RESUMEN
Single-cell analysis in living humans is essential for understanding disease mechanisms, but it is impractical in non-regenerative organs, such as the eye and brain, because tissue biopsies would cause serious damage. We resolve this problem by integrating proteomics of liquid biopsies with single-cell transcriptomics from all known ocular cell types to trace the cellular origin of 5,953 proteins detected in the aqueous humor. We identified hundreds of cell-specific protein markers, including for individual retinal cell types. Surprisingly, our results reveal that retinal degeneration occurs in Parkinson's disease, and the cells driving diabetic retinopathy switch with disease stage. Finally, we developed artificial intelligence (AI) models to assess individual cellular aging and found that many eye diseases not associated with chronological age undergo accelerated molecular aging of disease-specific cell types. Our approach, which can be applied to other organ systems, has the potential to transform molecular diagnostics and prognostics while uncovering new cellular disease and aging mechanisms.
Asunto(s)
Envejecimiento , Humor Acuoso , Inteligencia Artificial , Biopsia Líquida , Proteómica , Humanos , Envejecimiento/metabolismo , Humor Acuoso/química , Biopsia , Enfermedad de Parkinson/diagnósticoRESUMEN
Proper preimplantation development is essential to assemble a blastocyst capable of implantation. Live imaging has uncovered major events driving early development in mouse embryos; yet, studies in humans have been limited by restrictions on genetic manipulation and lack of imaging approaches. We have overcome this barrier by combining fluorescent dyes with live imaging to reveal the dynamics of chromosome segregation, compaction, polarization, blastocyst formation, and hatching in the human embryo. We also show that blastocyst expansion mechanically constrains trophectoderm cells, causing nuclear budding and DNA shedding into the cytoplasm. Furthermore, cells with lower perinuclear keratin levels are more prone to undergo DNA loss. Moreover, applying trophectoderm biopsy, a mechanical procedure performed clinically for genetic testing, increases DNA shedding. Thus, our work reveals distinct processes underlying human development compared with mouse and suggests that aneuploidies in human embryos may not only originate from chromosome segregation errors during mitosis but also from nuclear DNA shedding.
Asunto(s)
Diagnóstico Preimplantación , Embarazo , Femenino , Humanos , Animales , Ratones , Diagnóstico Preimplantación/métodos , Blastocisto , Implantación del Embrión , Pruebas Genéticas/métodos , Aneuploidia , Biopsia/métodosRESUMEN
Cellular perturbations underlying Alzheimer's disease (AD) are primarily studied in human postmortem samples and model organisms. Here, we generated a single-nucleus atlas from a rare cohort of cortical biopsies from living individuals with varying degrees of AD pathology. We next performed a systematic cross-disease and cross-species integrative analysis to identify a set of cell states that are specific to early AD pathology. These changes-which we refer to as the early cortical amyloid response-were prominent in neurons, wherein we identified a transitional hyperactive state preceding the loss of excitatory neurons, which we confirmed by acute slice physiology on independent biopsy specimens. Microglia overexpressing neuroinflammatory-related processes also expanded as AD pathology increased. Finally, both oligodendrocytes and pyramidal neurons upregulated genes associated with ß-amyloid production and processing during this early hyperactive phase. Our integrative analysis provides an organizing framework for targeting circuit dysfunction, neuroinflammation, and amyloid production early in AD pathogenesis.
Asunto(s)
Enfermedad de Alzheimer , Lóbulo Frontal , Microglía , Neuronas , Humanos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Amiloide , Péptidos beta-Amiloides/metabolismo , Microglía/patología , Neuronas/patología , Células Piramidales , Biopsia , Lóbulo Frontal/patología , Análisis de Expresión Génica de una Sola Célula , Núcleo Celular/metabolismo , Núcleo Celular/patologíaRESUMEN
The gut microbiome has been implicated in multiple human chronic gastrointestinal (GI) disorders. Determining its mechanistic role in disease has been difficult due to apparent disconnects between animal and human studies and lack of an integrated multi-omics view of disease-specific physiological changes. We integrated longitudinal multi-omics data from the gut microbiome, metabolome, host epigenome, and transcriptome in the context of irritable bowel syndrome (IBS) host physiology. We identified IBS subtype-specific and symptom-related variation in microbial composition and function. A subset of identified changes in microbial metabolites correspond to host physiological mechanisms that are relevant to IBS. By integrating multiple data layers, we identified purine metabolism as a novel host-microbial metabolic pathway in IBS with translational potential. Our study highlights the importance of longitudinal sampling and integrating complementary multi-omics data to identify functional mechanisms that can serve as therapeutic targets in a comprehensive treatment strategy for chronic GI diseases. VIDEO ABSTRACT.
Asunto(s)
Microbioma Gastrointestinal/genética , Regulación de la Expresión Génica/genética , Síndrome del Colon Irritable/metabolismo , Metaboloma , Purinas/metabolismo , Transcriptoma/genética , Animales , Ácidos y Sales Biliares/metabolismo , Biopsia , Butiratos/metabolismo , Cromatografía Liquida , Estudios Transversales , Epigenómica , Heces/microbiología , Femenino , Microbioma Gastrointestinal/fisiología , Regulación de la Expresión Génica/fisiología , Interacciones Microbiota-Huesped/genética , Humanos , Hipoxantina/metabolismo , Síndrome del Colon Irritable/genética , Síndrome del Colon Irritable/microbiología , Estudios Longitudinales , Masculino , Metaboloma/fisiología , Ratones , Estudios Observacionales como Asunto , Estudios Prospectivos , Programas Informáticos , Espectrometría de Masas en Tándem , Transcriptoma/fisiologíaRESUMEN
Biliary atresia (BA) is a severe cholangiopathy that leads to liver failure in infants, but its pathogenesis remains to be fully characterized. By single-cell RNA profiling, we observed macrophage hypo-inflammation, Kupffer cell scavenger function defects, cytotoxic T cell expansion, and deficiency of CX3CR1+effector T and natural killer (NK) cells in infants with BA. More importantly, we discovered that hepatic B cell lymphopoiesis did not cease after birth and that tolerance defects contributed to immunoglobulin G (IgG)-autoantibody accumulation in BA. In a rhesus-rotavirus induced BA model, depleting B cells or blocking antigen presentation ameliorated liver damage. In a pilot clinical study, we demonstrated that rituximab was effective in depleting hepatic B cells and restoring the functions of macrophages, Kupffer cells, and T cells to levels comparable to those of control subjects. In summary, our comprehensive immune profiling in infants with BA had educed that B-cell-modifying therapies may alleviate liver pathology.
Asunto(s)
Atresia Biliar/inmunología , Atresia Biliar/terapia , Hígado/inmunología , Animales , Antígenos CD20/metabolismo , Linfocitos B/inmunología , Atresia Biliar/sangre , Atresia Biliar/tratamiento farmacológico , Biopsia , Receptor 1 de Quimiocinas CX3C/metabolismo , Muerte Celular , Línea Celular , Proliferación Celular , Transdiferenciación Celular , Niño , Preescolar , Estudios de Cohortes , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunoglobulina G/metabolismo , Lactante , Inflamación/patología , Células Asesinas Naturales/inmunología , Macrófagos del Hígado/patología , Hígado/patología , Cirrosis Hepática/sangre , Cirrosis Hepática/complicaciones , Cirrosis Hepática/inmunología , Cirrosis Hepática/patología , Depleción Linfocítica , Linfopoyesis , Masculino , Ratones Endogámicos BALB C , Fagocitosis , ARN/metabolismo , Rituximab/administración & dosificación , Rituximab/farmacología , Rituximab/uso terapéutico , Rotavirus/fisiología , Análisis de la Célula Individual , Células TH1/inmunología , Células Th17/inmunologíaRESUMEN
A safe and controlled manipulation of endocytosis in vivo may have disruptive therapeutic potential. Here, we demonstrate that the anti-emetic/anti-psychotic prochlorperazine can be repurposed to reversibly inhibit the in vivo endocytosis of membrane proteins targeted by therapeutic monoclonal antibodies, as directly demonstrated by our human tumor ex vivo assay. Temporary endocytosis inhibition results in enhanced target availability and improved efficiency of natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), a mediator of clinical responses induced by IgG1 antibodies, demonstrated here for cetuximab, trastuzumab, and avelumab. Extensive analysis of downstream signaling pathways ruled out on-target toxicities. By overcoming the heterogeneity of drug target availability that frequently characterizes poorly responsive or resistant tumors, clinical application of reversible endocytosis inhibition may considerably improve the clinical benefit of ADCC-mediating therapeutic antibodies.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Resistencia a Antineoplásicos/inmunología , Neoplasias/tratamiento farmacológico , Proclorperazina/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados/farmacología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Presentación de Antígeno/efectos de los fármacos , Biopsia , Cetuximab/farmacología , Sistemas de Liberación de Medicamentos/métodos , Resistencia a Antineoplásicos/genética , Endocitosis/efectos de los fármacos , Endocitosis/inmunología , Xenoinjertos , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células MCF-7 , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Neoplasias/genética , Neoplasias/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Trastuzumab/farmacologíaRESUMEN
Clear-cell renal cell carcinoma (ccRCC) exhibits a broad range of metastatic phenotypes that have not been systematically studied to date. Here, we analyzed 575 primary and 335 metastatic biopsies across 100 patients with metastatic ccRCC, including two cases sampledat post-mortem. Metastatic competence was afforded by chromosome complexity, and we identify 9p loss as a highly selected event driving metastasis and ccRCC-related mortality (p = 0.0014). Distinct patterns of metastatic dissemination were observed, including rapid progression to multiple tissue sites seeded by primary tumors of monoclonal structure. By contrast, we observed attenuated progression in cases characterized by high primary tumor heterogeneity, with metastatic competence acquired gradually and initial progression to solitary metastasis. Finally, we observed early divergence of primitive ancestral clones and protracted latency of up to two decades as a feature of pancreatic metastases.
Asunto(s)
Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Neoplasias Renales/genética , Neoplasias Renales/patología , Mutación , Metástasis de la Neoplasia , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Biopsia , Mapeo Cromosómico , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 9 , Progresión de la Enfermedad , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Fenotipo , Estudios Prospectivos , Trombosis , Resultado del TratamientoRESUMEN
The molecular and cellular processes that lead to renal damage and to the heterogeneity of lupus nephritis (LN) are not well understood. We applied single-cell RNA sequencing (scRNA-seq) to renal biopsies from patients with LN and evaluated skin biopsies as a potential source of diagnostic and prognostic markers of renal disease. Type I interferon (IFN)-response signatures in tubular cells and keratinocytes distinguished patients with LN from healthy control subjects. Moreover, a high IFN-response signature and fibrotic signature in tubular cells were each associated with failure to respond to treatment. Analysis of tubular cells from patients with proliferative, membranous and mixed LN indicated pathways relevant to inflammation and fibrosis, which offer insight into their histologic differences. In summary, we applied scRNA-seq to LN to deconstruct its heterogeneity and identify novel targets for personalized approaches to therapy.
Asunto(s)
Perfilación de la Expresión Génica , Interferón Tipo I/metabolismo , Queratinocitos/metabolismo , Túbulos Renales/citología , Túbulos Renales/metabolismo , Nefritis Lúpica/genética , Nefritis Lúpica/metabolismo , Transcriptoma , Biopsia , Linaje de la Célula/genética , Biología Computacional/métodos , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Fibrosis , Perfilación de la Expresión Génica/métodos , Humanos , Nefritis Lúpica/patología , Unión Proteica , Transducción de Señal , Análisis de la Célula Individual , Piel/inmunología , Piel/metabolismo , Piel/patologíaRESUMEN
Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.
Asunto(s)
Riñón/inmunología , Nefritis Lúpica/inmunología , Biomarcadores , Biopsia , Análisis por Conglomerados , Biología Computacional/métodos , Células Epiteliales/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Inmunofenotipificación , Interferones/metabolismo , Riñón/metabolismo , Riñón/patología , Leucocitos/inmunología , Leucocitos/metabolismo , Nefritis Lúpica/genética , Nefritis Lúpica/metabolismo , Nefritis Lúpica/patología , Linfocitos/inmunología , Linfocitos/metabolismo , Anotación de Secuencia Molecular , Células Mieloides/inmunología , Células Mieloides/metabolismo , Análisis de la Célula Individual , TranscriptomaRESUMEN
Vaccine-associated enhanced respiratory disease (VAERD) was previously observed in some preclinical models of severe acute respiratory syndrome (SARS) and MERS coronavirus vaccines. We used the SARS coronavirus 2 (SARS-CoV-2) mouse-adapted, passage 10, lethal challenge virus (MA10) mouse model of acute lung injury to evaluate the immune response and potential for immunopathology in animals vaccinated with research-grade mRNA-1273. Whole-inactivated virus or heat-denatured spike protein subunit vaccines with alum designed to elicit low-potency antibodies and Th2-skewed CD4+ T cells resulted in reduced viral titers and weight loss post challenge but more severe pathological changes in the lung compared to saline-immunized animals. In contrast, a protective dose of mRNA-1273 induced favorable humoral and cellular immune responses that protected from viral replication in the upper and lower respiratory tract upon challenge. A subprotective dose of mRNA-1273 reduced viral replication and limited histopathological manifestations compared to animals given saline. Overall, our findings demonstrate an immunological signature associated with antiviral protection without disease enhancement following vaccination with mRNA-1273.
Asunto(s)
Vacunas contra la COVID-19/inmunología , COVID-19/inmunología , COVID-19/prevención & control , Interacciones Huésped-Patógeno/inmunología , SARS-CoV-2/inmunología , Vacunas Sintéticas/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Biopsia , Vacunas contra la COVID-19/administración & dosificación , Modelos Animales de Enfermedad , Humanos , Inmunoglobulina G , Inmunohistoquímica , Ratones , Evaluación de Resultado en la Atención de Salud , ARN Mensajero , Glicoproteína de la Espiga del Coronavirus/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Vacunas Sintéticas/administración & dosificación , Vacunas de ARNmRESUMEN
The emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant global morbidity, mortality, and societal disruption. A better understanding of virus-host interactions may potentiate therapeutic insights toward limiting this infection. Here we investigated the dynamics of the systemic response to SARS-CoV-2 in hamsters by histological analysis and transcriptional profiling. Infection resulted in consistently high levels of virus in the upper and lower respiratory tracts and sporadic occurrence in other distal tissues. A longitudinal cohort revealed a wave of inflammation, including a type I interferon (IFN-I) response, that was evident in all tissues regardless of viral presence but was insufficient to prevent disease progression. Bolstering the antiviral response with intranasal administration of recombinant IFN-I reduced viral disease, prevented transmission, and lowered inflammation in vivo. This study defines the systemic host response to SARS-CoV-2 infection and supports use of intranasal IFN-I as an effective means of early treatment.
Asunto(s)
COVID-19/metabolismo , COVID-19/virología , Interacciones Huésped-Patógeno , Interferón Tipo I/metabolismo , SARS-CoV-2/fisiología , Animales , Biopsia , COVID-19/genética , COVID-19/inmunología , Cricetinae , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata , Interferón Tipo I/genética , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Especificidad de Órganos/inmunología , Virulencia , Replicación Viral/inmunologíaRESUMEN
Guillain-Barré syndrome (GBS) is a rare heterogenous disorder of the peripheral nervous system, which is usually triggered by a preceding infection, and causes a potentially life-threatening progressive muscle weakness1. Although GBS is considered an autoimmune disease, the mechanisms that underlie its distinct clinical subtypes remain largely unknown. Here, by combining in vitro T cell screening, single-cell RNA sequencing and T cell receptor (TCR) sequencing, we identify autoreactive memory CD4+ cells, that show a cytotoxic T helper 1 (TH1)-like phenotype, and rare CD8+ T cells that target myelin antigens of the peripheral nerves in patients with the demyelinating disease variant. We characterized more than 1,000 autoreactive single T cell clones, which revealed a polyclonal TCR repertoire, short CDR3ß lengths, preferential HLA-DR restrictions and recognition of immunodominant epitopes. We found that autoreactive TCRß clonotypes were expanded in the blood of the same patient at distinct disease stages and, notably, that they were shared in the blood and the cerebrospinal fluid across different patients with GBS, but not in control individuals. Finally, we identified myelin-reactive T cells in the nerve biopsy from one patient, which indicates that these cells contribute directly to disease pathophysiology. Collectively, our data provide clear evidence of autoreactive T cell immunity in a subset of patients with GBS, and open new perspectives in the field of inflammatory peripheral neuropathies, with potential impact for biomedical applications.
Asunto(s)
Autoinmunidad , Linfocitos T CD8-positivos , Síndrome de Guillain-Barré , Nervios Periféricos , Enfermedades del Sistema Nervioso Periférico , Células TH1 , Humanos , Biopsia , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Síndrome de Guillain-Barré/sangre , Síndrome de Guillain-Barré/líquido cefalorraquídeo , Síndrome de Guillain-Barré/etiología , Síndrome de Guillain-Barré/inmunología , Antígenos HLA-DR/inmunología , Epítopos Inmunodominantes/inmunología , Vaina de Mielina/inmunología , Nervios Periféricos/inmunología , Nervios Periféricos/patología , Enfermedades del Sistema Nervioso Periférico/complicaciones , Enfermedades del Sistema Nervioso Periférico/inmunología , Enfermedades del Sistema Nervioso Periférico/patología , Receptores de Antígenos de Linfocitos T/inmunología , Células TH1/inmunología , Células TH1/patología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/patología , Memoria InmunológicaRESUMEN
For many adult human organs, tissue regeneration during chronic disease remains a controversial subject. Regenerative processes are easily observed in animal models, and their underlying mechanisms are becoming well characterized1-4, but technical challenges and ethical aspects are limiting the validation of these results in humans. We decided to address this difficulty with respect to the liver. This organ displays the remarkable ability to regenerate after acute injury, although liver regeneration in the context of recurring injury remains to be fully demonstrated. Here we performed single-nucleus RNA sequencing (snRNA-seq) on 47 liver biopsies from patients with different stages of metabolic dysfunction-associated steatotic liver disease to establish a cellular map of the liver during disease progression. We then combined these single-cell-level data with advanced 3D imaging to reveal profound changes in the liver architecture. Hepatocytes lose their zonation and considerable reorganization of the biliary tree takes place. More importantly, our study uncovers transdifferentiation events that occur between hepatocytes and cholangiocytes without the presence of adult stem cells or developmental progenitor activation. Detailed analyses and functional validations using cholangiocyte organoids confirm the importance of the PI3K-AKT-mTOR pathway in this process, thereby connecting this acquisition of plasticity to insulin signalling. Together, our data indicate that chronic injury creates an environment that induces cellular plasticity in human organs, and understanding the underlying mechanisms of this process could open new therapeutic avenues in the management of chronic diseases.
Asunto(s)
Transdiferenciación Celular , Hepatocitos , Hepatopatías , Hígado , Humanos , Sistema Biliar/citología , Sistema Biliar/metabolismo , Sistema Biliar/patología , Biopsia , Plasticidad de la Célula , Enfermedad Crónica , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/citología , Células Epiteliales/patología , Hepatocitos/metabolismo , Hepatocitos/citología , Hepatocitos/patología , Insulina/metabolismo , Hígado/patología , Hígado/metabolismo , Hígado/citología , Hepatopatías/patología , Hepatopatías/metabolismo , Regeneración Hepática , Organoides/metabolismo , Organoides/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , RNA-Seq , Transducción de Señal , Análisis de la Célula Individual , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury1-4. Here, we generated air-liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8+ T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity.
Asunto(s)
Enfermedad Celíaca , Duodeno , Interleucina-7 , Mucosa Intestinal , Modelos Biológicos , Organoides , Humanos , Autoanticuerpos/inmunología , Autoinmunidad , Linfocitos B/inmunología , Linfocitos B/metabolismo , Biopsia , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/patología , Enfermedad Celíaca/metabolismo , Duodeno/inmunología , Duodeno/patología , Duodeno/metabolismo , Epítopos/inmunología , Glútenes/inmunología , Glútenes/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/inmunología , Antígenos HLA-DQ/inmunología , Antígenos HLA-DQ/metabolismo , Interleucina-7/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Células Asesinas Naturales/inmunología , Células Mieloides/inmunología , Organoides/inmunología , Organoides/metabolismo , Organoides/patología , Proteína Glutamina Gamma Glutamiltransferasa 2/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Immune checkpoint blockade (ICB) benefits some patients with triple-negative breast cancer, but what distinguishes responders from non-responders is unclear1. Because ICB targets cell-cell interactions2, we investigated the impact of multicellular spatial organization on response, and explored how ICB remodels the tumour microenvironment. We show that cell phenotype, activation state and spatial location are intimately linked, influence ICB effect and differ in sensitive versus resistant tumours early on-treatment. We used imaging mass cytometry3 to profile the in situ expression of 43 proteins in tumours from patients in a randomized trial of neoadjuvant ICB, sampled at three timepoints (baseline, n = 243; early on-treatment, n = 207; post-treatment, n = 210). Multivariate modelling showed that the fractions of proliferating CD8+TCF1+T cells and MHCII+ cancer cells were dominant predictors of response, followed by cancer-immune interactions with B cells and granzyme B+ T cells. On-treatment, responsive tumours contained abundant granzyme B+ T cells, whereas resistant tumours were characterized by CD15+ cancer cells. Response was best predicted by combining tissue features before and on-treatment, pointing to a role for early biopsies in guiding adaptive therapy. Our findings show that multicellular spatial organization is a major determinant of ICB effect and suggest that its systematic enumeration in situ could help realize precision immuno-oncology.
Asunto(s)
Inmunoterapia , Linfocitos T , Neoplasias de la Mama Triple Negativas , Humanos , Linfocitos B/inmunología , Biopsia , Linfocitos T CD8-positivos/inmunología , Granzimas/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígeno Lewis X/metabolismo , Terapia Neoadyuvante , Medicina de Precisión , Pronóstico , Ensayos Clínicos Controlados Aleatorios como Asunto , Linfocitos T/inmunología , Neoplasias de la Mama Triple Negativas/inmunología , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/terapiaRESUMEN
Oncogene amplification on extrachromosomal DNA (ecDNA) drives the evolution of tumours and their resistance to treatment, and is associated with poor outcomes for patients with cancer1-6. At present, it is unclear whether ecDNA is a later manifestation of genomic instability, or whether it can be an early event in the transition from dysplasia to cancer. Here, to better understand the development of ecDNA, we analysed whole-genome sequencing (WGS) data from patients with oesophageal adenocarcinoma (EAC) or Barrett's oesophagus. These data included 206 biopsies in Barrett's oesophagus surveillance and EAC cohorts from Cambridge University. We also analysed WGS and histology data from biopsies that were collected across multiple regions at 2 time points from 80 patients in a case-control study at the Fred Hutchinson Cancer Center. In the Cambridge cohorts, the frequency of ecDNA increased between Barrett's-oesophagus-associated early-stage (24%) and late-stage (43%) EAC, suggesting that ecDNA is formed during cancer progression. In the cohort from the Fred Hutchinson Cancer Center, 33% of patients who developed EAC had at least one oesophageal biopsy with ecDNA before or at the diagnosis of EAC. In biopsies that were collected before cancer diagnosis, higher levels of ecDNA were present in samples from patients who later developed EAC than in samples from those who did not. We found that ecDNAs contained diverse collections of oncogenes and immunomodulatory genes. Furthermore, ecDNAs showed increases in copy number and structural complexity at more advanced stages of disease. Our findings show that ecDNA can develop early in the transition from high-grade dysplasia to cancer, and that ecDNAs progressively form and evolve under positive selection.
Asunto(s)
Adenocarcinoma , Esófago de Barrett , Carcinogénesis , ADN , Progresión de la Enfermedad , Detección Precoz del Cáncer , Neoplasias Esofágicas , Humanos , Adenocarcinoma/genética , Adenocarcinoma/patología , Esófago de Barrett/genética , Esófago de Barrett/patología , Estudios de Casos y Controles , ADN/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Carcinogénesis/genética , Secuenciación Completa del Genoma , Estudios de Cohortes , Biopsia , Oncogenes , Inmunomodulación , Variaciones en el Número de Copia de ADN , Amplificación de Genes , Detección Precoz del Cáncer/métodosRESUMEN
The peripheral T cell repertoire of healthy individuals contains self-reactive T cells1,2. Checkpoint receptors such as PD-1 are thought to enable the induction of peripheral tolerance by deletion or anergy of self-reactive CD8 T cells3-10. However, this model is challenged by the high frequency of immune-related adverse events in patients with cancer who have been treated with checkpoint inhibitors11. Here we developed a mouse model in which skin-specific expression of T cell antigens in the epidermis caused local infiltration of antigen-specific CD8 T cells with an effector gene-expression profile. In this setting, PD-1 enabled the maintenance of skin tolerance by preventing tissue-infiltrating antigen-specific effector CD8 T cells from (1) acquiring a fully functional, pathogenic differentiation state, (2) secreting significant amounts of effector molecules, and (3) gaining access to epidermal antigen-expressing cells. In the absence of PD-1, epidermal antigen-expressing cells were eliminated by antigen-specific CD8 T cells, resulting in local pathology. Transcriptomic analysis of skin biopsies from two patients with cutaneous lichenoid immune-related adverse events showed the presence of clonally expanded effector CD8 T cells in both lesional and non-lesional skin. Thus, our data support a model of peripheral T cell tolerance in which PD-1 allows antigen-specific effector CD8 T cells to co-exist with antigen-expressing cells in tissues without immunopathology.
Asunto(s)
Antígenos , Linfocitos T CD8-positivos , Tolerancia Inmunológica , Receptor de Muerte Celular Programada 1 , Piel , Animales , Humanos , Ratones , Antígenos/inmunología , Biopsia , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Epidermis/inmunología , Epidermis/metabolismo , Perfilación de la Expresión Génica , Liquen Plano/inmunología , Liquen Plano/patología , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Piel/citología , Piel/inmunología , Piel/metabolismo , Piel/patologíaRESUMEN
T cell receptors (TCRs) enable T cells to specifically recognize mutations in cancer cells1-3. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRß). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Edición Génica , Neoplasias , Medicina de Precisión , Receptores de Antígenos de Linfocitos T , Linfocitos T , Transgenes , Humanos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Biopsia , Tratamiento Basado en Trasplante de Células y Tejidos/efectos adversos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Síndrome de Liberación de Citoquinas/complicaciones , Progresión de la Enfermedad , Encefalitis/complicaciones , Técnicas de Sustitución del Gen , Técnicas de Inactivación de Genes , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Mutación , Neoplasias/complicaciones , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/terapia , Seguridad del Paciente , Medicina de Precisión/efectos adversos , Medicina de Precisión/métodos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Transgenes/genética , Antígenos HLA/inmunología , Sistemas CRISPR-CasRESUMEN
Gliomas synaptically integrate into neural circuits1,2. Previous research has demonstrated bidirectional interactions between neurons and glioma cells, with neuronal activity driving glioma growth1-4 and gliomas increasing neuronal excitability2,5-8. Here we sought to determine how glioma-induced neuronal changes influence neural circuits underlying cognition and whether these interactions influence patient survival. Using intracranial brain recordings during lexical retrieval language tasks in awake humans together with site-specific tumour tissue biopsies and cell biology experiments, we find that gliomas remodel functional neural circuitry such that task-relevant neural responses activate tumour-infiltrated cortex well beyond the cortical regions that are normally recruited in the healthy brain. Site-directed biopsies from regions within the tumour that exhibit high functional connectivity between the tumour and the rest of the brain are enriched for a glioblastoma subpopulation that exhibits a distinct synaptogenic and neuronotrophic phenotype. Tumour cells from functionally connected regions secrete the synaptogenic factor thrombospondin-1, which contributes to the differential neuron-glioma interactions observed in functionally connected tumour regions compared with tumour regions with less functional connectivity. Pharmacological inhibition of thrombospondin-1 using the FDA-approved drug gabapentin decreases glioblastoma proliferation. The degree of functional connectivity between glioblastoma and the normal brain negatively affects both patient survival and performance in language tasks. These data demonstrate that high-grade gliomas functionally remodel neural circuits in the human brain, which both promotes tumour progression and impairs cognition.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Vías Nerviosas , Humanos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , Trombospondina 1/antagonistas & inhibidores , Gabapentina/farmacología , Gabapentina/uso terapéutico , Progresión de la Enfermedad , Cognición , Tasa de Supervivencia , Vigilia , Biopsia , Proliferación Celular/efectos de los fármacosRESUMEN
Netrin-1 is upregulated in cancers as a protumoural mechanism1. Here we describe netrin-1 upregulation in a majority of human endometrial carcinomas (ECs) and demonstrate that netrin-1 blockade, using an anti-netrin-1 antibody (NP137), is effective in reduction of tumour progression in an EC mouse model. We next examined the efficacy of NP137, as a first-in-class single agent, in a Phase I trial comprising 14 patients with advanced EC. As best response we observed 8 stable disease (8 out of 14, 57.1%) and 1 objective response as RECIST v.1.1 (partial response, 1 out of 14 (7.1%), 51.16% reduction in target lesions at 6 weeks and up to 54.65% reduction during the following 6 months). To evaluate the NP137 mechanism of action, mouse tumour gene profiling was performed, and we observed, in addition to cell death induction, that NP137 inhibited epithelial-to-mesenchymal transition (EMT). By performing bulk RNA sequencing (RNA-seq), spatial transcriptomics and single-cell RNA-seq on paired pre- and on-treatment biopsies from patients with EC from the NP137 trial, we noted a net reduction in tumour EMT. This was associated with changes in immune infiltrate and increased interactions between cancer cells and the tumour microenvironment. Given the importance of EMT in resistance to current standards of care2, we show in the EC mouse model that a combination of NP137 with carboplatin-paclitaxel outperformed carboplatin-paclitaxel alone. Our results identify netrin-1 blockade as a clinical strategy triggering both tumour debulking and EMT inhibition, thus potentially alleviating resistance to standard treatments.