RESUMEN
Each of us is a story. Mine is a story of doing science for 60 years, and I am honored to be asked to tell it. Even though this autobiography was written for the Annual Review of Immunology, I have chosen to describe my whole career in science because the segment that was immunology is so intertwined with all else I was doing. This article is an elongation and modification of a talk I gave at my 80th birthday celebration at Caltech on March 23, 2018.
Asunto(s)
Alergia e Inmunología/historia , FN-kappa B/metabolismo , Virus ARN/fisiología , Virosis/inmunología , Animales , Modelos Animales de Enfermedad , Reordenamiento Génico , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Ratones , Proteínas Tirosina Quinasas/metabolismo , Transcripción Reversa , Estados UnidosRESUMEN
Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors for a wide range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed "DrugMap," an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF-κB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF-κB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription-factor activity.
Asunto(s)
Cisteína , Neoplasias , Animales , Humanos , Ratones , Línea Celular Tumoral , Cisteína/metabolismo , Cisteína/química , Ligandos , Melanoma/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , FN-kappa B/química , FN-kappa B/metabolismo , Oxidación-Reducción , Transducción de Señal , Factores de Transcripción SOXE/química , Factores de Transcripción SOXE/metabolismoRESUMEN
Ongoing, early-stage clinical trials illustrate the translational potential of human pluripotent stem cell (hPSC)-based cell therapies in Parkinson's disease (PD). However, an unresolved challenge is the extensive cell death following transplantation. Here, we performed a pooled CRISPR-Cas9 screen to enhance postmitotic dopamine neuron survival in vivo. We identified p53-mediated apoptotic cell death as a major contributor to dopamine neuron loss and uncovered a causal link of tumor necrosis factor alpha (TNF-α)-nuclear factor κB (NF-κB) signaling in limiting cell survival. As a translationally relevant strategy to purify postmitotic dopamine neurons, we identified cell surface markers that enable purification without the need for genetic reporters. Combining cell sorting and treatment with adalimumab, a clinically approved TNF-α inhibitor, enabled efficient engraftment of postmitotic dopamine neurons with extensive reinnervation and functional recovery in a preclinical PD mouse model. Thus, transient TNF-α inhibition presents a clinically relevant strategy to enhance survival and enable engraftment of postmitotic hPSC-derived dopamine neurons in PD.
Asunto(s)
Supervivencia Celular , Neuronas Dopaminérgicas , FN-kappa B , Factor de Necrosis Tumoral alfa , Proteína p53 Supresora de Tumor , Neuronas Dopaminérgicas/metabolismo , Animales , Humanos , FN-kappa B/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ratones , Supervivencia Celular/efectos de los fármacos , Transducción de Señal , Enfermedad de Parkinson/metabolismo , Células Madre Pluripotentes/metabolismo , Apoptosis , Modelos Animales de Enfermedad , Sistemas CRISPR-CasRESUMEN
Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo, but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc2-Man9-Glc3, suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action.
Asunto(s)
Sistemas CRISPR-Cas , Hexosiltransferasas , Lipopolisacáridos , Proteínas de la Membrana , FN-kappa B , Transducción de Señal , Receptor Toll-Like 4 , Hexosiltransferasas/metabolismo , Hexosiltransferasas/genética , FN-kappa B/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , Receptor Toll-Like 4/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Ratones , Células HEK293 , Inflamación/metabolismo , Inflamación/genética , Glicosilación , Microscopía por Crioelectrón , Dominio Catalítico , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genéticaRESUMEN
Cell proliferation and cell death are integral elements in maintaining homeostatic balance in metazoans. Disease pathologies ensue when these processes are disturbed. A plethora of evidence indicates that malfunction of cell death can lead to inflammation, autoimmunity, or immunodeficiency. Programmed necrosis or necroptosis is a form of nonapoptotic cell death driven by the receptor interacting protein kinase 3 (RIPK3) and its substrate, mixed lineage kinase domain-like (MLKL). RIPK3 partners with its upstream adaptors RIPK1, TRIF, or DAI to signal for necroptosis in response to death receptor or Toll-like receptor stimulation, pathogen infection, or sterile cell injury. Necroptosis promotes inflammation through leakage of cellular contents from damaged plasma membranes. Intriguingly, many of the signal adaptors of necroptosis have dual functions in innate immune signaling. This unique signature illustrates the cooperative nature of necroptosis and innate inflammatory signaling pathways in managing cell and organismal stresses from pathogen infection and sterile tissue injury.
Asunto(s)
Inflamación/metabolismo , Inflamación/patología , Necrosis/metabolismo , Transducción de Señal , Animales , Infecciones Bacterianas/genética , Infecciones Bacterianas/metabolismo , Infecciones Bacterianas/patología , Evolución Biológica , Muerte Celular , Humanos , Inflamasomas/metabolismo , Inflamación/genética , Interleucina-1beta/metabolismo , FN-kappa B/metabolismo , Enfermedades Parasitarias/genética , Enfermedades Parasitarias/metabolismo , Enfermedades Parasitarias/patología , Fosforilación , Unión Proteica , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Ubiquitinación , Virosis/genética , Virosis/metabolismo , Virosis/patologíaRESUMEN
Patients with autoinflammatory diseases present with noninfectious fever flares and systemic and/or disease-specific organ inflammation. Their excessive proinflammatory cytokine and chemokine responses can be life threatening and lead to organ damage over time. Studying such patients has revealed genetic defects that have helped unravel key innate immune pathways, including excessive IL-1 signaling, constitutive NF-κB activation, and, more recently, chronic type I IFN signaling. Discoveries of monogenic defects that lead to activation of proinflammatory cytokines have inspired the use of anticytokine-directed treatment approaches that have been life changing for many patients and have led to the approval of IL-1-blocking agents for a number of autoinflammatory conditions. In this review, we describe the genetically characterized autoinflammatory diseases, we summarize our understanding of the molecular pathways that drive clinical phenotypes and that continue to inspire the search for novel treatment targets, and we provide a conceptual framework for classification.
Asunto(s)
Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Predisposición Genética a la Enfermedad , Inflamación/genética , Inflamación/inmunología , Animales , Enfermedades Autoinmunes/metabolismo , Autoinmunidad , Modelos Animales de Enfermedad , Humanos , Inmunidad Innata , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Síndromes de Inmunodeficiencia/metabolismo , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Interferones/metabolismo , Interleucina-1/metabolismo , Trastornos Linfoproliferativos/genética , Trastornos Linfoproliferativos/inmunología , Trastornos Linfoproliferativos/metabolismo , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , FN-kappa B/metabolismo , Transducción de SeñalRESUMEN
The evolution of T cell molecular signatures in the distal lung of patients with severe pneumonia is understudied. Here, we analyzed T cell subsets in longitudinal bronchoalveolar lavage fluid samples from 273 patients with severe pneumonia, including unvaccinated patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or with respiratory failure not linked to pneumonia. In patients with SARS-CoV-2 pneumonia, activation of interferon signaling pathways, low activation of the NF-κB pathway and preferential targeting of spike and nucleocapsid proteins early after intubation were associated with favorable outcomes, whereas loss of interferon signaling, activation of NF-κB-driven programs and specificity for the ORF1ab complex late in disease were associated with mortality. These results suggest that in patients with severe SARS-CoV-2 pneumonia, alveolar T cell interferon responses targeting structural SARS-CoV-2 proteins characterize individuals who recover, whereas responses against nonstructural proteins and activation of NF-κB are associated with poor outcomes.
Asunto(s)
COVID-19 , FN-kappa B , SARS-CoV-2 , Humanos , COVID-19/inmunología , SARS-CoV-2/inmunología , Masculino , Femenino , Persona de Mediana Edad , FN-kappa B/metabolismo , Anciano , Líquido del Lavado Bronquioalveolar/inmunología , Adulto , Transducción de Señal/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Interferones/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Alveolos Pulmonares/inmunología , Alveolos Pulmonares/patologíaRESUMEN
Rare genetic variants in toll-like receptor 7 (TLR7) are known to cause lupus in humans and mice. UNC93B1 is a transmembrane protein that regulates TLR7 localization into endosomes. In the present study, we identify two new variants in UNC93B1 (T314A, located proximally to the TLR7 transmembrane domain, and V117L) in a cohort of east Asian patients with childhood-onset systemic lupus erythematosus. The V117L variant was associated with increased expression of type I interferons and NF-κB-dependent cytokines in patient plasma and immortalized B cells. THP-1 cells expressing the variant UNC93B1 alleles exhibited exaggerated responses to stimulation of TLR7/-8, but not TLR3 or TLR9, which could be inhibited by targeting the downstream signaling molecules, IRAK1/-4. Heterozygous mice expressing the orthologous Unc93b1V117L variant developed a spontaneous lupus-like disease that was more severe in homozygotes and again hyperresponsive to TLR7 stimulation. Together, this work formally identifies genetic variants in UNC93B1 that can predispose to childhood-onset systemic lupus erythematosus.
Asunto(s)
Predisposición Genética a la Enfermedad , Lupus Eritematoso Sistémico , Receptor Toll-Like 7 , Lupus Eritematoso Sistémico/genética , Humanos , Animales , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismo , Ratones , Niño , Femenino , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Masculino , Edad de Inicio , Variación Genética , FN-kappa B/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Adolescente , Células THP-1 , Interferón Tipo I/metabolismoRESUMEN
The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans.
Asunto(s)
Síndromes de Inmunodeficiencia , Proteínas del Tejido Nervioso , Ubiquitinas , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Femenino , Masculino , FN-kappa B/metabolismo , Ubiquitina-Proteína Ligasas/genética , Inflamación/inmunología , Inflamación/genética , Linfocitos B/inmunología , Mutación con Pérdida de Función , Fibroblastos/metabolismo , Fibroblastos/inmunología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Animales , Ratones , AlelosRESUMEN
Immune cells experience large cell shape changes during environmental patrolling because of the physical constraints that they encounter while migrating through tissues. These cells can adapt to such deformation events using dedicated shape-sensing pathways. However, how shape sensing affects immune cell function is mostly unknown. Here, we identify a shape-sensing mechanism that increases the expression of the chemokine receptor CCR7 and guides dendritic cell migration from peripheral tissues to lymph nodes at steady state. This mechanism relies on the lipid metabolism enzyme cPLA2, requires nuclear envelope tensioning and is finely tuned by the ARP2/3 actin nucleation complex. We also show that this shape-sensing axis reprograms dendritic cell transcription by activating an IKKß-NF-κB-dependent pathway known to control their tolerogenic potential. These results indicate that cell shape changes experienced by immune cells can define their migratory behavior and immunoregulatory properties and reveal a contribution of the physical properties of tissues to adaptive immunity.
Asunto(s)
Movimiento Celular , Células Dendríticas , Homeostasis , Ganglios Linfáticos , Ratones Endogámicos C57BL , Receptores CCR7 , Animales , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/citología , Receptores CCR7/metabolismo , Ratones , Movimiento Celular/inmunología , Forma de la Célula , FN-kappa B/metabolismo , Ratones Noqueados , Transducción de Señal/inmunología , Quinasa I-kappa B/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismoRESUMEN
Viral infection makes us feel sick as the immune system alters systemic metabolism to better fight the pathogen. The extent of these changes is relative to the severity of disease. Whether blood glucose is subject to infection-induced modulation is mostly unknown. Here we show that strong, nonlethal infection restricts systemic glucose availability, which promotes the antiviral type I interferon (IFN-I) response. Following viral infection, we find that IFNγ produced by γδ T cells stimulates pancreatic ß cells to increase glucose-induced insulin release. Subsequently, hyperinsulinemia lessens hepatic glucose output. Glucose restriction enhances IFN-I production by curtailing lactate-mediated inhibition of IRF3 and NF-κB signaling. Induced hyperglycemia constrained IFN-I production and increased mortality upon infection. Our findings identify glucose restriction as a physiological mechanism to bring the body into a heightened state of responsiveness to viral pathogens. This immune-endocrine circuit is disrupted in hyperglycemia, possibly explaining why patients with diabetes are more susceptible to viral infection.
Asunto(s)
Glucemia , Inmunidad Innata , Interferón gamma , Animales , Interferón gamma/metabolismo , Interferón gamma/inmunología , Ratones , Glucemia/metabolismo , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Ratones Endogámicos C57BL , Transducción de Señal/inmunología , Insulina/metabolismo , Insulina/inmunología , Ratones Noqueados , Hiperglucemia/inmunología , Factor 3 Regulador del Interferón/metabolismo , FN-kappa B/metabolismo , Humanos , Hígado/inmunología , Hígado/virología , Hígado/metabolismo , MasculinoRESUMEN
The 2021 Laskerâ¼Koshland Special Achievement Award will be presented to David Baltimore for an extraordinary career that has personified the combination of outstanding biomedical research and exemplary scientific statesmanship.
Asunto(s)
Distinciones y Premios , Investigación Biomédica/historia , Animales , Historia del Siglo XX , Historia del Siglo XXI , Humanos , FN-kappa B/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismoRESUMEN
The nuclear factor kappa B (NF-κB) family of transcription factors orchestrates signal-induced gene expression in diverse cell types. Cellular responses to NF-κB activation are regulated at the level of cell and signal specificity, as well as differential use of family members (subunit specificity). Here we used time-dependent multi-omics to investigate the selective functions of Rel and RelA, two closely related NF-κB proteins, in primary B lymphocytes activated via the B cell receptor. Despite large numbers of shared binding sites genome wide, Rel and RelA directed kinetically distinct cascades of gene expression in activated B cells. Single-cell RNA sequencing revealed marked heterogeneity of Rel- and RelA-specific responses, and sequential binding of these factors was not a major mechanism of protracted transcription. Moreover, nuclear co-expression of Rel and RelA led to functional antagonism between the factors. By rigorously identifying the target genes of each NF-κB subunit, these studies provide insights into exclusive functions of Rel and RelA in immunity and cancer.
Asunto(s)
FN-kappa B , Factor de Transcripción ReIA , FN-kappa B/metabolismo , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Linfocitos B/metabolismo , Sitios de Unión , Receptores de Antígenos/metabolismoRESUMEN
Cytoplasmic accumulation of TDP-43 is a disease hallmark for many cases of amyotrophic lateral sclerosis (ALS), associated with a neuroinflammatory cytokine profile related to upregulation of nuclear factor κB (NF-κB) and type I interferon (IFN) pathways. Here we show that this inflammation is driven by the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) when TDP-43 invades mitochondria and releases DNA via the permeability transition pore. Pharmacologic inhibition or genetic deletion of cGAS and its downstream signaling partner STING prevents upregulation of NF-κB and type I IFN induced by TDP-43 in induced pluripotent stem cell (iPSC)-derived motor neurons and in TDP-43 mutant mice. Finally, we document elevated levels of the specific cGAS signaling metabolite cGAMP in spinal cord samples from patients, which may be a biomarker of mtDNA release and cGAS/STING activation in ALS. Our results identify mtDNA release and cGAS/STING activation as critical determinants of TDP-43-associated pathology and demonstrate the potential for targeting this pathway in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de la Membrana/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , Nucleotidiltransferasas/metabolismo , Alarminas/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Citoplasma/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Inflamación/metabolismo , Interferón Tipo I/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Degeneración Nerviosa/patología , Fosfotransferasas (Aceptor de Grupo Alcohol) , Subunidades de Proteína/metabolismo , Transducción de SeñalRESUMEN
Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.
Asunto(s)
Pruebas Genéticas , Genómica , FN-kappa B/genética , Factor de Transcripción ReIA/genética , Animales , Sistemas CRISPR-Cas , Línea Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Humanos , Complejo Mediador/genética , ARN Guía de Kinetoplastida/genéticaRESUMEN
The signaling organelles of the innate immune system consist of oligomeric protein complexes known as supramolecular organizing centers (SMOCs). Examples of SMOCs include myddosomes and inflammasomes, which respectively induce transcription-dependent and -independent inflammatory responses. The common use of oligomeric structures as signaling platforms suggests multifunctionality, but each SMOC has a singular biochemically defined function. Here, we report that the myddosome is a multifunctional organizing center. In addition to promoting inflammatory transcription factor activation, the myddosome drives the rapid induction of glycolysis. We identify the kinase TBK1 as a myddosome component that promotes glycolysis, but not nuclear factor κB (NF-κB) activation. Synthetic immunology approaches further diversified SMOC activities, as we created interferon- or necroptosis-inducing myddosomes, inflammasomes that induce interferon responses instead of pyroptosis, and a SMOC-like nanomachine that induces interferon expression in response to a chemical ligand. These discoveries demonstrate the flexibility of immune signaling organelles, which permits the design of user-defined innate immune responses.
Asunto(s)
Inmunidad Innata/inmunología , Inmunidad Innata/fisiología , Transducción de Señal/inmunología , Animales , Glucólisis/inmunología , Inflamasomas , Ratones , Ratones Endogámicos C57BL , Enzimas Multifuncionales/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/metabolismo , Orgánulos/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Toll-LikeRESUMEN
ROCK-Myosin II drives fast rounded-amoeboid migration in cancer cells during metastatic dissemination. Analysis of human melanoma biopsies revealed that amoeboid melanoma cells with high Myosin II activity are predominant in the invasive fronts of primary tumors in proximity to CD206+CD163+ tumor-associated macrophages and vessels. Proteomic analysis shows that ROCK-Myosin II activity in amoeboid cancer cells controls an immunomodulatory secretome, enabling the recruitment of monocytes and their differentiation into tumor-promoting macrophages. Both amoeboid cancer cells and their associated macrophages support an abnormal vasculature, which ultimately facilitates tumor progression. Mechanistically, amoeboid cancer cells perpetuate their behavior via ROCK-Myosin II-driven IL-1α secretion and NF-κB activation. Using an array of tumor models, we show that high Myosin II activity in tumor cells reprograms the innate immune microenvironment to support tumor growth. We describe an unexpected role for Myosin II dynamics in cancer cells controlling myeloid function via secreted factors.
Asunto(s)
Movimiento Celular/fisiología , Miosina Tipo II/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular/inmunología , Proteínas del Citoesqueleto , Femenino , Humanos , Interleucina-1alfa/metabolismo , Masculino , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Persona de Mediana Edad , FN-kappa B/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Fosforilación , Proteómica , Receptor Cross-Talk/fisiología , Transducción de Señal , Microambiente Tumoral/inmunologíaRESUMEN
CD8 T cells play essential roles in anti-tumor immune responses. Here, we performed genome-scale CRISPR screens in CD8 T cells directly under cancer immunotherapy settings and identified regulators of tumor infiltration and degranulation. The in vivo screen robustly re-identified canonical immunotherapy targets such as PD-1 and Tim-3, along with genes that have not been characterized in T cells. The infiltration and degranulation screens converged on an RNA helicase Dhx37. Dhx37 knockout enhanced the efficacy of antigen-specific CD8 T cells against triple-negative breast cancer in vivo. Immunological characterization in mouse and human CD8 T cells revealed that DHX37 suppresses effector functions, cytokine production, and T cell activation. Transcriptomic profiling and biochemical interrogation revealed a role for DHX37 in modulating NF-κB. These data demonstrate high-throughput in vivo genetic screens for immunotherapy target discovery and establishes DHX37 as a functional regulator of CD8 T cells.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , ARN Helicasas/genética , Animales , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Citocinas/genética , Citocinas/metabolismo , Femenino , Humanos , Memoria Inmunológica , Inmunoterapia , Masculino , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , ARN Helicasas/deficiencia , ARN Guía de Kinetoplastida/metabolismo , TranscriptomaRESUMEN
Metabolism has been shown to control peripheral immunity, but little is known about its role in central nervous system (CNS) inflammation. Through a combination of proteomic, metabolomic, transcriptomic, and perturbation studies, we found that sphingolipid metabolism in astrocytes triggers the interaction of the C2 domain in cytosolic phospholipase A2 (cPLA2) with the CARD domain in mitochondrial antiviral signaling protein (MAVS), boosting NF-κB-driven transcriptional programs that promote CNS inflammation in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis. cPLA2 recruitment to MAVS also disrupts MAVS-hexokinase 2 (HK2) interactions, decreasing HK enzymatic activity and the production of lactate involved in the metabolic support of neurons. Miglustat, a drug used to treat Gaucher and Niemann-Pick disease, suppresses astrocyte pathogenic activities and ameliorates EAE. Collectively, these findings define a novel immunometabolic mechanism that drives pro-inflammatory astrocyte activities, outlines a new role for MAVS in CNS inflammation, and identifies candidate targets for therapeutic intervention.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Astrocitos/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Fosfolipasas A2 Secretoras/metabolismo , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacología , 1-Desoxinojirimicina/uso terapéutico , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Astrocitos/efectos de los fármacos , Astrocitos/patología , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Femenino , Hexoquinasa/metabolismo , Humanos , Ácido Láctico/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Fosfolipasas A2 Secretoras/genéticaRESUMEN
Inducible nucleosome remodeling at hundreds of latent enhancers and several promoters shapes the transcriptional response to Toll-like receptor 4 (TLR4) signaling in macrophages. We aimed to define the identities of the transcription factors that promote TLR-induced remodeling. An analysis strategy based on ATAC-seq and single-cell ATAC-seq that enriched for genomic regions most likely to undergo remodeling revealed that the transcription factor nuclear factor κB (NF-κB) bound to all high-confidence peaks marking remodeling during the primary response to the TLR4 ligand, lipid A. Deletion of NF-κB subunits RelA and c-Rel resulted in the loss of remodeling at high-confidence ATAC-seq peaks, and CRISPR-Cas9 mutagenesis of NF-κB-binding motifs impaired remodeling. Remodeling selectivity at defined regions was conferred by collaboration with other inducible factors, including IRF3- and MAP-kinase-induced factors. Thus, NF-κB is unique among TLR4-activated transcription factors in its broad contribution to inducible nucleosome remodeling, alongside its ability to activate poised enhancers and promoters assembled into open chromatin.