RESUMEN
Single-cell RNA sequencing is a powerful tool to examine cellular heterogeneity, novel markers and target genes, and therapeutic mechanisms in human cancers and animal models. Here, we analyzed single-cell RNA sequencing data of T cells obtained from multiple mouse tumor models by PCA-based subclustering coupled with TCR tracking using the STARTRAC algorithm. This approach revealed various differentiated T cell subsets and activation states, and a correspondence of T cell subsets between human and mouse tumors. STARTRAC analyses demonstrated peripheral T cell subsets that were developmentally connected with tumor-infiltrating CD8+ cells, CD4+ Th1 cells, and T reg cells. In addition, large amounts of paired TCRα/ß sequences enabled us to identify a specific enrichment of paired public TCR clones in tumor. Finally, we identified CCR8 as a tumor-associated T reg cell marker that could preferentially deplete tumor-associated T reg cells. We showed that CCR8-depleting antibody treatment provided therapeutic benefit in CT26 tumors and synergized with anti-PD-1 treatment in MC38 and B16F10 tumor models.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Neoplasias/inmunología , Linfocitos T Reguladores/inmunología , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/inmunología , Células TH1/inmunologíaRESUMEN
COVID-19 presents with a wide range of severity, from asymptomatic in some individuals to fatal in others. Based on a study of 1,051,032 23andMe research participants, we report genetic and nongenetic associations with testing positive for SARS-CoV-2, respiratory symptoms and hospitalization. Using trans-ancestry genome-wide association studies, we identified a strong association between blood type and COVID-19 diagnosis, as well as a gene-rich locus on chromosome 3p21.31 that is more strongly associated with outcome severity. Hospitalization risk factors include advancing age, male sex, obesity, lower socioeconomic status, non-European ancestry and preexisting cardiometabolic conditions. While non-European ancestry was a significant risk factor for hospitalization after adjusting for sociodemographics and preexisting health conditions, we did not find evidence that these two primary genetic associations explain risk differences between populations for severe COVID-19 outcomes.
Asunto(s)
COVID-19/genética , Predisposición Genética a la Enfermedad , Sistema del Grupo Sanguíneo ABO/genética , Tipificación y Pruebas Cruzadas Sanguíneas , Cromosomas Humanos Par 3 , Bases de Datos Genéticas , Susceptibilidad a Enfermedades , Femenino , Galactosiltransferasas/genética , Estudio de Asociación del Genoma Completo , Hospitalización , Humanos , Masculino , Persona de Mediana Edad , Gravedad del Paciente , Grupos Raciales , Factores de RiesgoRESUMEN
The retinoic acid receptor-related orphan nuclear receptor gamma t (RORγt), which is a promising therapeutic target for immune diseases, is a major transcription factor of genes related to psoriasis pathogenesis, such as interleukin (IL)-17A, IL-22, and IL-23R. Inspired by the co-crystal structure of RORγt, a 6-oxo-4-phenyl-hexanoic acid derivative 6a was designed, synthesized, and identified as a ligand of RORγt. The structure-activity relationship (SAR) studies in 6a, which focus on the improvement of its membrane permeability profile by introducing chlorine atoms, led to finding 12a, which has a potent RORγt inhibitory activity and a favorable pharmacokinetic profile.
Asunto(s)
Caproatos/farmacología , Descubrimiento de Drogas , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Animales , Caproatos/química , Caproatos/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Relación Estructura-ActividadRESUMEN
The retinoic acid receptor-related orphan nuclear receptor γt (RORγt), a promising therapeutic target, is a major transcription factor of genes related to psoriasis pathogenesis such as interleukin (IL)-17A, IL-22, and IL-23R. On the basis of the X-ray cocrystal structure of RORγt with 1a, an analogue of the known piperazine RORγt inverse agonist 1, triazolopyridine derivatives of 1 were designed and synthesized, and analogue 3a was found to be a potent RORγt inverse agonist. Structure-activity relationship studies on 3a, focusing on the treatment of its metabolically unstable cyclopentyl ring and the central piperazine core, led to a novel analogue, namely, 6-methyl-N-(7-methyl-8-(((2S,4S)-2-methyl-1-(4,4,4-trifluoro-3-(trifluoromethyl)butanoyl)piperidin-4-yl)oxy)[1,2,4]triazolo[1,5-a]pyridin-6-yl)nicotinamide (5a), which exhibited strong RORγt inhibitory activity and a favorable pharmacokinetic profile. Moreover, the in vitro and in vivo evaluation of 5a in a human whole-blood assay and a mouse IL-18/23-induced cytokine expression model revealed its robust and dose-dependent inhibitory effect on IL-17A production.
RESUMEN
The real-time quantification of target engagement (TE) by small-molecule ligands in living cells remains technically challenging. Systematic quantification of such interactions in a high-throughput setting holds promise for identification of target-specific, potent small molecules within a pathophysiological and biologically relevant cellular context. The salt-inducible kinases (SIKs) belong to a subfamily of the AMP-activated protein kinase (AMPK) family and are composed of three isoforms in humans (SIK1, SIK2, and SIK3). They modulate the production of pro- and anti-inflammatory cytokines in immune cells. Although pan-SIK inhibitors are sufficient to reverse SIK-dependent inflammatory responses, the apparent toxicity associated with SIK3 inhibition suggests that isoform-specific inhibition is required to realize therapeutic benefit with acceptable safety margins. Here, we used the NanoBRET TE intracellular kinase assay, a sensitive energy transfer technique, to directly measure molecular proximity and quantify TE in HEK293T cells overexpressing SIK2 or SIK3. Our 384-well high-throughput screening of 530 compounds demonstrates that the NanoBRET TE intracellular kinase assay was sensitive and robust enough to reveal differential engagement of candidate compounds with the two SIK isoforms and further highlights the feasibility of high-throughput implementation of NanoBRET TE intracellular kinase assays for target-driven small-molecule screening.
Asunto(s)
Fosfotransferasas/aislamiento & purificación , Isoformas de Proteínas/genética , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Transferencia Resonante de Energía de Fluorescencia , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Fosfotransferasas/genética , Isoformas de Proteínas/antagonistas & inhibidores , Proteínas Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidoresRESUMEN
Th17 cells, which express the cytokine IL-17A, and master regulator RORγt, are important in the inflammatory response to fungal and bacterial pathogens, but also have a pathogenic role in many inflammatory disorders. In contrast, regulatory T cells (Treg), expressing the Foxp3 transcription factor, have a suppressive function and can dampen an immune response. The appropriate balance of these distinct effector functions is critical for an effective immune response and autoimmunity can arise if this process goes awry. In this issue, Wang et al. demonstrate a critical role for the transcription factor CREB (cyclic AMP-responsive element binding protein) in regulating the balance between inflammatory Th17 and suppressive Treg cells with implications for autoimmunity.
Asunto(s)
Linfocitos T Reguladores , Células Th17 , Autoinmunidad , Factores de Transcripción Forkhead , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores NuclearesRESUMEN
Interleukin-23 (IL-23) and IL-17 are cytokines currently being targeted in clinical trials. Although inhibition of both of these cytokines is effective for treating psoriasis, IL-12 and IL-23 p40 inhibition attenuates Crohn's disease, whereas IL-17A or IL-17 receptor A (IL-17RA) inhibition exacerbates Crohn's disease. This dichotomy between IL-23 and IL-17 was effectively modeled in the multidrug resistance-1a-ablated (Abcb1a(-/-)) mouse model of colitis. IL-23 inhibition attenuated disease by decreasing colonic inflammation while enhancing regulatory T (Treg) cell accumulation. Exacerbation of colitis by IL-17A or IL-17RA inhibition was associated with severe weakening of the intestinal epithelial barrier, culminating in increased colonic inflammation and accelerated mortality. These data show that IL-17A acts on intestinal epithelium to promote barrier function and provide insight into mechanisms underlying exacerbation of Crohn's disease when IL-17A or IL-17RA is inhibited.
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Colitis/inmunología , Interleucina-17/fisiología , Interleucina-23/fisiología , Receptores de Interleucina-17/fisiología , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Animales , Colitis/tratamiento farmacológico , Colitis/etiología , Colitis/microbiología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Epitelio/fisiopatología , Femenino , Factores de Transcripción Forkhead/análisis , Regulación de la Expresión Génica/inmunología , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/inmunología , Inmunización Pasiva , Inmunoglobulina G/uso terapéutico , Subunidad p40 de la Interleucina-12/antagonistas & inhibidores , Interleucina-17/inmunología , Interleucina-23/inmunología , Subunidad p19 de la Interleucina-23/antagonistas & inhibidores , Subunidad p19 de la Interleucina-23/inmunología , Mucosa Intestinal/fisiopatología , Ratones , Ratones Noqueados , Permeabilidad , Receptores de Interleucina-17/antagonistas & inhibidores , Receptores de Interleucina-17/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , TranscriptomaRESUMEN
Activation of the TPL2-MKK1/2-ERK1/2 signalling pathway is essential for lipopolysaccharide (LPS)-stimulated production of TNF alpha in macrophages. Here, we demonstrate that, unexpectedly, TPL2-deficient or MKK1-inhibited macrophages produce near normal levels of pre-TNF alpha when TLR2, TLR4 and TLR6 are activated by their respective agonists, but fail to secrete TNFalpha. We show that LPS stimulates the appearance of pre-TNFalpha at the cell surface and that this is prevented by inhibition of MAPK kinases 1 and 2 (MKK1/2) or in TPL2-deficient macrophages. However, the transport of pre-TNF alpha from the Golgi to the plasma membrane is unaffected by inhibition of the TPL2-MKK1/2-ERK1/2 pathway. Finally, we show that TACE, the protease that cleaves pre-TNF alpha to secreted TNFalpha, is phosphorylated by ERK1 and ERK2 (ERK1/2) at Thr735 in LPS-stimulated macrophages. Therefore, although TACE activity per se is not required for the LPS-stimulated cell surface expression of pre-TNF alpha, the phosphorylation of this protease might contribute to, or be required for, the cell surface expression of the pre-TNF alpha-TACE complex.
Asunto(s)
Membrana Celular/metabolismo , Regulación Enzimológica de la Expresión Génica , Aparato de Golgi/metabolismo , Lipopolisacáridos/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Macrófagos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Modelos BiológicosRESUMEN
The TPL-2 MEK kinase is essential for activation of the Erk MAP kinase pathway during innate immune responses. TPL-2 is found in complex with ABIN-2 (A20-binding inhibitor of NF-kappaB 2). Here, using antigen-presenting cells from ABIN-2-deficient mice, we show that ABIN-2 was required for optimal activation of Erk induced by receptors that signal via TPL-2, including Toll-like receptor 4 and tumor necrosis factor receptor 1 in macrophages, and CD40 in B cells. ABIN-2 was necessary for the maintenance of TPL-2 protein stability. In contrast, ABIN-2 deficiency did not affect agonist-induced regulation of transcription factor NF-kappaB. Stimulation of ABIN-2-deficient macrophages via Toll-like receptor 4 showed that different thresholds of Erk signaling were required for optimal induction of tumor necrosis factor and interleukin 1beta. Thus, ABIN-2 acts to positively regulate the Erk signaling potential by stabilizing TPL-2.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Inmunidad Innata , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Linfocitos B/inmunología , Antígenos CD40/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Activación Enzimática , Interleucina-1/metabolismo , Lipopolisacáridos/inmunología , Macrófagos/inmunología , Ratones , Ratones Mutantes , FN-kappa B/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
Toll-like receptors, which respond to invariant microbial molecules, and receptors for the proinflammatory cytokines tumour necrosis factor and interleukin-1 are crucial for initiation and regulation of innate immune responses. These receptors activate each of the major mitogen-activated protein (MAP) kinase subtypes, extracellular signal-regulated protein kinases, c-Jun amino-terminal kinases and p38 MAP kinases, which are crucial for cell survival and controlling the expression of immune mediators. Here we discuss recent studies characterizing the specific MAP kinase kinase kinases (MAP 3-kinases) that link MAP kinases to receptors involved in innate immunity and the mechanisms by which the activity of MAP 3-kinases is regulated by such receptors.
Asunto(s)
Inmunidad Innata/inmunología , Quinasas Quinasa Quinasa PAM/metabolismo , Animales , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Humanos , Quinasas Quinasa Quinasa PAM/química , Quinasas Quinasa Quinasa PAM/genética , Sistema de Señalización de MAP Quinasas , FosforilaciónRESUMEN
The transmembrane phosphatase CD45 regulates both Lck activity and T cell receptor (TCR) signaling. Here we have tested whether the large ectodomain of CD45 has a role in this regulation. A CD45 chimera containing the large ectodomain of CD43 efficiently rescues TCR signaling in CD45-null T cells, whereas CD45 chimeras containing small ectodomains from other phosphatases do not. Both basal Lck activity in unstimulated cells and the TCR-induced increase in tyrosine phosphorylation of the TCR zeta-chain and in Lck activity depend on the expression of CD45 with a large ectodomain. Unlike CD45 chimeras containing small ectodomains, both the CD45 chimera with a large ectodomain and wild-type CD45 itself are partially localized to glycosphingolipid-enriched membranes (GEMs). Taken together, these data show that the large CD45 ectodomain is required for optimal TCR signaling.