RESUMEN
To investigate the role of the kinase Zap70 in T cells, we generated mice expressing a Zap70 mutant whose catalytic activity can be selectively blocked by a small-molecule inhibitor. We found that conventional naive, effector and memory T cells were dependent on the kinase activity of Zap70 for their activation, which demonstrated a nonredundant role for Zap70 in signals induced by the T cell antigen receptor (TCR). In contrast, the catalytic activity of Zap70 was not required for activation of the GTPase Rap1 and inside-out signals that promote integrin adhesion. This Zap70 kinase-independent pathway was sufficient for the suppressive activity of regulatory T cells (T(reg) cells), which was unperturbed by inhibition of the catalytic activity of Zap70. Our results indicate Zap70 is a likely therapeutic target.
Asunto(s)
Biocatálisis , Activación de Linfocitos/inmunología , Transducción de Señal/inmunología , Linfocitos T Reguladores/enzimología , Proteína Tirosina Quinasa ZAP-70/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Separación Celular , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Immunoblotting , Inmunoprecipitación , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Transgénicos , Pirazoles/química , Pirazoles/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
How pathogenic cluster of differentiation 4 (CD4) T cells in rheumatoid arthritis (RA) develop remains poorly understood. We used Nur77-a marker of T cell antigen receptor (TCR) signaling-to identify antigen-activated CD4 T cells in the SKG mouse model of autoimmune arthritis and in patients with RA. Using a fluorescent reporter of Nur77 expression in SKG mice, we found that higher levels of Nur77-eGFP in SKG CD4 T cells marked their autoreactivity, arthritogenic potential, and ability to more readily differentiate into interleukin-17 (IL-17)-producing cells. The T cells with increased autoreactivity, nonetheless had diminished ex vivo inducible TCR signaling, perhaps reflective of adaptive inhibitory mechanisms induced by chronic autoantigen exposure in vivo. The enhanced autoreactivity was associated with up-regulation of IL-6 cytokine signaling machinery, which might be attributable, in part, to a reduced amount of expression of suppressor of cytokine signaling 3 (SOCS3)-a key negative regulator of IL-6 signaling. As a result, the more autoreactive GFPhi CD4 T cells from SKGNur mice were hyperresponsive to IL-6 receptor signaling. Consistent with findings from SKGNur mice, SOCS3 expression was similarly down-regulated in RA synovium. This suggests that despite impaired TCR signaling, autoreactive T cells exposed to chronic antigen stimulation exhibit heightened sensitivity to IL-6, which contributes to the arthritogenicity in SKG mice, and perhaps in patients with RA.
Asunto(s)
Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Linfocitos T CD4-Positivos/inmunología , Membrana Sinovial/inmunología , Células Th17/inmunología , Adulto , Anciano , Animales , Artritis Experimental/patología , Artritis Reumatoide/patología , Artritis Reumatoide/cirugía , Biopsia , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/inmunología , Regulación hacia Abajo , Femenino , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Humanos , Interleucina-17/metabolismo , Interleucina-6/inmunología , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/química , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Proteína 3 Supresora de la Señalización de Citocinas/inmunología , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Sinovectomía , Membrana Sinovial/citología , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Células Th17/metabolismo , Zimosan/administración & dosificación , Zimosan/inmunologíaRESUMEN
Survival of naive T cells requires engagement of TCR with self-peptide major histocompatibility Ags. The signaling pathways required to transmit this survival signal are poorly understood. In this study, we asked whether the tyrosine kinase Zap70 is required to transmit survival signals in naive CD8 T cells. In the absence of Zap70 expression, thymic development is completely blocked. Using a tetracycline-inducible Zap70 transgene (TetZap70), thymic development of Zap70-deficient TCR transgenic F5 mice was restored. Feeding mice doxycycline to induce Zap70 expression resulted in repopulation of the peripheral naive compartment. Zap70 transgene expression was then ablated by withdrawal of doxycycline. Survival of Zap70-deficient naive CD8 T cells depended on host environment. In hosts with a replete T cell compartment, naive T cells died rapidly in the absence of Zap70 expression. In lymphopenic hosts, Zap70-deficient T cells survived far longer, in an IL-7-dependent manner, but failed to undergo lymphopenia-induced proliferation. Analyzing mixed bone marrow chimeras revealed that intact Zap70-dependent signaling was important for integration of recent thymic emigrants into the mature naive compartment. Finally, we asked whether adaptor function conferred by Zap70 tyrosines 315 and 319 was necessary for transmission of homeostatic TCR signals. This was done by analyzing F5 mice expressing mutant Zap70 in which these residues had been mutated to alanines (Zap70(YYAA)). Inducible Zap70 expression rescued thymic development in F5 TetZap70 Zap70(YYAA) mice. However, in the absence of wild-type Zap70 expression, the Zap70(YYAA) mutant failed to transmit either survival or proliferative homeostatic signals.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfopoyesis/genética , Receptores de Antígenos de Linfocitos T/inmunología , Proteína Tirosina Quinasa ZAP-70/genética , Animales , Proliferación Celular , Supervivencia Celular/genética , Doxiciclina/farmacología , Técnicas de Sustitución del Gen , Proteínas de Homeodominio/genética , Receptores de Hialuranos/biosíntesis , Receptores de Hialuranos/genética , Interleucina-7/inmunología , Linfopenia/inmunología , Ratones , Ratones Noqueados , Receptores de Interleucina-7/biosíntesis , Receptores de Interleucina-7/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Transgenes/genética , Proteína Tirosina Quinasa ZAP-70/biosíntesis , Proteína Tirosina Quinasa ZAP-70/inmunologíaRESUMEN
The tyrosine ZAP-70 (zeta-associated protein of 70 kDa) kinase plays a critical role in activating many downstream signal transduction pathways in T cells following T-cell receptor (TCR) engagement. The importance of ZAP-70 is evidenced by the severe combined immunodeficiency that occurs in ZAP-70-deficient mice and humans. In this review, we describe recent analyses of the ZAP-70 crystal structure, revealing a complex regulatory mechanism of ZAP-70 activity, the differential requirements for ZAP-70 and spleen tyrosine kinase (SyK) in early T-cell development, as well as the role of ZAP-70 in chronic lymphocytic leukemia and autoimmunity. Thus, the critical importance of ZAP-70 in TCR signaling and its predominantly T-cell-restricted expression pattern make ZAP-70 an attractive drug target for the inhibition of pathological T-cell responses in disease.
Asunto(s)
Proteína Tirosina Quinasa ZAP-70/química , Proteína Tirosina Quinasa ZAP-70/inmunología , Animales , Humanos , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
Mice deficient for the B cell-restricted transcription factor Pax5 show a defect in the VH to DJH rearrangement step of immunoglobulin heavy chain gene assembly even though the expression of the V(D)J recombinase is not diminished in Pax5-/- pro-B cells. To investigate whether Pax5 is limiting for VH to DJH rearrangement, we generated transgenic mice which express Pax5 in developing thymocytes. We show that enforced expression of Pax5 in thymocytes results in a partial block in T cell development due to defective pre-TCR signaling in beta-selection. Moreover, our results demonstrate that expression of Pax5 in early thymocytes is sufficient to induce VH to DJH rearrangements in CD4+CD8+ T cells and lead us to suggest that Pax5 may play a direct role in the lineage-specific regulation of immunoglobulin heavy chain gene rearrangement.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Expresión Génica , Reordenamiento Génico de Linfocito B/fisiología , Genes de Inmunoglobulinas/fisiología , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Citometría de Flujo , Ratones , Ratones Transgénicos , Factor de Transcripción PAX5 , Reacción en Cadena de la Polimerasa/métodos , Receptores de Antígenos de Linfocitos T/metabolismo , Selectinas/metabolismo , Transducción de Señal/fisiología , Linfocitos T/fisiología , Timo/citología , Timo/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , VDJ Recombinasas/metabolismoRESUMEN
Expression of V(D)J recombinase activity in developing lymphocytes is absolutely required for initiation of V(D)J recombination at antigen receptor loci. However, little is known about when during hematopoietic development the V(D)J recombinase is first active, nor is it known what elements activate the recombinase in multipotent hematopoietic progenitors. Using mice that express a fluorescent transgenic V(D)J recombination reporter, we show that the V(D)J recombinase is active as early as common lymphoid progenitors (CLPs) but not in the upstream progenitors that retain myeloid lineage potential. Evidence of this recombinase activity is detectable in all four progeny lineages (B, T, and NK, and DC), and rag2 levels are the highest in progenitor subsets immediately downstream of the CLP. By single cell PCR, we demonstrate that V(D)J rearrangements are detectable at IgH loci in approximately 5% of splenic natural killer cells. Finally, we show that recombinase activity in CLPs is largely controlled by the Erag enhancer. As activity of the Erag enhancer is restricted to the B cell lineage, this provides the first molecular evidence for establishment of a lineage-specific transcription program in multipotent progenitors.
Asunto(s)
Linfocitos B/inmunología , Células Madre Hematopoyéticas/inmunología , Linfopoyesis/inmunología , VDJ Recombinasas/metabolismo , Animales , Linfocitos B/enzimología , Proteínas Fluorescentes Verdes , Células Madre Hematopoyéticas/enzimología , Proteínas de Homeodominio/genética , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , VDJ Recombinasas/genéticaRESUMEN
Zap70 plays a critical role in normal T cell development and T cell function. However, little is known about how perturbation of allosteric autoinhibitory mechanisms in Zap70 impacts T cell biology. Here, we analyze mice with a hypermorphic Zap70 mutation, W131A, which destabilizes the autoinhibitory conformation of Zap70, rendering the kinase in a semiactive state. W131A mutant mice with wild-type T cell receptor (TCR) repertoires exhibited relatively normal T cell development. However, crossing the W131A mutant mice to OTII TCR transgenic mice resulted in increased negative selection of OTII+ thymocytes and in increased thymic and peripheral T regulatory cells. Strikingly, increased basal TCR signaling was associated with a marked increase in inhibitory receptor expression and with T cells that were relatively refractory to TCR stimulation. PD-1 inhibitory receptor blockade partially reversed T cell unresponsiveness. Collectively, disruption of normal Zap70 autoinhibition engaged negative feedback mechanisms by which negative selection and inhibitory receptors restrain TCR signaling to enforce both central and peripheral tolerance.
Asunto(s)
Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/fisiología , Proteína Tirosina Quinasa ZAP-70/fisiología , Animales , Apoptosis , Interleucina-2/biosíntesis , Ratones , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/análisis , Receptor de Muerte Celular Programada 1/fisiología , Conformación Proteica , Estabilidad Proteica , Transducción de Señal/fisiología , Linfocitos T Reguladores/fisiología , Regulación hacia Arriba , Proteína Tirosina Quinasa ZAP-70/químicaRESUMEN
Increased osteoclastic bone resorption leads to periarticular erosions and systemic osteoporosis in RA patients. Although a great deal is known about how osteoclasts differentiate from precursors and resorb bone, the identity of an osteoclast precursor (OCP) population in vivo and its regulatory role in RA remains elusive. Here, we report the identification of a CD11b(-/lo)Ly6C(hi) BM population with OCP activity in vitro and in vivo. These cells, which can be distinguished from previously characterized precursors in the myeloid lineage, display features of both M1 and M2 monocytes and expand in inflammatory arthritis models. Surprisingly, in one mouse model of RA (adoptive transfer of SKG arthritis), cotransfer of OCP with SKG CD4+ T cells diminished inflammatory arthritis. Similar to monocytic myeloid-derived suppressor cells (M-MDSCs), OCPs suppressed CD4+ and CD8+ T cell proliferation in vitro through the production of NO. This study identifies a BM myeloid precursor population with osteoclastic and T cell-suppressive activity that is expanded in inflammatory arthritis. Therapeutic strategies that prevent the development of OCPs into mature bone-resorbing cells could simultaneously prevent bone resorption and generate an antiinflammatory milieu in the RA joint.
Asunto(s)
Artritis/patología , Células Progenitoras Mieloides/fisiología , Osteoclastos/patología , Traslado Adoptivo , Animales , Antígenos de Diferenciación/metabolismo , Antígenos Ly/metabolismo , Artritis/inducido químicamente , Artritis/complicaciones , Enfermedades Óseas Metabólicas/etiología , Médula Ósea/patología , Antígeno CD11b/metabolismo , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD4-Positivos/trasplante , Receptor 1 de Quimiocinas CX3C , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Femenino , Humanos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/trasplante , Osteoclastos/metabolismo , Osteoclastos/trasplante , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Receptores de Quimiocina/metabolismo , ZimosanRESUMEN
ZAP-70 is a cytoplasmic protein tyrosine kinase that plays a critical role in the events involved in initiating T-cell responses by the antigen receptor. Here we review the structure of ZAP-70, its regulation, its role in development and in disease. We also describe a model experimental system in which ZAP-70 function can be interrupted by a small chemical inhibitor.
Asunto(s)
Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/enzimología , Proteína Tirosina Quinasa ZAP-70/metabolismo , Animales , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Modelos Moleculares , Fosforilación , Conformación Proteica , Linfocitos T/citología , Proteína Tirosina Quinasa ZAP-70/antagonistas & inhibidores , Proteína Tirosina Quinasa ZAP-70/químicaRESUMEN
ZAP-70 is critical for T cell receptor (TCR) signaling. Tyrosine to phenylalanine mutations of Y315 and Y319 in ZAP-70 suggest these residues function to recruit downstream effector molecules, but mutagenesis and crystallization studies reveal that these residues also play an important role in autoinhibition ZAP-70. To address the importance of the scaffolding function, we generated a zap70 mutant mouse (YYAA mouse) with Y315 and Y319 both mutated to alanines. These YYAA mice reveal that the scaffolding function is important for normal development and function. Moreover, the YYAA mice have many similarities to a previously identified ZAP-70 mutant mouse, SKG, which harbors a distinct hypomorphic mutation. Both YYAA and SKG mice have impaired T cell development and hyporesponsiveness to TCR stimulation, markedly reduced numbers of thymic T regulatory cells and defective positive and negative selection. YYAA mice, like SKG mice, develop rheumatoid factor antibodies, but fail to develop autoimmune arthritis. Signaling differences that result from ZAP-70 mutations appear to skew the TCR repertoire in ways that differentially influence propensity to autoimmunity versus autoimmune disease susceptibility. By uncoupling the relative contribution from T regulatory cells and TCR repertoire during thymic selection, our data help to identify events that may be important, but alone are insufficient, for the development of autoimmune disease.
Asunto(s)
Alelos , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Autoinmunidad/genética , Autoinmunidad/inmunología , Timo/inmunología , Proteína Tirosina Quinasa ZAP-70/genética , Animales , Proliferación Celular , Susceptibilidad a Enfermedades/inmunología , Eliminación de Gen , Técnicas de Sustitución del Gen , Interleucina-17/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Mutación/genética , Fenotipo , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Superantígenos/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Timo/patologíaRESUMEN
Regulated gene rearrangement is thought to underlie allelic exclusion, the observation that an individual B cell expresses only a single immunoglobulin molecule. Previous data has implicated transcriptional activation of rearranging loci in the regulation of their accessibility to the V(D)J recombinase. Using homologous recombination in ES cells, we have generated "knockin" mice which express a GFP cDNA from an unrearranged immunoglobulin kappa light-chain allele. Surprisingly, we find that only a small fraction of kappa alleles are highly transcribed in a population of pre-B cells, that such transcription is monoallelic, and that these highly transcribed alleles account for the vast majority of kappa light-chain gene rearrangement. These data lead us to suggest that probabilistic enhancer activation and allelic competition are part of the mechanism of kappa locus allelic exclusion and may be a general mechanism contributing to cellular differentiation during development.
Asunto(s)
Alelos , Linfocitos B/inmunología , Cadenas Ligeras de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/genética , Células Madre/inmunología , Activación Transcripcional , Animales , Linfocitos B/citología , Biomarcadores , Células de la Médula Ósea/inmunología , Linaje de la Célula , Células Cultivadas , Elementos de Facilitación Genéticos , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Frecuencia de los Genes , Reordenamiento Génico de Cadena Ligera de Linfocito B/inmunología , Genes de Inmunoglobulinas , Genes Reporteros , Proteínas Fluorescentes Verdes , Heterocigoto , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Mutantes , Regiones Promotoras Genéticas , Recombinación Genética , Bazo/citologíaRESUMEN
Previous in vitro studies defined the minimal regions of RAG1 and RAG2 essential for V(D)J recombination. In order to characterize the role of the C-terminal "dispensable" portion of RAG2, we generated core-RAG2 knock-in mice. We found that the core-RAG2-containing recombinase complex is selectively defective in catalyzing V-to-DJ rearrangement at the IgH and TCRbeta loci, resulting in partial developmental blocks in B and T lymphopoiesis. Analysis of recombination intermediates showed defects at the cleavage phase of the reaction. We also observed a reduction in overall recombinase activity in core-RAG2-expressing thymocytes, leading us to suggest that the interaction of a defective recombinase with RSS sequences unique to VH and Vbeta gene segments may underlie the specific V-to-DJ rearrangement defect in core-RAG2 mice.
Asunto(s)
Linfocitos B/inmunología , Diferenciación Celular/inmunología , Proteínas de Unión al ADN/genética , Recombinación Genética/inmunología , Linfocitos T/inmunología , Animales , Diferenciación Celular/genética , Línea Celular , ADN Nucleotidiltransferasas/inmunología , Proteínas de Unión al ADN/inmunología , Reordenamiento Génico de Cadena Pesada de Linfocito B/inmunología , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/inmunología , Ratones , VDJ RecombinasasRESUMEN
Although expression of the RAG1 and RAG2 genes is essential for lymphocyte development, the mechanisms responsible for the lymphoid- and developmental stage-specific regulation of these genes are poorly understood. We have identified a novel, evolutionarily conserved transcriptional enhancer in the RAG locus, called Erag, which was essential for the expression of a chromosomal reporter gene driven by either RAG promoter. Targeted deletion of Erag in the mouse germline results in a partial block in B cell development associated with deficient V(D)J recombination, whereas T cell development appears unaffected. We found that E2A transcription factors bind to Erag in vivo and can transactivate Erag-dependent reporter constructs in cotransfected cell lines. These findings lead us to conclude that RAG transcription is regulated by distinct elements in developing B and T cells and that Erag is required for optimal levels of RAG expression in early B cell precursors but not in T cells.