor , Genes Virais , Herpesvirus Humano 4/fisiologia , Linfoma de Células B/virologia , Transtornos Linfoproliferativos/virologia , Complicações Pós-Operatórias/virologia , Proteínas Supressoras de Tumor/fisiologia , Infecções Tumorais por Vírus/virologia , Animais , Linhagem Celular Transformada/transplante , Linhagem Celular Transformada/virologia , Quimiocina CXCL10/biossíntese , Quimiocina CXCL10/deficiência , Quimiocina CXCL10/genética , Quimera , Análise Mutacional de DNA , Infecções por Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/genética , Deleção de Genes , Transplante de Células-Tronco Hematopoéticas , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/patogenicidade , Humanos , Interferon gama/deficiência , Interferon gama/genética , Linfoma de Células B/genética , Transtornos Linfoproliferativos/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Mutação , Complicações Pós-Operatórias/genética , Transplante Heterólogo , Proteínas Supressoras de Tumor/genética , Infecções Tumorais por Vírus/genéticaRESUMO
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.
Assuntos
Subpopulações de Linfócitos B/citologia , Antígenos CD79/fisiologia , Diferenciação Celular/imunologia , Proliferação de Células , Células-Tronco/citologia , Células-Tronco/imunologia , Tirosina , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos/genética , Animais , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Antígenos CD79/deficiência , Antígenos CD79/genética , Diferenciação Celular/genética , Células Cultivadas , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Fosforilação , Receptores de Células Precursoras de Linfócitos B , Receptores de Antígenos de Linfócitos B/biossíntese , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células-Tronco/metabolismo , Tirosina/genéticaRESUMO
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.
Assuntos
Linfócitos B/imunologia , Cadeias Leves de Imunoglobulina/metabolismo , Receptores de Antígenos de Linfócitos B/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Humanos , Cadeias Leves Substitutas da Imunoglobulina , Ligantes , Glicoproteínas de Membrana/metabolismo , Camundongos , Fosforilação , Transdução de SinaisRESUMO
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.