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1.
Nat Methods ; 14(5): 521-530, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28369043

RESUMEN

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCR-αß+ single-positive CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vß expression, consistent with allelic exclusion of Vß-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.


Asunto(s)
Órganos Artificiales , Diferenciación Celular , Células Madre Hematopoyéticas/citología , Organoides/citología , Linfocitos T/citología , Timo/citología , Biotecnología/métodos , Células Madre Hematopoyéticas/inmunología , Humanos , Organoides/inmunología , Células Madre/citología , Células Madre/inmunología , Linfocitos T/inmunología , Timo/inmunología
2.
Nucleic Acids Res ; 43(1): 682-90, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25520191

RESUMEN

Lentiviral vectors almost universally use heterologous internal promoters to express transgenes. One of the most commonly used promoter fragments is a 1.2-kb sequence from the human ubiquitin C (UBC) gene, encompassing the promoter, some enhancers, first exon, first intron and a small part of the second exon of UBC. Because splicing can occur after transcription of the vector genome during vector production, we investigated whether the intron within the UBC promoter fragment is faithfully transmitted to target cells. Genetic analysis revealed that more than 80% of proviral forms lack the intron of the UBC promoter. The human elongation factor 1 alpha (EEF1A1) promoter fragment intron was not lost during lentiviral packaging, and this difference between the UBC and EEF1A1 promoter introns was conferred by promoter exonic sequences. UBC promoter intron loss caused a 4-fold reduction in transgene expression. Movement of the expression cassette to the opposite strand prevented intron loss and restored full expression. This increase in expression was mostly due to non-classical enhancer activity within the intron, and movement of putative intronic enhancer sequences to multiple promoter-proximal sites actually repressed expression. Reversal of the UBC promoter also prevented intron loss and restored full expression in bidirectional lentiviral vectors.


Asunto(s)
Vectores Genéticos , Intrones , Lentivirus/genética , Regiones Promotoras Genéticas , Ubiquitina C/genética , Elementos de Facilitación Genéticos , Exones , Expresión Génica , Células HEK293 , Humanos , Células K562 , Factor 1 de Elongación Peptídica/genética , Empalme del ARN
3.
Proc Natl Acad Sci U S A ; 110(5): 1857-62, 2013 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-23319634

RESUMEN

Positron emission tomography (PET) reporter genes allow noninvasive whole-body imaging of transplanted cells by detection with radiolabeled probes. We used a human deoxycytidine kinase containing three amino acid substitutions within the active site (hdCK3mut) as a reporter gene in combination with the PET probe [(18)F]-L-FMAU (1-(2-deoxy-2-(18)fluoro-ß-L-arabinofuranosyl)-5-methyluracil) to monitor models of mouse and human hematopoietic stem cell (HSC) transplantation. These mutations in hdCK3mut expanded the substrate capacity allowing for reporter-specific detection with a thymidine analog probe. Measurements of long-term engrafted cells (up to 32 wk) demonstrated that hdCK3mut expression is maintained in vivo with no counter selection against reporter-labeled cells. Reporter cells retained equivalent engraftment and differentiation capacity being detected in all major hematopoietic lineages and tissues. This reporter gene and probe should be applicable to noninvasively monitor therapeutic cell transplants in multiple tissues.


Asunto(s)
Desoxicitidina Quinasa/metabolismo , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Animales , Arabinofuranosil Uracilo/análogos & derivados , Arabinofuranosil Uracilo/química , Arabinofuranosil Uracilo/metabolismo , Western Blotting , Línea Celular Tumoral , Desoxicitidina Quinasa/genética , Femenino , Radioisótopos de Flúor/química , Células Madre Hematopoyéticas/metabolismo , Inmunohistoquímica , Subunidad gamma Común de Receptores de Interleucina/deficiencia , Subunidad gamma Común de Receptores de Interleucina/genética , Estimación de Kaplan-Meier , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Endogámicos , Ratones Noqueados , Ratones SCID , Mutación , Timo/diagnóstico por imagen , Timo/metabolismo , Factores de Tiempo , Trasplante Heterólogo
4.
Proc Natl Acad Sci U S A ; 105(38): 14459-64, 2008 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-18791068

RESUMEN

With their unique ability to differentiate into all cell types, embryonic stem (ES) cells hold great therapeutic promise. To improve the efficiency of embryoid body (EB)-mediated ES cell differentiation, we studied murine EBs on the basis of their size and found that EBs with an intermediate size (diameter 100-300 microm) are the most proliferative, hold the greatest differentiation potential, and have the lowest rate of cell death. In an attempt to promote the formation of this subpopulation, we surveyed several biocompatible substrates with different surface chemical parameters and identified a strong correlation between hydrophobicity and EB development. Using self-assembled monolayers of various lengths of alkanethiolates on gold substrates, we directly tested this correlation and found that surfaces that exhibit increasing hydrophobicity enrich for the intermediate-size EBs. When this approach was applied to the human ES cell system, similar phenomena were observed. Our data demonstrate that hydrophobic surfaces serve as a platform to deliver uniform EB populations and may significantly improve the efficiency of ES cell differentiation.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Madre Embrionarias/citología , Interacciones Hidrofóbicas e Hidrofílicas , Animales , Proliferación Celular , Supervivencia Celular , Medio de Cultivo Libre de Suero , Dimetilpolisiloxanos/farmacología , Células Madre Embrionarias/efectos de los fármacos , Humanos , Ratones
5.
J Immunother Cancer ; 8(2)2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32737142

RESUMEN

BACKGROUND: Dendritic cells (DCs) are crucial for the efficacy of cancer vaccines, but current vaccines do not harness the key cDC1 subtype required for effective CD8+ T-cell-mediated tumor immune responses. Vaccine immunogenicity could be enhanced by specific delivery of immunogenic tumor antigens to CD141+ DCs, the human cDC1 equivalent. CD141+ DCs exclusively express the C-type-lectin-like receptor CLEC9A, which is important for the regulation of CD8+ T cell responses. This study developed a new vaccine that harnesses a human anti-CLEC9A antibody to specifically deliver the immunogenic tumor antigen, NY-ESO-1 (New York esophageal squamous cell carcinoma 1), to human CD141+ DCs. The ability of the CLEC9A-NY-ESO-1 antibody to activate NY-ESO-1-specific naïve and memory CD8+ T cells was examined and compared with a vaccine comprised of a human DEC-205-NY-ESO-1 antibody that targets all human DCs. METHODS: Human anti-CLEC9A, anti-DEC-205 and isotype control IgG4 antibodies were genetically fused to NY-ESO-1 polypeptide. Cross-presentation to NY-ESO-1-epitope-specific CD8+ T cells and reactivity of T cell responses in patients with melanoma were assessed by interferon γ (IFNγ) production following incubation of CD141+ DCs and patient peripheral blood mononuclear cells with targeting antibodies. Humanized mice containing human DC subsets and a repertoire of naïve NY-ESO-1-specific CD8+ T cells were used to investigate naïve T cell priming. T cell effector function was measured by expression of IFNγ, MIP-1ß, tumor necrosis factor and CD107a and by lysis of target tumor cells. RESULTS: CLEC9A-NY-ESO-1 antibodies (Abs) were effective at mediating delivery and cross-presentation of multiple NY-ESO-1 epitopes by CD141+ DCs for activation of NY-ESO-1-specific CD8+ T cells. When benchmarked to NY-ESO-1 conjugated to an untargeted control antibody or to anti-human DEC-205, CLEC9A-NY-ESO-1 was superior at ex vivo reactivation of NY-ESO-1-specific T cell responses in patients with melanoma. Moreover, CLEC9A-NY-ESO-1 induced priming of naïve NY-ESO-1-specific CD8+ T cells with polyclonal effector function and potent tumor killing capacity in vitro. CONCLUSIONS: These data advocate human CLEC9A-NY-ESO-1 Ab as an attractive strategy for specific targeting of CD141+ DCs to enhance tumor immunogenicity in NY-ESO-1-expressing malignancies.


Asunto(s)
Antígenos de Neoplasias/metabolismo , Linfocitos T CD8-positivos/metabolismo , Células Dendríticas/inmunología , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Mitogénicos/metabolismo , Trombomodulina/metabolismo , Animales , Femenino , Voluntarios Sanos , Humanos , Ratones
6.
Clin Cancer Res ; 25(3): 1000-1011, 2019 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-30409823

RESUMEN

PURPOSE: To improve persistence of adoptively transferred T-cell receptor (TCR)-engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application. EXPERIMENTAL DESIGN: HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/Kb mice within a formal Good Laboratory Practice (GLP)-compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)-compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use. RESULTS: TCR genetically modified and ex vivo-cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality. CONCLUSIONS: Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471.


Asunto(s)
Terapia Genética/métodos , Células Madre Hematopoyéticas/inmunología , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Antígenos de Neoplasias/genética , Células Cultivadas , Ensayos Clínicos como Asunto , Drogas en Investigación/uso terapéutico , Antígeno HLA-A2/genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias/genética , Neoplasias/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo
7.
Elife ; 52016 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-27823582

RESUMEN

T cells engineered to express a tumor-specific αß T cell receptor (TCR) mediate anti-tumor immunity. However, mispairing of the therapeutic αß chains with endogenous αß chains reduces therapeutic TCR surface expression and generates self-reactive TCRs. We report a general strategy to prevent TCR mispairing: swapping constant domains between the α and ß chains of a therapeutic TCR. When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses. By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity. We validate this approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease. We also validate a related approach whereby replacement of αß TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair. This work enables the design of safer TCR gene therapies for cancer immunotherapy.


Asunto(s)
Genes Codificadores de los Receptores de Linfocitos T , Terapia Genética/efectos adversos , Terapia Genética/métodos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Animales , Modelos Animales de Enfermedad , Enfermedad Injerto contra Huésped , Ratones , Dominios Proteicos , Recombinación Genética
8.
Stem Cells Transl Med ; 4(2): 136-45, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25575527

RESUMEN

Autologous human induced pluripotent stem cells (hiPSCs) should allow cellular therapeutics without an associated immune response. This concept has been controversial since the original report that syngeneic mouse iPSCs elicited an immune response after transplantation. However, an investigative analysis of any potential acute immune responses in hiPSCs and their derivatives has yet to be conducted. In the present study, we used correlative gene expression analysis of two putative mouse "immunogenicity" genes, ZG16 and HORMAD1, to assay their human homologous expression levels in human pluripotent stem cells and their derivatives. We found that ZG16 expression is heterogeneous across multiple human embryonic stem cell and hiPSC-derived cell types. Additionally, ectopic expression of ZG16 in antigen-presenting cells is insufficient to trigger a detectable response in a peripheral blood mononuclear cell coculture assay. Neither of the previous immunogenicity-associated genes in the mouse currently appears to be relevant in a human context.


Asunto(s)
Células Madre Embrionarias/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/inmunología , Células Madre Pluripotentes/inmunología , Animales , Línea Celular , Células Madre Embrionarias/citología , Humanos , Lectinas/inmunología , Proteínas de la Membrana/inmunología , Ratones , Células Madre Pluripotentes/citología , Especificidad de la Especie
9.
Cancer Res ; 74(18): 5173-83, 2014 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-25038231

RESUMEN

Engineering immunity against cancer by the adoptive transfer of hematopoietic stem cells (HSC) modified to express antigen-specific T-cell receptors (TCR) or chimeric antigen receptors generates a continual supply of effector T cells, potentially providing superior anticancer efficacy compared with the infusion of terminally differentiated T cells. Here, we demonstrate the in vivo generation of functional effector T cells from CD34-enriched human peripheral blood stem cells modified with a lentiviral vector designed for clinical use encoding a TCR recognizing the cancer/testes antigen NY-ESO-1, coexpressing the PET/suicide gene sr39TK. Ex vivo analysis of T cells showed antigen- and HLA-restricted effector function against melanoma. Robust engraftment of gene-modified human cells was demonstrated with PET reporter imaging in hematopoietic niches such as femurs, humeri, vertebrae, and the thymus. Safety was demonstrated by the in vivo ablation of PET signal, NY-ESO-1-TCR-bearing cells, and integrated lentiviral vector genomes upon treatment with ganciclovir, but not with vehicle control. Our study provides support for the efficacy and safety of gene-modified HSCs as a therapeutic modality for engineered cancer immunotherapy. Cancer Res; 74(18); 5173-83. ©2014 AACR.


Asunto(s)
Genes Transgénicos Suicidas , Células Madre Hematopoyéticas/fisiología , Herpesvirus Humano 1/genética , Inmunoterapia/métodos , Tomografía de Emisión de Positrones/métodos , Linfocitos T/inmunología , Animales , Antígenos CD34/sangre , Antígenos CD34/inmunología , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Modelos Animales de Enfermedad , Terapia Genética , Vectores Genéticos/genética , Células Madre Hematopoyéticas/diagnóstico por imagen , Células Madre Hematopoyéticas/inmunología , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Transducción Genética
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