RESUMO
The kinase LCK and CD4/CD8 co-receptors are crucial components of the T cell antigen receptor (TCR) signaling machinery, leading to key T cell fate decisions. Despite decades of research, the roles of CD4-LCK and CD8-LCK interactions in TCR triggering in vivo remain unknown. In this study, we created animal models expressing endogenous levels of modified LCK to resolve whether and how co-receptor-bound LCK drives TCR signaling. We demonstrated that the role of LCK depends on the co-receptor to which it is bound. The CD8-bound LCK is largely dispensable for antiviral and antitumor activity of cytotoxic T cells in mice; however, it facilitates CD8+ T cell responses to suboptimal antigens in a kinase-dependent manner. By contrast, the CD4-bound LCK is required for efficient development and function of helper T cells via a kinase-independent stabilization of surface CD4. Overall, our findings reveal the role of co-receptor-bound LCK in T cell biology, show that CD4- and CD8-bound LCK drive T cell development and effector immune responses using qualitatively different mechanisms and identify the co-receptor-LCK interactions as promising targets for immunomodulation.
Assuntos
Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Linfócitos T Citotóxicos , Camundongos , Animais , Linfócitos T Citotóxicos/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Antígenos CD4 , Transdução de Sinais , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos CD8/metabolismoRESUMO
Self-non-self discrimination is central to T cell-mediated immunity. The kinetic proofreading model can explain T cell antigen receptor (TCR) ligand discrimination; however, the rate-limiting steps have not been identified. Here, we show that tyrosine phosphorylation of the T cell adapter protein LAT at position Y132 is a critical kinetic bottleneck for ligand discrimination. LAT phosphorylation at Y132, mediated by the kinase ZAP-70, leads to the recruitment and activation of phospholipase C-γ1 (PLC-γ1), an important effector molecule for T cell activation. The slow phosphorylation of Y132, relative to other phosphosites on LAT, is governed by a preceding glycine residue (G131) but can be accelerated by substituting this glycine with aspartate or glutamate. Acceleration of Y132 phosphorylation increases the speed and magnitude of PLC-γ1 activation and enhances T cell sensitivity to weaker stimuli, including weak agonists and self-peptides. These observations suggest that the slow phosphorylation of Y132 acts as a proofreading step to facilitate T cell ligand discrimination.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ativação Linfocitária , Proteínas de Membrana/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Feminino , Ligantes , Masculino , Proteínas de Membrana/imunologia , Camundongos , Fosfolipase C gama/metabolismo , Fosforilação/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/metabolismo , Tirosina/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
T cell-antigen receptor (TCR) signaling requires the sequential activities of the kinases Lck and Zap70. Upon TCR stimulation, Lck phosphorylates the TCR, thus leading to the recruitment, phosphorylation, and activation of Zap70. Lck binds and stabilizes phosho-Zap70 by using its SH2 domain, and Zap70 phosphorylates the critical adaptors LAT and SLP76, which coordinate downstream signaling. It is unclear whether phosphorylation of these adaptors occurs through passive diffusion or active recruitment. We report the discovery of a conserved proline-rich motif in LAT that mediates efficient LAT phosphorylation. Lck associates with this motif via its SH3 domain, and with phospho-Zap70 via its SH2 domain, thereby acting as a molecular bridge that facilitates the colocalization of Zap70 and LAT. Elimination of this proline-rich motif compromises TCR signaling and T cell development. These results demonstrate the remarkable multifunctionality of Lck, wherein each of its domains has evolved to orchestrate a distinct step in TCR signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas de Membrana/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Motivos de Aminoácidos , Animais , Células HEK293 , Humanos , Células Jurkat , Proteínas de Membrana/química , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Prolina/análise , Receptores de Antígenos de Linfócitos T/metabolismo , Timo/imunologiaRESUMO
Virtual memory T cells are foreign antigen-inexperienced T cells that have acquired memory-like phenotype and constitute 10-20% of all peripheral CD8+ T cells in mice. Their origin, biological roles, and relationship to naïve and foreign antigen-experienced memory T cells are incompletely understood. By analyzing T-cell receptor repertoires and using retrogenic monoclonal T-cell populations, we demonstrate that the virtual memory T-cell formation is a so far unappreciated cell fate decision checkpoint. We describe two molecular mechanisms driving the formation of virtual memory T cells. First, virtual memory T cells originate exclusively from strongly self-reactive T cells. Second, the stoichiometry of the CD8 interaction with Lck regulates the size of the virtual memory T-cell compartment via modulating the self-reactivity of individual T cells. Although virtual memory T cells descend from the highly self-reactive clones and acquire a partial memory program, they are not more potent in inducing experimental autoimmune diabetes than naïve T cells. These data underline the importance of the variable level of self-reactivity in polyclonal T cells for the generation of functional T-cell diversity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Memória Imunológica , Receptores de Antígenos de Linfócitos T/análise , Animais , Homeostase , CamundongosRESUMO
The intestinal tract generates significant reactive oxygen species (ROS), but the role of T cell antioxidant mechanisms in maintaining intestinal homeostasis is poorly understood. We used T cell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), which impaired glutathione (GSH) production, crucially reducing IL-22 production by Th17 cells in the lamina propria, which is critical for gut protection. Under steady-state conditions, Gclc deficiency did not alter cytokine secretion; however, C. rodentium infection induced increased ROS and disrupted mitochondrial function and TFAM-driven mitochondrial gene expression, resulting in decreased cellular ATP. These changes impaired the PI3K/AKT/mTOR pathway, reducing phosphorylation of 4E-BP1 and consequently limiting IL-22 translation. The resultant low IL-22 levels led to poor bacterial clearance, severe intestinal damage, and high mortality. Our findings highlight a previously unrecognized, essential role of Th17 cell-intrinsic GSH in promoting mitochondrial function and cellular signaling for IL-22 protein synthesis, which is critical for intestinal integrity and defense against gastrointestinal infections.
Assuntos
Glutationa , Interleucina 22 , Interleucinas , Mitocôndrias , Células Th17 , Animais , Interleucinas/metabolismo , Mitocôndrias/metabolismo , Glutationa/metabolismo , Células Th17/metabolismo , Células Th17/imunologia , Camundongos , Transdução de Sinais , Espécies Reativas de Oxigênio/metabolismo , Camundongos Endogâmicos C57BL , Citrobacter rodentium , Intestinos/patologia , Intestinos/imunologia , Inflamação/metabolismo , Inflamação/patologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/metabolismo , Infecções por Enterobacteriaceae/patologia , Camundongos Knockout , Serina-Treonina Quinases TOR/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologiaRESUMO
Pyruvate dehydrogenase (PDH) is the central enzyme connecting glycolysis and the tricarboxylic acid (TCA) cycle. The importance of PDH function in T helper 17 (Th17) cells still remains to be studied. Here, we show that PDH is essential for the generation of a glucose-derived citrate pool needed for Th17 cell proliferation, survival, and effector function. In vivo, mice harboring a T cell-specific deletion of PDH are less susceptible to developing experimental autoimmune encephalomyelitis. Mechanistically, the absence of PDH in Th17 cells increases glutaminolysis, glycolysis, and lipid uptake in a mammalian target of rapamycin (mTOR)-dependent manner. However, cellular citrate remains critically low in mutant Th17 cells, which interferes with oxidative phosphorylation (OXPHOS), lipid synthesis, and histone acetylation, crucial for transcription of Th17 signature genes. Increasing cellular citrate in PDH-deficient Th17 cells restores their metabolism and function, identifying a metabolic feedback loop within the central carbon metabolism that may offer possibilities for therapeutically targeting Th17 cell-driven autoimmunity.
Assuntos
Ácido Cítrico , Células Th17 , Camundongos , Animais , Citratos , Oxirredutases , Lipídeos , Piruvatos , MamíferosRESUMO
Although the intestinal tract is a major site of reactive oxygen species (ROS) generation, the mechanisms by which antioxidant defense in gut T cells contribute to intestinal homeostasis are currently unknown. Here we show, using T cell-specific ablation of the catalytic subunit of glutamate cysteine ligase (Gclc), that the ensuing loss of glutathione (GSH) impairs the production of gut-protective IL-22 by Th17 cells within the lamina propria. Although Gclc ablation does not affect T cell cytokine secretion in the gut of mice at steady-state, infection with C. rodentium increases ROS, inhibits mitochondrial gene expression and mitochondrial function in Gclc-deficient Th17 cells. These mitochondrial deficits affect the PI3K/AKT/mTOR pathway, leading to reduced phosphorylation of the translation repressor 4E-BP1. As a consequence, the initiation of translation is restricted, resulting in decreased protein synthesis of IL-22. Loss of IL-22 results in poor bacterial clearance, enhanced intestinal damage, and high mortality. ROS-scavenging, reconstitution of IL-22 expression or IL-22 supplementation in vivo prevent the appearance of these pathologies. Our results demonstrate the existence of a previously unappreciated role for Th17 cell-intrinsic GSH coupling to promote mitochondrial function, IL-22 translation and signaling. These data reveal an axis that is essential for maintaining the integrity of the intestinal barrier and protecting it from damage caused by gastrointestinal infection.
RESUMO
Mature T cells are selected for recognizing self-antigens with low to intermediate affinity in the thymus. Recently, the relative differences in self-reactivity among individual T-cell clones were appreciated as important factors regulating their fate and immune response, but the role of self-reactivity in T-cell biology is incompletely understood. We addressed the role of self-reactivity in T-cell diversity by generating an atlas of mouse peripheral CD8+ T cells, which revealed two unconventional populations of antigen-inexperienced T cells. In the next step, we examined the steady-state phenotype of monoclonal T cells with various levels of self-reactivity. Highly self-reactive clones preferentially differentiate into antigen-inexperienced memory-like cells, but do not form a population expressing type I interferon-induced genes, showing that these two subsets have unrelated origins. The functional comparison of naïve monoclonal CD8+ T cells specific to the identical model antigen did not show any correlation between the level of self-reactivity and the magnitude of the immune response.
Assuntos
Linfócitos T CD8-Positivos , Interferon Tipo I , Camundongos , Animais , Timo , Células Clonais , AutoantígenosRESUMO
CD4 and CD8 mark helper and cytotoxic T cell lineages, respectively, and serve as coreceptors for MHC-restricted TCR recognition. How coreceptor expression is matched with TCR specificity is central to understanding CD4/CD8 lineage choice, but visualising coreceptor gene activity in individual selection intermediates has been technically challenging. It therefore remains unclear whether the sequence of coreceptor gene expression in selection intermediates follows a stereotypic pattern, or is responsive to signaling. Here we use single cell RNA sequencing (scRNA-seq) to classify mouse thymocyte selection intermediates by coreceptor gene expression. In the unperturbed thymus, Cd4+Cd8a- selection intermediates appear before Cd4-Cd8a+ selection intermediates, but the timing of these subsets is flexible according to the strength of TCR signals. Our data show that selection intermediates discriminate MHC class prior to the loss of coreceptor expression and suggest a model where signal strength informs the timing of coreceptor gene activity and ultimately CD4/CD8 lineage choice.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Timo/citologia , Timo/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Antígenos de Histocompatibilidade/metabolismo , Ativação Linfocitária/genética , Camundongos Endogâmicos C57BL , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Overtly self-reactive T cells are removed during thymic selection. However, it has been recently established that T cell self-reactivity promotes protective immune responses. Apparently, the level of self-reactivity of mature T cells must be tightly balanced. Our mathematical model and experimental data show that the dynamic regulation of CD4- and CD8-LCK coupling establish the self-reactivity of the peripheral T cell pool. The stoichiometry of the interaction between CD8 and LCK, but not between CD4 and LCK, substantially increases upon T cell maturation. As a result, peripheral CD8+ T cells are more self-reactive than CD4+ T cells. The different levels of self-reactivity of mature CD8+ and CD4+ T cells likely reflect the unique roles of these subsets in immunity. These results indicate that the evolutionary selection pressure tuned the CD4-LCK and CD8-LCK stoichiometries, as they represent the unique parts of the proximal T cell receptor (TCR) signaling pathway, which differ between CD4+ and CD8+ T cells.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Animais , Antígenos/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular , Homeostase , Camundongos Endogâmicos C57BL , Ligação Proteica , Transdução de SinaisRESUMO
T cells require the protein tyrosine phosphatase CD45 to detect and respond to antigen because it activates the Src family kinase Lck, which phosphorylates the T cell antigen receptor (TCR) complex. CD45 activates Lck by opposing the negative regulatory kinase Csk. Paradoxically, CD45 has also been implicated in suppressing TCR signaling by dephosphorylating the same signaling motifs within the TCR complex upon which Lck acts. We sought to reconcile these observations using chemical and genetic perturbations of the Csk/CD45 regulatory axis incorporated with computational analyses. Specifically, we titrated the activities of Csk and CD45 and assessed their influence on Lck activation, TCR-associated ζ-chain phosphorylation, and more downstream signaling events. Acute inhibition of Csk revealed that CD45 suppressed ζ-chain phosphorylation and was necessary for a regulatable pool of active Lck, thereby interconnecting the activating and suppressive roles of CD45 that tune antigen discrimination. CD45 suppressed signaling events that were antigen independent or induced by low-affinity antigen but not those initiated by high-affinity antigen. Together, our findings reveal that CD45 acts as a signaling "gatekeeper," enabling graded signaling outputs while filtering weak or spurious signaling events.
Assuntos
Antígenos Comuns de Leucócito/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Proteína Tirosina Quinase CSK/genética , Humanos , Células Jurkat , Antígenos Comuns de Leucócito/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/genética , Linfócitos T/citologiaRESUMO
Polymer carriers based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers with incorporated organic nitrates as nitric oxide (NO) donors were designed with the aim to localise NO generation in solid tumours, thus highly increasing the enhanced permeability and retention (EPR) effect. The NO donors were coupled to the polymer carrier either through a stable bond or through a hydrolytically degradable, pH sensitive, bond. In vivo, the co-administration of the polymer NO donor and HPMA copolymer-bound cytotoxic drug (doxorubicin; Dox) resulted in an improvement in the treatment of murine EL4 T-cell lymphoma. The polymer NO donors neither potentiated the in vitro toxicity of the cytotoxic drug nor exerted any effect on in vivo model without the EPR effect, such as BCL1 leukaemia. Thus, an increase in passive accumulation of the nanomedicine carrying cytotoxic drug via NO-enhanced EPR effect was the operative mechanism of action. The most significant improvement in the therapy was observed in a combination treatment with such a polymer conjugate of Dox, which is characterised by increased circulation in the blood and efficient accumulation in solid tumours. Notably, the combination treatment enabled the development of an anti-tumour immune response, which was previously demonstrated as an important feature of HPMA-based polymer cytotoxic drugs.