RESUMEN
Expansion of antigen-experienced CD8+ T cells is critical for the success of tumour-infiltrating lymphocyte (TIL)-adoptive cell therapy (ACT) in patients with cancer1. Interleukin-2 (IL-2) acts as a key regulator of CD8+ cytotoxic T lymphocyte functions by promoting expansion and cytotoxic capability2,3. Therefore, it is essential to comprehend mechanistic barriers to IL-2 sensing in the tumour microenvironment to implement strategies to reinvigorate IL-2 responsiveness and T cell antitumour responses. Here we report that prostaglandin E2 (PGE2), a known negative regulator of immune response in the tumour microenvironment4,5, is present at high concentrations in tumour tissue from patients and leads to impaired IL-2 sensing in human CD8+ TILs via the PGE2 receptors EP2 and EP4. Mechanistically, PGE2 inhibits IL-2 sensing in TILs by downregulating the IL-2Rγc chain, resulting in defective assembly of IL-2Rß-IL2Rγc membrane dimers. This results in impaired IL-2-mTOR adaptation and PGC1α transcriptional repression, causing oxidative stress and ferroptotic cell death in tumour-reactive TILs. Inhibition of PGE2 signalling to EP2 and EP4 during TIL expansion for ACT resulted in increased IL-2 sensing, leading to enhanced proliferation of tumour-reactive TILs and enhanced tumour control once the cells were transferred in vivo. Our study reveals fundamental features that underlie impairment of human TILs mediated by PGE2 in the tumour microenvironment. These findings have therapeutic implications for cancer immunotherapy and cell therapy, and enable the development of targeted strategies to enhance IL-2 sensing and amplify the IL-2 response in TILs, thereby promoting the expansion of effector T cells with enhanced therapeutic potential.
Asunto(s)
Linfocitos T CD8-positivos , Proliferación Celular , Dinoprostona , Interleucina-2 , Linfocitos Infiltrantes de Tumor , Mitocondrias , Transducción de Señal , Animales , Humanos , Ratones , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Dinoprostona/metabolismo , Regulación hacia Abajo , Ferroptosis , Subunidad gamma Común de Receptores de Interleucina/biosíntesis , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Interleucina-2/antagonistas & inhibidores , Interleucina-2/inmunología , Interleucina-2/metabolismo , Subunidad beta del Receptor de Interleucina-2/metabolismo , Linfocitos Infiltrantes de Tumor/citología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Microambiente Tumoral/inmunologíaRESUMEN
Innate lymphoid cells (ILCs) have potent immunological functions in experimental conditions in mice, but their contributions to immunity in natural conditions in humans have remained unclear. We investigated the presence of ILCs in a cohort of patients with severe combined immunodeficiency (SCID). All ILC subsets were absent in patients with SCID who had mutation of the gene encoding the common γ-chain cytokine receptor subunit IL-2Rγ or the gene encoding the tyrosine kinase JAK3. T cell reconstitution was observed in patients with SCID after hematopoietic stem cell transplantation (HSCT), but the patients still had considerably fewer ILCs in the absence of myeloablation than did healthy control subjects, with the exception of rare cases of reconstitution of the ILC1 subset of ILCs. Notably, the ILC deficiencies observed were not associated with any particular susceptibility to disease, with follow-up extending from 7 years to 39 years after HSCT. We thus report here selective ILC deficiency in humans and show that ILCs might be dispensable in natural conditions, if T cells are present and B cell function is preserved.
Asunto(s)
Inmunidad Innata , Linfocitos/inmunología , Adolescente , Adulto , Animales , Biomarcadores , Niño , Modelos Animales de Enfermedad , Supervivencia de Injerto , Trasplante de Células Madre Hematopoyéticas , Humanos , Sistema Inmunológico/citología , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismo , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Janus Quinasa 3/deficiencia , Recuento de Linfocitos , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Linfocitos/metabolismo , Linfopenia/sangre , Linfopenia/etiología , Ratones , Ratones Noqueados , Fenotipo , Inmunodeficiencia Combinada Grave/sangre , Inmunodeficiencia Combinada Grave/inmunología , Inmunodeficiencia Combinada Grave/metabolismo , Inmunodeficiencia Combinada Grave/terapia , Piel/inmunología , Piel/patologíaRESUMEN
Aire is a transcriptional regulator that induces the promiscuous expression of thousands of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs), a step critical for the induction of immunological self-tolerance. Studies have offered molecular insights into how Aire operates, but more comprehensive understanding of this process still remains elusive. Here we found abundant expression of the protein deacetylase Sirtuin-1 (Sirt1) in mature Aire(+) mTECs, wherein it was required for the expression of Aire-dependent TRA-encoding genes and the subsequent induction of immunological self-tolerance. Our study elucidates a previously unknown molecular mechanism for Aire-mediated transcriptional regulation and identifies a unique function for Sirt1 in preventing organ-specific autoimmunity.
Asunto(s)
Tolerancia Central/inmunología , Sirtuina 1/inmunología , Factores de Transcripción/inmunología , Activación Transcripcional/inmunología , Acetilación , Animales , Antígenos/inmunología , Tolerancia Central/genética , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Citometría de Flujo , Células HEK293 , Humanos , Immunoblotting , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Especificidad de Órganos/inmunología , Unión Proteica/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirtuina 1/genética , Sirtuina 1/metabolismo , Timo/citología , Timo/inmunología , Timo/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma/inmunología , Proteína AIRERESUMEN
The classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127- and CD127+ early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127- and CD127+ ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127- ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127+ ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis.
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Linfocitos B/metabolismo , Células Asesinas Naturales/metabolismo , Células Progenitoras Linfoides/metabolismo , Linfopoyesis/genética , Linfocitos T/metabolismo , Adolescente , Adulto , Animales , Linfocitos B/citología , Diferenciación Celular/genética , Linaje de la Célula/genética , Células Cultivadas , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad alfa del Receptor de Interleucina-7/genética , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Células Asesinas Naturales/citología , Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/trasplante , Masculino , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Persona de Mediana Edad , Trasplante de Células Madre , Linfocitos T/citología , Trasplante Heterólogo , Adulto JovenRESUMEN
CD4dim CD8bright T cells are a mature population of CD8+ T cells that upon activation upregulate CD4 dimly on their surface. Expression of CD4 on these cells suggests that they can be an additional source of HIV neuroinvasion and persistence in the brain. We used HIV-infected NOD/SCID/IL-2rcγ-/- (NSG) humanized mice to track CD4dim CD8bright T cell homing to the brain and define their role in HIV dissemination into the brain. We report here that CD4dim CD8bright T cells are found in the brain at a median frequency of 2.6% and in the spleen at median frequency of 7.6% of CD3+ T cells. In the brain, 10 to 20% of CD4dim CD8bright T cells contain integrated provirus, which is infectious as demonstrated by viral outgrowth assay. CD4dim CD8bright T cells in the brain exhibited significantly higher expression of the brain homing receptors CX3CR1 and CXCR3 in comparison to their single-positive CD8+ T cell counterpart. Blocking lymphocyte trafficking into the brain of humanized mice via anti-VLA4 and anti-LFA1 antibodies reduced CD4dim CD8bright T cell trafficking into the brain by 60% and diminished brain HIV proviral DNA by 72%. Collectively, our findings demonstrate that CD4dim CD8bright T cells can home to the brain and support productive HIV replication. These studies also reveal for the first time that CD4dim CD8bright T cells are capable of HIV neuroinvasion and are a reservoir for HIV. IMPORTANCE We report here a seminal finding of a novel population of T cells, termed CD4dim CD8bright T cells, that plays a role in HIV neuroinvasion and is a reservoir for HIV in the brain.
Asunto(s)
Encéfalo , Antígenos CD4 , Antígenos CD8 , Linfocitos T CD8-positivos , Movimiento Celular , Infecciones por VIH , VIH-1 , Tropismo Viral , Animales , Encéfalo/inmunología , Encéfalo/metabolismo , Encéfalo/virología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Receptor 1 de Quimiocinas CX3C/metabolismo , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/patogenicidad , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Ratones , Ratones Endogámicos NOD , Ratones SCID , Provirus/genética , Provirus/aislamiento & purificación , Receptores CXCR3/metabolismo , Receptores Mensajeros de Linfocitos/metabolismoRESUMEN
Erythropoiesis is a complex multistage process that involves differentiation of early erythroid progenitors to enucleated mature red blood cells, in which lineage-specific transcription factors play essential roles. Erythroid Krüppel-like factor (EKLF/KLF1) is a pleiotropic erythroid transcription factor that is required for the proper maturation of the erythroid cells, whose expression and activation are tightly controlled in a temporal and differentiation stage-specific manner. Here, we uncover a novel role of G-protein pathway suppressor 2 (GPS2), a subunit of the nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid hormone receptor corepressor complex, in erythrocyte differentiation. Our study demonstrates that knockdown of GPS2 significantly suppresses erythroid differentiation of human CD34+ cells cultured in vitro and xenotransplanted in nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor γ-chain null mice. Moreover, global deletion of GPS2 in mice causes impaired erythropoiesis in the fetal liver and leads to severe anemia. Flow cytometric analysis and Wright-Giemsa staining show a defective differentiation at late stages of erythropoiesis in Gps2-/- embryos. Mechanistically, GPS2 interacts with EKLF and prevents proteasome-mediated degradation of EKLF, thereby increasing EKLF stability and transcriptional activity. Moreover, we identify the amino acids 191-230 region in EKLF protein, responsible for GPS2 binding, that is highly conserved in mammals and essential for EKLF protein stability. Collectively, our study uncovers a previously unknown role of GPS2 as a posttranslational regulator that enhances the stability of EKLF protein and thereby promotes erythroid differentiation.
Asunto(s)
Eritropoyesis/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Factores de Transcripción de Tipo Kruppel/fisiología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Secuencia Conservada , Células Precursoras Eritroides/citología , Técnicas de Silenciamiento del Gen , Trasplante de Células Madre Hematopoyéticas , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Factores de Transcripción de Tipo Kruppel/química , Hígado/embriología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Proteolisis , Interferencia de ARN , ARN Interferente Pequeño/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética , Trasplante Heterólogo , Ubiquitinación , Regulación hacia ArribaRESUMEN
Lymph nodes (LNs) facilitate the cellular interactions that orchestrate immune responses. Human immune system (HIS) mice are powerful tools for interrogation of human immunity but lack secondary lymphoid tissue (SLT) as a result of a deficiency in Il2rg-dependent lymphoid tissue inducer cells. To restore LN development, we induced expression of thymic-stromal-cell-derived lymphopoietin (TSLP) in a Balb/c Rag2-/-Il2rg-/-SirpaNOD (BRGS) HIS mouse model. The resulting BRGST HIS mice developed a full array of LNs with compartmentalized human B and T cells. Compared with BRGS HIS mice, BRGST HIS mice have a larger thymus, more mature B cells, and abundant IL-21-producing follicular helper T (TFH) cells, and show enhanced antigen-specific responses. Using BRGST HIS mice, we demonstrated that LN TFH cells are targets of acute HIV infection and represent a reservoir for latent HIV. In summary, BRGST HIS mice reflect the effects of SLT development on human immune responses and provide a model for visualization and interrogation of regulators of immunity.
Asunto(s)
Ganglios Linfáticos/crecimiento & desarrollo , Ganglios Linfáticos/inmunología , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Citocinas/genética , Citocinas/inmunología , Femenino , Infecciones por VIH/inmunología , VIH-1 , Humanos , Cambio de Clase de Inmunoglobulina , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Ganglios Linfáticos/citología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Inmunológicos , Linfocitos T/citología , Linfocitos T/inmunología , Latencia del Virus/inmunología , Linfopoyetina del Estroma TímicoAsunto(s)
Edición Génica , Subunidad gamma Común de Receptores de Interleucina , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X , Humanos , Subunidad gamma Común de Receptores de Interleucina/genética , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Edición Génica/métodos , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/genética , Enfermedades por Inmunodeficiencia Combinada Ligada al Cromosoma X/terapia , Marcación de Gen , Terapia Genética/métodos , Adenina/análogos & derivados , Animales , Sistemas CRISPR-CasRESUMEN
MicroRNA-138 (miR-138) is a pro-survival oncomiR for glioma stem cells. In malignant gliomas, dysregulated expression of microRNAs, such as miR-138, promotes Tumour initiation and progression. Here, we identify the ancillary role of the CCAAT/enhancer binding protein ß (C/EBPß) as a transcriptional activator of miR-138. We demonstrate that a short 158 bp DNA sequence encoding the precursor of miR-138-2 is essential and sufficient for transcription of miR-138. This short sequence includes the A-box and B-box elements characteristic of RNA Polymerase III (Pol III) promoters, and is also directly bound by C/EBPß via an embedded 'C/EBPß responsive element' (CRE). CRE and the Pol III B-box element overlap, suggesting that C/EBPß and transcription factor 3C (TFIIIC) interact at the miR-138-2 locus. We propose that this interaction is essential for the recruitment of the RNA Pol III initiation complex and associated transcription of the oncomiR, miR-138 in malignant gliomas.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/genética , Glioma/genética , MicroARNs/genética , ARN Polimerasa III/metabolismo , Transcripción Genética , Animales , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Glioma/metabolismo , Glioma/terapia , Células HEK293 , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Oncogenes/genética , Unión Proteica , Interferencia de ARN , Tratamiento con ARN de Interferencia/métodos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
BACKGROUND: Loss of monoubiquitination of histone H2B (H2Bub1) was found to be associated with poor differentiation, cancer stemness, and enhanced malignancy of non-small cell lung cancer (NSCLC). Herein, we investigated the biological significance and therapeutic implications of ubiquitin-specific protease 22 (USP22), an H2Bub1 deubiquitinase, in non-small cell lung cancer (NSCLC). METHODS: USP22 expression and its clinical relevance were assessed in NSCLC patients. The effects of USP22 knockout on sensitivity to cisplatin and irradiation, and growth, metastasis of NSCLC xenografts, and survival of cancer-bearing mice were investigated. The underlying mechanisms of targeting USP22 were explored. RESULTS: Overexpression of USP22 was observed in 49.0% (99/202) of NSCLC tissues; higher USP22 immunostaining was found to be associated with enhanced angiogenesis and recurrence of NSCLC. Notably, USP22 knockout dramatically suppressed in vitro proliferation, colony formation; and angiogenesis, growth, metastasis of A549 and H1299 in mouse xenograft model, and significantly prolonged survival of metastatic cancer-bearing mice. Furthermore, USP22 knockout significantly impaired non-homologous DNA damage repair capacity, enhanced cisplatin and irradiation-induced apoptosis in these cells. In terms of underlying mechanisms, RNA sequencing and gene ontology enrichment analysis demonstrated that USP22 knockout significantly suppressed angiogenesis, proliferation, EMT, RAS, c-Myc pathways, concurrently enhanced oxidative phosphorylation and tight junction pathways in A549 and H1299 NSCLC cells. Immunoblot analysis confirmed that USP22 knockout upregulated E-cadherin, p16; reduced ALDH1A3, Cyclin E1, c-Myc, and attenuated activation of AKT and ERK pathways in these cells. CONCLUSIONS: Our findings suggest USP22 plays critical roles in the malignancy and progression of NSCLC and provide rationales for targeting USP22, which induces broad anti-cancer activities, as a novel therapeutic strategy for NSCLC patient.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neovascularización Patológica/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/terapia , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Reparación del ADN , ADN de Neoplasias/análisis , Modelos Animales de Enfermedad , Femenino , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas Experimentales/secundario , Neoplasias Pulmonares/terapia , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Persona de Mediana Edad , Metástasis de la Neoplasia , Neovascularización Patológica/patología , Neovascularización Patológica/terapia , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/deficienciaRESUMEN
IFN-γ is known to be predominantly produced by lymphoid cells such as certain subsets of T cells, NK cells, and other group 1 innate lymphoid cells. In this study, we used IFN-γ reporter mouse models to search for additional cells capable of secreting this cytokine. We identified a novel and rare population of nonconventional IFN-γ-producing cells of hematopoietic origin that were characterized by the expression of Thy1.2 and the lack of lymphoid, myeloid, and NK lineage markers. The expression of IFN-γ by this population was higher in the liver and lower in the spleen. Furthermore, these cells were present in mice lacking both the Rag2 and the common γ-chain (γc) genes (Rag2-/-γc-/-), indicating their innate nature and their γc cytokine independence. Rag2-/-γc-/- mice are as resistant to Mycobacterium avium as Rag2-/- mice, whereas Rag2-/- mice lacking IFN-γ are more susceptible than either Rag2-/- or Rag2-/-γc-/- These lineage-negative CD45+/Thy1.2+ cells are found within the mycobacterially induced granulomatous structure in the livers of infected Rag2-/-γc-/- animals and are adjacent to macrophages that expressed inducible NO synthase, suggesting a potential protective role for these IFN-γ-producing cells. Accordingly, Thy1.2-specific mAb administration to infected Rag2-/-γc-/- animals increased M. avium growth in the liver. Overall, our results demonstrate that a population of Thy1.2+ non-NK innate-like cells present in the liver expresses IFN-γ and can confer protection against M. avium infection in immunocompromised mice.
Asunto(s)
Células Madre Hematopoyéticas/inmunología , Inmunidad Innata , Interferón gamma/biosíntesis , Interferón gamma/genética , Subunidad gamma Común de Receptores de Interleucina/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Granuloma/inmunología , Granuloma/microbiología , Huésped Inmunocomprometido/inmunología , Interferón gamma/inmunología , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Células Asesinas Naturales/inmunología , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/inmunología , Hígado/citología , Hígado/inmunología , Hígado/microbiología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Mycobacterium avium/crecimiento & desarrollo , Mycobacterium avium/inmunología , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/inmunología , Bazo/citología , Bazo/inmunología , Antígenos Thy-1/genética , Antígenos Thy-1/inmunologíaRESUMEN
BACKGROUND & AIMS: Hepatitis B virus (HBV) infects hepatocytes, but the mechanisms of the immune response against the virus and how it affects disease progression are unclear. METHODS: We performed studies with BALB/c Rag2-/-Il2rg-/-SirpaNODAlb-uPAtg/tg mice, stably engrafted with human hepatocytes (HUHEP) with or without a human immune system (HIS). HUHEP and HIS-HUHEP mice were given an intraperitoneal injection of HBV. Mononuclear cells were isolated from spleen and liver for analysis by flow cytometry. Liver was analyzed by immunohistochemistry and mRNA levels were measured by quantitative reverse transcription polymerase chain reaction (PCR). Plasma levels of HBV DNA were quantified by PCR reaction, and antigen-specific antibodies were detected by immunocytochemistry of HBV-transfected BHK-21 cells. RESULTS: Following HBV infection, a complete viral life cycle, with production of HBV DNA, hepatitis B e (HBe), core (HBc) and surface (HBs) antigens, and covalently closed circular DNA, was observed in HUHEP and HIS-HUHEP mice. HBV replicated unrestricted in HUHEP mice resulting in high viral titers without pathologic effects. In contrast, HBV-infected HIS-HUHEP mice developed chronic hepatitis with 10-fold lower titers and antigen-specific IgGs, (anti-HBs, anti-HBc), consistent with partial immune control. HBV-infected HIS-HUHEP livers contained infiltrating Kupffer cells, mature activated natural killer cells (CD69+), and PD-1+ effector memory T cells (CD45RO+). Reducing the viral inoculum resulted in more efficient immune control. Plasma from HBV-infected HIS-HUHEP mice had increased levels of inflammatory and immune-suppressive cytokines (C-X-C motif chemokine ligand 10 and interleukin 10), which correlated with populations of intrahepatic CD4+ T cells (CD45RO+PD-1+). Mice with high levels of viremia had HBV-infected liver progenitor cells. Giving the mice the nucleoside analogue entecavir reduced viral loads and decreased liver inflammation. CONCLUSION: In HIS-HUHEP mice, HBV infection completes a full life cycle and recapitulates some of the immunopathology observed in patients with chronic infection. Inoculation with different viral loads led to different immune responses and levels of virus control. We found HBV to infect liver progenitor cells, which could be involved in hepatocellular carcinogenesis. This is an important new system to study anti-HBV immune responses and screen for combination therapies against hepatotropic viruses.
Asunto(s)
Virus de la Hepatitis B/crecimiento & desarrollo , Hepatitis B Crónica/virología , Hepatocitos/virología , Hígado/virología , Bazo/virología , Carga Viral , Replicación Viral , Animales , ADN Viral/sangre , ADN Viral/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Femenino , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/genética , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/metabolismo , Hepatocitos/inmunología , Hepatocitos/metabolismo , Interacciones Huésped-Patógeno , Humanos , Inmunidad Celular , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Hígado/inmunología , Hígado/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Inmunológicos/deficiencia , Receptores Inmunológicos/genética , Albúmina Sérica Humana/genética , Albúmina Sérica Humana/metabolismo , Bazo/inmunología , Bazo/metabolismo , Factores de Tiempo , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
RNA-binding proteins (RBPs) are increasingly identified as post-transcriptional drivers of cancer progression. The RBP LARP1 is an mRNA stability regulator, and elevated expression of the protein in hepatocellular and lung cancers is correlated with adverse prognosis. LARP1 associates with an mRNA interactome that is enriched for oncogenic transcripts. Here we explore the role of LARP1 in epithelial ovarian cancer, a disease characterized by the rapid acquisition of resistance to chemotherapy through the induction of pro-survival signalling. We show, using ovarian cell lines and xenografts, that LARP1 is required for cancer cell survival and chemotherapy resistance. LARP1 promotes tumour formation in vivo and maintains cancer stem cell-like populations. Using transcriptomic analysis following LARP1 knockdown, cross-referenced against the LARP1 interactome, we identify BCL2 and BIK as LARP1 mRNA targets. We demonstrate that, through an interaction with the 3' untranslated regions (3' UTRs) of BCL2 and BIK, LARP1 stabilizes BCL2 but destabilizes BIK with the net effect of resisting apoptosis. Together, our data indicate that by differentially regulating the stability of a selection of mRNAs, LARP1 promotes ovarian cancer progression and chemotherapy resistance.
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Autoantígenos/genética , Carcinogénesis/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Ováricas/genética , Ribonucleoproteínas/genética , Animales , Antineoplásicos/farmacología , Autoantígenos/metabolismo , Western Blotting , Carcinogénesis/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Microscopía Confocal , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleoproteínas/metabolismo , Análisis de Supervivencia , Trasplante Heterólogo , Antígeno SS-BRESUMEN
Nonresolving chronic inflammation at the neoplastic site is consistently associated with promoting tumor progression and poor patient outcomes. However, many aspects behind the mechanisms that establish this tumor-promoting inflammatory microenvironment remain undefined. Using bladder cancer (BC) as a model, we found that CD14-high cancer cells express higher levels of numerous inflammation mediators and form larger tumors compared with CD14-low cells. CD14 antigen is a glycosyl-phosphatidylinositol (GPI)-linked glycoprotein and has been shown to be critically important in the signaling pathways of Toll-like receptor (TLR). CD14 expression in this BC subpopulation of cancer cells is required for increased cytokine production and increased tumor growth. Furthermore, tumors formed by CD14-high cells are more highly vascularized with higher myeloid cell infiltration. Inflammatory factors produced by CD14-high BC cells recruit and polarize monocytes and macrophages to acquire immune-suppressive characteristics. In contrast, CD14-low BC cells have a higher baseline cell division rate than CD14-high cells. Importantly, CD14-high cells produce factors that further increase the proliferation of CD14-low cells. Collectively, we demonstrate that CD14-high BC cells may orchestrate tumor-promoting inflammation and drive tumor cell proliferation to promote tumor growth.
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Proliferación Celular/genética , Citocinas/genética , Receptores de Lipopolisacáridos/genética , Microambiente Tumoral/genética , Neoplasias de la Vejiga Urinaria/genética , Animales , Línea Celular Tumoral , Citocinas/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación Neoplásica de la Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Queratina-14/genética , Queratina-14/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Trasplante de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante Heterólogo , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. Recent high-throughput analyses of genomic alterations revealed several driver genes and altered pathways in GC. However, therapeutic applications from genomic data are limited, largely as a result of the lack of druggable molecular targets and preclinical models for drug selection. To identify new therapeutic targets for GC, we performed array comparative genomic hybridization (aCGH) of DNA from 103 patients with GC for copy number alteration (CNA) analysis, and whole-exome sequencing from 55 GCs from the same patients for mutation profiling. Pathway analysis showed recurrent alterations in the Wnt signaling [APC, CTNNB1, and DLC1 (deleted in liver cancer 1)], ErbB signaling (ERBB2, PIK3CA, and KRAS), and p53 signaling/apoptosis [TP53 and BCL2L1 (BCL2-like 1)] pathways. In 18.4% of GC cases (19/103), amplification of the antiapoptotic gene BCL2L1 was observed, and subsequently a BCL2L1 inhibitor was shown to markedly decrease cell viability in BCL2L1-amplified cell lines and in similarly altered patient-derived GC xenografts, especially when combined with other chemotherapeutic agents. In 10.9% of cases (6/55), mutations in DLC1 were found and were also shown to confer a growth advantage for these cells via activation of Rho-ROCK signaling, rendering these cells more susceptible to a ROCK inhibitor. Taken together, our study implicates BCL2L1 and DLC1 as potential druggable targets for specific subsets of GC cases.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Proteínas Activadoras de GTPasa/genética , Estudio de Asociación del Genoma Completo/métodos , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Proteínas Supresoras de Tumor/genética , Proteína bcl-X/genética , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Supervivencia Celular/genética , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN , Exoma/genética , Femenino , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Masculino , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Persona de Mediana Edad , Mutación , Interferencia de ARN , Análisis de Secuencia de ADN/métodos , Neoplasias Gástricas/patología , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína bcl-X/metabolismoRESUMEN
Adult T-cell leukemia (ATL) develops in individuals infected with human T-cell lymphotropic virus-1 (HTLV-1). Presently there is no curative therapy for ATL. HTLV-1-encoded protein Tax (transactivator from the X-gene region) up-regulates Bcl-xL (B-cell lymphoma-extra large) expression and activates interleukin-2 (IL-2), IL-9, and IL-15 autocrine/paracrine systems, resulting in amplified JAK/STAT signaling. Inhibition of JAK signaling reduces cytokine-dependent ex vivo proliferation of peripheral blood mononuclear cells (PBMCs) from ATL patients in smoldering/chronic stages. Currently, two JAK inhibitors are approved for human use. In this study, we examined activity of multiple JAK inhibitors in ATL cell lines. The selective JAK inhibitor ruxolitinib was examined in a high-throughput matrix screen combined with >450 potential therapeutic agents, and Bcl-2/Bcl-xL inhibitor navitoclax was identified as a strong candidate for multicomponent therapy. The combination was noted to strongly activate BAX (Bcl-2-associated X protein), effect mitochondrial depolarization, and increase caspase 3/7 activities that lead to cleavage of PARP (poly ADP ribose polymerase) and Mcl-1 (myeloid cell leukemia 1). Ruxolitinib and navitoclax independently demonstrated modest antitumor efficacy, whereas the combination dramatically lowered tumor burden and prolonged survival in an ATL murine model. This combination strongly blocked ex vivo proliferation of five ATL patients' PBMCs. These studies provide support for a therapeutic trial in patients with smoldering/chronic ATL using a drug combination that inhibits JAK signaling and antiapoptotic protein Bcl-xL.
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Interleucina-2/metabolismo , Quinasas Janus/metabolismo , Leucemia-Linfoma de Células T del Adulto/metabolismo , Factores de Transcripción STAT/metabolismo , Proteína bcl-X/metabolismo , Compuestos de Anilina/farmacología , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Células Cultivadas , Femenino , Citometría de Flujo , Humanos , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Quinasas Janus/antagonistas & inhibidores , Leucemia-Linfoma de Células T del Adulto/tratamiento farmacológico , Leucemia-Linfoma de Células T del Adulto/patología , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Nitrilos , Pirazoles/farmacología , Pirimidinas , Factores de Transcripción STAT/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína bcl-X/antagonistas & inhibidoresRESUMEN
OBJECTIVE: Although early proof-of-concept studies of somatic in vivo genome editing of the mouse ortholog of proprotein convertase subtilisin/kexin type 9 (Pcsk9) in mice have established its therapeutic potential for the prevention of cardiovascular disease, the unique nature of genome-editing technology-permanent alteration of genomic DNA sequences-mandates that it be tested in vivo against human genes in normal human cells with human genomes to give reliable preclinical insights into the efficacy (on-target mutagenesis) and safety (lack of off-target mutagenesis) of genome-editing therapy before it can be used in patients. APPROACH AND RESULTS: We used a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) 9 genome-editing system to target the human PCSK9 gene in chimeric liver-humanized mice bearing human hepatocytes. We demonstrated high on-target mutagenesis (approaching 50%), greatly reduced blood levels of human PCSK9 protein, and minimal off-target mutagenesis. CONCLUSIONS: This work yields important information on the efficacy and safety of CRISPR-Cas9 therapy targeting the human PCSK9 gene in human hepatocytes in vivo, and it establishes humanized mice as a useful platform for the preclinical assessment of applications of somatic in vivo genome editing.
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Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Edición Génica/métodos , Marcación de Gen/métodos , Hepatocitos/enzimología , Proproteína Convertasa 9/genética , Animales , Proteínas Asociadas a CRISPR/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Genotipo , Hepatocitos/trasplante , Humanos , Hidrolasas/deficiencia , Hidrolasas/genética , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Proproteína Convertasa 9/biosíntesis , Proproteína Convertasa 9/sangreRESUMEN
Little is known of the genetic architecture of cancer at the subclonal and single-cell level or in the cells responsible for cancer clone maintenance and propagation. Here we have examined this issue in childhood acute lymphoblastic leukaemia in which the ETV6-RUNX1 gene fusion is an early or initiating genetic lesion followed by a modest number of recurrent or 'driver' copy number alterations. By multiplexing fluorescence in situ hybridization probes for these mutations, up to eight genetic abnormalities can be detected in single cells, a genetic signature of subclones identified and a composite picture of subclonal architecture and putative ancestral trees assembled. Subclones in acute lymphoblastic leukaemia have variegated genetics and complex, nonlinear or branching evolutionary histories. Copy number alterations are independently and reiteratively acquired in subclones of individual patients, and in no preferential order. Clonal architecture is dynamic and is subject to change in the lead-up to a diagnosis and in relapse. Leukaemia propagating cells, assayed by serial transplantation in NOD/SCID IL2Rγ(null) mice, are also genetically variegated, mirroring subclonal patterns, and vary in competitive regenerative capacity in vivo. These data have implications for cancer genomics and for the targeted therapy of cancer.
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Células Clonales/patología , Variación Genética/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Animales , Células Clonales/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Variaciones en el Número de Copia de ADN/genética , Análisis Mutacional de ADN , Progresión de la Enfermedad , Genotipo , Humanos , Inmunofenotipificación , Hibridación Fluorescente in Situ , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Proteínas de Fusión Oncogénica/genéticaRESUMEN
Inhalation exposures to ozone commonly encountered in photochemical smog cause airway injury and inflammation. Elevated ambient ozone concentrations have been epidemiologically associated with nasal airway activation of neutrophils and eosinophils. In the present study, we elucidated the temporal onset and lymphoid cell dependency of eosinophilic rhinitis and associated epithelial changes in mice repeatedly exposed to ozone. Lymphoid cell-sufficient C57BL/6 mice were exposed to 0 or 0.5 parts per million (ppm) ozone for 1, 2, 4, or 9 consecutive weekdays (4 h/d). Lymphoid cell-deficient, Rag2(-/-)Il2rg(-/-) mice were similarly exposed for 9 weekdays. Nasal tissues were taken at 2 or 24 hours after exposure for morphometric and gene expression analyses. C57BL/6 mice exposed to ozone for 1 day had acute neutrophilic rhinitis, with airway epithelial necrosis and overexpression of mucosal Ccl2 (MCP-1), Ccl11 (eotaxin), Cxcl1 (KC), Cxcl2 (MIP-2), Hmox1, Il1b, Il5, Il6, Il13, and Tnf mRNA. In contrast, 9-day ozone exposure elicited type 2 immune responses in C57BL/6 mice, with mucosal mRNA overexpression of Arg1, Ccl8 (MCP-2), Ccl11, Chil4 (Ym2), Clca1 (Gob5), Il5, Il10, and Il13; increased density of mucosal eosinophils; and nasal epithelial remodeling (e.g., hyperplasia/hypertrophy, mucous cell metaplasia, hyalinosis, and increased YM1/YM2 proteins). Rag2(-/-)Il2rg(-/-) mice exposed to ozone for 9 days, however, had no nasal pathology or overexpression of transcripts related to type 2 immunity. These results provide a plausible paradigm for the activation of eosinophilic inflammation and type 2 immunity found in the nasal airways of nonatopic individuals subjected to episodic exposures to high ambient ozone.
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Eosinofilia/inmunología , Inmunidad Mucosa , Linfocitos/inmunología , Mucosa Nasal/inmunología , Ozono , Rinitis/inmunología , Animales , Citocinas/genética , Citocinas/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Eosinofilia/inducido químicamente , Eosinofilia/genética , Eosinofilia/metabolismo , Regulación de la Expresión Génica , Genotipo , Mediadores de Inflamación/metabolismo , Exposición por Inhalación , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Linfocitos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mucosa Nasal/metabolismo , Fenotipo , ARN Mensajero/metabolismo , Rinitis/inducido químicamente , Rinitis/genética , Rinitis/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although mechanisms of BCR/ABL1-induced transformation are well-defined, little is known about effector-molecules contributing to malignant expansion and the extramedullary spread of leukemic SC (LSC) in CML. We have identified the cytokine-targeting surface enzyme dipeptidylpeptidase-IV (DPPIV/CD26) as a novel, specific and pathogenetically relevant biomarker of CD34(+)/CD38(â) CML LSC. In functional assays, CD26 was identified as target enzyme disrupting the SDF-1-CXCR4-axis by cleaving SDF-1, a chemotaxin recruiting CXCR4(+) SC. CD26 was not detected on normal SC or LSC in other hematopoietic malignancies. Correspondingly, CD26(+) LSC decreased to low or undetectable levels during successful treatment with imatinib. CD26(+) CML LSC engrafted NOD-SCID-IL-2Rγ(-/-) (NSG) mice with BCR/ABL1(+) cells, whereas CD26(â) SC from the same patients produced multilineage BCR/ABL1(-) engraftment. Finally, targeting of CD26 by gliptins suppressed the expansion of BCR/ABL1(+) cells. Together, CD26 is a new biomarker and target of CML LSC. CD26 expression may explain the abnormal extramedullary spread of CML LSC, and inhibition of CD26 may revert abnormal LSC function and support curative treatment approaches in this malignancy.