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1.
Nature ; 612(7939): 338-346, 2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36385526

RESUMEN

Ferroptosis is a non-apoptotic form of regulated cell death that is triggered by the discoordination of regulatory redox mechanisms culminating in massive peroxidation of polyunsaturated phospholipids. Ferroptosis inducers have shown considerable effectiveness in killing tumour cells in vitro, yet there has been no obvious success in experimental animal models, with the notable exception of immunodeficient mice1,2. This suggests that the effect of ferroptosis on immune cells remains poorly understood. Pathologically activated neutrophils (PMNs), termed myeloid-derived suppressor cells (PMN-MDSCs), are major negative regulators of anti-tumour immunity3-5. Here we found that PMN-MDSCs in the tumour microenvironment spontaneously die by ferroptosis. Although decreasing the presence of PMN-MDSCs, ferroptosis induces the release of oxygenated lipids and limits the activity of human and mouse T cells. In immunocompetent mice, genetic and pharmacological inhibition of ferroptosis abrogates suppressive activity of PMN-MDSCs, reduces tumour progression and synergizes with immune checkpoint blockade to suppress the tumour growth. By contrast, induction of ferroptosis in immunocompetent mice promotes tumour growth. Thus, ferroptosis is a unique and targetable immunosuppressive mechanism of PMN-MDSCs in the tumour microenvironment that can be pharmacologically modulated to limit tumour progression.


Asunto(s)
Neoplasias , Humanos , Ratones , Animales , Microambiente Tumoral
2.
Cancer Biol Ther ; 25(1): 2296048, 2024 12 31.
Artículo en Inglés | MEDLINE | ID: mdl-38206570

RESUMEN

CD73 is a cell surface 5'nucleotidase (NT5E) and key node in the catabolic process generating immunosuppressive adenosine in cancer. Using a murine monoclonal antibody surrogate of Oleclumab, we investigated the effect of CD73 inhibition in concert with cytotoxic therapies (chemotherapies as well as fractionated radiotherapy) and PD-L1 blockade. Our results highlight improved survival in syngeneic tumor models of colorectal cancer (CT26 and MC38) and sarcoma (MCA205). This therapeutic outcome was in part driven by cytotoxic CD8 T-cells, as evidenced by the detrimental effect of CD8 depleting antibody treatment of MCA205 tumor bearing mice treated with anti-CD73, anti-PD-L1 and 5-Fluorouracil+Oxaliplatin (5FU+OHP). We hypothesize that the improved responses are tumor microenvironment (TME)-driven, as suggested by the lack of anti-CD73 enhanced cytopathic effects mediated by 5FU+OHP on cell lines in vitro. Pharmacodynamic analysis, using imaging mass cytometry and RNA-sequencing, revealed noteworthy changes in specific cell populations like cytotoxic T cells, B cells and NK cells in the CT26 TME. Transcriptomic analysis highlighted treatment-related modulation of gene profiles associated with an immune response, NK and T-cell activation, T cell receptor signaling and interferon (types 1 & 2) pathways. Inclusion of comparator groups representing the various components of the combination allowed deconvolution of contribution of the individual therapeutic elements; highlighting specific effects mediated by the anti-CD73 antibody with respect to immune-cell representation, chemotaxis and myeloid biology. These pre-clinical data reflect complementarity of adenosine blockade with cytotoxic therapy, and T-cell checkpoint inhibition, and provides new mechanistic insights in support of combination therapy.


Asunto(s)
Anticuerpos Monoclonales , Sarcoma , Animales , Ratones , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Inmunosupresores , Adenosina , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Microambiente Tumoral
3.
Nat Commun ; 15(1): 1700, 2024 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-38402224

RESUMEN

The Ataxia telangiectasia and Rad3-related (ATR) inhibitor ceralasertib in combination with the PD-L1 antibody durvalumab demonstrated encouraging clinical benefit in melanoma and lung cancer patients who progressed on immunotherapy. Here we show that modelling of intermittent ceralasertib treatment in mouse tumor models reveals CD8+ T-cell dependent antitumor activity, which is separate from the effects on tumor cells. Ceralasertib suppresses proliferating CD8+ T-cells on treatment which is rapidly reversed off-treatment. Ceralasertib causes up-regulation of type I interferon (IFNI) pathway in cancer patients and in tumor-bearing mice. IFNI is experimentally found to be a major mediator of antitumor activity of ceralasertib in combination with PD-L1 antibody. Improvement of T-cell function after ceralasertib treatment is linked to changes in myeloid cells in the tumor microenvironment. IFNI also promotes anti-proliferative effects of ceralasertib on tumor cells. Here, we report that broad immunomodulatory changes following intermittent ATR inhibition underpins the clinical therapeutic benefit and indicates its wider impact on antitumor immunity.


Asunto(s)
Linfocitos T CD8-positivos , Indoles , Morfolinas , Neoplasias , Pirimidinas , Sulfonamidas , Humanos , Animales , Ratones , Antígeno B7-H1 , Microambiente Tumoral , Línea Celular Tumoral , Inmunoterapia , Modelos Animales de Enfermedad , Proteínas de la Ataxia Telangiectasia Mutada
4.
J Thorac Oncol ; 18(5): 650-656, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36641093

RESUMEN

INTRODUCTION: CD73 is overexpressed in EGFR-mutated NSCLC and may promote immune evasion, suggesting potential for combining CD73 blockers with EGFR tyrosine kinase inhibitors (TKIs). This phase 1b-2 study (NCT03381274) evaluated the anti-CD73 antibody oleclumab plus the third-generation EGFR TKI osimertinib in advanced EGFR-mutated NSCLC. METHODS: Patients had tissue T790M-negative NSCLC with TKI-sensitive EGFR mutations after progression on a first- or second-generation EGFR TKI and were osimertinib naive. They received osimertinib 80 mg orally once daily plus oleclumab 1500 mg (dose level 1 [DL1]) or 3000 mg (DL2) intravenously every 2 weeks. Primary end points included safety and objective response rate by Response Evaluation Criteria in Solid Tumors version 1.1. RESULTS: By July 9, 2021, five patients received DL1 and 21 received DL2. Of these patients, 60.0% and 85.7% had any-grade treatment-related adverse events (TRAEs) and 20.0% and 14.3% had grade 3 TRAEs, respectively. No dose-limiting toxicities, serious TRAEs, or deaths occurred. Four patients were T790M positive on retrospective circulating tumor DNA (ctDNA) testing; three had objective partial responses. In patients who were T790M negative in tumor and ctDNA, objective response rate was 25.0% at DL1 and 11.8% at DL2 (all partial responses); response durations at DL2 were 14.8 and 16.6 months. In patients receiving DL2, excluding those who were T790M positive by ctDNA, median progression-free survival was 7.4 months, and median overall survival was 24.8 months. DL2 was the recommended phase 2 dose. CONCLUSIONS: Oleclumab plus osimertinib was found to have moderate activity with acceptable tolerability in previously treated patients with advanced EGFR-mutated NSCLC.


Asunto(s)
Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Compuestos de Anilina , Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Receptores ErbB , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Estudios Retrospectivos , 5'-Nucleotidasa/antagonistas & inhibidores
5.
Cancer Discov ; 13(11): 2394-2411, 2023 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-37707791

RESUMEN

Neoadjuvant chemoimmunotherapy improves pathologic complete response rate and event-free survival in patients with resectable non-small cell lung cancer (NSCLC) versus chemotherapy alone. NeoCOAST was the first randomized, multidrug platform trial to examine novel neoadjuvant immuno-oncology combinations for patients with resectable NSCLC, using major pathologic response (MPR) rate as the primary endpoint. Eighty-three patients received a single cycle of treatment: 26 received durvalumab (anti-PD-L1) monotherapy, 21 received durvalumab plus oleclumab (anti-CD73), 20 received durvalumab plus monalizumab (anti-NKG2A), and 16 received durvalumab plus danvatirsen (anti-STAT3 antisense oligonucleotide). MPR rates were higher for patients in the combination arms versus durvalumab alone. Safety profiles for the combinations were similar to those of durvalumab alone. Multiplatform immune profiling suggested that improved MPR rates in the durvalumab plus oleclumab and durvalumab plus monalizumab arms were associated with enhanced effector immune infiltration of tumors, interferon responses and markers of tertiary lymphoid structure formation, and systemic functional immune cell activation. SIGNIFICANCE: A neoadjuvant platform trial can rapidly generate clinical and translational data using candidate surrogate endpoints like MPR. In NeoCOAST, patients with resectable NSCLC had improved MPR rates after durvalumab plus oleclumab or monalizumab versus durvalumab alone and tumoral transcriptomic signatures indicative of augmented immune cell activation and function. See related commentary by Cooper and Yu, p. 2306. This article is featured in Selected Articles from This Issue, p. 2293.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Anticuerpos Monoclonales/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Terapia Neoadyuvante
6.
J Proteome Res ; 11(2): 1196-207, 2012 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-22149024

RESUMEN

Ubiquitination is crucial for many cellular processes such as protein degradation, DNA repair, transcription regulation, and cell signaling. Ubiquitin attachment takes place via a sequential enzymatic cascade involving ubiquitin activation (by E1 enzymes), ubiquitin conjugation (by E2 enzymes), and ubiquitin substrate tagging (by E3 enzymes). E3 ligases mediate ubiquitin transfer from E2s to substrates and as such confer substrate specificity. Although E3s can interact and function with numerous E2s, it is still unclear how they choose which E2 to use. Identifying all E2 partners of an E3 is essential for inferring the principles guiding E2 selection by an E3. Here we model the interactions of E3 and E2 proteins in a large, proteome-scale strategy based on interface structural motifs, which allows elucidation of (1) which E3s interact with which E2s in the human ubiquitination pathway and (2) how they interact with each other. Interface analysis of E2-E3 complexes reveals that loop L1 of E2s is critical for binding; the residue in the sixth position in loop L1 is widely utilized as an interface hot spot and appears indispensible for E2 interactions. Other loop L1 residues also confer specificity on the E2-E3 interactions: HECT E3s are in contact with the residue in the second position in loop L1 of E2s, but this is not the case for the RING finger type E3s. Our modeled E2-E3 complexes illuminate how slight sequence variations in E2 residues may contribute to specificity in E3 binding. These findings may be important for discovering drug candidates targeting E3s, which have been implicated in many diseases.


Asunto(s)
Proteómica/métodos , Enzimas Ubiquitina-Conjugadoras/química , Ubiquitina-Proteína Ligasas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Simulación por Computador , Bases de Datos de Proteínas , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Alineación de Secuencia , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
7.
J Immunother Cancer ; 10(4)2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35387780

RESUMEN

BACKGROUND: The Regulatory T cell (Treg) lineage is defined by the transcription factor FOXP3, which controls immune-suppressive gene expression profiles. Tregs are often recruited in high frequencies to the tumor microenvironment where they can suppress antitumor immunity. We hypothesized that pharmacological inhibition of FOXP3 by systemically delivered, unformulated constrained ethyl-modified antisense oligonucleotides could modulate the activity of Tregs and augment antitumor immunity providing therapeutic benefit in cancer models and potentially in man. METHODS: We have identified murine Foxp3 antisense oligonucleotides (ASOs) and clinical candidate human FOXP3 ASO AZD8701. Pharmacology and biological effects of FOXP3 inhibitors on Treg function and antitumor immunity were tested in cultured Tregs and mouse syngeneic tumor models. Experiments were controlled by vehicle and non-targeting control ASO groups as well as by use of multiple independent FOXP3 ASOs. Statistical significance of biological effects was evaluated by one or two-way analysis of variance with multiple comparisons. RESULTS: AZD8701 demonstrated a dose-dependent knockdown of FOXP3 in primary Tregs, reduction of suppressive function and efficient target downregulation in humanized mice at clinically relevant doses. Surrogate murine FOXP3 ASO, which efficiently downregulated Foxp3 messenger RNA and protein levels in primary Tregs, reduced Treg suppressive function in immune suppression assays in vitro. FOXP3 ASO promoted more than 70% reduction in FOXP3 levels in Tregs in vitro and in vivo, strongly modulated Treg effector molecules (eg, ICOS, CTLA-4, CD25 and 4-1BB), and augmented CD8+ T cell activation and produced antitumor activity in syngeneic tumor models. The combination of FOXP3 ASOs with immune checkpoint blockade further enhanced antitumor efficacy. CONCLUSIONS: Antisense inhibitors of FOXP3 offer a promising novel cancer immunotherapy approach. AZD8701 is being developed clinically as a first-in-class FOXP3 inhibitor for the treatment of cancer currently in Ph1a/b clinical trial (NCT04504669).


Asunto(s)
Neoplasias , Oligonucleótidos Antisentido , Animales , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Terapia de Inmunosupresión , Inmunoterapia , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Linfocitos T Reguladores , Microambiente Tumoral
8.
Brief Bioinform ; 10(3): 217-32, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19240123

RESUMEN

The unanimous agreement that cellular processes are (largely) governed by interactions between proteins has led to enormous community efforts culminating in overwhelming information relating to these proteins; to the regulation of their interactions, to the way in which they interact and to the function which is determined by these interactions. These data have been organized in databases and servers. However, to make these really useful, it is essential not only to be aware of these, but in particular to have a working knowledge of which tools to use for a given problem; what are the tool advantages and drawbacks; and no less important how to combine these for a particular goal since usually it is not one tool, but some combination of tool-modules that is needed. This is the goal of this review.


Asunto(s)
Bases de Datos de Proteínas , Internet , Mapeo de Interacción de Proteínas , Proteínas/química , Animales , Sitios de Unión , Humanos , Modelos Moleculares , Conformación Proteica , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal/fisiología , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
9.
J Clin Invest ; 131(16)2021 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-34228641

RESUMEN

Myeloid-derived suppressor cells (MDSCs) are major negative regulators of immune responses in cancer and chronic infections. It remains unclear if regulation of MDSC activity in different conditions is controlled by similar mechanisms. We compared MDSCs in mice with cancer and lymphocytic choriomeningitis virus (LCMV) infection. Chronic LCMV infection caused the development of monocytic MDSCs (M-MDSCs) but did not induce polymorphonuclear MDSCs (PMN-MDSCs). In contrast, both MDSC populations were present in cancer models. An acquisition of immune-suppressive activity by PMN-MDSCs in cancer was controlled by IRE1α and ATF6 pathways of the endoplasmic reticulum (ER) stress response. Abrogation of PMN-MDSC activity by blockade of the ER stress response resulted in an increase in tumor-specific immune response and reduced tumor progression. In contrast, the ER stress response was dispensable for suppressive activity of M-MDSCs in cancer and LCMV infection. Acquisition of immune-suppressive activity by M-MDSCs in spleens was mediated by IFN-γ signaling. However, it was dispensable for suppressive activity of M-MDSCs in tumor tissues. Suppressive activity of M-MDSCs in tumors was retained due to the effect of IL-6 present at high concentrations in the tumor site. These results demonstrate disease- and population-specific mechanisms of MDSC accumulation and the need for targeting different pathways to achieve inactivation of these cells.


Asunto(s)
Células Supresoras de Origen Mieloide/inmunología , Neoplasias/inmunología , Virosis/inmunología , Animales , Línea Celular Tumoral , Enfermedad Crónica , Estrés del Retículo Endoplásmico/genética , Estrés del Retículo Endoplásmico/inmunología , Femenino , Humanos , Tolerancia Inmunológica/genética , Interferón gamma/inmunología , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/clasificación , Virus de la Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/patogenicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Supresoras de Origen Mieloide/clasificación , Células Supresoras de Origen Mieloide/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/metabolismo , Transcriptoma , Virosis/genética , Virosis/metabolismo
10.
PLoS One ; 16(5): e0251233, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34003838

RESUMEN

The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells in general, but with an emphasis on Th2 cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Macrófagos/inmunología , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Neumonía/inmunología , Células Th2/inmunología , Animales , Antígenos Helmínticos/inmunología , Antígenos Helmínticos/metabolismo , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Nippostrongylus/inmunología , Neumonía/parasitología , Neumonía/patología , Infecciones por Strongylida/inmunología , Infecciones por Strongylida/parasitología
11.
PLoS Comput Biol ; 5(12): e1000601, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-20011507

RESUMEN

Protein-protein interaction networks provide a global picture of cellular function and biological processes. Some proteins act as hub proteins, highly connected to others, whereas some others have few interactions. The dysfunction of some interactions causes many diseases, including cancer. Proteins interact through their interfaces. Therefore, studying the interface properties of cancer-related proteins will help explain their role in the interaction networks. Similar or overlapping binding sites should be used repeatedly in single interface hub proteins, making them promiscuous. Alternatively, multi-interface hub proteins make use of several distinct binding sites to bind to different partners. We propose a methodology to integrate protein interfaces into cancer interaction networks (ciSPIN, cancer structural protein interface network). The interactions in the human protein interaction network are replaced by interfaces, coming from either known or predicted complexes. We provide a detailed analysis of cancer related human protein-protein interfaces and the topological properties of the cancer network. The results reveal that cancer-related proteins have smaller, more planar, more charged and less hydrophobic binding sites than non-cancer proteins, which may indicate low affinity and high specificity of the cancer-related interactions. We also classified the genes in ciSPIN according to phenotypes. Within phenotypes, for breast cancer, colorectal cancer and leukemia, interface properties were found to be discriminating from non-cancer interfaces with an accuracy of 71%, 67%, 61%, respectively. In addition, cancer-related proteins tend to interact with their partners through distinct interfaces, corresponding mostly to multi-interface hubs, which comprise 56% of cancer-related proteins, and constituting the nodes with higher essentiality in the network (76%). We illustrate the interface related affinity properties of two cancer-related hub proteins: Erbb3, a multi interface, and Raf1, a single interface hub. The results reveal that affinity of interactions of the multi-interface hub tends to be higher than that of the single-interface hub. These findings might be important in obtaining new targets in cancer as well as finding the details of specific binding regions of putative cancer drug candidates.


Asunto(s)
Biología Computacional/métodos , Neoplasias/química , Neoplasias/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas/análisis , Animales , Sitios de Unión , Regulación Neoplásica de la Expresión Génica , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Unión Proteica , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Transducción de Señal
12.
Cell Rep ; 31(7): 107628, 2020 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-32433953

RESUMEN

Here, using single-cell RNA sequencing, we examine the stromal compartment in murine melanoma and draining lymph nodes (LNs) at points across tumor development, providing data at http://www.teichlab.org/data/. Naive lymphocytes from LNs undergo activation and clonal expansion within the tumor, before PD1 and Lag3 expression, while tumor-associated myeloid cells promote the formation of a suppressive niche. We identify three temporally distinct stromal populations displaying unique functional signatures, conserved across mouse and human tumors. Whereas "immune" stromal cells are observed in early tumors, "contractile" cells become more prevalent at later time points. Complement component C3 is specifically expressed in the immune population. Its cleavage product C3a supports the recruitment of C3aR+ macrophages, and perturbation of C3a and C3aR disrupts immune infiltration, slowing tumor growth. Our results highlight the power of scRNA-seq to identify complex interplays and increase stromal diversity as a tumor develops, revealing that stromal cells acquire the capacity to modulate immune landscapes from early disease.


Asunto(s)
Melanoma/inmunología , Análisis de Secuencia de ARN/métodos , Células del Estroma/metabolismo , Microambiente Tumoral/inmunología , Animales , Humanos , Ratones
13.
Nat Commun ; 11(1): 3588, 2020 07 17.
Artículo en Inglés | MEDLINE | ID: mdl-32680985

RESUMEN

Tumors subvert immune cell function to evade immune responses, yet the complex mechanisms driving immune evasion remain poorly understood. Here we show that tumors induce de novo steroidogenesis in T lymphocytes to evade anti-tumor immunity. Using a transgenic steroidogenesis-reporter mouse line we identify and characterize de novo steroidogenic immune cells, defining the global gene expression identity of these steroid-producing immune cells and gene regulatory networks by using single-cell transcriptomics. Genetic ablation of T cell steroidogenesis restricts primary tumor growth and metastatic dissemination in mouse models. Steroidogenic T cells dysregulate anti-tumor immunity, and inhibition of the steroidogenesis pathway is sufficient to restore anti-tumor immunity. This study demonstrates T cell de novo steroidogenesis as a mechanism of anti-tumor immunosuppression and a potential druggable target.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Melanoma/inmunología , Esteroides/inmunología , Animales , Linfocitos T CD4-Positivos/metabolismo , Línea Celular Tumoral , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/inmunología , Humanos , Evasión Inmune , Melanoma/genética , Melanoma/metabolismo , Ratones , Ratones Noqueados , Esteroides/biosíntesis
14.
Nat Commun ; 10(1): 3350, 2019 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-31350390

RESUMEN

The liver parenchyma is composed of hepatocytes and bile duct epithelial cells (BECs). Controversy exists regarding the cellular origin of human liver parenchymal tissue generation during embryonic development, homeostasis or repair. Here we report the existence of a hepatobiliary hybrid progenitor (HHyP) population in human foetal liver using single-cell RNA sequencing. HHyPs are anatomically restricted to the ductal plate of foetal liver and maintain a transcriptional profile distinct from foetal hepatocytes, mature hepatocytes and mature BECs. In addition, molecular heterogeneity within the EpCAM+ population of freshly isolated foetal and adult human liver identifies diverse gene expression signatures of hepatic and biliary lineage potential. Finally, we FACS isolate foetal HHyPs and confirm their hybrid progenitor phenotype in vivo. Our study suggests that hepatobiliary progenitor cells previously identified in mice also exist in humans, and can be distinguished from other parenchymal populations, including mature BECs, by distinct gene expression profiles.


Asunto(s)
Hígado/citología , Transcripción Genética , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Molécula de Adhesión Celular Epitelial/genética , Molécula de Adhesión Celular Epitelial/metabolismo , Feto/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Análisis de la Célula Individual , Células Madre/citología , Células Madre/metabolismo
15.
Nat Med ; 25(7): 1153-1163, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31209336

RESUMEN

Human lungs enable efficient gas exchange and form an interface with the environment, which depends on mucosal immunity for protection against infectious agents. Tightly controlled interactions between structural and immune cells are required to maintain lung homeostasis. Here, we use single-cell transcriptomics to chart the cellular landscape of upper and lower airways and lung parenchyma in healthy lungs, and lower airways in asthmatic lungs. We report location-dependent airway epithelial cell states and a novel subset of tissue-resident memory T cells. In the lower airways of patients with asthma, mucous cell hyperplasia is shown to stem from a novel mucous ciliated cell state, as well as goblet cell hyperplasia. We report the presence of pathogenic effector type 2 helper T cells (TH2) in asthmatic lungs and find evidence for type 2 cytokines in maintaining the altered epithelial cell states. Unbiased analysis of cell-cell interactions identifies a shift from airway structural cell communication in healthy lungs to a TH2-dominated interactome in asthmatic lungs.


Asunto(s)
Asma/patología , Pulmón/citología , Adulto , Anciano , Linfocitos T CD4-Positivos/fisiología , Comunicación Celular , Células Epiteliales/inmunología , Células Epiteliales/fisiología , Femenino , Estudio de Asociación del Genoma Completo , Células Caliciformes/metabolismo , Humanos , Pulmón/inmunología , Pulmón/patología , Masculino , Metaplasia , Persona de Mediana Edad , Células Th2/fisiología , Transcriptoma
16.
Sci Rep ; 8(1): 685, 2018 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-29330484

RESUMEN

The crucial capability of T cells for discrimination between self and non-self peptides is based on negative selection of developing thymocytes by medullary thymic epithelial cells (mTECs). The mTECs purge autoreactive T cells by expression of cell-type specific genes referred to as tissue-restricted antigens (TRAs). Although the autoimmune regulator (AIRE) protein is known to promote the expression of a subset of TRAs, its mechanism of action is still not fully understood. The expression of TRAs that are not under the control of AIRE also needs further characterization. Furthermore, expression patterns of TRA genes have been suggested to change over the course of mTEC development. Herein we have used single-cell RNA-sequencing to resolve patterns of TRA expression during mTEC development. Our data indicated that mTEC development consists of three distinct stages, correlating with previously described jTEC, mTEChi and mTEClo phenotypes. For each subpopulation, we have identified marker genes useful in future studies. Aire-induced TRAs were switched on during jTEC-mTEC transition and were expressed in genomic clusters, while otherwise the subsets expressed largely overlapping sets of TRAs. Moreover, population-level analysis of TRA expression frequencies suggested that such differences might not be necessary to achieve efficient thymocyte selection.


Asunto(s)
Autoantígenos/genética , Células Epiteliales/metabolismo , ARN/metabolismo , Animales , Autoantígenos/metabolismo , Diferenciación Celular , Células Epiteliales/citología , Femenino , Fase G1 , Redes Reguladoras de Genes/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Componente Principal , ARN/química , ARN/aislamiento & purificación , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Timo/citología , Factores de Transcripción/metabolismo , Transcriptoma , Proteína AIRE
17.
Genome Med ; 10(1): 76, 2018 10 24.
Artículo en Inglés | MEDLINE | ID: mdl-30355343

RESUMEN

BACKGROUND: The IRE1a-XBP1 pathway is a conserved adaptive mediator of the unfolded protein response. The pathway is indispensable for the development of secretory cells by facilitating protein folding and enhancing secretory capacity. In the immune system, it is known to function in dendritic cells, plasma cells, and eosinophil development and differentiation, while its role in T helper cell is unexplored. Here, we investigated the role of the IRE1a-XBP1 pathway in regulating activation and differentiation of type-2 T helper cell (Th2), a major T helper cell type involved in allergy, asthma, helminth infection, pregnancy, and tumor immunosuppression. METHODS: We perturbed the IRE1a-XBP1 pathway and interrogated its role in Th2 cell differentiation. We performed genome-wide transcriptomic analysis of differential gene expression to reveal IRE1a-XBP1 pathway-regulated genes and predict their biological role. To identify direct target genes of XBP1 and define XBP1's regulatory network, we performed XBP1 ChIPmentation (ChIP-seq). We validated our predictions by flow cytometry, ELISA, and qPCR. We also used a fluorescent ubiquitin cell cycle indicator mouse to demonstrate the role of XBP1 in the cell cycle. RESULTS: We show that Th2 lymphocytes induce the IRE1a-XBP1 pathway during in vitro and in vivo activation. Genome-wide transcriptomic analysis of differential gene expression by perturbing the IRE1a-XBP1 pathway reveals XBP1-controlled genes and biological pathways. Performing XBP1 ChIPmentation (ChIP-seq) and integrating with transcriptomic data, we identify XBP1-controlled direct target genes and its transcriptional regulatory network. We observed that the IRE1a-XBP1 pathway controls cytokine secretion and the expression of two Th2 signature cytokines, IL13 and IL5. We also discovered that the IRE1a-XBP1 pathway facilitates activation-dependent Th2 cell proliferation by facilitating cell cycle progression through S and G2/M phase. CONCLUSIONS: We confirm and detail the critical role of the IRE1a-XBP1 pathway during Th2 lymphocyte activation in regulating cytokine expression, secretion, and cell proliferation. Our high-quality genome-wide XBP1 ChIP and gene expression data provide a rich resource for investigating XBP1-regulated genes. We provide a browsable online database available at http://data.teichlab.org .


Asunto(s)
Diferenciación Celular , Endorribonucleasas/metabolismo , Estudio de Asociación del Genoma Completo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Estrés Fisiológico , Células Th2/citología , Proteína 1 de Unión a la X-Box/metabolismo , Animales , Secuencia de Bases , Ciclo Celular , Proliferación Celular , Citocinas/metabolismo , Endorribonucleasas/genética , Femenino , Regulación de la Expresión Génica , Activación de Linfocitos/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/genética , Regulación hacia Arriba/genética , Proteína 1 de Unión a la X-Box/genética
18.
FEBS Lett ; 591(15): 2213-2225, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28524227

RESUMEN

The recent developments in high-throughput single-cell RNA sequencing technology (scRNA-seq) have enabled the generation of vast amounts of transcriptomic data at cellular resolution. With these advances come new modes of data analysis, building on high-dimensional data mining techniques. Here, we consider biological questions for which scRNA-seq data is used, both at a cell and gene level, and describe tools available for these types of analyses. This is an exciting and rapidly evolving field, where clustering, pseudotime inference, branching inference and gene-level analyses are particularly informative areas of computational analysis.


Asunto(s)
Biología Computacional/métodos , Expresión Génica , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Animales , Análisis por Conglomerados , Humanos
19.
Nat Cell Biol ; 19(6): 603-613, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28504705

RESUMEN

The epidermis is maintained by multiple stem cell populations whose progeny differentiate along diverse, and spatially distinct, lineages. Here we show that the transcription factor Gata6 controls the identity of the previously uncharacterized sebaceous duct (SD) lineage and identify the Gata6 downstream transcription factor network that specifies a lineage switch between sebocytes and SD cells. During wound healing differentiated Gata6+ cells migrate from the SD into the interfollicular epidermis and dedifferentiate, acquiring the ability to undergo long-term self-renewal and differentiate into a much wider range of epidermal lineages than in undamaged tissue. Our data not only demonstrate that the structural and functional complexity of the junctional zone is regulated by Gata6, but also reveal that dedifferentiation is a previously unrecognized property of post-mitotic, terminally differentiated cells that have lost contact with the basement membrane. This resolves the long-standing debate about the contribution of terminally differentiated cells to epidermal wound repair.


Asunto(s)
Desdiferenciación Celular , Epidermis/metabolismo , Factor de Transcripción GATA6/metabolismo , Glándulas Sebáceas/metabolismo , Células Madre/metabolismo , Cicatrización de Heridas , Heridas y Lesiones/metabolismo , Animales , Linaje de la Célula , Movimiento Celular , Plasticidad de la Célula , Autorrenovación de las Células , Células Cultivadas , Modelos Animales de Enfermedad , Epidermis/patología , Femenino , Factor de Transcripción GATA6/deficiencia , Factor de Transcripción GATA6/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Glándulas Sebáceas/patología , Transducción de Señal , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Heridas y Lesiones/genética , Heridas y Lesiones/patología
20.
Nat Commun ; 8(1): 36, 2017 06 26.
Artículo en Inglés | MEDLINE | ID: mdl-28652613

RESUMEN

Polycomb repressive complexes (PRCs) are important histone modifiers, which silence gene expression; yet, there exists a subset of PRC-bound genes actively transcribed by RNA polymerase II (RNAPII). It is likely that the role of Polycomb repressive complex is to dampen expression of these PRC-active genes. However, it is unclear how this flipping between chromatin states alters the kinetics of transcription. Here, we integrate histone modifications and RNAPII states derived from bulk ChIP-seq data with single-cell RNA-sequencing data. We find that Polycomb repressive complex-active genes have greater cell-to-cell variation in expression than active genes, and these results are validated by knockout experiments. We also show that PRC-active genes are clustered on chromosomes in both two and three dimensions, and interactions with active enhancers promote a stabilization of gene expression noise. These findings provide new insights into how chromatin regulation modulates stochastic gene expression and transcriptional bursting, with implications for regulation of pluripotency and development.Polycomb repressive complexes modify histones but it is unclear how changes in chromatin states alter kinetics of transcription. Here, the authors use single-cell RNAseq and ChIPseq to find that actively transcribed genes with Polycomb marks have greater cell-to-cell variation in expression.


Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteínas del Grupo Polycomb/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Marcadores Genéticos , Ratones , Ratones Noqueados , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Proteínas del Grupo Polycomb/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
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