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1.
Cell ; 165(7): 1672-1685, 2016 Jun 16.
Artículo en Inglés | MEDLINE | ID: mdl-27315481

RESUMEN

Long intergenic noncoding RNAs (lincRNAs) are important regulators of gene expression. Although lincRNAs are expressed in immune cells, their functions in immunity are largely unexplored. Here, we identify an immunoregulatory lincRNA, lincRNA-EPS, that is precisely regulated in macrophages to control the expression of immune response genes (IRGs). Transcriptome analysis of macrophages from lincRNA-EPS-deficient mice, combined with gain-of-function and rescue experiments, revealed a specific role for this lincRNA in restraining IRG expression. Consistently, lincRNA-EPS-deficient mice manifest enhanced inflammation and lethality following endotoxin challenge in vivo. lincRNA-EPS localizes at regulatory regions of IRGs to control nucleosome positioning and repress transcription. Further, lincRNA-EPS mediates these effects by interacting with heterogeneous nuclear ribonucleoprotein L via a CANACA motif located in its 3' end. Together, these findings identify lincRNA-EPS as a repressor of inflammatory responses, highlighting the importance of lincRNAs in the immune system.


Asunto(s)
Regulación de la Expresión Génica , Inflamación/genética , Macrófagos/inmunología , ARN Largo no Codificante/metabolismo , Animales , Cromátides/metabolismo , Eliminación de Gen , Humanos , Listeria monocytogenes/fisiología , Listeriosis/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Macrófagos/virología , Ratones , Ratones Endogámicos C57BL , ARN Largo no Codificante/genética , Infecciones por Respirovirus/inmunología , Virus Sendai/fisiología , Receptores Toll-Like/metabolismo , Transcriptoma
2.
Mol Ther ; 31(7): 2132-2153, 2023 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-37194236

RESUMEN

To leverage complementary mechanisms for cancer cell removal, we developed a novel cell engineering and therapeutic strategy co-opting phagocytic clearance and antigen presentation activity into T cells. We engineered a chimeric engulfment receptor (CER)-1236, which combines the extracellular domain of TIM-4, a phagocytic receptor recognizing the "eat me" signal phosphatidylserine, with intracellular signaling domains (TLR2/TIR, CD28, and CD3ζ) to enhance both TIM-4-mediated phagocytosis and T cell cytotoxic function. CER-1236 T cells demonstrate target-dependent phagocytic function and induce transcriptional signatures of key regulators responsible for phagocytic recognition and uptake, along with cytotoxic mediators. Pre-clinical models of mantle cell lymphoma (MCL) and EGFR mutation-positive non-small cell lung cancer (NSCLC) demonstrate collaborative innate-adaptive anti-tumor immune responses both in vitro and in vivo. Treatment with BTK (MCL) and EGFR (NSCLC) inhibitors increased target ligand, conditionally driving CER-1236 function to augment anti-tumor responses. We also show that activated CER-1236 T cells exhibit superior cross-presentation ability compared with conventional T cells, triggering E7-specific TCR T responses in an HLA class I- and TLR-2-dependent manner, thereby overcoming the limited antigen presentation capacity of conventional T cells. Therefore, CER-1236 T cells have the potential to achieve tumor control by eliciting both direct cytotoxic effects and indirect-mediated cross-priming.


Asunto(s)
Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Adulto , Linfocitos T , Reactividad Cruzada , Fosfatidilserinas , Antígenos de Neoplasias , Receptores ErbB , Inmunoterapia Adoptiva , Receptores de Antígenos de Linfocitos T/genética
3.
J Immunol ; 204(2): 428-437, 2020 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-31836654

RESUMEN

Functional peptides encoded by short open reading frames are emerging as important mediators of fundamental biological processes. In this study, we identified a micropeptide produced from a putative long noncoding RNA (lncRNAs) that is important in controlling innate immunity. By studying lncRNAs in mice macrophages, we identified lncRNA 1810058I24Rik, which was downregulated in both human and murine myeloid cells exposed to LPS as well as other TLR ligands and inflammatory cytokines. Analysis of lncRNA 1810058I24Rik subcellular localization revealed that this transcript was localized in the cytosol, prompting us to evaluate its coding potential. In vitro translation with 35S-labeled methionine resulted in translation of a 47 aa micropeptide. Microscopy and subcellular fractionation studies in macrophages demonstrated endogenous expression of this peptide on the mitochondrion. We thus named this gene mitochondrial micropeptide-47 (Mm47). Crispr-Cas9-mediated deletion of Mm47, as well as small interfering RNA studies in mice primary macrophages, showed that the transcriptional response downstream of TLR4 was intact in cells lacking Mm47. In contrast, Mm47-deficient or knockdown cells were compromised for Nlrp3 inflammasome responses. Activation of Nlrc4 or Aim2 inflammasomes were intact in cells lacking Mm47. This study therefore identifies, to our knowledge, a novel mitochondrial micropeptide Mm47 that is required for the activation of the Nlrp3 inflammasome. This work further highlights the functional activity of short open reading frame-encoded peptides and underscores their importance in innate immunity.


Asunto(s)
Citosol/metabolismo , Inflamasomas/metabolismo , Macrófagos/fisiología , Mitocondrias/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Fragmentos de Péptidos/metabolismo , ARN Largo no Codificante/genética , Animales , Sistemas CRISPR-Cas , Células Cultivadas , Humanos , Inmunidad Innata/genética , Lipopolisacáridos/inmunología , Ratones , Mitocondrias/genética , Fragmentos de Péptidos/genética , ARN Interferente Pequeño/genética
4.
Proc Natl Acad Sci U S A ; 111(33): 12193-8, 2014 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-25092305

RESUMEN

Hepatitis B virus (HBV) chronically infects 400 million people worldwide and is a leading driver of end-stage liver disease and liver cancer. Research into the biology and treatment of HBV requires an in vitro cell-culture system that supports the infection of human hepatocytes, and accurately recapitulates virus-host interactions. Here, we report that micropatterned cocultures of primary human hepatocytes with stromal cells (MPCCs) reliably support productive HBV infection, and infection can be enhanced by blocking elements of the hepatocyte innate immune response associated with the induction of IFN-stimulated genes. MPCCs maintain prolonged, productive infection and represent a facile platform for studying virus-host interactions and for developing antiviral interventions. Hepatocytes obtained from different human donors vary dramatically in their permissiveness to HBV infection, suggesting that factors--such as divergence in genetic susceptibility to infection--may influence infection in vitro. To establish a complementary, renewable system on an isogenic background in which candidate genetics can be interrogated, we show that inducible pluripotent stem cells differentiated into hepatocyte-like cells (iHeps) support HBV infection that can also be enhanced by blocking interferon-stimulated gene induction. Notably, the emergence of the capacity to support HBV transcriptional activity and initial permissiveness for infection are marked by distinct stages of iHep differentiation, suggesting that infection of iHeps can be used both to study HBV, and conversely to assess the degree of iHep differentiation. Our work demonstrates the utility of these infectious systems for studying HBV biology and the virus' interactions with host hepatocyte genetics and physiology.


Asunto(s)
Virus de la Hepatitis B/fisiología , Hepatocitos/virología , Interacciones Huésped-Patógeno , Células Madre Pluripotentes Inducidas/citología , Modelos Biológicos , Antivirales/farmacología , Diferenciación Celular , Hepatocitos/citología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos
5.
J Hepatol ; 65(2): 334-43, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27151182

RESUMEN

BACKGROUND & AIMS: Human liver chimeric mice are useful models of human hepatitis virus infection, including hepatitis B and C virus infections. Independently, immunodeficient mice reconstituted with CD34(+) hematopoietic stem cells (HSC) derived from fetal liver reliably develop human T and B lymphocytes. Combining these systems has long been hampered by inefficient liver reconstitution of human fetal hepatoblasts. Our study aimed to enhance hepatoblast engraftment in order to create a mouse model with syngeneic human liver and immune cells. METHODS: The effects of human oncostatin-M administration on fetal hepatoblast engraftment into immunodeficient fah(-/-) mice was tested. Mice were then transplanted with syngeneic human hepatoblasts and HSC after which human leukocyte chimerism and functionality were analyzed by flow cytometry, and mice were challenged with HBV. RESULTS: Addition of human oncostatin-M enhanced human hepatoblast engraftment in immunodeficient fah(-/-) mice by 5-100 fold. In contrast to mice singly engrafted with HSC, which predominantly developed human T and B lymphocytes, mice co-transplanted with syngeneic hepatoblasts also contained physiological levels of human monocytes and natural killer cells. Upon infection with HBV, these mice displayed rapid and sustained viremia. CONCLUSIONS: Our study provides a new mouse model with improved human fetal hepatoblast engraftment and an expanded human immune cell repertoire. With further improvements, this model may become useful for studying human immunity against viral hepatitis. LAY SUMMARY: Important human pathogens such as hepatitis B virus, hepatitis C virus and human immunodeficiency virus only infect human cells which complicates the development of mouse models for the study of these pathogens. One way to make mice permissive for human pathogens is the transplantation of human cells into immune-compromised mice. For instance, the transplantation of human liver cells will allow the infection of these so-called "liver chimeric mice" with hepatitis B virus and hepatitis C virus. The co-transplantation of human immune cells into liver chimeric mice will further allow the study of human immune responses to hepatitis B virus or hepatitis C virus. However, for immunological studies it will be crucial that the transplanted human liver and immune cells are derived from the same human donor. In our study we describe the efficient engraftment of human fetal liver cells and immune cells derived from the same donor into mice. We show that liver co-engraftment resulted in an expanded human immune cell repertoire, including monocytes and natural killer cells in the liver. We further demonstrate that these mice could be infected with hepatitis B virus, which lead to an expansion of natural killer cells. In conclusion we have developed a new mouse model that could be useful to study human immune responses to human liver pathogens.


Asunto(s)
Células Asesinas Naturales , Monocitos , Animales , Hepatitis B , Hepatocitos , Humanos , Ratones , Ratones SCID
6.
Clin Cancer Res ; 30(9): 1878-1888, 2024 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-38451195

RESUMEN

PURPOSE: Disruption of lipid bilayer asymmetry is a common feature observed in cancer cells and offers novel routes for therapeutic targeting. We used the natural immune receptor TIM-4 to interrogate for loss of plasma membrane phospholipid polarity in primary acute myelogenous leukemia (AML) samples and evaluated the anti-leukemic activity of TIM-4-L-directed T-cell therapy in preclinical AML models. EXPERIMENTAL DESIGN: We performed FACS analysis on 33 primary AML bone marrow specimens and correlated TIM-4-L expression frequency and intensity with molecular disease characteristics. Using Kasumi-1 and MV-4-11 AML cell lines, we further tested the anti-leukemic effects of TIM-4-L-directed engineered T cells in vitro and in vivo. RESULTS: We found that 86% of untreated AML blasts displayed upregulation of cell surface TIM-4-L. These observations were agnostic to AML genetic classification, as samples with mutations in TP53, ASXL1, and RUNX1 displayed TIM-4-L upregulation similar to that seen in favorable and intermediate subtypes. TIM-4-L dysregulation was also stably present in AML cell lines. To evaluate the potential of targeting upregulated TIM-4-L with adoptive T-cell therapy, we constructed TIM-4-L-directed engineered T cells, which demonstrated potent anti-leukemic effects, effectively eliminating AML cell lines with a range of endogenous TIM-4-L expression levels both in vitro and in vivo. CONCLUSIONS: These results highlight TIM-4-L as a highly prevalent target on AML across a range of genetic classifications and novel target for T-cell-based therapy in AML. Further investigations into the role of TIM-4-L in AML pathogenesis and its potential as an anti-leukemic target for clinical development are warranted.


Asunto(s)
Leucemia Mieloide Aguda , Proteínas de la Membrana , Linfocitos T , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/metabolismo , Ratones , Animales , Linfocitos T/inmunología , Linfocitos T/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Femenino , Masculino , Persona de Mediana Edad , Adulto , Anciano , Inmunoterapia Adoptiva/métodos
7.
Anal Biochem ; 422(2): 66-73, 2012 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-22281394

RESUMEN

Therapeutic drugs and environmental pollutants may exhibit high reactivity toward DNA bases and backbone. Understanding the mechanisms of drug-DNA binding is crucial for predicting their potential genotoxicity. We developed a fluorescence analytical method for the determination of the preferential binding mode for drug-DNA interactions. Two nucleic acid dyes were employed in the method: TO-PRO-3 iodide (TP3) and 4',6-diamidino-2-phenylindole (DAPI). TP3 binds DNA by intercalation, whereas DAPI exhibits minor groove binding. Both dyes exhibit significant fluorescence magnification on binding to DNA. We evaluated the DNA binding constant, K(b), for each dye. We also performed fluorescence quenching experiments with 11 molecules of various structures and measured a C(50) value for each compound. We determined preferential binding modes for the aforementioned molecules and found that they bound to DNA consistently, as indicated by other studies. The values of the likelihood of DNA intercalation were correlated with the partition coefficients of the molecules. In addition, we performed nuclear magnetic resonance (NMR) studies of the interactions with calf thymus DNA for the three molecules. The results were consistent with the fluorescence method described above. Thus, we conclude that the fluorescence method we developed provides a reliable determination of the likelihoods of the two different DNA binding modes.


Asunto(s)
ADN/química , Colorantes Fluorescentes/química , Sustancias Intercalantes/química , Animales , Sitios de Unión , Bioensayo , Carbocianinas/química , Bovinos , Contaminantes Ambientales/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Indicadores y Reactivos/química , Indoles/química , Cinética , Espectroscopía de Resonancia Magnética , Medicamentos bajo Prescripción/química , Espectrometría de Fluorescencia
8.
J Immunother ; 41(9): 399-405, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29757889

RESUMEN

In this study, we address one of the major critiques for tumor-infiltrating lymphocyte (TIL) therapy-the time needed for proper expansion of a suitable product. We postulated that T-cell receptor activation in the first phase of expansion combined with an agonistic stimulation of CD137/4-1BB and interleukin-2 would favor preferential expansion of CD8 TIL. Indeed, this novel 3-signal approach for optimal T-cell activation resulted in faster and more consistent expansion of CD8CD3 TIL. This new method allowed for successful expansion of TIL from cutaneous and uveal melanoma tumors in 100% of the cultures in <3 weeks. Finally, providing the 3 signals attributed to optimal T-cell activation led to expansion of TIL capable of recognizing their tumor counterpart in cutaneous and uveal melanoma. This new methodology for the initial phase of TIL expansion brings a new opportunity for translation of TIL therapy in challenging malignancies such as uveal melanoma.


Asunto(s)
Interleucina-2/uso terapéutico , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/trasplante , Melanoma/terapia , Neoplasias Cutáneas/terapia , Linfocitos T/inmunología , Neoplasias de la Úvea/terapia , Femenino , Humanos , Inmunoterapia Adoptiva , Masculino , Melanoma/inmunología , Neoplasias Cutáneas/inmunología , Neoplasias de la Úvea/inmunología
9.
Clin Cancer Res ; 24(18): 4416-4428, 2018 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-29848573

RESUMEN

Purpose: Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) has consistently demonstrated clinical efficacy in metastatic melanoma. Recent widespread use of checkpoint blockade has shifted the treatment landscape, raising questions regarding impact of these therapies on response to TIL and appropriate immunotherapy sequence.Patients and Methods: Seventy-four metastatic melanoma patients were treated with autologous TIL and evaluated for clinical response according to irRC, overall survival, and progression-free survival. Immunologic factors associated with response were also evaluated.Results: Best overall response for the entire cohort was 42%; 47% in 43 checkpoint-naïve patients, 38% when patients were exposed to anti-CTLA4 alone (21 patients) and 33% if also exposed to anti-PD1 (9 patients) prior to TIL ACT. Median overall survival was 17.3 months; 24.6 months in CTLA4-naïve patients and 8.6 months in patients with prior CTLA4 blockade. The latter patients were infused with fewer TIL and experienced a shorter duration of response. Infusion of higher numbers of TIL with CD8 predominance and expression of BTLA correlated with improved response in anti-CTLA4 naïve patients, but not in anti-CTLA4 refractory patients. Baseline serum levels of IL9 predicted response to TIL ACT, while TIL persistence, tumor recognition, and mutation burden did not correlate with outcome.Conclusions: This study demonstrates the deleterious effects of prior exposure to anti-CTLA4 on TIL ACT response and shows that baseline IL9 levels can potentially serve as a predictive tool to select the appropriate sequence of immunotherapies. Clin Cancer Res; 24(18); 4416-28. ©2018 AACR.


Asunto(s)
Antígeno CTLA-4/antagonistas & inhibidores , Interleucina-9/sangre , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma/terapia , Adulto , Anciano , Antígeno CTLA-4/inmunología , Terapia Combinada , Femenino , Humanos , Inmunoterapia Adoptiva , Masculino , Melanoma/sangre , Melanoma/inmunología , Melanoma/patología , Persona de Mediana Edad , Metástasis de la Neoplasia , Neoplasias Primarias Secundarias/inmunología , Neoplasias Primarias Secundarias/patología , Neoplasias Primarias Secundarias/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/inmunología , Supervivencia sin Progresión
10.
Sci Rep ; 5: 10833, 2015 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-26035283

RESUMEN

Chronic hepatitis B virus (HBV) infection is prevalent, deadly, and seldom cured due to the persistence of viral episomal DNA (cccDNA) in infected cells. Newly developed genome engineering tools may offer the ability to directly cleave viral DNA, thereby promoting viral clearance. Here, we show that the CRISPR/Cas9 system can specifically target and cleave conserved regions in the HBV genome, resulting in robust suppression of viral gene expression and replication. Upon sustained expression of Cas9 and appropriately chosen guide RNAs, we demonstrate cleavage of cccDNA by Cas9 and a dramatic reduction in both cccDNA and other parameters of viral gene expression and replication. Thus, we show that directly targeting viral episomal DNA is a novel therapeutic approach to control the virus and possibly cure patients.


Asunto(s)
Sistemas CRISPR-Cas , ADN Viral/genética , ADN Viral/metabolismo , Virus de la Hepatitis B/genética , Replicación Viral/genética , Animales , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Línea Celular Tumoral , Células Cultivadas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ADN Circular , Modelos Animales de Enfermedad , Regulación Viral de la Expresión Génica , Marcación de Gen , Hepatitis B/virología , Humanos , Ratones , ARN Guía de Kinetoplastida
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