RESUMEN
In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.
Asunto(s)
Citometría de Flujo , Trasplante Heterólogo , Pez Cebra , Animales , Pez Cebra/embriología , Citometría de Flujo/métodos , Trasplante Heterólogo/métodos , Trasplante de Neoplasias/métodos , Embrión no MamíferoRESUMEN
T cell receptor (TCR) signaling regulates important developmental transitions, partly through induction of the E protein antagonist, Id3. Although normal γδ T cell development depends on Id3, Id3 deficiency produces different phenotypes in distinct γδ T cell subsets. Here, we show that Id3 deficiency impairs development of the Vγ3+ subset, while markedly enhancing development of NKγδT cells expressing the invariant Vγ1Vδ6.3 TCR. These effects result from Id3 regulating both the generation of the Vγ1Vδ6.3 TCR and its capacity to support development. Indeed, the Trav15 segment, which encodes the Vδ6.3 TCR subunit, is directly bound by E proteins that control its expression. Once expressed, the Vγ1Vδ6.3 TCR specifies the innate-like NKγδT cell fate, even in progenitors beyond the normally permissive perinatal window, and this is enhanced by Id3-deficiency. These data indicate that the paradoxical behavior of NKγδT cells in Id3-deficient mice is determined by its stereotypic Vγ1Vδ6.3 TCR complex.
Asunto(s)
Proteínas Inhibidoras de la Diferenciación , Receptores de Antígenos de Linfocitos T gamma-delta , Animales , Proteínas Inhibidoras de la Diferenciación/metabolismo , Proteínas Inhibidoras de la Diferenciación/genética , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Ratones , Ratones Noqueados , Ratones Endogámicos C57BL , Diferenciación Celular , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Transducción de SeñalRESUMEN
We report here that expression of the ribosomal protein, RPL22, is frequently reduced in human myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML); reduced RPL22 expression is associated with worse outcomes. Mice null for Rpl22 display characteristics of an MDS-like syndrome and develop leukemia at an accelerated rate. Rpl22-deficient mice also display enhanced hematopoietic stem cell (HSC) self-renewal and obstructed differentiation potential, which arises not from reduced protein synthesis but from increased expression of the Rpl22 target, ALOX12, an upstream regulator of fatty acid oxidation (FAO). The increased FAO mediated by Rpl22-deficiency also persists in leukemia cells and promotes their survival. Altogether, these findings reveal that Rpl22 insufficiency enhances the leukemia potential of HSC via non-canonical de-repression of its target, ALOX12, which enhances FAO, a process that may serve as a therapeutic vulnerability of Rpl22 low MDS and AML leukemia cells. Highlights: RPL22 insufficiency is observed in MDS/AML and is associated with reduced survivalRpl22-deficiency produces an MDS-like syndrome and facilitates leukemogenesisRpl22-deficiency does not impair global protein synthesis by HSCRpl22 controls leukemia cell survival by non-canonical regulation of lipid oxidation eTOC: Rpl22 controls the function and transformation potential of hematopoietic stem cells through effects on ALOX12 expression, a regulator of fatty acid oxidation.
RESUMEN
We have previously demonstrated that loss of the tumor suppressive activity of ribosomal protein (RP) RPL22 predisposes to development of leukemia in mouse models and aggressive disease in human patients; however, the role of RPL22 in solid tumors, specifically colorectal cancer (CRC), had not been explored. We report here that RPL22 is either deleted or mutated in 36% of CRC and provide new insights into its mechanism of action. Indeed, Rpl22 inactivation causes the induction of its highly homologous paralog, RPL22L1, which serves as a driver of cell proliferation and anchorage-independent growth in CRC cells. Moreover, RPL22L1 protein is highly expressed in patient CRC samples and correlates with poor survival. Interestingly, the association of high RPL22L1 expression with poor prognosis appears to be linked to resistance to 5-Fluorouracil, which is a core component of most CRC therapeutic regimens. Indeed, in an avatar trial, we found that human CRC samples that were unresponsive to 5-Fluorouracil in patient-derived xenografts exhibited elevated expression levels of RPL22L1. This link between RPL22L1 induction and 5-Fluorouracil resistance appears to be causal, because ectopic expression or knockdown of RPL22L1 in cell lines increases and decreases 5-Fluorouracil resistance, respectively, and this is associated with changes in expression of the DNA-repair genes, MGMT and MLH1. In summary, our data suggest that RPL22L1 might be a prognostic marker in CRC and predict 5-FU responsiveness.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Resistencia a Antineoplásicos , Fluorouracilo/uso terapéutico , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/metabolismo , Animales , Proliferación Celular , Neoplasias Colorrectales/patología , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Humanos , Ratones , Homólogo 1 de la Proteína MutL/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Regulation of the actin cytoskeleton is crucial for normal development and function of the immune system, as evidenced by the severe immune abnormalities exhibited by patients bearing inactivating mutations in the Wiskott-Aldrich syndrome protein (WASP), a key regulator of actin dynamics. WASP exerts its effects on actin dynamics through a multisubunit complex termed Arp2/3. Despite the critical role played by Arp2/3 as an effector of WASP-mediated control over actin polymerization, mutations in protein components of the Arp2/3 complex had not previously been identified as a cause of immunodeficiency. Here, we describe two brothers with hematopoietic and immunologic symptoms reminiscent of Wiskott-Aldrich syndrome (WAS). However, these patients lacked mutations in any of the genes previously associated with WAS. Whole-exome sequencing revealed a homozygous 2 bp deletion, n.c.G623DEL-TC (p.V208VfsX20), in Arp2/3 complex component ARPC1B that causes a frame shift resulting in premature termination. Modeling of the disease in zebrafish revealed that ARPC1B plays a critical role in supporting T cell and thrombocyte development. Moreover, the defects in development caused by ARPC1B loss could be rescued by the intact human ARPC1B ortholog, but not by the p.V208VfsX20 variant identified in the patients. Moreover, we found that the expression of ARPC1B is restricted to hematopoietic cells, potentially explaining why a mutation in ARPC1B has now been observed as a cause of WAS, whereas mutations in other, more widely expressed, components of the Arp2/3 complex have not been observed.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/genética , Plaquetas/patología , Mutación del Sistema de Lectura , Síndromes de Inmunodeficiencia/genética , Linfopoyesis/genética , Linfocitos T/patología , Trombopoyesis/genética , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/deficiencia , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/fisiología , Preescolar , Codón sin Sentido , Consanguinidad , Resultado Fatal , Humanos , Lactante , Masculino , Complejos Multiproteicos , Linaje , Polimerizacion , Recombinación V(D)J , Síndrome de Wiskott-Aldrich/genética , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genéticaRESUMEN
The analysis of individuals with telomere defects may shed light on the delicate interplay of factors controlling genome stability, premature aging, and cancer. We herein describe two Coats plus patients with telomere and genomic defects; both harbor distinct, novel mutations in STN1, a member of the human CTC1-STN1-TEN1 (CST) complex, thus linking this gene for the first time to a human telomeropathy. We characterized the patients' phenotype, recapitulated it in a zebrafish model and rescued cellular and clinical aspects by the ectopic expression of wild-type STN1 or by thalidomide treatment. Interestingly, a significant lengthy control of the gastrointestinal bleeding in one of our patients was achieved by thalidomide treatment, exemplifying a successful bed-to-bench-and-back approach.
Asunto(s)
Ataxia , Neoplasias Encefálicas , Calcinosis , Quistes del Sistema Nervioso Central , Regulación de la Expresión Génica/efectos de los fármacos , Leucoencefalopatías , Espasticidad Muscular , Mutación , Enfermedades de la Retina , Convulsiones , Proteínas de Unión a Telómeros , Telómero , Talidomida/administración & dosificación , Animales , Ataxia/tratamiento farmacológico , Ataxia/genética , Ataxia/metabolismo , Ataxia/patología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Calcinosis/tratamiento farmacológico , Calcinosis/genética , Calcinosis/metabolismo , Calcinosis/patología , Quistes del Sistema Nervioso Central/tratamiento farmacológico , Quistes del Sistema Nervioso Central/genética , Quistes del Sistema Nervioso Central/metabolismo , Quistes del Sistema Nervioso Central/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Leucoencefalopatías/tratamiento farmacológico , Leucoencefalopatías/genética , Leucoencefalopatías/metabolismo , Leucoencefalopatías/patología , Masculino , Espasticidad Muscular/tratamiento farmacológico , Espasticidad Muscular/genética , Espasticidad Muscular/metabolismo , Espasticidad Muscular/patología , Enfermedades de la Retina/tratamiento farmacológico , Enfermedades de la Retina/genética , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Convulsiones/tratamiento farmacológico , Convulsiones/genética , Convulsiones/metabolismo , Convulsiones/patología , Telómero/genética , Telómero/metabolismo , Telómero/patología , Proteínas de Unión a Telómeros/biosíntesis , Proteínas de Unión a Telómeros/genética , Talidomida/efectos adversos , Pez CebraRESUMEN
Gradations in extracellular regulated kinase (ERK) signaling have been implicated in essentially every developmental checkpoint or differentiation process encountered by lymphocytes. Yet, despite intensive effort, the molecular basis by which differences in ERK activation specify alternative cell fates remains poorly understood. We report here that differential ERK signaling controls lymphoid-fate specification through an alternative mode of action. While ERK phosphorylates most substrates, such as RSK, by targeting them through its D-domain, this well-studied mode of ERK action was dispensable for development of γδ T cells. Instead, development of γδ T cells was dependent upon an alternative mode of action mediated by the DEF-binding pocket (DBP) of ERK. This domain enabled ERK to bind a distinct and select set of proteins required for specification of the γδ fate. These data provide the first in vivo demonstration for the role of DBP-mediated interactions in orchestrating alternate ERK-dependent developmental outcomes.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T/inmunología , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Células Cultivadas , Activación Enzimática/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Ratones , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Estabilidad Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Transducción de Señal/genética , Especificidad por Sustrato/genéticaRESUMEN
It remains controversial whether the highly homologous ribosomal protein (RP) paralogs found in lower eukaryotes have distinct functions and this has not been explored in vertebrates. Here we demonstrate that despite ubiquitous expression, the RP paralogs, Rpl22 and Rpl22-like1 (Rpl22l1) play essential, distinct, and antagonistic roles in hematopoietic development. Knockdown of Rpl22 in zebrafish embryos selectively blocks the development of T lineage progenitors after they have seeded the thymus. In contrast, knockdown of the Rpl22 paralog, Rpl22l1, impairs the emergence of hematopoietic stem cells (HSC) in the aorta-gonad-mesonephros by abrogating Smad1 expression and the consequent induction of essential transcriptional regulator, Runx1. Indeed, despite the ability of both paralogs to bind smad1 RNA, Rpl22 and Rpl22l1 have opposing effects on Smad1 expression. Accordingly, circumstances that tip the balance of these paralogs in favor of Rpl22 (e.g., Rpl22l1 knockdown or Rpl22 overexpression) result in repression of Smad1 and blockade of HSC emergence.
Asunto(s)
Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , Células Madre Hematopoyéticas/citología , Proteínas Ribosómicas/metabolismo , Linfocitos T/citología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Animales , Western Blotting , Diferenciación Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Células Madre Hematopoyéticas/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ribosómicas/genética , Ribosomas/metabolismo , Proteína Smad1/metabolismo , Linfocitos T/metabolismo , Timo/citología , Timo/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Ribosomal protein (RP) mutations in diseases such as 5q- syndrome both disrupt hematopoiesis and increase the risk of developing hematologic malignancy. However, the mechanism by which RP mutations increase cancer risk has remained an important unanswered question. We show here that monoallelic, germline inactivation of the ribosomal protein L22 (Rpl22) predisposes T-lineage progenitors to transformation. Indeed, RPL22 was found to be inactivated in â¼ 10% of human T-acute lymphoblastic leukemias. Moreover, monoallelic loss of Rpl22 accelerates development of thymic lymphoma in both a mouse model of T-cell malignancy and in acute transformation assays in vitro. We show that Rpl22 inactivation enhances transformation potential through induction of the stemness factor, Lin28B. Our finding that Rpl22 inactivation promotes transformation by inducing expression of Lin28B provides the first insight into the mechanistic basis by which mutations in Rpl22, and perhaps some other RP genes, increases cancer risk.
Asunto(s)
Transformación Celular Neoplásica/genética , Proteínas de Unión al ADN/metabolismo , Neoplasias Hematológicas/genética , Proteínas de Unión al ARN/genética , Proteínas Ribosómicas/genética , Linfocitos T/metabolismo , Animales , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Silenciador del Gen , Neoplasias Hematológicas/metabolismo , Humanos , Immunoblotting , Ratones , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
alphabeta and gammadelta T cells arise from a common thymocyte progenitor during development in the thymus. Emerging evidence suggests that the pre-T cell receptor (pre-TCR) and gammadelta T cell receptor (gammadeltaTCR) play instructional roles in specifying the alphabeta and gammadelta T-lineage fates, respectively. Nevertheless, the signaling pathways differentially engaged to specify fate and promote the development of these lineages remain poorly understood. Here, we show that differential activation of the extracellular signal-related kinase (ERK)-early growth response gene (Egr)-inhibitor of DNA binding 3 (Id3) pathway plays a defining role in this process. In particular, Id3 expression served to regulate adoption of the gammadelta fate. Moreover, Id3 was both necessary and sufficient to enable gammadelta-lineage cells to differentiate independently of Notch signaling and become competent IFNgamma-producing effectors. Taken together, these findings identify Id3 as a central player that controls both adoption of the gammadelta fate and its maturation in the thymus.
Asunto(s)
Proteínas Inhibidoras de la Diferenciación/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Proteína 1 de la Respuesta de Crecimiento Precoz/inmunología , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/inmunología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Inhibidoras de la Diferenciación/genética , Proteínas Inhibidoras de la Diferenciación/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas RGS/inmunología , Proteínas RGS/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Receptores Notch/inmunología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/metabolismo , Timo/inmunologíaRESUMEN
The repertoire of TCR specificities is established by a selection process in the thymus, during which precursor survival and maturation is dictated by the nature of the TCR signals. The differences in signals that determine whether precursors will survive and mature or be induced to die remain poorly understood. Among the molecular effectors involved in executing the differentiation process initiated by TCR-ligand interactions is a family of Zn-finger transcription factors termed early growth response genes (Egr). Indeed, ablation of the Egr1 gene impairs ligand-induced maturation (positive selection) but not ligand-induced deletion (negative selection). The partial impairment of positive selection by Egr1 deficiency is not enhanced by simultaneous deletion of another Egr family member, Egr3. Accordingly, we asked whether this results from compensation by another family member, Egr2. In this manuscript, we demonstrate that deletion of Egr2 impairs positive selection of both CD4 and CD8 single-positive thymocytes. Interestingly, many of the genes involved in positive selection and T cell differentiation are up-regulated normally in the Egr2-deficient thymocytes. However, Bcl-2 up-regulation is not sustained during late stages of positive selection. This defect is at least partially responsible for the developmental blockade in Egr2-deficient thymocytes, as enforced expression of Bcl-2 rescues T cell development in Egr2(-/-) thymocytes. Taken together, these data suggest that Egr2 plays a central role in the up-regulation of the survival molecule Bcl-2 during positive selection.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Proteína 2 de la Respuesta de Crecimiento Precoz/inmunología , Células Progenitoras Linfoides/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Timo/inmunología , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/genética , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Supresión Clonal/genética , Supresión Clonal/inmunología , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/inmunología , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 2 de la Respuesta de Crecimiento Precoz/biosíntesis , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/metabolismo , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Timo/citología , Timo/metabolismo , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
The role of the T cell antigen receptor complex (TCR) in alphabeta/gammadelta lineage commitment remains controversial, in particular whether different TCR isoforms intrinsically favor adoption of a certain lineage. Here, we demonstrate that impairing the signaling capacity of a gammadeltaTCR complex enables it to efficiently direct thymocytes to the alphabeta lineage. In the presence of a ligand, a transgenic gammadeltaTCR mediates almost exclusive adoption of the gammadelta lineage, while in the absence of ligand, the same gammadeltaTCR promotes alphabeta lineage development with efficiency comparable to the pre-TCR. Importantly, attenuating gammadeltaTCR signaling through Lck deficiency causes reduced ERK1/2 activation and Egr expression and diverts thymocytes to the alphabeta lineage even in the presence of ligand. Conversely, ectopic Egr overexpression favors gammadelta T cell development. Our data support a model whereby gammadelta versus alphabeta lineage commitment is controlled by TCR signal strength, which depends critically on the ERK MAPK-Egr pathway.
Asunto(s)
Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Animales , Diferenciación Celular , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Inmediatas-Precoces/biosíntesis , Proteínas Inmediatas-Precoces/genética , Proteínas Inhibidoras de la Diferenciación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosforilación , Proteínas/genética , Proteínas/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Transducción de Señal , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
The molecular changes that restrict multipotent murine thymocytes to the T cell lineage and render them responsive to Ag receptor signals remain poorly understood. In this study, we report our analysis of the role of the Ets transcription factor, Spi-B, in this process. Spi-B expression is acutely induced coincident with T cell lineage commitment at the CD4(-)CD8(-)CD44(-)CD25(+) (DN3) stage of thymocyte development and is then down-regulated as thymocytes respond to pre-TCR signals and develop beyond the beta-selection checkpoint to the CD4(-)CD8(-)CD44(-)CD25(-) (DN4) stage. We found that dysregulation of Spi-B expression in DN3 thymocytes resulted in a dose-dependent perturbation of thymocyte development. Indeed, DN3 thymocytes expressing approximately five times the endogenous level of Spi-B were arrested at the beta-selection checkpoint, due to impaired induction of Egr proteins, which are important molecular effectors of the beta-selection checkpoint. T lineage-committed DN3 thymocytes expressing even higher levels of Spi-B were diverted to the dendritic cell lineage. Thus, we demonstrate that the prescribed modulation of Spi-B expression is important for T lineage commitment and differentiation beyond the beta-selection checkpoint; and we provide insight into the mechanism underlying perturbation of development when that expression pattern is disrupted.
Asunto(s)
Diferenciación Celular/genética , Diferenciación Celular/inmunología , Regulación hacia Abajo/inmunología , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T/inmunología , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/metabolismo , Transactivadores/biosíntesis , Transactivadores/genética , Animales , Línea Celular Tumoral , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Células Dendríticas/citología , Células Dendríticas/inmunología , Regulación hacia Abajo/genética , Desarrollo Fetal/genética , Desarrollo Fetal/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Técnicas de Cultivo de Órganos , Proteínas Proto-Oncogénicas/deficiencia , Subgrupos de Linfocitos T/inmunología , Timoma/genética , Timoma/inmunología , Timo/citología , Timo/inmunología , Timo/metabolismo , Neoplasias del Timo/genética , Neoplasias del Timo/inmunología , Transactivadores/deficiencia , Factores de Transcripción/biosíntesis , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transducción GenéticaRESUMEN
A hallmark of T cell activation is the ligation-induced down-modulation of the TCR:CD3 complex. However, little is known about the molecular events that drive this process. The CD3 zeta-chain has been shown to play a unique role in regulating the assembly, transport, and cell surface expression of the TCR:CD3 complex. In this study we have investigated the relationship between CD3zeta and the TCRalphabetaCD3epsilondeltagamma complex after ligation by MHC:peptide complexes. Our results show that there is a significant increase in free surface CD3zeta, which is not associated with the TCR:CD3 complex, after T cell stimulation. This may reflect dissociation of CD3zeta from the TCRalphabetaCD3epsilondeltagamma complex or transport of intracellular CD3zeta directly to the cell surface. We also show that MHC:peptide ligation also results in exposure of the TCR-associated CD3zeta NH2 terminus, which is ordinarily buried in the complex. These observations appears to be dependent on Src family protein tyrosine kinases, which are known to be critical for efficient T cell activation. These data suggest a mechanism by which ligated TCR may be differentiated from unligated TCR and selectively down-modulated.
Asunto(s)
Complejo Mayor de Histocompatibilidad/inmunología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismo , Secuencia de Aminoácidos , Animales , Biotinilación , Complejo CD3/metabolismo , Complejo CD3/fisiología , Espacio Extracelular/inmunología , Espacio Extracelular/metabolismo , Humanos , Hibridomas , Ligandos , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/fisiología , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/fisiología , Complejo Receptor-CD3 del Antígeno de Linfocito T/antagonistas & inhibidores , Complejo Receptor-CD3 del Antígeno de Linfocito T/biosíntesis , Complejo Receptor-CD3 del Antígeno de Linfocito T/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Familia-src Quinasas/fisiologíaRESUMEN
Pre-TCR complexes are thought to signal in a ligand-independent manner because they are constitutively targeted to lipid rafts. We report that ligand-independent signaling is not a unique capability of the pre-TCR complex. Indeed, the TCR alpha subunit restores development of pT alpha-deficient thymocytes to the CD4(+)CD8(+) stage even in the absence of conventional MHC class I and class II ligands. Moreover, we found that pre-TCR and alpha beta TCR complexes exhibit no appreciable difference in their association with lipid rafts, suggesting that ligand-independence is a function of the CD4(-)CD8(-) (DN) thymocytes in which pre-TCR signaling occurs. In agreement, we found that only CD44(-)CD25(+) DN thymocytes (DN3) enabled activation of extracellular signal-regulated kinases by the pre-TCR complex. DN thymocytes also exhibited a lower signaling threshold relative to CD4(+)CD8(+) thymocytes, which was associated with both the markedly elevated lipid raft content of their plasma membranes and more robust capacitative Ca(2+) entry. Taken together these data suggest that cell-autonomous, ligand-independent signaling is primarily a property of the thymocytes in which pre-TCR signaling occurs.