RESUMEN
Type I and III interferons (IFNs) are robustly induced during infections and protect cells against viral infection. Both type I and III IFNs are also produced at low levels in the thymus at steady state; however, their role in T cell development and immune tolerance is unclear. Here, we found that both type I and III IFNs were constitutively produced by a very small number of AIRE+ murine thymic epithelial cells, independent of microbial stimulation. Antigen-presenting cells were highly responsive to thymic IFNs, and IFNs were required for the activation and maturation of thymic type 1 conventional dendritic cells, macrophages, and B cells. Loss of IFN sensing led to reduced regulatory T cell selection, reduced T cell receptor (TCR) repertoire diversity, and enhanced autoreactive T cell responses to self-antigens expressed during peripheral IFN signaling. Thus, constitutive exposure to IFNs in the thymus is required for generating a tolerant and diverse TCR repertoire.
Asunto(s)
Interferones , Ratones Endogámicos C57BL , Timo , Animales , Timo/inmunología , Ratones , Interferones/inmunología , Ratones Noqueados , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunologíaRESUMEN
Mertk, a type I receptor tyrosine kinase and member of the TAM family of receptors, has important functions in promoting efferocytosis and resolving inflammation under physiological conditions. In recent years, Mertk has also been linked to pathophysiological roles in cancer, whereby, in several cancer types, including solid cancers and leukemia/lymphomas. Mertk contributes to oncogenic features of proliferation and cell survival as an oncogenic tyrosine kinase. In addition, Mertk expressed on macrophages, including tumor-associated macrophages, promotes immune evasion in cancer and is suggested to act akin to a myeloid checkpoint inhibitor that skews macrophages towards inhibitory phenotypes that suppress host T-cell anti-tumor immunity. In the present study, to better understand the post-translational regulation mechanisms controlling Mertk expression in monocytes/macrophages, we used a PMA-differentiated THP-1 cell model to interrogate the regulation of Mertk expression and developed a novel Mertk reporter cell line to study the intracellular trafficking of Mertk. We show that PMA treatment potently up-regulates Mertk as well as components of the ectodomain proteolytic processing platform ADAM17, whereas PMA differentially regulates the canonical Mertk ligands Gas6 and Pros1 (Gas6 is down-regulated and Pros1 is up-regulated). Under non-stimulated homeostatic conditions, Mertk in PMA-differentiated THP1 cells shows active constitutive proteolytic cleavage by the sequential activities of ADAM17 and the Presenilin/γ-secretase complex, indicating that Mertk is cleaved homeostatically by the combined sequential action of ADAM17 and γ-secretase, after which the cleaved intracellular fragment of Mertk is degraded in a proteasome-dependent mechanism. Using chimeric Flag-Mertk-EGFP-Myc reporter receptors, we confirm that inhibitors of γ-secretase and MG132, which inhibits the 26S proteasome, stabilize the intracellular fragment of Mertk without evidence of nuclear translocation. Finally, the treatment of cells with active γ-carboxylated Gas6, but not inactive Warfarin-treated non-γ-carboxylated Gas6, regulates a distinct proteolytic itinerary-involved receptor clearance and lysosomal proteolysis. Together, these results indicate that pleotropic and complex proteolytic activities regulate Mertk ectodomain cleavage as a homeostatic negative regulatory event to safeguard against the overactivation of Mertk.
Asunto(s)
Proteína ADAM17 , Secretasas de la Proteína Precursora del Amiloide , Proteolisis , Tirosina Quinasa c-Mer , Humanos , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa c-Mer/genética , Proteína ADAM17/metabolismo , Proteína ADAM17/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células THP-1 , Macrófagos/metabolismo , Proteína S/metabolismo , Monocitos/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The activation of thymic B cells is critical for their licensing as antigen presenting cells and resulting ability to mediate T cell central tolerance. The processes leading to licensing are still not fully understood. By comparing thymic B cells to activated Peyer's patch B cells at steady state, we found that thymic B cell activation starts during the neonatal period and is characterized by TCR/CD40-dependent activation, followed by immunoglobulin class switch recombination (CSR) without forming germinal centers. Transcriptional analysis also demonstrated a strong interferon signature, which was not apparent in the periphery. Thymic B cell activation and CSR were primarily dependent on type III IFN signaling, and loss of type III IFN receptor in thymic B cells resulted in reduced thymocyte regulatory T cell (Treg) development. Finally, from TCR deep sequencing, we estimate that licensed B cells induce development of a substantial fraction of the Treg cell repertoire. Together, these findings reveal the importance of steady-state type III IFN in generating licensed thymic B cells that induce T cell tolerance to activated B cells.
Asunto(s)
Interferón lambda , Linfocitos T Reguladores , Humanos , Recién Nacido , Timo , Timocitos , Receptores de Antígenos de Linfocitos TRESUMEN
Neuroimmune interactions are crucial for regulating immunity and inflammation. Recent studies have revealed that the central nervous system (CNS) senses peripheral inflammation and responds by releasing molecules that limit immune cell activation, thereby promoting tolerance and tissue integrity. However, the extent to which this is a bidirectional process, and whether peripheral immune cells also promote tolerance mechanisms in the CNS remains poorly defined. Here we report that helminth-induced type 2 inflammation promotes monocyte responses in the brain that are required to inhibit excessive microglial activation and host death. Mechanistically, infection-induced monocytes express YM1 that is sufficient to inhibit tumor necrosis factor production from activated microglia. Importantly, neuroprotective monocytes persist in the brain, and infected mice are protected from subsequent lipopolysaccharide-induced neuroinflammation months after infection-induced inflammation has resolved. These studies demonstrate that infiltrating monocytes promote CNS homeostasis in response to inflammation in the periphery and demonstrate that a peripheral infection can alter the immunologic landscape of the host brain.
Asunto(s)
Encéfalo , Encefalitis , Homeostasis , Monocitos , Neuroinmunomodulación , Trichinella spiralis , Triquinelosis , Animales , Encéfalo/inmunología , Encéfalo/parasitología , Encefalitis/inmunología , Encefalitis/parasitología , Homeostasis/inmunología , Lectinas/metabolismo , Ratones , Microglía/inmunología , Monocitos/inmunología , Trichinella spiralis/inmunología , Triquinelosis/inmunología , Triquinelosis/patología , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Infants with attenuated type III IFN (IFN-λ) responses are at increased risk of severe lower respiratory tract infection (sLRI). The IL-28Rα-chain and IL-10Rß-chain form a heterodimeric receptor complex, necessary for IFN-λ signaling. Therefore, to better understand the immunopathogenic mechanisms through which an IFN-λlo microenvironment predisposes to a sLRI, we inoculated neonatal wild-type and IL-28R-deficient (IL-28R -/-) mice with pneumonia virus of mice, a rodent-specific pneumovirus. Infected IL-28R -/- neonates displayed an early, pronounced, and persistent neutrophilia that was associated with enhanced reactive oxygen species (ROS) production, NETosis, and mucus hypersecretion. Targeted deletion of the IL-28R in neutrophils was sufficient to increase neutrophil activation, ROS production, NET formation, and mucus production in the airways. Inhibition of protein-arginine deiminase type 4 (PAD4), a regulator of NETosis, had no effect on myeloperoxidase expression, citrullinated histones, and the magnitude of the inflammatory response in the lungs of infected IL-28R -/- mice. In contrast, inhibition of ROS production decreased NET formation, cellular inflammation, and mucus hypersecretion. These data suggest that IFN-λ signaling in neutrophils dampens ROS-induced NETosis, limiting the magnitude of the inflammatory response and mucus production. Therapeutics that promote IFN-λ signaling may confer protection against sLRI.
Asunto(s)
Bronquiolitis Viral , Trampas Extracelulares , Interferones/metabolismo , Animales , Animales Recién Nacidos , Bronquiolitis Viral/metabolismo , Bronquiolitis Viral/patología , Trampas Extracelulares/metabolismo , Humanos , Ratones , NADPH Oxidasas/metabolismo , Neutrófilos/metabolismo , Arginina Deiminasa Proteína-Tipo 4 , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Emerging viruses threaten global health, but few experimental models can characterize the virus and host factors necessary for within- and cross-species transmission. Here, we leverage a model whereby pet store mice or rats-which harbor natural rodent pathogens-are cohoused with laboratory mice. This "dirty" mouse model offers a platform for studying acute transmission of viruses between and within hosts via natural mechanisms. We identified numerous viruses and other microbial species that transmit to cohoused mice, including prospective new members of the Coronaviridae, Astroviridae, Picornaviridae, and Narnaviridae families, and uncovered pathogen interactions that promote or prevent virus transmission. We also evaluated transmission dynamics of murine astroviruses during transmission and spread within a new host. Finally, by cohousing our laboratory mice with the bedding of pet store rats, we identified cross-species transmission of a rat astrovirus. Overall, this model system allows for the analysis of transmission of natural rodent viruses and is a platform to further characterize barriers to zoonosis.
Asunto(s)
Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Virosis/etiología , Virosis/transmisión , Enfermedades de los Animales/transmisión , Enfermedades de los Animales/virología , Animales , Biomarcadores , Interacciones Huésped-Patógeno , Humanos , Interferones/metabolismo , Ratones , Ratones Noqueados , Interacciones Microbianas , Roedores , Virosis/metabolismoRESUMEN
Immunopathology and intestinal stem cell (ISC) loss in the gastrointestinal (GI) tract is the prima facie manifestation of graft-versus-host disease (GVHD) and is responsible for significant mortality after allogeneic bone marrow transplantation (BMT). Approaches to prevent GVHD to date focus on immune suppression. Here, we identify interferon-λ (IFN-λ; interleukin-28 [IL-28]/IL-29) as a key protector of GI GVHD immunopathology, notably within the ISC compartment. Ifnlr1-/- mice displayed exaggerated GI GVHD and mortality independent of Paneth cells and alterations to the microbiome. Ifnlr1-/- intestinal organoid growth was significantly impaired, and targeted Ifnlr1 deficiency exhibited effects intrinsic to recipient Lgr5+ ISCs and natural killer cells. PEGylated recombinant IL-29 (PEG-rIL-29) treatment of naive mice enhanced Lgr5+ ISC numbers and organoid growth independent of both IL-22 and type I IFN and modulated proliferative and apoptosis gene sets in Lgr5+ ISCs. PEG-rIL-29 treatment improved survival, reduced GVHD severity, and enhanced epithelial proliferation and ISC-derived organoid growth after BMT. The preservation of ISC numbers in response to PEG-rIL-29 after BMT occurred both in the presence and absence of IFN-λ-signaling in recipient natural killer cells. IFN-λ is therefore an attractive and rapidly testable approach to prevent ISC loss and immunopathology during GVHD.
Asunto(s)
Trasplante de Médula Ósea , Citocinas/farmacología , Enfermedades Gastrointestinales , Enfermedad Injerto contra Huésped , Interleucinas/farmacocinética , Transducción de Señal , Animales , Citocinas/inmunología , Enfermedades Gastrointestinales/tratamiento farmacológico , Enfermedades Gastrointestinales/genética , Enfermedades Gastrointestinales/inmunología , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Interleucinas/inmunología , Ratones , Ratones Noqueados , Receptores de Interferón/genética , Receptores de Interferón/inmunología , Índice de Severidad de la Enfermedad , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Trasplante HomólogoRESUMEN
Phosphatidylserine (PS)-targeting monoclonal Abs (mAbs) that directly target PS and target PS via ß2-gp1 (ß2GP1) have been in preclinical and clinical development for over 10 y for the treatment of infectious diseases and cancer. Although the intended targets of PS-binding mAbs have traditionally included pathogens as well as stressed tumor cells and its associated vasculature in oncology, the effects of PS-targeting mAbs on activated immune cells, notably T cells, which externalize PS upon Ag stimulation, is not well understood. Using human T cells from healthy donor PBMCs activated with an anti-CD3 + anti-CD28 Ab mixture (anti-CD3/CD28) as a model for TCR-mediated PS externalization and T cell stimulation, we investigated effects of two different PS-targeting mAbs, 11.31 and bavituximab (Bavi), on TCR activation and TCR-mediated cytokine production in an ex vivo paradigm. Although 11.31 and Bavi bind selectivity to anti-CD3/28 activated T cells in a PS-dependent manner, surprisingly, they display distinct functional activities in their effect on IFN-γ and TNF-É production, whereby 11.31, but not Bavi, suppressed cytokine production. This inhibitory effect on anti-CD3/28 activated T cells was observed on both CD4+ and CD8+ cells and independently of monocytes, suggesting the effects of 11.31 were directly mediated by binding to externalized PS on activated T cells. Imaging showed 11.31 and Bavi bind at distinct focal depots on the cell membrane. Collectively, our findings indicate that PS-targeting mAb 11.31 suppresses cytokine production by anti-CD3/28 activated T cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD28/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Interferón gamma/inmunología , Muromonab-CD3/inmunología , Fosfatidilserinas/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Complejo CD3/inmunología , Línea Celular , Células HEK293 , Humanos , Leucocitos Mononucleares/inmunología , Activación de Linfocitos/inmunologíaRESUMEN
The etiology of ulcerative colitis is poorly understood and is likely to involve perturbation of the complex interactions between the mucosal immune system and the commensal bacteria of the gut, with cytokines acting as important cross-regulators. Here we use IFN receptor-deficient mice in a dextran sulfate sodium (DSS) model of acute intestinal injury to study the contributions of type I and III interferons (IFN) to the initiation, progression and resolution of acute colitis. We find that mice lacking both types of IFN receptors exhibit enhanced barrier destruction, extensive loss of goblet cells and diminished proliferation of epithelial cells in the colon following DSS-induced damage. Impaired mucosal healing in double IFN receptor-deficient mice is driven by decreased amphiregulin expression, which IFN signaling can up-regulate in either the epithelial or hematopoietic compartment. Together, these data underscore the pleiotropic functions of IFNs and demonstrate that these critical antiviral cytokines also support epithelial regeneration following acute colonic injury.
Asunto(s)
Colitis Ulcerosa/inmunología , Interferones/metabolismo , Mucosa Intestinal/patología , Repitelización/inmunología , Animales , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/patología , Sulfato de Dextran/administración & dosificación , Sulfato de Dextran/toxicidad , Modelos Animales de Enfermedad , Células Epiteliales , Femenino , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Masculino , Ratones , Ratones Noqueados , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Receptores de Interferón/genética , Receptores de Interferón/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Organismos Libres de Patógenos EspecíficosRESUMEN
Interferons are potent antiviral cytokines that modulate immunity in response to infection or other danger signals. In addition to their antiviral functions, type I interferons (IFNα and IFNß) are important in the pathogenesis of autoimmune diseases. Type III interferons (IFNλs) were initially described as a specialized system that inhibits viral replication at epithelial barrier surfaces while limiting inflammatory damage. However, evidence now suggests that type III interferons have complex effects on both innate and adaptive immune responses and might also be pathogenic in systemic autoimmune diseases. Concentrations of IFNλs are increased in blood and tissues in a number of autoimmune rheumatic diseases, including systemic lupus erythematosus, and are further associated with specific clinical and laboratory parameters. This Review is aimed at providing a critical evaluation of the current literature on IFNλ biology and how type III interferons might contribute to immune dysregulation and tissue damage in autoimmunity. The potential effects of type III interferons on treatment strategies for autoimmune rheumatic diseases, such as interferon blockade, are also considered.
Asunto(s)
Inflamación/inmunología , Interferones/inmunología , Enfermedades Reumáticas/inmunología , Animales , Antivirales/inmunología , Antivirales/farmacología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/fisiopatología , Ensayos Clínicos como Asunto , Citocinas , Humanos , Inmunidad/inmunología , Inmunidad Innata/inmunología , Inflamación/tratamiento farmacológico , Interferón Tipo I/inmunología , Interferones/farmacología , Lupus Eritematoso Sistémico/inmunología , Ratones , Enfermedades Reumáticas/metabolismo , Enfermedades Reumáticas/patología , Transducción de Señal/inmunología , Interferón lambdaRESUMEN
Despite the promising clinical benefit of targeted and immune checkpoint blocking therapeutics, current strategies have limited success in breast cancer, indicating that additional inhibitory pathways are required to complement existing therapeutics. TAM receptors (Tyro-3, Axl, and Mertk) are often correlated with poor prognosis because of their capacities to sustain an immunosuppressive environment. Here, we ablate Axl on tumor cells using CRISPR/Cas9 gene editing, and by targeting Mertk in the tumor microenvironment (TME), we observed distinct functions of TAM as oncogenic kinases, as well as inhibitory immune receptors. Depletion of Axl suppressed cell intrinsic oncogenic properties, decreased tumor growth, reduced the incidence of lung metastasis and increased overall survival of mice when injected into mammary fat pad of syngeneic mice, and demonstrated synergy when combined with anti-PD-1 therapy. Blockade of Mertk function on macrophages decreased efferocytosis, altered the cytokine milieu, and resulted in suppressed macrophage gene expression patterns. Mertk-knockout mice or treatment with anti-Mertk-neutralizing mAb also altered the cellular immune profile, resulting in a more inflamed tumor environment with enhanced T-cell infiltration into tumors and T-cell-mediated cytotoxicity. The antitumor activity from Mertk inhibition was abrogated by depletion of cytotoxic CD8α T cells by using anti-CD8α mAb or by transplantation of tumor cells into B6.CB17-Prkdc SCID mice. Our data indicate that targeting Axl expressed on tumor cells and Mertk in the TME is predicted to have a combinatorial benefit to enhance current immunotherapies and that Axl and Mertk have distinct functional activities that impair host antitumor response. SIGNIFICANCE: This study demonstrates how TAM receptors act both as oncogenic tyrosine kinases and as receptors that mediate immune evasion in cancer progression.
Asunto(s)
Evasión Inmune/inmunología , Neoplasias Mamarias Experimentales/inmunología , Proteínas Proto-Oncogénicas/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Transducción de Señal/inmunología , Tirosina Quinasa c-Mer/inmunología , Animales , Línea Celular Tumoral , Células Cultivadas , Femenino , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Evasión Inmune/genética , Inmunoterapia/métodos , Estimación de Kaplan-Meier , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/terapia , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/genética , Tirosina Quinasa c-Mer/genética , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa del Receptor AxlRESUMEN
With the first reports on coronavirus disease 2019 (COVID-19), which is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the scientific community working in the field of type III IFNs (IFN-λ) realized that this class of IFNs could play an important role in this and other emerging viral infections. In this Viewpoint, we present our opinion on the benefits and potential limitations of using IFN-λ to prevent, limit, and treat these dangerous viral infections.
Asunto(s)
Betacoronavirus/fisiología , Infecciones por Coronavirus/metabolismo , Interferones/metabolismo , Neumonía Viral/metabolismo , COVID-19 , Humanos , Pandemias , SARS-CoV-2 , Internalización del VirusRESUMEN
Type III IFN lambdas (IFN-λ) have recently been described as important mediators of immune responses at barrier surfaces. However, their role in autoimmune diseases such as systemic lupus erythematosus (SLE), a condition characterized by aberrant type I IFN signaling, has not been determined. Here, we identify a nonredundant role for IFN-λ in immune dysregulation and tissue inflammation in a model of TLR7-induced lupus. IFN-λ protein is increased in murine lupus and IFN-λ receptor (Ifnlr1) deficiency significantly reduces immune cell activation and associated organ damage in the skin and kidneys without effects on autoantibody production. Single-cell RNA sequencing in mouse spleen and human peripheral blood revealed that only mouse neutrophils and human B cells are directly responsive to this cytokine. Rather, IFN-λ activates keratinocytes and mesangial cells to produce chemokines that induce immune cell recruitment and promote tissue inflammation. These data provide insights into the immunobiology of SLE and identify type III IFNs as important factors for tissue-specific pathology in this disease.
Asunto(s)
Interferones/fisiología , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Animales , Linfocitos B/inmunología , Línea Celular , Eliminación de Gen , Humanos , Imiquimod/farmacología , Inflamación/inmunología , Inflamación/patología , Inductores de Interferón/farmacología , Interferón Tipo I/fisiología , Interferones/farmacología , Queratinocitos/efectos de los fármacos , Queratinocitos/inmunología , Queratinocitos/patología , Células Mesangiales/efectos de los fármacos , Células Mesangiales/inmunología , Células Mesangiales/patología , Ratones Endogámicos C57BL , Ratones Mutantes , Receptores de Interferón/genética , Transducción de Señal , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 7/fisiología , Interferón lambdaRESUMEN
The unexpected discovery of a novel family of antiviral mediators, type III IFNs or IFN-λs, challenged the widely accepted primacy of type I IFNs in antiviral immunity, and it is now well recognized that the IFN-λ-based antiviral system plays a major role in antiviral protection of epithelial barriers. The recent characterization of previously unknown IFN-λ-mediated activities has prompted further reassessment of the role of type I IFNs in innate and adaptive immune and inflammatory responses. Since type I and type III IFNs are co-produced in response to a variety of stimuli, it is likely that many physiological processes are simultaneously and coordinately regulated by these cytokines in pathological conditions, and likely at steady state, as baseline expression of both IFN types is maintained by microbiota. In this review, we discuss emerging differences in the production and signaling of type I and type III IFNs, and summarize results of recent studies describing the involvement of type III IFNs in anti-bacterial and anti-fungal, as well as antiviral, defenses.
Asunto(s)
Infecciones Bacterianas/inmunología , Interferón Tipo I/metabolismo , Interferones/metabolismo , Microbiota/inmunología , Micosis/inmunología , Virosis/inmunología , Animales , Humanos , Inmunidad , Inflamación , Transducción de Señal , Interferón lambdaRESUMEN
Molecular events activating the PI3K pathway are frequently detected in human tumors and the activation of PI3K signaling alters numerous cellular processes including tumor cell proliferation, survival, and motility. More recent studies have highlighted the impact of PI3K signaling on the cellular response to interferons and other immunologic processes relevant to antitumor immunity. Given the ability of IFNγ to regulate antigen processing and presentation and the pivotal role of MHC class I (MHCI) and II (MHCII) expression in T-cell-mediated antitumor immunity, we sought to determine the impact of PI3K signaling on MHCI and MHCII induction by IFNγ. We found that the induction of cell surface MHCI and MHCII molecules by IFNγ is enhanced by the clinical grade PI3K inhibitors dactolisib and pictilisib. We also found that PI3K inhibition increases STAT1 protein levels following IFNγ treatment and increases accessibility at genomic STAT1-binding motifs. Conversely, we found that pharmacologic activation of PI3K signaling can repress the induction of MHCI and MHCII molecules by IFNγ, and likewise, the loss of PTEN attenuates the induction of MHCI, MHCII, and STAT1 by IFNγ. Consistent with these in vitro studies, we found that within human head and neck squamous cell carcinomas, intratumoral regions with high phospho-AKT IHC staining had reduced MHCI IHC staining. IMPLICATIONS: Collectively, these findings demonstrate that MHC expression can be modulated by PI3K signaling and suggest that activation of PI3K signaling may promote immune escape via effects on antigen presentation.
Asunto(s)
Interferón gamma/farmacología , Fosfatidilinositol 3-Quinasa/genética , Factor de Transcripción STAT1/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Presentación de Antígeno/genética , Presentación de Antígeno/inmunología , Sitios de Unión/genética , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica/genética , Regulación Neoplásica de la Expresión Génica/inmunología , Genes MHC Clase I/genética , Genes MHC Clase I/inmunología , Genes MHC Clase II/genética , Genes MHC Clase II/inmunología , Genómica , Humanos , Interferón gamma/genética , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasa/inmunología , Unión Proteica/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/inmunología , Carcinoma de Células Escamosas de Cabeza y Cuello/patologíaRESUMEN
Tyro3, Axl, and Mertk (TAM) represent a family of homologous tyrosine kinase receptors known for their functional role in phosphatidylserine (PS)-dependent clearance of apoptotic cells and also for their immune modulatory functions in the resolution of inflammation. Previous studies in our laboratory have shown that Gas6/PS-mediated activation of TAM receptors on tumor cells leads to subsequent upregulation of PD-L1, defining a putative PSâTAM receptorâPD-L1 inhibitory signaling axis in the cancer microenvironment that may promote tolerance. In this study, we tested combinations of TAM inhibitors and PD-1 mAbs in a syngeneic orthotopic E0771 murine triple-negative breast cancer model, whereby tumor-bearing mice were treated with pan-TAM kinase inhibitor (BMS-777607) or anti-PD-1 alone or in combination. Tyro3, Axl, and Mertk were differentially expressed on multiple cell subtypes in the tumor microenvironment. Although monotherapeutic administration of either pan-TAM kinase inhibitor (BMS-777607) or anti-PD-1 mAb therapy showed partial antitumor activity, combined treatment of BMS-777607 with anti-PD-1 significantly decreased tumor growth and incidence of lung metastasis. Moreover, combined treatment with BMS-777607 and anti-PD-1 showed increased infiltration of immune stimulatory T cells versus either monotherapy treatment alone. RNA NanoString profiling showed enhanced infiltration of antitumor effector T cells and a skewed immunogenic immune profile. Proinflammatory cytokines increased with combinational treatment. Together, these studies indicate that pan-TAM inhibitor BMS-777607 cooperates with anti-PD-1 in a syngeneic mouse model for triple-negative breast cancer and highlights the clinical potential for this combined therapy. SIGNIFICANCE: These findings show that pan-inhibition of TAM receptors in combination with anti-PD-1 may have clinical value as cancer therapeutics to promote an inflammatory tumor microenvironment and improve host antitumor immunity.
Asunto(s)
Aminopiridinas/farmacología , Anticuerpos Monoclonales/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Piridonas/farmacología , Neoplasias de la Mama Triple Negativas/terapia , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos C57BL , Neoplasias de la Mama Triple Negativas/inmunología , Microambiente Tumoral/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cells of all mammals recognize double-stranded RNA (dsRNA) as a foreign material. In response, they release interferons (IFNs) and activate a ubiquitously expressed pseudokinase/endoribonuclease RNase L. RNase L executes regulated RNA decay and halts global translation. Here, we developed a biosensor for 2',5'-oligoadenylate (2-5A), the natural activator of RNase L. Using this biosensor, we found that 2-5A was acutely synthesized by cells in response to dsRNA sensing, which immediately triggered cellular RNA cleavage by RNase L and arrested host protein synthesis. However, translation-arrested cells still transcribed IFN-stimulated genes and secreted IFNs of types I and III (IFN-ß and IFN-λ). Our data suggest that IFNs escape from the action of RNase L on translation. We propose that the 2-5A/RNase L pathway serves to rapidly and accurately suppress basal protein synthesis, preserving privileged production of defense proteins of the innate immune system.
Asunto(s)
Técnicas Biosensibles , Endorribonucleasas/química , Interferón beta/química , Interferones/química , Biosíntesis de Proteínas , Línea Celular , Endorribonucleasas/metabolismo , Humanos , Interferón beta/metabolismo , Interferones/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-ActividadRESUMEN
Crystal structure of the ternary complex of human IL-24 with two receptors, IL-22R1 and IL-20R2, has been determined at 2.15 Å resolution. A crystallizable complex was created by a novel approach involving fusing the ligand with a flexible linker to the presumed low-affinity receptor, and coexpression of this construct in Drosophila S2 cells together with the presumed high-affinity receptor. This approach, which may be generally applicable to other multiprotein complexes with low-affinity components, was necessitated by the instability of IL-24 expressed by itself in either bacteria or insect cells. Although IL-24 expressed in Escherichia coli was unstable and precipitated almost immediately upon its refolding and purification, a small fraction of IL-24 remaining in the folded state was shown to be active in a cell-based assay. In the crystal structure presented here, we found that two cysteine residues in IL-24 do not form a predicted disulfide bond. Lack of structural restraint by disulfides, present in other related cytokines, is most likely reason for the low stability of IL-24. Although the contact area between IL-24 and IL-22R1 is larger than between the cytokine and IL-20R2, calculations show the latter interaction to be slightly more stable, suggesting that the shared receptor (IL-20R2) might be the higher-affinity receptor.
Asunto(s)
Interleucinas/metabolismo , Complejos Multiproteicos/metabolismo , Receptores de Interleucina/metabolismo , Animales , Línea Celular , Cristalografía por Rayos X , Citocinas , Drosophila , Humanos , Unión Proteica , Conformación Proteica , Dominios Proteicos/genética , Receptores de Interleucina/genética , Transducción de SeñalRESUMEN
The Tyro3, Axl, and Mertk (TAM) receptors are homologous type I receptor tyrosine kinases that have critical functions in the clearance of apoptotic cells in multicellular organisms. TAMs are activated by their endogenous ligands, growth arrest-specific 6 (Gas6), and protein S (Pros1), that function as bridging molecules between externalized phosphatidylserine (PS) on apoptotic cells and the TAM ectodomains. However, the molecular mechanisms by which Gas6/Pros1 promote TAM activation remains elusive. Using TAM/IFNγR1 reporter cell lines to monitor functional TAM activity, we found that Gas6 activity was exquisitely dependent on vitamin K-mediated γ-carboxylation, whereby replacing vitamin K with anticoagulant warfarin, or by substituting glutamic acid residues involved in PS binding, completely abrogated Gas6 activity as a TAM ligand. Furthermore, using domain and point mutagenesis, Gas6 activity also required both an intact Gla domain and intact EGF-like domains, suggesting these domains function cooperatively in order to achieve TAM activation. Despite the requirement of γ-carboxylation and the functional Gla domain, non-γ-carboxylated Gas6 and Gla deletion/EGF-like domain deletion mutants still retained their ability to bind TAMs and acted as blocking decoy ligands. Finally, we found that distinct sources of PS-positive cells/vesicles (including apoptotic cells, calcium-induced stressed cells, and exosomes) bound Gas6 and acted as cell-derived or exosome-derived ligands to activate TAMs. Taken together, our findings indicate that PS is indispensable for TAM activation by Gas6, and by inference, provides new perspectives on how PS, regulates TAM receptors and efferocytosis.