RESUMEN
Natural killer (NK) cells have high intrinsic cytotoxic capacity, and clinical trials have demonstrated their safety and efficacy for adoptive cancer therapy. Expression of chimeric antigen receptors (CARs) enhances NK cell target specificity, with these cells applicable as off-the-shelf products generated from allogeneic donors. Here, we present for the first time an innovative approach for CAR NK cell engineering employing a non-viral Sleeping Beauty (SB) transposon/transposase-based system and minimized DNA vectors termed minicircles. SB-modified peripheral blood-derived primary NK cells displayed high and stable CAR expression and more frequent vector integration into genomic safe harbors than lentiviral vectors. Importantly, SB-generated CAR NK cells demonstrated enhanced cytotoxicity compared with non-transfected NK cells. A strong antileukemic potential was confirmed using established acute lymphocytic leukemia cells and patient-derived primary acute B cell leukemia and lymphoma samples as targets in vitro and in vivo in a xenograft leukemia mouse model. Our data suggest that the SB-transposon system is an efficient, safe, and cost-effective approach to non-viral engineering of highly functional CAR NK cells, which may be suitable for cancer immunotherapy of leukemia as well as many other malignancies.
Asunto(s)
Vectores Genéticos , Inmunoterapia Adoptiva , Células Asesinas Naturales , Receptores Quiméricos de Antígenos , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Animales , Ratones , Vectores Genéticos/genética , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Inmunoterapia Adoptiva/métodos , Ensayos Antitumor por Modelo de Xenoinjerto , Transposasas/genética , Transposasas/metabolismo , Línea Celular Tumoral , Elementos Transponibles de ADN , Citotoxicidad Inmunológica , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Ingeniería Celular/métodosRESUMEN
Respiratory tract infections are among the deadliest communicable diseases worldwide. Severe cases of viral lung infections are often associated with a cytokine storm and alternating platelet numbers. We report that hematopoietic stem and progenitor cells (HSPCs) sense a non-systemic influenza A virus (IAV) infection via inflammatory cytokines. Irrespective of antiviral treatment or vaccination, at a certain threshold of IAV titer in the lung, CD41-positive hematopoietic stem cells (HSCs) enter the cell cycle while endothelial protein C receptor-positive CD41-negative HSCs remain quiescent. Active CD41-positive HSCs represent the source of megakaryocytes, while their multi-lineage reconstitution potential is reduced. This emergency megakaryopoiesis is thrombopoietin independent and attenuated in IAV-infected interleukin-1 receptor-deficient mice. Newly produced platelets during IAV infection are immature and hyper-reactive. After viral clearance, HSC quiescence is re-established. Our study reveals that non-systemic viral respiratory infection has an acute impact on HSCs via inflammatory cytokines to counteract IAV-induced thrombocytopenia.
Asunto(s)
Virus de la Influenza A , Gripe Humana , Animales , Antivirales/metabolismo , Citocinas/metabolismo , Receptor de Proteína C Endotelial/metabolismo , Hematopoyesis , Humanos , Gripe Humana/metabolismo , Megacariocitos/metabolismo , Ratones , Receptores de Interleucina-1/metabolismo , Trombopoyetina/metabolismoRESUMEN
Human T lymphocytes that transgenically express a chimeric antigen receptor (CAR) have proven efficacy and safety in gene- and cell-based immunotherapy of certain hematological cancers. Appropriate gene vectors and methods of genetic engineering are required for therapeutic cell products to be biologically potent and their manufacturing to be economically viable. Transposon-based gene transfer satisfies these needs, and is currently being evaluated in clinical trials. In this protocol we describe the basic Sleeping Beauty (SB) transposon vector components required for stable gene integration in human cells, with special emphasis on minicircle DNA vectors and the use of synthetic mRNA. We provide a protocol for functional validation of the vector components in cultured human cell lines on the basis of fluorescent reporter gene expression. Finally, we provide a protocol for CAR-T cell engineering and describe assays that address transgene expression, biological potency and genomic vector copy numbers in polyclonal cell populations. Because transposons allow virus-free gene transfer with naked nucleic acids, the protocol can be adopted by any laboratory equipped with biological safety level S1 facilities.
Asunto(s)
Elementos Transponibles de ADN , Técnicas de Transferencia de Gen , Receptores de Antígenos de Linfocitos T , Receptores Quiméricos de Antígenos , Terapia Genética/métodos , Vectores Genéticos/genética , Humanos , Inmunoterapia Adoptiva/métodos , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T , Transposasas/genética , Transposasas/metabolismoRESUMEN
Metastasis, a complex, multistep process, is responsible for the overwhelming majority of cancer-related deaths. Despite its devastating consequences, it is not possible to effectively treat cancer that has spread to vital organs, the mechanisms leading to metastasis are still poorly understood, and the catalog of metastasis promoting genes is still incomprehensive. To identify new driver genes of metastasis development, we performed an in vitro Sleeping Beauty transposon-based forward genetic screen in nonmetastatic SKBR3 human breast cancer cells. Boyden chamber-based matrix invasion assays were used to harvest cells that acquired a de novo invasive phenotype. Using targeted RNA sequencing data from 18 pools of invasive cells, we carried out a gene-centric candidate gene prediction and identified established and novel metastasis driver genes. Analysis of these genes revealed their association with metastasis related processes and we further established their clinical relevance in metastatic breast cancer. Two novel candidate genes, G protein-coupled receptor kinase interacting ArfGAP 2 (GIT2) and muscle-associated receptor tyrosine kinase (MUSK), were functionally validated as metastasis driver genes in a series of in vitro and in vivo experimental metastasis models. We propose that our robust and scalable approach will be a useful addition to the toolkit of methodologic resources used to identify genes driving cancer metastasis. IMPLICATIONS: Novel metastasis drivers were identified in a human breast cancer cell line by performing an in vitro, Sleeping Beauty transposon-based forward genetic screen and an RNA fusion-based candidate gene prediction.
Asunto(s)
Neoplasias de la Mama , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Elementos Transponibles de ADN/genética , Femenino , Humanos , Mutagénesis , Mutagénesis Insercional , Proteínas Tirosina Quinasas/genética , ARN , Receptores Acoplados a Proteínas G/genéticaAsunto(s)
Proteínas de Unión al ADN/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteína 1 Compañera de Translocación de RUNX1/genética , Proteína 1 Compañera de Translocación de RUNX1/metabolismo , Factores de Transcripción/genética , Biomarcadores de Tumor , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Pronóstico , Factores de Transcripción/metabolismoRESUMEN
Differentiation of hematopoietic stem cells is regulated by a concert of different transcription factors. Disturbed transcription factor function can be the basis of (pre)malignancies such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Growth factor independence 1b (Gfi1b) is a repressing transcription factor regulating quiescence of hematopoietic stem cells and differentiation of erythrocytes and platelets. Here, we show that low expression of Gfi1b in blast cells is associated with an inferior prognosis of MDS and AML patients. Using different models of human MDS or AML, we demonstrate that AML development was accelerated with heterozygous loss of Gfi1b, and latency was further decreased when Gfi1b was conditionally deleted. Loss of Gfi1b significantly increased the number of leukemic stem cells with upregulation of genes involved in leukemia development. On a molecular level, we found that loss of Gfi1b led to epigenetic changes, increased levels of reactive oxygen species, as well as alteration in the p38/Akt/FoXO pathways. These results demonstrate that Gfi1b functions as an oncosuppressor in MDS and AML development.
Asunto(s)
Leucemia Mieloide Aguda/etiología , Síndromes Mielodisplásicos/etiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Represoras/fisiología , Animales , Epigenómica , Proteína Forkhead Box O1/metabolismo , Eliminación de Gen , Heterocigoto , Homocigoto , Humanos , Ratones , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The differentiation of haematopoietic cells is regulated by a plethora of so-called transcription factors (TFs). Mutations in genes encoding TFs or graded reduction in their expression levels can induce the development of various malignant diseases such as acute myeloid leukaemia (AML). Growth Factor Independence 1 (GFI1) is a transcriptional repressor with key roles in haematopoiesis, including regulating self-renewal of haematopoietic stem cells (HSCs) as well as myeloid and lymphoid differentiation. Analysis of AML patients and different AML mouse models with reduced GFI1 gene expression levels revealed a direct link between low GFI1 protein level and accelerated AML development and inferior prognosis. Here, we report that upregulated expression of GFI1 in several widely used leukemic cell lines inhibits their growth and decreases the ability to generate colonies in vitro. Similarly, elevated expression of GFI1 impedes the in vitro expansion of murine pre-leukemic cells. Using a humanized AML model, we demonstrate that upregulation of GFI1 expression leads to myeloid differentiation morphologically and immunophenotypically, increased level of apoptosis and reduction in number of cKit+ cells. These results suggest that increasing GFI1 level in leukemic cells with low GFI1 expression level could be a therapeutic approach.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Factores de Transcripción/metabolismo , Animales , Apoptosis , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones Endogámicos C57BL , Ensayo de Tumor de Célula Madre , Regulación hacia ArribaRESUMEN
The growth of malignant cells is not only driven by cell-intrinsic factors, but also by the surrounding stroma. Monocytes/Macrophages play an important role in the onset and progression of solid cancers. However, little is known about their role in the development of acute myeloid leukemia, a malignant disease characterized by an aberrant development of the myeloid compartment of the hematopoietic system. It is also unclear which factors are responsible for changing the status of macrophage polarization, thus supporting the growth of malignant cells instead of inhibiting it. We report herein that acute myeloid leukemia leads to the invasion of acute myeloid leukemia-associated macrophages into the bone marrow and spleen of leukemic patients and mice. In different leukemic mouse models, these macrophages support the in vitro expansion of acute myeloid leukemia cell lines better than macrophages from non-leukemic mice. The grade of macrophage infiltration correlates in vivo with the survival of the mice. We found that the transcriptional repressor Growth factor independence 1 is crucial in the process of macrophage polarization, since its absence impedes macrophage polarization towards a leukemia supporting state and favors an anti-tumor state both in vitro and in vivo These results not only suggest that acute myeloid leukemia-associated macrophages play an important role in the progression of acute myeloid leukemia, but also implicate Growth factor independence 1 as a pivotal factor in macrophage polarization. These data may provide new insights and opportunities for novel therapies for acute myeloid leukemia.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Leucemia Mieloide Aguda/patología , Macrófagos/patología , Factores de Transcripción/fisiología , Animales , Médula Ósea/patología , Línea Celular Tumoral , Movimiento Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Ratones , Ratones Transgénicos , Bazo/patologíaRESUMEN
Epigenetic changes can contribute to development of acute myeloid leukemia (AML), a malignant disease of the bone marrow. A single-nucleotide polymorphism of transcription factor growth factor independence 1 (GFI1) generates a protein with an asparagine at position 36 (GFI1(36N)) instead of a serine at position 36 (GFI1(36S)), which is associated with de novo AML in humans. However, how GFI1(36N) predisposes to AML is poorly understood. To explore the mechanism, we used knock-in mouse strains expressing GFI1(36N) or GFI1(36S). Presence of GFI1(36N) shortened the latency and increased the incidence of AML in different murine models of myelodysplastic syndrome/AML. On a molecular level, GFI1(36N) induced genomewide epigenetic changes, leading to expression of AML-associated genes. On a therapeutic level, use of histone acetyltransferase inhibitors specifically impeded growth of GFI1(36N)-expressing human and murine AML cells in vitro and in vivo. These results establish, as a proof of principle, how epigenetic changes in GFI1(36N)-induced AML can be targeted.
Asunto(s)
Proteínas de Unión al ADN/genética , Epigénesis Genética , Leucemia Mieloide Aguda/genética , Mutación , Factores de Transcripción/genética , Sustitución de Aminoácidos , Animales , Médula Ósea/patología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Transformación Celular Neoplásica/genética , Codón , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Epigénesis Genética/efectos de los fármacos , Regulación Leucémica de la Expresión Génica , Predisposición Genética a la Enfermedad , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Humanos , Inmunofenotipificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Ratones , Ratones Transgénicos , Modelos Biológicos , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/mortalidadRESUMEN
Inherited gene variants play an important role in malignant diseases. The transcriptional repressor growth factor independence 1 (GFI1) regulates hematopoietic stem cell (HSC) self-renewal and differentiation. A single-nucleotide polymorphism of GFI1 (rs34631763) generates a protein with an asparagine (N) instead of a serine (S) at position 36 (GFI1(36N)) and has a prevalence of 3%-5% among Caucasians. Because GFI1 regulates myeloid development, we examined the role of GFI1(36N) on the course of MDS disease. To this end, we determined allele frequencies of GFI1(36N) in four independent MDS cohorts from the Netherlands and Belgium, Germany, the ICGC consortium, and the United States. The GFI1(36N) allele frequency in the 723 MDS patients genotyped ranged between 9% and 12%. GFI1(36N) was an independent adverse prognostic factor for overall survival, acute myeloid leukemia-free survival, and event-free survival in a univariate analysis. After adjustment for age, bone marrow blast percentage, IPSS score, mutational status, and cytogenetic findings, GFI1(36N) remained an independent adverse prognostic marker. GFI1(36S) homozygous patients exhibited a sustained response to treatment with hypomethylating agents, whereas GFI1(36N) patients had a poor sustained response to this therapy. Because allele status of GFI1(36N) is readily determined using basic molecular techniques, we propose inclusion of GFI1(36N) status in future prospective studies for MDS patients to better predict prognosis and guide therapeutic decisions.
Asunto(s)
Alelos , Proteínas de Unión al ADN/genética , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/mortalidad , Polimorfismo de Nucleótido Simple , Factores de Transcripción/genética , Adulto , Anciano , Sustitución de Aminoácidos , Biomarcadores , Médula Ósea/patología , Estudios de Cohortes , Análisis Citogenético , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/tratamiento farmacológico , Pancitopenia , Fenotipo , Pronóstico , Análisis de SupervivenciaRESUMEN
BACKGROUND: Graft versus host disease (GvHD) occurs in 20% of cases with patients having an MHC I matched bone marrow transplantation (BMT). Mechanisms causing this disease remain to be studied. METHODS: Here we used a CD8+ T cell transgenic mouse line (P14/CD45.1+) and transgenic DEE mice bearing ubiquitously the glycoprotein 33-41 (GP33) antigen derived from the major lymphocytic choriomeningitis virus (LCMV) epitope to study mechanisms of tolerance in anti-host reactive CD8+ T cells after BMT. RESULTS: We found that anti-host reactive CD8+ T cells (P14 T cells) were not negatively selected in the thymus and that they were present in wild type (WT) recipient mice as well as in DEE recipient mice. Anti-host reactive CD8+ T cells ignored the GP33 antigen expressed ubiquitously by host cells but they could be activated ex vivo via LCMV-infection. Lipopolysaccharides (LPS) induced transient cell damage in DEE mice bearing anti-host reactive CD8+ T cells after BMT, suggesting that induction of host inflammatory response could break antigen ignorance. Introducing the GP33 antigen into BM cells led to deletion of anti-host reactive CD8+ T cells. CONCLUSION: We found that after BMT anti-host reactive CD8+ T cells ignored host antigen in recipients and that they were only deleted when host antigen was present in hematopoietic cells. Moreover, LPS-induced immune activation contributed to induction of alloreactivity of anti-host reactive CD8+ T cells after BMT.
Asunto(s)
Trasplante de Médula Ósea , Linfocitos T CD8-positivos/inmunología , Tolerancia Inmunológica , Alanina Transaminasa/metabolismo , Animales , Anticuerpos/inmunología , Antígenos Virales/genética , Antígenos Virales/inmunología , Antígenos Virales/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Epítopos/inmunología , Citometría de Flujo , Glicoproteínas/genética , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Lipopolisacáridos/toxicidad , Virus de la Coriomeningitis Linfocítica/genética , Virus de la Coriomeningitis Linfocítica/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Trasplante Homólogo , Proteínas Virales/genética , Proteínas Virales/inmunología , Proteínas Virales/metabolismoRESUMEN
Clinically used human vaccination aims to induce specific antibodies that can guarantee long-term protection against a pathogen. The reasons that other immune components often fail to induce protective immunity are still debated. Recently we found that enforced viral replication in secondary lymphoid organs is essential for immune activation. In this study we used the lymphocytic choriomeningitis virus (LCMV) to determine whether enforced virus replication occurs in the presence of virus-specific antibodies or virus-specific CD8(+) T cells. We found that after systemic recall infection with LCMV-WE the presence of virus-specific antibodies allowed intracellular replication of virus in the marginal zone of spleen. In contrast, specific antibodies limited viral replication in liver, lung, and kidney. Upon recall infection with the persistent virus strain LCMV-Docile, viral replication in spleen was essential for the priming of CD8(+) T cells and for viral control. In contrast to specific antibodies, memory CD8(+) T cells inhibited viral replication in marginal zone but failed to protect mice from persistent viral infection. We conclude that virus-specific antibodies limit viral infection in peripheral organs but still allow replication of LCMV in the marginal zone, a mechanism that allows immune boosting during recall infection and thereby guarantees control of persistent virus.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Virus de la Coriomeningitis Linfocítica/genética , Virosis/inmunología , Replicación Viral/inmunología , Animales , Anticuerpos Antivirales/genética , Anticuerpos Antivirales/inmunología , Linfocitos T CD8-positivos/virología , Humanos , Virus de la Coriomeningitis Linfocítica/inmunología , Tejido Linfoide/inmunología , Tejido Linfoide/virología , Ratones , Bazo/inmunología , Bazo/virología , Virosis/genética , Replicación Viral/genéticaRESUMEN
Currently, lentiviral vectors for research and gene therapy are produced from 293-T cells that are transiently transfected with plasmids encoding the vector and helper functions. However, transiently transfected vectors as well as the presence of SV40 virus large T-antigen (T-Ag) cause serious technical and safety considerations. We aimed to exploit single copy integration sites in the HEK293 genome supporting lentiviral vector production. We found that lentiviral vectors result in minimal infectious particle production from single copy integrants in HEK293. Moreover, once this cell line harbors single copy integrations of lentiviral vectors, its ability to transiently produce lentiviral vectors becomes strongly impaired. T-Ag has a dramatic effect on virus production. Low levels of constitutive T-Ag expression can overcome the production restriction imposed by integrated lentiviral vectors copies. Interestingly, T-Ag does not exert its role at the level of transcriptional activity of the vector; rather, it seems to impose an indirect effect on the cell thereby enabling lentiviral vector production. Altogether, our study highlights the restrictions for integrated lentiviral vectors that are relevant for the establishment of stable and safe producer cell lines.