RESUMEN
Blimp-1 is a master regulator of terminal B cell differentiation and plays a pivotal role in various developmental processes. In addition to full length Blimp-1, a Blimp-1 mRNA lacking exon 7 (Blimp-1Delta7) has been described to occur in murine B cells. The activity and function of the mutant mRNA-encoded protein (Blimp-1Delta7), lacking three crucial zinc fingers necessary for DNA interaction, is completely unknown. Since isoforms of other prdm family proteins affect each other's functions, we wondered whether Blimp-1Delta7 still plays a role in B cells, independent of direct DNA binding. In this study, we found that Blimp-1Delta7 is preferentially expressed in naïve CD19(+) B cells. A fraction of Blimp-1Delta7 migrates to the nucleus, colocalizes with HDAC2 and is found at sites of repressed chromatin, although it does not bind to the Blimp-1 DNA consensus site. Unexpectedly, Blimp-1 and Blimp-1Delta7 homodimerize as well as heterodimerize with each other. Ectopic expression of Blimp-1Delta7 in WEHI 231 cells, a Blimp-1-negative murine lymphoma line, leads to cessation of proliferation and enhancement of apoptosis. Importantly, LPS-induced differentiation is suppressed in the presence of Blimp-1Delta7. This is in agreement with our finding that Blimp-1Delta7 interferes with endogenous Blimp-1 expression. Thus, our data suggest an auto-regulatory mechanism of Blimp-1 activation.
Asunto(s)
Eliminación de Gen , Regulación de la Expresión Génica , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Animales , Antígenos CD19/metabolismo , Apoptosis/genética , Comunicación Autocrina/genética , Linfocitos B/metabolismo , Ciclo Celular/genética , Células Cultivadas , Exones , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiología , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
In multiple sclerosis, CD8 T-cells are thought play a key pathogenetic role, but mechanistic evidence from rodent models is limited. Here, we have tested the encephalitogenic potential of CD8 T-cells specific for the model antigen ovalbumin (OVA) sequestered in oligodendrocytes as a cytosolic molecule. We show that in these 'ODC-OVA' mice, the neo-self antigen remains invisible to CD4 cells expressing the OVA-specific OT-II receptor. In contrast, OVA is accessible to naïve CD8 T-cells expressing the OT-I T-cell receptor, during the first 10 days of life, resulting in antigen release into the periphery. Introduction of OT-I as a second transgene leads to fulminant demyelinating experimental autoimmune encephalomyelitis with multiple sclerosis-like lesions, affecting cerebellum, brainstem, optic nerve and spinal cord. OVA-transgenic oligodendrocytes activate naïve OT-I cells in vitro, and both major histocompatibility complex class I expression and the OT-I response are further up-regulated by interferon-gamma (IFN-gamma). Release of IFN-gamma into the circulation of ODC-OVA/OT-I double transgenic mice precedes disease manifestation, and pathogenicity of OT-I cells transferred into ODC-OVA mice is largely IFN-gamma dependent. In conclusion, naïve CD8 T-cells gaining access to an 'immune-privileged' organ can initiate autoimmunity via an IFN-gamma-assisted amplification loop even if the self-antigen in question is not spontaneously released for presentation by professional antigen presenting cells.
Asunto(s)
Autoinmunidad , Linfocitos T CD8-positivos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Traslado Adoptivo , Animales , Presentación de Antígeno/inmunología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Cerebelo/inmunología , Cerebelo/patología , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/patología , Interferón gamma/inmunología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oligodendroglía/inmunología , Ovalbúmina/inmunología , Médula Espinal/inmunología , Médula Espinal/patologíaRESUMEN
The transcription factor C/EBPbeta transactivates the IL-4 gene in murine T lymphocytes and facilitates Th2 cell responses. In this study, we demonstrate that C/EBPbeta also acts as a repressor of T cell proliferation. By binding to the c-myc promoter(s), C/EBPbeta represses c-Myc expression and, therefore, arrests T cells in the G1 phase of the cell cycle. For C/EBPbeta-mediated repression, the integrity of its N-terminal transactivation domain is essential whereas the central regulatory domain is dispensable. This central regulatory domain is sumoylated in vivo which leads to an alteration of the activity of C/EBPbeta. Whereas sumoylation does not affect the C/EBPbeta-mediated activation of the IL-4 gene, it relieves its repressive effect on c-Myc expression and T cell proliferation. Similar to several other transcription factors, sumoylation redistributes nuclear C/EBPbeta and targets it to pericentric heterochromatin. These results suggest an important role of sumoylation in adjusting the finely tuned balance between proliferation and differentiation in peripheral T cells which is controlled by C/EBPbeta.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Genes myc , Interleucina-4/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular , Proliferación Celular , ADN/genética , ADN/metabolismo , Fase G1 , Técnicas In Vitro , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Modelos Inmunológicos , Regiones Promotoras Genéticas , Supresión Genética , Linfocitos T/citología , Activación TranscripcionalRESUMEN
We have used the 5' flanking sequence of the myelin basic protein gene known to include the core promoter and a strong oligodendrocyte (ODC)-specific enhancer to target expression of the well-studied model antigen ovalbumin (OVA) to ODC in transgenic mice. OVA protein was detected in a tissue- and cell-specific manner in these "ODC-OVA" mice. Without immunization, CD4 T cells and B cells remained ignorant of the neo-self antigen expressed in the central nervous system (CNS), as indicated by unimpaired development and lack of activation of OVA/IA(b)-specific TCR transgenic T cells in these mice, and the ability to mount normal OVA-specific recall and antibody responses. Upon immunization with OVA in complete Freund's adjuvant, about half of the transgenic mice developed neurological symptoms characteristic of experimental autoimmune encephalomyelitis (EAE). Mononuclear infiltrates in the brain and spinal cord contained both macrophages and T cells, similar to classical models of EAE induced by immunization with CNS antigens in adjuvant. The wealth of immunological reagents available to study and manipulate the OVA-specific response should make this new model useful for the investigation of components and mechanisms involved in CNS-specific autoimmunity.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Oligodendroglía/inmunología , Ovalbúmina/inmunología , Animales , Linfocitos B/inmunología , Western Blotting , Encéfalo/inmunología , Encéfalo/patología , Electroforesis en Gel de Poliacrilamida , Elementos de Facilitación Genéticos , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Inmunohistoquímica , Masculino , Ratones , Ratones Transgénicos , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/inmunología , Ovalbúmina/genética , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Receptores de Antígenos de Linfocitos T/inmunología , Médula Espinal/inmunología , Médula Espinal/patologíaRESUMEN
Dendritic cells (DC) are unique antigen-presenting cells capable of triggering NK cell effector functions and priming naive T cells in vivo. Microbial stimulation induces early IL-2 production by mouse DC. Previous reports demonstrated that IL-2 is enriched at the site of DC/T cell interaction and promotes allogeneic T cell proliferation. However, the direct role of DC-derived IL-2 in the differentiation of cytotoxic T lymphocytes and in NK cell triggering in vivo has not been investigated. Lipopolysaccharide (LPS) stimulation of mouse bone marrow-derived DC results in early IL-2 production unless IL-4 is introduced in DC cultures. Here we show that IL-2 produced by LPS-activated DC is dispensable for cognate T cell responses since IL-2 loss of function DC elicit OVA-specific Tc1 effector and memory lymphocytes in draining lymph nodes in a setting where ex vivo cultured DC do not transfer antigens to host DC. Moreover, adoptively transferred IL-2 loss of function DC maintain their capacity to trigger NK cell proliferation/recruitment in lymph nodes. Therefore, immediate inducible IL-2 production by DC following microbial infection might play a regulatory role at ports of entry rather than in secondary lymphoid organs.
Asunto(s)
Células Dendríticas/inmunología , Interleucina-2/inmunología , Ganglios Linfáticos/inmunología , Linfocitos T Citotóxicos/inmunología , Traslado Adoptivo , Animales , Médula Ósea/inmunología , Células de la Médula Ósea/inmunología , Femenino , Interleucina-2/deficiencia , Células Asesinas Naturales/inmunología , Lipopolisacáridos/inmunología , Ganglios Linfáticos/citología , Activación de Linfocitos/inmunología , RatonesRESUMEN
Treatment of Th cells with compounds that elevate cAMP levels augments Th2-type lymphokine expression, in particular the synthesis of IL-5. Using primary murine CD4(+) T lymphocytes, we show in this study that inhibition of protein kinase A (PKA) activity in Th2 effector cells impairs IL-5 synthesis, whereas the expression of PKA catalytic subunit alpha enhances IL-5 synthesis in Th0 cells. In addition, we observed by coexpression of PKA catalytic subunit and GATA-3 in Th1 cells that the stimulatory effect of PKA is dependent on GATA-3 activity. These data demonstrate that activation of PKA in Th effector cells induces the IL-5 gene expression in a GATA-3-dependent manner.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/inmunología , Interleucina-5/biosíntesis , Interleucina-5/genética , Sulfonamidas , Células Th2/inmunología , Células Th2/metabolismo , Transactivadores/fisiología , Animales , Dominio Catalítico/genética , Dominio Catalítico/inmunología , Línea Celular , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Factor de Transcripción GATA3 , Humanos , Interleucina-4/biosíntesis , Isoquinolinas/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Mutación , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/genética , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/enzimología , Transfección , Células Tumorales Cultivadas , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
A decade after the first description of IL-2-deficient mice, the redundancy of IL-2 as a T cell growth factor is well accepted and the focus of research has shifted to the unexpected multiorgan autoimmunity and inflammation observed in mice lacking components of the IL-2/IL-2R system. So far, a set of defects at the levels of repertoire selection, the generation of suppressive regulatory T cells, T cell homing and clonal contraction via activation induced cell death (AICD) have been documented. We propose that these individual defects jointly contribute to the severe disturbance of T cell homeostasis and self-tolerance underlying the immunopathology of the IL-2 deficiency syndrome.
Asunto(s)
Enfermedades Autoinmunes/fisiopatología , Interleucina-2/fisiología , Animales , Apoptosis , Enfermedades Autoinmunes/genética , Movimiento Celular , Supresión Clonal , Células Dendríticas/inmunología , Homeostasis , Humanos , Tolerancia Inmunológica/fisiología , Interleucina-2/deficiencia , Interleucina-2/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos , Ratones Noqueados , Receptores de Interleucina-2/deficiencia , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/fisiología , Síndrome , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Receptor fas/fisiologíaRESUMEN
Threshold levels of individual NFAT factors appear to be critical for apoptosis induction in effector T cells. In these cells, the short isoform A of NFATc1 is induced to high levels due to the autoregulation of the NFATc1 promoter P1 by NFATs. P1 is located within a CpG island in front of exon 1, represents a DNase I hypersensitive chromatin site, and harbors several sites for binding of inducible transcription factors, including a tandemly arranged NFAT site. A second promoter, P2, before exon 2, is not controlled by NFATs and directs synthesis of the longer NFATc1/B+C isoforms. Contrary to other NFATs, NFATc1/A is unable to promote apoptosis, suggesting that NFATc1/A enhances effector functions without promoting apoptosis of effector T cells.
Asunto(s)
Apoptosis , Proteínas de Unión al ADN/biosíntesis , Proteínas Nucleares , Linfocitos T Reguladores/fisiología , Factores de Transcripción/biosíntesis , Empalme Alternativo , Animales , Secuencia de Bases , Metilación de ADN , Proteínas de Unión al ADN/genética , Desoxirribonucleasa I/metabolismo , Electroforesis en Gel de Poliacrilamida , Homeostasis , Humanos , Células Jurkat , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Factores de Transcripción NFATC , Poli A/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Hematopoietic progenitor kinase 1 (HPK1) is a member of germinal center kinases that is predominantly expressed in hematopoietic cells and transiently activated by T-cell receptor (TCR) triggering. We show here that HPK1 supports apoptosis of T cells. When HPK1 was overexpressed in murine CD4(+) T cells, a substantial increase was observed in spontaneous and TCR/CD3-mediated apoptosis as well as in Fas ligand (FasL) expression. In H2O2-treated EL-4 thymoma cells, which show an increase in reactive oxygen species (ROS) and apoptosis, overexpression of HPK1 enhanced ROS-mediated apoptosis, whereas expression of HPK1 antisense (AS) RNA impaired apoptosis. HPK1 expression also led to a sustained increase in c-Jun N-terminal kinase (JNK) activity, suggesting that JNK activation contributes to the HPK1-mediated apoptosis in H2O2-treated EL-4 cells. Under the same conditions, a rapid cleavage of HPK1 was observed, and overexpression of N- and C-terminal cleavage products in CD4(+) T cells resulted in, similar to full-length HPK1, an increase in apoptosis. In agreement with published data, we show that the C-terminal portion of HPK1 suppresses IkappaBalpha degradation, thereby inhibiting nuclear factor (NF)-kappaB activation. These findings suggest that by inhibiting the antiapoptotic action of NF-kappaB and inducing the proapoptotic activity of JNK, OHPK1 supports apoptosis in T cells.