RESUMEN
The current paradigm about the function of T cell immune checkpoints is that these receptors switch on inhibitory signals upon cognate ligand interaction. We here revisit this simple switch model and provide evidence that the T cell lineage protein THEMIS enhances the signaling threshold at which the immune checkpoint BTLA (B- and T-lymphocyte attenuator) represses T cell responses. THEMIS is recruited to the cytoplasmic domain of BTLA and blocks its signaling capacity by promoting/stabilizing the oxidation of the catalytic cysteine of the tyrosine phosphatase SHP-1. In contrast, THEMIS has no detectable effect on signaling pathways regulated by PD-1 (Programmed cell death protein 1), which depend mainly on the tyrosine phosphatase SHP-2. BTLA inhibitory signaling is tuned according to the THEMIS expression level, making CD8+ T cells more resistant to BTLA-mediated inhibition than CD4+ T cells. In the absence of THEMIS, the signaling capacity of BTLA is exacerbated, which results in the attenuation of signals driven by the T cell antigen receptor and by receptors for IL-2 and IL-15, consequently hampering thymocyte positive selection and peripheral CD8+ T cell maintenance. By characterizing the pivotal role of THEMIS in restricting the transmission of BTLA signals, our study suggests that immune checkpoint operability is conditioned by intracellular signal attenuators.
Asunto(s)
Linfocitos T CD8-positivos , Péptidos y Proteínas de Señalización Intercelular , Receptores Inmunológicos , Transducción de Señal , Animales , Humanos , Ratones , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Receptor de Muerte Celular Programada 1/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Receptores Inmunológicos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismoRESUMEN
The susceptibility to autoimmune diseases is conditioned by the association of modest genetic alterations which altogether weaken self-tolerance. The mechanism whereby these genetic interactions modulate T-cell pathogenicity remains largely uncovered. Here, we investigated the epistatic interaction of two interacting proteins involved in T Cell Receptor signaling and which were previously associated with the development of Multiple Sclerosis. To this aim, we used mice expressing an hypomorphic variant of Vav1 (Vav1R63W), combined with a T cell-conditional deletion of Themis. We show that the combined mutations in Vav1 and Themis induce a strong attenuation of the severity of Experimental Autoimmune Encephalomyelitis (EAE), contrasting with the moderate effect of the single mutation in each of those two proteins. This genotype-dependent gradual decrease of EAE severity correlates with decreased quantity of phosphorylated Vav1 in CD4 T cells, establishing that Themis promotes the development of encephalitogenic Tconv response by enhancing Vav1 activity. We also show that the cooperative effect of Themis and Vav1 on EAE severity is independent of regulatory T cells and unrelated to the impact of Themis on thymic selection. Rather, it results from decreased production of pro-inflammatory cytokines (IFN-γ, IL-17, TNF and GM-CSF) and reduced T cell infiltration in the CNS. Together, our results provide a rationale to study combination of related genes, in addition to single gene association, to better understand the genetic bases of human diseases.
Asunto(s)
Linfocitos T CD4-Positivos , Encefalomielitis Autoinmune Experimental , Animales , Humanos , Ratones , Linfocitos T CD4-Positivos/metabolismo , Sistema Nervioso Central/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Inflamación , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas Proto-Oncogénicas c-vav/metabolismo , VirulenciaRESUMEN
The ability to proliferate is a common feature of most T-cell populations. However, proliferation follows different cell-cycle dynamics and is coupled to different functional outcomes according to T-cell subsets. Whether the mitotic machineries supporting these qualitatively distinct proliferative responses are identical remains unknown. Here, we show that disruption of the microtubule-associated protein LIS1 in mouse models leads to proliferative defects associated with a blockade of T-cell development after ß-selection and of peripheral CD4+ T-cell expansion after antigen priming. In contrast, cell divisions in CD8+ T cells occurred independently of LIS1 following T-cell antigen receptor stimulation, although LIS1 was required for proliferation elicited by pharmacological activation. In thymocytes and CD4+ T cells, LIS1 deficiency did not affect signaling events leading to activation but led to an interruption of proliferation after the initial round of division and to p53-induced cell death. Proliferative defects resulted from a mitotic failure, characterized by the presence of extra-centrosomes and the formation of multipolar spindles, causing abnormal chromosomes congression during metaphase and separation during telophase. LIS1 was required to stabilize dynein/dynactin complexes, which promote chromosome attachment to mitotic spindles and ensure centrosome integrity. Together, these results suggest that proliferative responses are supported by distinct mitotic machineries across T-cell subsets.
Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa , Proteínas Asociadas a Microtúbulos , Linfocitos T , Animales , Ratones , Linaje de la Célula , Centrosoma/metabolismo , Segregación Cromosómica , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitosis , Huso Acromático/metabolismoRESUMEN
Signals that determine the differentiation of naïve CD4+ T helper (TH) cells into specific effector cell subsets are primarily stimulated by cytokines, but additional signals are required to adjust the magnitude of TH cell responses and set the balance between effective immunity and immunological tolerance. By inducing the post-thymic deletion of the T cell lineage signaling protein THEMIS, we showed that THEMIS promoted the development of optimal type 1 immune responses to foreign antigens but stimulated signals that favored encephalitogenic responses to self-neuroantigens. THEMIS was required to stimulate the expression of the gene encoding the transcriptional regulator T-BET and the production of the cytokine interferon-γ (IFN-γ), and it enhanced the ability of encephalitogenic CD4+ T cells to migrate into the central nervous system. Consistently, analysis of THEMIS expression in polarized CD4+ T cells showed that THEMIS was selectively increased in abundance in TH1 cells. The stimulation of predifferentiated effector CD4+ T cells with antigen-presenting cells revealed a stimulatory function for THEMIS on type 1 cytokine responses, similar to those observed ex vivo after immunization. In contrast, THEMIS exerted opposing effects on naïve CD4+ T cells in vitro by inhibiting the T cell receptor (TCR)-mediated signals that lead to TH1 cell responses. These data suggest that THEMIS exerts TCR-independent functions in effector T cells, which increase the magnitude of normal and pathogenic TH1 cell-mediated responses.
Asunto(s)
Receptores de Antígenos de Linfocitos T , Linfocitos T , Células Presentadoras de Antígenos , Citocinas , Inmunidad , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Células TH1RESUMEN
The activity of T cells is finely controlled by a set of negative regulators of T-cell antigen receptor (TCR)-mediated signaling. However, how those negative regulators are themselves controlled to prevent ineffective TCR-mediated responses remain poorly understood. Thymocyte-expressed molecule involved in selection (THEMIS) has been characterized over a decade ago as an important player of T cell development. Although the molecular function of THEMIS has long remained puzzling and subject to controversies, latest investigations suggest that THEMIS stimulates TCR-mediated signaling by repressing the tyrosine phosphatases SHP-1 and SHP-2 which exert regulatory function on T cell activation. Recent evidences also point to a role for THEMIS in peripheral T cells beyond its role on thymic selection. Here, we present an overview of the past research on THEMIS in the context of T cell development and peripheral T cell function and discuss the possible implication of THEMIS-based mechanisms on TCR-dependent and independent signaling outcomes.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Linfocitos T , Timo , Diferenciación Celular , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal , Linfocitos T/inmunología , Timo/inmunologíaRESUMEN
CD5 is characterized as an inhibitory coreceptor with an important regulatory role during T cell development. The molecular mechanism by which CD5 operates has been puzzling and its function in mature T cells suggests promoting rather than repressing effects on immune responses. Here, we combined quantitative mass spectrometry and genetic studies to analyze the components and the activity of the CD5 signaling machinery in primary T cells. We found that T cell receptor (TCR) engagement induces the selective phosphorylation of CD5 tyrosine 429, which serves as a docking site for proteins with adaptor functions (c-Cbl, CIN85, CRKL), connecting CD5 to positive (PI3K) and negative (UBASH3A, SHIP1) regulators of TCR signaling. c-CBL acts as a coordinator in this complex enabling CD5 to synchronize positive and negative feedbacks on TCR signaling through the other components. Disruption of CD5 signalosome in mutant mice reveals that it modulates TCR signal outputs to selectively repress the transactivation of Foxp3 and limit the inopportune induction of peripherally induced regulatory T cells during immune responses against foreign antigen. Our findings bring insights into the paradigm of coreceptor signaling, suggesting that, in addition to providing dualistic enhancing or dampening inputs, coreceptors can engage concomitant stimulatory and inhibitory signaling events, which act together to promote specific functional outcomes.
Asunto(s)
Antígenos/inmunología , Antígenos CD5/metabolismo , Diferenciación Celular/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Reguladores/fisiología , Animales , Antígenos CD5/genética , Diferenciación Celular/genética , Regulación de la Expresión Génica/inmunología , Activación de Linfocitos/genética , Espectrometría de Masas , Ratones , Ratones Transgénicos , Cultivo Primario de Células , Receptores de Antígenos de Linfocitos T/antagonistas & inhibidores , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
The remarkable T cell receptor (TCR) performs essential functions in the initiation of intracellular signals required for T cell development, repertoire selection and effector responses to foreign antigens. How TCR signals elicit such diverse cellular responses and outcomes remains a major question for investigation. Recent years have witnessed important advances in our understanding of the regulatory processes that control and modulate the TCR signalling response. Here, we review newly identified mechanisms for the regulation of TCR signalling and then discuss how the TCR signalling response is regulated to control two critical cellular processes - namely, positive selection and T cell homeostasis.
Asunto(s)
Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Antígenos CD/inmunología , Calcineurina/inmunología , Diferenciación Celular/inmunología , Retroalimentación Fisiológica , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/inmunología , Modelos Inmunológicos , Transducción de Señal/inmunología , Linfocitos T/citología , Linfocitos T/metabolismo , Ubiquitinación , Proteína Tirosina Quinasa ZAP-70/inmunologíaRESUMEN
Themis is a new component of the TCR signaling machinery that plays a critical role during T cell development. The positive selection of immature CD4+CD8+ double-positive thymocytes and their commitment to the CD4+CD8- single-positive stage are impaired in Themis-/- mice, suggesting that Themis might be important to sustain TCR signals during these key developmental processes. However, the analysis of Themis mRNA levels revealed that Themis gene expression is rapidly extinguished during positive selection. We show in this article that Themis protein expression is increased in double-positive thymocytes undergoing positive selection and is sustained in immature single-positive thymocytes, despite the strong decrease in Themis mRNA levels in these subsets. We found that Themis stability is controlled by the ubiquitin-specific protease USP9X, which removes ubiquitin K48-linked chains on Themis following TCR engagement. Biochemical analyses indicate that USP9X binds directly to the N-terminal CABIT domain of Themis and indirectly to the adaptor protein Grb2, with the latter interaction enabling recruitment of Themis/USP9X complexes to LAT, thereby sustaining Themis expression following positive selection. Together, these data suggest that TCR-mediated signals enhance Themis stability upon T cell development and identify USP9X as a key regulator of Themis protein turnover.
Asunto(s)
Endopeptidasas/metabolismo , Células Precursoras de Linfocitos T/fisiología , Proteínas/metabolismo , Linfocitos T/fisiología , Timo/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Selección Clonal Mediada por Antígenos , Proteína Adaptadora GRB2/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Activación de Linfocitos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfoproteínas/metabolismo , Unión Proteica , Estabilidad Proteica , Proteínas/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Ubiquitina TiolesterasaRESUMEN
THEMIS, a recently identified T-lineage-restricted protein, is the founding member of a large metazoan protein family. Gene inactivation studies have revealed a critical requirement for THEMIS during thymocyte positive selection, implicating THEMIS in signaling downstream of the T cell antigen receptor (TCR), but the mechanistic underpinnings of THEMIS function have remained elusive. A previous model posited that THEMIS prevents thymocytes from inappropriately crossing the positive/negative selection threshold by dampening TCR signaling. However, new data suggest an alternative model where THEMIS enhances TCR signaling enabling thymocytes to reach the threshold for positive selection, avoiding death by neglect. We review the data supporting each model and conclude that the preponderance of evidence favors an enhancing function for THEMIS in TCR signaling.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Inmunológicos , Linfocitos T/fisiología , Timocitos/fisiología , Animales , Diferenciación Celular , Cisteína/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Activación de Linfocitos , Dominios Proteicos/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de SeñalRESUMEN
Regulatory T cells (Treg) represent a minor subpopulation of T lymphocytes that is crucial for the maintenance of immune homeostasis. Here, we present a large-scale quantitative mass spectrometry study that defines a specific proteomic "signature" of Treg. Treg and conventional T lymphocyte (Tconv) subpopulations were sorted by flow cytometry and subjected to global proteomic analysis by single-run nanoLC-MS/MS on a fast-sequencing Q-Exactive mass spectrometer. Besides "historical" proteins that characterize Treg, our study identified numerous new proteins that are up- or downregulated in Treg versus Tconv. We focused on Themis1, a protein particularly under-represented in Treg, and recently described as being involved in the pathogenesis of immune diseases. Using a transgenic mouse model overexpressing Themis1, we provided in vivo and in vitro evidence of its importance for Treg suppressive functions, in an animal model of inflammatory bowel disease and in coculture assays. We showed that this enhanced suppressive activity in vitro is associated with an accumulation of Tregs. Thus, our study highlights the usefulness of label free quantitative methods to better characterize the Treg cell lineage and demonstrates the potential role of Themis1 in the suppressive functions of these cells.
Asunto(s)
Tolerancia Inmunológica , Proteínas/metabolismo , Proteómica/métodos , Linfocitos T Reguladores/inmunología , Animales , Cromatografía Liquida , Modelos Animales de Enfermedad , Citometría de Flujo , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos C57BL , Proteínas/análisis , Proteínas/genética , Linfocitos T Reguladores/química , Espectrometría de Masas en TándemRESUMEN
THEMIS, a T cell-specific protein with high expression in CD4+CD8+ thymocytes, has a crucial role in positive selection and T cell development. THEMIS lacks defined catalytic domains but contains two tandem repeats of a distinctive module of unknown function (CABIT). Here we found that THEMIS directly regulated the catalytic activity of the tyrosine phosphatase SHP-1. This action was mediated by the CABIT modules, which bound to the phosphatase domain of SHP-1 and promoted or stabilized oxidation of SHP-1's catalytic cysteine residue, which inhibited the tyrosine-phosphatase activity of SHP-1. Deletion of SHP-1 alleviated the developmental block in Themis-/- thymocytes. Thus, THEMIS facilitates thymocyte positive selection by enhancing the T cell antigen receptor signaling response to low-affinity ligands.
Asunto(s)
Selección Clonal Mediada por Antígenos/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Eliminación de Gen , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados , Oxidación-Reducción , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína Tirosina Fosfatasa no Receptora Tipo 6/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 6/química , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Especies Reactivas de Oxígeno/metabolismo , Linfocitos T/citología , Timocitos/citología , Timocitos/inmunología , Timocitos/metabolismoRESUMEN
The positive and negative selection of lymphocytes by antigen is central to adaptive immunity and self-tolerance, yet how this is determined by different antigens is not completely understood. We found that thymocyte-selection-associated family member 2 (Themis2) increased the positive selection of B1 cells and germinal center B cells by self and foreign antigens. Themis2 lowered the threshold for B-cell activation by low-avidity, but not high-avidity, antigens. Themis2 constitutively bound the adaptor protein Grb2, src-kinase Lyn and signal transducer phospholipase γ2 (PLC-γ2), and increased activation of PLC-γ2 and its downstream pathways following B cell receptor stimulation. Our findings identify a unique function for Themis2 in differential signaling and provide insight into how B cells discriminate between antigens of different quantity and quality.
Asunto(s)
Linfocitos B/fisiología , Selección Clonal Mediada por Antígenos , Centro Germinal/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Activación de Linfocitos , Inmunidad Adaptativa , Animales , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Proteína Adaptadora GRB2/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosfolipasa C gamma/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Autotolerancia , Familia-src Quinasas/metabolismoRESUMEN
The T cell signaling protein Themis1 is essential for the positive and negative selection of thymocytes in the thymus. Although the developmental defect that results from the loss of Themis1 suggests that it enhances T cell receptor (TCR) signaling, Themis1 also recruits Src homology 2 domain-containing phosphatase-1 (SHP-1) to the vicinity of TCR signaling complexes, suggesting that it has an inhibitory role in TCR signaling. We used TCR signaling reporter mice and quantitative proteomics to explore the role of Themis1 in developing T cells. We found that Themis1 acted mostly as a positive regulator of TCR signaling in vivo when receptors were activated by positively selecting ligands. Proteomic analysis of the Themis1 interactome identified SHP-1, the TCR-associated adaptor protein Grb2, and the guanine nucleotide exchange factor Vav1 as the principal interacting partners of Themis1 in isolated mouse thymocytes. Analysis of TCR signaling in Themis1-deficient and Themis1-overexpressing mouse thymocytes demonstrated that Themis1 promoted Vav1 activity both in vitro and in vivo. The reduced activity of Vav1 and the impaired T cell development in Themis1(-/-) mice were due in part to increased degradation of Grb2, which suggests that Themis1 is required to maintain the steady-state abundance of Grb2 in thymocytes. Together, these data suggest that Themis1 acts as a positive regulator of TCR signaling in developing T cells, and identify a mechanism by which Themis1 regulates thymic selection.
Asunto(s)
Proteína Adaptadora GRB2/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Timocitos/citología , Animales , Diferenciación Celular , Péptidos y Proteínas de Señalización Intercelular , Activación de Linfocitos , Ratones , Ratones Transgénicos , Neuropéptidos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteómica/métodos , Transducción de Señal , Transgenes , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The development of inflammatory diseases depends on complex interactions between several genes and various environmental factors. Discovering new genetic risk factors and understanding the mechanisms whereby they influence disease development is of paramount importance. We previously reported that deficiency in Themis1, a new actor of TCR signaling, impairs regulatory T cell (Treg) function and predisposes Brown-Norway (BN) rats to spontaneous inflammatory bowel disease (IBD). In this study, we reveal that the epistasis between Themis1 and Vav1 controls the occurrence of these phenotypes. Indeed, by contrast with BN rats, Themis1 deficiency in Lewis rats neither impairs Treg suppressive functions nor induces pathological manifestations. By using congenic lines on the BN genomic background, we show that the impact of Themis1 deficiency on Treg suppressive functions depends on a 117-kb interval coding for a R63W polymorphism that impacts Vav1 expression and functions. Indeed, the introduction of a 117-kb interval containing the Lewis Vav1-R63 variant restores Treg function and protects Themis1-deficient BN rats from spontaneous IBD development. We further show that Themis1 binds more efficiently to the BN Vav1-W63 variant and is required to stabilize its recruitment to the transmembrane adaptor LAT and to fully promote the activation of Erk kinases. Together, these results highlight the importance of the signaling pathway involving epistasis between Themis1 and Vav1 in the control of Treg suppressive function and susceptibility to IBD development.
Asunto(s)
Epistasis Genética , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/inmunología , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Proto-Oncogénicas c-vav/genética , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Citocinas/biosíntesis , Modelos Animales de Enfermedad , Femenino , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Mutación , Proteínas Proto-Oncogénicas c-vav/metabolismo , Ratas , Ratas Transgénicas , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Timocitos/inmunología , Timocitos/metabolismoRESUMEN
The T-cell antigen receptor (TCR) complex contains 10 copies of a di-tyrosine Immunoreceptor-Tyrosine-based-Activation-Motif (ITAM) that initiates TCR signalling by recruiting protein tyrosine kinases. ITAM multiplicity amplifies TCR signals, but the importance of this capability for T-cell responses remains undefined. Most TCR ITAMs (6 of 10) are contributed by the CD3ζ subunits. We generated 'knock-in' mice that express non-signalling CD3ζ chains in lieu of wild-type CD3ζ. Here we demonstrate that ITAM multiplicity is important for the development of innate-like T-cells and follicular helper T-cells, events that are known to require strong/sustained TCR-ligand interactions, but is not essential for 'general' T-cell responses including proliferation and cytokine production or for the generation of a diverse antigen-reactive TCR repertoire.
Asunto(s)
Motivo de Activación del Inmunorreceptor Basado en Tirosina , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/citología , Animales , Antígenos/inmunología , Proliferación Celular , Células Clonales , Femenino , Memoria Inmunológica , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Células T Asesinas Naturales/citología , Células T Asesinas Naturales/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/química , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Transducción de Señal , Relación Estructura-ActividadRESUMEN
CD4(+) helper T cells are essential for immune responses and differentiate in the thymus from CD4(+) CD8(+) "double-positive" (DP) thymocytes. The transcription factor Runx3 inhibits CD4(+) T-cell differentiation by repressing Cd4 gene expression; accordingly, Runx3 is not expressed in DP thymocytes or developing CD4(+) T cells. The transcription factor Thpok is upregulated in CD4-differentiating thymocytes and required to repress Runx3. However, how Runx3 is controlled at early stages of CD4(+) T-cell differentiation, before the onset of Thpok expression, remains unknown. Here we show that Gata3, a transcription factor preferentially and transiently upregulated by CD4(+) T-cell precursors, represses Runx3 and binds the Runx3 locus in vivo. Accordingly, we show that high-level Gata3 expression and expression of Runx3 are mutually exclusive. Furthermore, whereas Runx3 represses Cd4, we show that Gata3 promotes Cd4 expression in Thpok-deficient thymocytes. Thus, in addition to its previously documented role in promoting CD4-lineage gene-expression, Gata3 represses CD8-lineage gene expression. These findings identify Gata3 as a critical pivot of CD4-CD8 lineage differentiation.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Subunidad alfa 3 del Factor de Unión al Sitio Principal/metabolismo , Factor de Transcripción GATA3/metabolismo , Timo/inmunología , Timo/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Regulación de la Expresión Génica , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Factores de Transcripción/genéticaRESUMEN
Negative selection and regulatory T (T reg) cell development are two thymus-dependent processes necessary for the enforcement of self-tolerance, and both require high-affinity interactions between the T cell receptor (TCR) and self-ligands. However, it remains unclear if they are similarly impacted by alterations in TCR signaling potential. We generated a knock-in allele (6F) of the TCR ζ chain gene encoding a mutant protein lacking signaling capability whose expression is controlled by endogenous ζ regulatory sequences. Although negative selection was defective in 6F/6F mice, leading to the survival of autoreactive T cells, 6F/6F mice did not develop autoimmune disease. We found that 6F/6F mice generated increased numbers of thymus-derived T reg cells. We show that attenuation of TCR signaling potential selectively impacts downstream signaling responses and that this differential effect favors Foxp3 expression and T reg cell lineage commitment. These results identify a potential compensatory pathway for the enforcement of immune tolerance in response to defective negative selection caused by reduced TCR signaling capability.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Selección Clonal Mediada por Antígenos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal , Alelos , Animales , Enfermedades Autoinmunes/genética , Trasplante de Médula Ósea/inmunología , Selección Clonal Mediada por Antígenos/genética , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Expresión Génica , Orden Génico , Proteínas de Homeodominio/genética , Memoria Inmunológica , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Transgénicos , Mutación , Fenotipo , Receptores de Antígenos de Linfocitos T/genética , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Timocitos/inmunología , Timocitos/metabolismoRESUMEN
Themis1, a recently identified T cell protein, has a critical function in the generation of mature CD4(+)CD8(-) and CD4(-)CD8(+) (CD4 and CD8 single-positive [SP]) thymocytes and T cells. Although Themis1 has been shown to bind to the adaptor proteins LAT and Grb2, previous studies have yielded conflicting results regarding whether thymocytes from Themis1(-/-) mice exhibit TCR-mediated signaling defects. In this study, we demonstrate that, in the absence of Themis1, TCR-mediated signaling is selectively impaired in CD4 SP and CD8 SP thymocytes but is not affected in CD4(+)CD8(+) double-positive thymocytes despite high expression of Themis1 in double-positive thymocytes. Like Themis1, Themis2, a related member of the Themis family, which is expressed in B cells and macrophages, contains two conserved cysteine-based domains, a proline-rich region, and a nuclear localization signal. To determine whether Themis1 and Themis2 can perform similar functions in vivo, we analyzed T cell development and TCR-mediated signaling in Themis1(-/-) mice reconstituted with either Themis1 or Themis2 transgenes. Notably, Themis1 and Themis2 exhibited the same potential to restore T cell development and TCR-mediated signaling in Themis1(-/-) mice. Both proteins were tyrosine phosphorylated and were recruited within Grb2 signaling complexes to LAT following TCR engagement. These results suggest that conserved molecular features of the Themis1 and Themis2 proteins are important for their biological activity and predict that Themis1 and Themis2 may perform similar functions in T and B cells, respectively.
Asunto(s)
Diferenciación Celular/inmunología , Secuencia Conservada/inmunología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Células Jurkat , Ratones , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Conejos , Linfocitos T/citologíaRESUMEN
During positive selection, thymocytes transition through a stage during which T cell antigen receptor (TCR) signaling controls CD4-versus-CD8 lineage 'choice' and subsequent maturation. Here we describe a previously unknown T cell-specific protein, Themis, that serves a distinct function during this stage. In Themis(-/-) mice, thymocyte selection was impaired and the number of transitional CD4(+)CD8(int) thymocytes as well as CD4(+) or CD8(+) single-positive thymocytes was lower. Notably, although we detected no overt TCR-proximal signaling deficiencies, Themis(-/-) CD4(+)CD8(int) thymocytes showed developmental defects consistent with attenuated signaling that were reversible by TCR stimulation. Our results identify Themis as a critical component of the T cell developmental program and suggest that Themis functions to sustain and/or integrate signals required for proper lineage commitment and maturation.