RESUMEN
While CRISPR screens are helping uncover genes regulating many cell-intrinsic processes, existing approaches are suboptimal for identifying extracellular gene functions, particularly in the tissue context. Here, we developed an approach for spatial functional genomics called Perturb-map. We applied Perturb-map to knock out dozens of genes in parallel in a mouse model of lung cancer and simultaneously assessed how each knockout influenced tumor growth, histopathology, and immune composition. Moreover, we paired Perturb-map and spatial transcriptomics for unbiased analysis of CRISPR-edited tumors. We found that in Tgfbr2 knockout tumors, the tumor microenvironment (TME) was converted to a fibro-mucinous state, and T cells excluded, concomitant with upregulated TGFß and TGFß-mediated fibroblast activation, indicating that TGFß-receptor loss on cancer cells increased TGFß bioavailability and its immunosuppressive effects on the TME. These studies establish Perturb-map for functional genomics within the tissue at single-cell resolution with spatial architecture preserved and provide insight into how TGFß responsiveness of cancer cells can affect the TME.
Asunto(s)
Neoplasias , Microambiente Tumoral , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Genómica , Ratones , Neoplasias/genética , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Cytokine expression during T cell differentiation is a highly regulated process that involves long-range promoter-enhancer and CTCF-CTCF contacts at cytokine loci. Here, we investigated the impact of dynamic chromatin loop formation within the topologically associating domain (TAD) in regulating the expression of interferon gamma (IFN-γ) and interleukin-22 (IL-22); these cytokine loci are closely located in the genome and are associated with complex enhancer landscapes, which are selectively active in type 1 and type 3 lymphocytes. In situ Hi-C analyses revealed inducible TADs that insulated Ifng and Il22 enhancers during Th1 cell differentiation. Targeted deletion of a 17 bp boundary motif of these TADs imbalanced Th1- and Th17-associated immunity, both in vitro and in vivo, upon Toxoplasma gondii infection. In contrast, this boundary element was dispensable for cytokine regulation in natural killer cells. Our findings suggest that precise cytokine regulation relies on lineage- and developmental stage-specific interactions of 3D chromatin architectures and enhancer landscapes.
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Factor de Unión a CCCTC , Diferenciación Celular , Interferón gamma , Interleucina-22 , Interleucinas , Células TH1 , Animales , Factor de Unión a CCCTC/metabolismo , Factor de Unión a CCCTC/genética , Células TH1/inmunología , Ratones , Diferenciación Celular/inmunología , Interferón gamma/metabolismo , Sitios de Unión , Interleucinas/metabolismo , Interleucinas/genética , Elementos de Facilitación Genéticos/genética , Ratones Endogámicos C57BL , Cromatina/metabolismo , Toxoplasmosis/inmunología , Toxoplasmosis/parasitología , Toxoplasmosis/genética , Regulación de la Expresión Génica , Toxoplasma/inmunología , Citocinas/metabolismo , Linaje de la Célula , Células Th17/inmunologíaRESUMEN
CRISPR pools are being widely employed to identify gene functions. However, current technology, which utilizes DNA as barcodes, permits limited phenotyping and bulk-cell resolution. To enable novel screening capabilities, we developed a barcoding system operating at the protein level. We synthesized modules encoding triplet combinations of linear epitopes to generate >100 unique protein barcodes (Pro-Codes). Pro-Code-expressing vectors were introduced into cells and analyzed by CyTOF mass cytometry. Using just 14 antibodies, we detected 364 Pro-Code populations; establishing the largest set of protein-based reporters. By pairing each Pro-Code with a different CRISPR, we simultaneously analyzed multiple phenotypic markers, including phospho-signaling, on dozens of knockouts. Pro-Code/CRISPR screens found two interferon-stimulated genes, the immunoproteasome component Psmb8 and a chaperone Rtp4, are important for antigen-dependent immune editing of cancer cells and identified Socs1 as a negative regulator of Pd-l1. The Pro-Code technology enables simultaneous high-dimensional protein-level phenotyping of 100s of genes with single-cell resolution.
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Sistemas CRISPR-Cas , Citometría de Flujo/métodos , Genómica/métodos , Espectrometría de Masas/métodos , Análisis de la Célula Individual/métodos , Animales , Epítopos/química , Epítopos/clasificación , Epítopos/genética , Células HEK293 , Humanos , Inmunofenotipificación/métodos , Células Jurkat , Ratones Endogámicos BALB C , Proteoma/química , Proteoma/clasificación , Proteoma/genética , Células THP-1RESUMEN
Aberrant tissue-immune interactions are the hallmark of diverse chronic lung diseases. Here, we sought to define these interactions in emphysema, a progressive disease characterized by infectious exacerbations and loss of alveolar epithelium. Single-cell analysis of human emphysema lungs revealed the expansion of tissue-resident lymphocytes (TRLs). Murine studies identified a stromal niche for TRLs that expresses Hhip, a disease-variant gene downregulated in emphysema. Stromal-specific deletion of Hhip induced the topographic expansion of TRLs in the lung that was mediated by a hyperactive hedgehog-IL-7 axis. 3D immune-stem cell organoids and animal models of viral exacerbations demonstrated that expanded TRLs suppressed alveolar stem cell growth through interferon gamma (IFNγ). Finally, we uncovered an IFNγ-sensitive subset of human alveolar stem cells that was preferentially lost in emphysema. Thus, we delineate a stromal-lymphocyte-epithelial stem cell axis in the lung that is modified by a disease-variant gene and confers host susceptibility to emphysema.
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Enfisema , Enfermedad Pulmonar Obstructiva Crónica , Enfisema Pulmonar , Humanos , Ratones , Animales , Enfisema Pulmonar/genética , Pulmón , Linfocitos , Células MadreRESUMEN
Local environmental factors influence CD8+ T cell priming in lymph nodes (LNs). Here, we sought to understand how factors unique to the tumor-draining mediastinal LN (mLN) impact CD8+ T cell responses toward lung cancer. Type 1 conventional dendritic cells (DC1s) showed a mLN-specific failure to induce robust cytotoxic T cells responses. Using regulatory T (Treg) cell depletion strategies, we found that Treg cells suppressed DC1s in a spatially coordinated manner within tissue-specific microniches within the mLN. Treg cell suppression required MHC II-dependent contact between DC1s and Treg cells. Elevated levels of IFN-γ drove differentiation Treg cells into Th1-like effector Treg cells in the mLN. In patients with cancer, Treg cell Th1 polarization, but not CD8+/Treg cell ratios, correlated with poor responses to checkpoint blockade immunotherapy. Thus, IFN-γ in the mLN skews Treg cells to be Th1-like effector Treg cells, driving their close interaction with DC1s and subsequent suppression of cytotoxic T cell responses.
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Neoplasias Pulmonares , Linfocitos T Reguladores , Humanos , Linfocitos T CD8-positivos , Interferón gamma , Linfocitos T CitotóxicosRESUMEN
Here we report a phase 1b clinical trial testing the impact of oncolytic virotherapy with talimogene laherparepvec on cytotoxic T cell infiltration and therapeutic efficacy of the anti-PD-1 antibody pembrolizumab. Twenty-one patients with advanced melanoma were treated with talimogene laherparepvec followed by combination therapy with pembrolizumab. Therapy was generally well tolerated, with fatigue, fevers, and chills as the most common adverse events. No dose-limiting toxicities occurred. Confirmed objective response rate was 62%, with a complete response rate of 33% per immune-related response criteria. Patients who responded to combination therapy had increased CD8+ T cells, elevated PD-L1 protein expression, as well as IFN-γ gene expression on several cell subsets in tumors after talimogene laherparepvec treatment. Response to combination therapy did not appear to be associated with baseline CD8+ T cell infiltration or baseline IFN-γ signature. These findings suggest that oncolytic virotherapy may improve the efficacy of anti-PD-1 therapy by changing the tumor microenvironment. VIDEO ABSTRACT.
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Anticuerpos Monoclonales Humanizados/administración & dosificación , Melanoma/terapia , Viroterapia Oncolítica/efectos adversos , Terapia Combinada , Herpesviridae/genética , Humanos , Inmunoterapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Microambiente TumoralRESUMEN
Allergic immunity is orchestrated by group 2 innate lymphoid cells (ILC2s) and type 2 helper T (Th2) cells prominently arrayed at epithelial- and microbial-rich barriers. However, ILC2s and Th2 cells are also present in fibroblast-rich niches within the adventitial layer of larger vessels and similar boundary structures in sterile deep tissues, and it remains unclear whether they undergo dynamic repositioning during immune perturbations. Here, we used thick-section quantitative imaging to show that allergic inflammation drives invasion of lung and liver non-adventitial parenchyma by ILC2s and Th2 cells. However, during concurrent type 1 and type 2 mixed inflammation, IFNγ from broadly distributed type 1 lymphocytes directly blocked both ILC2 parenchymal trafficking and subsequent cell survival. ILC2 and Th2 cell confinement to adventitia limited mortality by the type 1 pathogen Listeria monocytogenes. Our results suggest that the topography of tissue lymphocyte subsets is tightly regulated to promote appropriately timed and balanced immunity.
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Inflamación/inmunología , Interferón gamma/inmunología , Subgrupos Linfocitarios/inmunología , Células Th2/inmunología , Animales , Muerte Celular/inmunología , Movimiento Celular/inmunología , Hipersensibilidad/inmunología , Inmunidad Innata , Interleucina-33/inmunología , Interleucina-5/metabolismo , Listeria monocytogenes , Listeriosis/inmunología , Listeriosis/mortalidad , Hígado/inmunología , Pulmón/inmunología , Subgrupos Linfocitarios/metabolismo , Lisofosfolípidos/inmunología , Ratones , Tejido Parenquimatoso/inmunología , Esfingosina/análogos & derivados , Esfingosina/inmunología , Células TH1/inmunología , Células Th2/metabolismoRESUMEN
Type I and II interferons (IFNs) stimulate pro-inflammatory programs that are critical for immune activation, but also induce immune-suppressive feedback circuits that impede control of cancer growth. Here, we sought to determine how these opposing programs are differentially induced. We demonstrated that the transcription factor interferon regulatory factor 2 (IRF2) was expressed by many immune cells in the tumor in response to sustained IFN signaling. CD8+ T cell-specific deletion of IRF2 prevented acquisition of the T cell exhaustion program within the tumor and instead enabled sustained effector functions that promoted long-term tumor control and increased responsiveness to immune checkpoint and adoptive cell therapies. The long-term tumor control by IRF2-deficient CD8+ T cells required continuous integration of both IFN-I and IFN-II signals. Thus, IRF2 is a foundational feedback molecule that redirects IFN signals to suppress T cell responses and represents a potential target to enhance cancer control.
Asunto(s)
Interferón Tipo I , Neoplasias , Humanos , Factor 2 Regulador del Interferón/genética , Linfocitos T CD8-positivos , Factores de Transcripción , Agotamiento de Células T , Neoplasias/patologíaRESUMEN
Inflammation can support or restrain cancer progression and the response to therapy. Here, we searched for primary regulators of cancer-inhibitory inflammation through deep profiling of inflammatory tumor microenvironments (TMEs) linked to immune-dependent control in mice. We found that early intratumoral accumulation of interferon gamma (IFN-γ)-producing natural killer (NK) cells induced a profound remodeling of the TME and unleashed cytotoxic T cell (CTL)-mediated tumor eradication. Mechanistically, tumor-derived prostaglandin E2 (PGE2) acted selectively on EP2 and EP4 receptors on NK cells, hampered the TME switch, and enabled immune evasion. Analysis of patient datasets across human cancers revealed distinct inflammatory TME phenotypes resembling those associated with cancer immune control versus escape in mice. This allowed us to generate a gene-expression signature that integrated opposing inflammatory factors and predicted patient survival and response to immune checkpoint blockade. Our findings identify features of the tumor inflammatory milieu associated with immune control of cancer and establish a strategy to predict immunotherapy outcomes.
Asunto(s)
Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inflamación/inmunología , Neoplasias/inmunología , Escape del Tumor/inmunología , Animales , Dinoprostona/metabolismo , Humanos , Inmunoterapia , Inflamación/genética , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Ratones , Neoplasias/terapia , Fenotipo , Pronóstico , Prostaglandina-Endoperóxido Sintasas/genética , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Linfocitos T Citotóxicos/inmunología , Microambiente Tumoral/inmunologíaRESUMEN
Graft-versus-host disease (GVHD) in the gastrointestinal (GI) tract is the principal determinant of lethality following allogeneic bone marrow transplantation (BMT). Here, we examined the mechanisms that initiate GVHD, including the relevant antigen-presenting cells. MHC class II was expressed on intestinal epithelial cells (IECs) within the ileum at steady state but was absent from the IECs of germ-free mice. IEC-specific deletion of MHC class II prevented the initiation of lethal GVHD in the GI tract. MHC class II expression on IECs was absent from mice deficient in the TLR adaptors MyD88 and TRIF and required IFNγ secretion by lamina propria lymphocytes. IFNγ responses are characteristically driven by IL-12 secretion from myeloid cells. Antibiotic-mediated depletion of the microbiota inhibited IL-12/23p40 production by ileal macrophages. IL-12/23p40 neutralization prevented MHC class II upregulation on IECs and initiation of lethal GVHD in the GI tract. Thus, MHC class II expression by IECs in the ileum initiates lethal GVHD, and blockade of IL-12/23p40 may represent a readily translatable therapeutic strategy.
Asunto(s)
Presentación de Antígeno/inmunología , Células Presentadoras de Antígenos/inmunología , Microbioma Gastrointestinal/inmunología , Enfermedad Injerto contra Huésped/etiología , Antígenos de Histocompatibilidad Clase II/inmunología , Mucosa Intestinal/inmunología , Animales , Células Presentadoras de Antígenos/metabolismo , Biomarcadores , Citocinas/metabolismo , Susceptibilidad a Enfermedades , Femenino , Expresión Génica , Enfermedad Injerto contra Huésped/mortalidad , Antígenos de Histocompatibilidad Clase II/genética , Íleon/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Estimación de Kaplan-Meier , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Masculino , Ratones , Ratones Transgénicos , Pronóstico , Regiones Promotoras Genéticas , Transducción de SeñalRESUMEN
Hemophagocytic lymphohistiocytosis (HLH) is a disorder that has been recognized since the middle of the last century. In recent decades, increasing understanding of the genetic roots and pathophysiology of HLH has led to improved diagnosis and treatment of this once universally fatal disorder. HLH is best conceptualized as a maladaptive state of excessive T cell activation driving life-threatening myeloid cell activation, largely via interferon-gamma (IFN-γ). In familial forms of HLH (F-HLH), inherited defects of lymphocyte cytotoxic biology underlie excessive T cell activation, demonstrating the importance of the perforin/granzyme pathway as a negative feedback loop limiting acute T cell activation in response to environmental factors. HLH occurring in other contexts and without apparent inherited genetic predisposition remains poorly understood, though it may share some downstream aspects of pathophysiology including excessive IFN-γ action and activation of innate immune effectors. Iatrogenic forms of HLH occurring after immune-activating therapies for cancer are providing new insights into the potential toxicities of inadequately controlled T cell activation. Diagnosing HLH increasingly relies on context-specific measures of T cell activation, IFN-γ activity, and inflammation. Treatment of HLH largely relies on cytotoxic chemotherapy, though targeted therapies against T cells, IFN-γ, and other cytokines are increasingly utilized.
Asunto(s)
Linfohistiocitosis Hemofagocítica , Humanos , Linfohistiocitosis Hemofagocítica/diagnóstico , Linfohistiocitosis Hemofagocítica/genética , Linfohistiocitosis Hemofagocítica/terapia , Linfocitos T/metabolismo , Citocinas , Interferón gamma/metabolismo , InflamaciónRESUMEN
Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs), which is a highly heterogeneous process. Here we report the cell fate continuum during somatic cell reprogramming at single-cell resolution. We first develop SOT to analyze cell fate continuum from Oct4/Sox2/Klf4- or OSK-mediated reprogramming and show that cells bifurcate into two categories, reprogramming potential (RP) or non-reprogramming (NR). We further show that Klf4 contributes to Cd34+/Fxyd5+/Psca+ keratinocyte-like NR fate and that IFN-γ impedes the final transition to chimera-competent pluripotency along the RP cells. We analyze more than 150,000 single cells from both OSK and chemical reprograming and identify additional NR/RP bifurcation points. Our work reveals a generic bifurcation model for cell fate decisions during somatic cell reprogramming that may be applicable to other systems and inspire further improvements for reprogramming.
Asunto(s)
Diferenciación Celular/genética , Linaje de la Célula/genética , Técnicas de Reprogramación Celular , Reprogramación Celular/genética , Células Madre Pluripotentes Inducidas/fisiología , Células Madre Embrionarias de Ratones/fisiología , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica , Células Madre Pluripotentes Inducidas/metabolismo , Interferón gamma/genética , Interferón gamma/metabolismo , Factor 4 Similar a Kruppel , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Células Madre Embrionarias de Ratones/metabolismo , Fenotipo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We developed a highly sensitive assay for detecting protein-protein interaction using chimeric receptors comprising two molecules of interest in the extracellular domain and interferon alpha and beta receptor subunit 1 or 2 (IFNAR1/2) in the intracellular domain. This intracellular IFNAR1/2 reconstitution system (IFNARRS) proved markedly more sensitive than the NanoBiT system, currently considered one of the best detection systems for protein interaction. Employing chimeric receptors with extracellular domains from the IFNγ or IL-2 receptor and the intracellular domains of IFNAR1/2, the IFNARRS system effectively identifies low IFNγ or IL-2 levels. Cells stably expressing these chimeric receptors responded to IFNγ secreted by activated T cells following various stimuli, including a specific peptide-antigen. The activation signals were further enhanced by the expression of relevant genes, such as costimulators, via IFN-stimulated response elements in the promoters. Besides IFNγ or IL-2, the IFNARRS system demonstrated the capability to detect other cytokines by using the corresponding extracellular domains from these target cytokine receptors.
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Interferón gamma , Interleucina-2 , Receptor de Interferón alfa y beta , Linfocitos T , Humanos , Receptor de Interferón alfa y beta/metabolismo , Receptor de Interferón alfa y beta/genética , Linfocitos T/metabolismo , Linfocitos T/inmunología , Interleucina-2/metabolismo , Interferón gamma/metabolismo , Receptores de Interleucina-2/metabolismo , Receptores de Interleucina-2/genética , Unión Proteica , Activación de Linfocitos , Células HEK293RESUMEN
Mechanisms by which interferon (IFN)-γ activates genes to promote macrophage activation are well studied, but little is known about mechanisms and functions of IFN-γ-mediated gene repression. We used an integrated transcriptomic and epigenomic approach to analyze chromatin accessibility, histone modifications, transcription-factor binding, and gene expression in IFN-γ-primed human macrophages. IFN-γ suppressed basal expression of genes corresponding to an "M2"-like homeostatic and reparative phenotype. IFN-γ repressed genes by suppressing the function of enhancers enriched for binding by transcription factor MAF. Mechanistically, IFN-γ disassembled a subset of enhancers by inducing coordinate suppression of binding by MAF, lineage-determining transcription factors, and chromatin accessibility. Genes associated with MAF-binding enhancers were suppressed in macrophages isolated from rheumatoid-arthritis patients, revealing a disease-associated signature of IFN-γ-mediated repression. These results identify enhancer inactivation and disassembly as a mechanism of IFN-γ-mediated gene repression and reveal that MAF regulates the macrophage enhancer landscape and is suppressed by IFN-γ to augment macrophage activation.
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Artritis Reumatoide/inmunología , Ensamble y Desensamble de Cromatina , Interferón gamma/metabolismo , Macrófagos/inmunología , Proteínas Proto-Oncogénicas c-maf/metabolismo , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Citocinas/metabolismo , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica , Histonas/metabolismo , Humanos , Unión Proteica , Proteínas Proto-Oncogénicas c-maf/genética , TranscriptomaRESUMEN
Host defense requires the specification of CD4+ helper T (Th) cells into distinct fates, including Th1 cells that preferentially produce interferon-γ (IFN-γ). IFN-γ, a member of a large family of anti-pathogenic and anti-tumor IFNs, induces T-bet, a lineage-defining transcription factor for Th1 cells, which in turn supports IFN-γ production in a feed-forward manner. Herein, we show that a cell-intrinsic role of T-bet influences how T cells perceive their secreted product in the environment. In the absence of T-bet, IFN-γ aberrantly induced a type I IFN transcriptomic program. T-bet preferentially repressed genes and pathways ordinarily activated by type I IFNs to ensure that its transcriptional response did not evoke an aberrant amplification of type I IFN signaling circuitry, otherwise triggered by its own product. Thus, in addition to promoting Th1 effector commitment, T-bet acts as a repressor in differentiated Th1 cells to prevent abberant autocrine type I IFN and downstream signaling.
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Comunicación Autocrina , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Proteínas de Dominio T Box/metabolismo , Células TH1/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Humanos , Interferón Tipo I/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Proteínas de Dominio T Box/genética , Células TH1/microbiología , Células TH1/virología , TranscriptomaRESUMEN
The IFN-γ/STAT1 immune signaling pathway impacts many homeostatic and pathological aspects of neurons, beyond its canonical role in controlling intracellular pathogens. Well known for its potent pro-inflammatory and anti-viral functions in the periphery, the IFN-γ/STAT1 pathway is rapidly activated then deactivated to prevent excessive inflammation; however, neurons utilize unique IFN-γ/STAT1 activation patterns, which may contribute to the non-canonical neuron-specific downstream effects. Though it is now well-established that the immune system interacts and supports the CNS in health and disease, many aspects regarding IFN-γ production in the CNS and how neurons respond to IFN-γ are unclear. Additionally, it is not well understood how the diversity of the IFN-γ/STAT1 pathway is regulated in neurons to control homeostatic functions, support immune surveillance, and prevent pathologies. In this review, we discuss the neuron-specific mechanisms and kinetics of IFN-γ/STAT1 activation, the potential sources and entry sites of IFN-γ in the CNS, and the diverse set of homeostatic and pathological effects IFN-γ/STAT1 signaling in neurons has on CNS health and disease. We will also highlight the different contexts and conditions under which IFN-γ-induced STAT1 activation has been studied in neurons, and how various factors might contribute to the vast array of downstream effects observed.
Asunto(s)
Neuronas , Transducción de Señal , Humanos , Interferón gamma/metabolismo , Neuronas/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/farmacologíaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder marked by progressive motor neuron degeneration and muscle denervation. A recent transcriptomic study integrating a wide range of human ALS samples revealed that the upregulation of p53, a downstream target of inflammatory stress, is commonly detected in familial and sporadic ALS cases by a mechanism linked to a transactive response DNA-binding protein 43 (TDP-43) dysfunction. In this study, we show that prolonged interferon-gamma (IFNγ) treatment of human induced pluripotent stem cell-derived spinal motor neurons results in a severe cytoplasmic aggregation of TDP-43. TDP-43 dysfunction resulting from either IFNγ exposure or an ALS-associated TDP-43 mutation was associated with the activation of the p53 pathway. This was accompanied by the hyperactivation of neuronal firing, followed by the complete loss of their electrophysiological function. Through a comparative single-cell transcriptome analysis, we have identified significant alterations in ALS-associated genes in motor neurons exposed to IFNγ, implicating their direct involvement in ALS pathology. Interestingly, IFNγ was found to induce significant levels of programmed death-ligand 1 (PD-L1) expression in motor neurons without affecting the levels of any other immune checkpoint proteins. This finding suggests a potential role of excessive PD-L1 expression in ALS development, given that PD-L1 was recently reported to impair neuronal firing ability in mice. Our findings suggest that exposing motor neurons to IFNγ could directly derive ALS pathogenesis, even without the presence of the inherent genetic mutation or functional glia component. Furthermore, this study provides a comprehensive list of potential candidate genes for future immunotherapeutic targets with which to treat sporadic forms of ALS, which account for 90% of all reported cases.
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Esclerosis Amiotrófica Lateral , Células Madre Pluripotentes Inducidas , Animales , Humanos , Ratones , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Antígeno B7-H1/metabolismo , Biomarcadores , Proteínas de Unión al ADN/genética , Células Madre Pluripotentes Inducidas/metabolismo , Interferón gamma/metabolismo , Interferón gamma/farmacología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
OBJECTIVES: In the post-SARS-CoV-2 pandemic era, "breakthrough infections" are still documented, due to variants of concerns (VoCs) emergence and waning humoral immunity. Despite widespread utilization, the definition of the anti-Spike (S) immunoglobulin-G (IgG) threshold to define protection has unveiled several limitations. Here, we explore the advantages of incorporating T-cell response assessment to enhance the definition of immune memory profile. METHODS: SARS-CoV-2 interferon-gamma release assay test (IGRA) was performed on samples collected longitudinally from immunocompetent healthcare workers throughout their immunization by infection and/or vaccination, anti-receptor-binding domain IgG levels were assessed in parallel. The risk of symptomatic infection according to cellular/humoral immune capacities during Omicron BA.1 wave was then estimated. RESULTS: Close to 40% of our samples were exclusively IGRA-positive, largely due to time elapsed since their last immunization. This suggests that individuals have sustained long-lasting cellular immunity, while they would have been classified as lacking protective immunity based solely on IgG threshold. Moreover, the Cox regression model highlighted that Omicron BA.1 circulation raises the risk of symptomatic infection while increased anti-receptor-binding domain IgG and IGRA levels tended to reduce it. CONCLUSION: The discrepancy between humoral and cellular responses highlights the significance of assessing the overall adaptive immune response. This integrated approach allows the identification of vulnerable subjects and can be of interest to guide antiviral prophylaxis at an individual level.
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Anticuerpos Antivirales , COVID-19 , Inmunidad Humoral , Inmunoglobulina G , Memoria Inmunológica , Ensayos de Liberación de Interferón gamma , SARS-CoV-2 , Humanos , COVID-19/inmunología , SARS-CoV-2/inmunología , Memoria Inmunológica/inmunología , Inmunidad Humoral/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina G/sangre , Masculino , Femenino , Adulto , Persona de Mediana Edad , Ensayos de Liberación de Interferón gamma/métodos , Glicoproteína de la Espiga del Coronavirus/inmunología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Linfocitos T/inmunología , Personal de Salud , Vacunas contra la COVID-19/inmunologíaRESUMEN
CD4+ T cells play a key role in γ-herpesvirus infection control. However, the mechanisms involved are unclear. Murine herpesvirus type 4 (MuHV-4) allows relevant immune pathways to be dissected experimentally in mice. In the lungs, it colonizes myeloid cells, which can express MHC class II (MHCII), and type 1 alveolar epithelial cells (AEC1), which lack it. Nevertheless, CD4+ T cells can control AEC1 infection, and this control depends on MHCII expression in myeloid cells. Interferon-gamma (IFNγ) is a major component of CD4+ T cell-dependent MuHV-4 control. Here, we show that the action of IFNγ is also indirect, as CD4+ T cell-mediated control of AEC1 infection depended on IFNγ receptor (IFNγR1) expression in CD11c+ cells. Indirect control also depended on natural killer (NK) cells. Together, the data suggest that the activation of MHCII+ CD11c+ antigen-presenting cells is key to the CD4+ T cell/NK cell protection axis. By contrast, CD8+ T cell control of AEC1 infection appeared to operate independently. IMPORTANCE: CD4+ T cells are critical for the control of gamma-herpesvirus infection; they act indirectly, by recruiting natural killer (NK) cells to attack infected target cells. Here, we report that the CD4+ T cell/NK cell axis of gamma-herpesvirus control requires interferon-γ engagement of CD11c+ dendritic cells. This mechanism of CD4+ T cell control releases the need for the direct engagement of CD4+ T cells with virus-infected cells and may be a common strategy for host control of immune-evasive pathogens.
Asunto(s)
Linfocitos T CD4-Positivos , Infecciones por Herpesviridae , Interferón gamma , Células Asesinas Naturales , Receptores de Interferón , Rhadinovirus , Animales , Linfocitos T CD4-Positivos/inmunología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Ratones , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Células Asesinas Naturales/inmunología , Receptores de Interferón/genética , Receptores de Interferón/metabolismo , Rhadinovirus/inmunología , Ratones Endogámicos C57BL , Receptor de Interferón gamma , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Células Epiteliales Alveolares/inmunología , Células Epiteliales Alveolares/virología , Linfocitos T CD8-positivos/inmunología , Antígeno CD11c/metabolismo , Antígeno CD11c/inmunología , Pulmón/inmunología , Pulmón/virologíaRESUMEN
The antiviral role of the tripartite motif-containing (TRIM) protein family , a member of the E3-ubiquitin ligase family, has recently been actively studied. Hepatitis B virus (HBV) infection is a major contributor to liver diseases; however, the host factors regulated by cytokine-inducible TRIM21 to suppress HBV remain unclear. In this study, we showed the antiviral efficacy of TRIM21 against HBV in hepatoma cell lines, primary human hepatocytes isolated from patient liver tissues, and mouse model. Using TRIM21 knock-out cells, we confirmed that the antiviral effects of interferon-gamma, which suppress HBV replication, are diminished when TRIM21 is deficient. Northern blot analysis confirmed a reduction of HBV RNA levels by TRIM21. Using Luciferase reporter assay, we also discovered that TRIM21 decreases the activity of HBV enhancers, which play a crucial role in covalently closed circular DNA transcription. The participation of the RING domain and PRY-SPRY domain in the anti-HBV effect of TRIM21 was demonstrated through experiments using deletion mutants. We identified a novel interaction between TRIM21 and hepatocyte nuclear factor 4α (HNF4α) through co-immunoprecipitation assay. More specifically, ubiquitination assay revealed that TRIM21 promotes ubiquitin-mediated proteasomal degradation of HNF4α. HNF1α transcription is down-regulated as a result of the degradation of HNF4α, an activator for the HNF1α promoter. Therefore, the reduction of key HBV enhancer activators, HNF4α and HNF1α, by TRIM21 resulted in a decline in HBV transcription, ultimately leading to the inhibition of HBV replication.IMPORTANCEDespite extensive research efforts, a definitive cure for chronic hepatitis B remains elusive, emphasizing the persistent importance of this viral infection as a substantial public health concern. Although the risks associated with hepatitis B virus (HBV) infection are well known, host factors capable of suppressing HBV are largely uncharacterized. This study elucidates that tripartite motif-containing protein 21 (TRIM21) suppresses HBV transcription and consequently inhibits HBV replication by downregulating the hepatocyte nuclear factors, which are host factors associated with the HBV enhancers. Our findings demonstrate a novel anti-HBV mechanism of TRIM21 in interferon-gamma-induced anti-HBV activity. These findings may contribute to new strategies to block HBV.