Los autores desean agradecer a Michael Chou, Bingbing Dai, y Liang Xiao para aportar datos de este manuscrito. También se agradece al Dr. John Gray por las generosas donaciones de los reactivos utilizados en este estudio. PB es apoyado por una beca postdoctoral del Centro Nacional del Cáncer. Esta investigación fue apoyada por subvenciones del Instituto Nacional de Salud (R01AI68978, P01CA132681 y RCA170820A), una subvención de la Fundación Bill y Melinda Gates, una subvención aceleración traslacional del Centro Conjunto para la Medicina Traslacional y una subvención de la epidemia de VIH / SIDA de California Programa de Investigación.

DC-LV células productoras estables se construyen sobre la base de la línea de envasado 11 células GPR que contiene los componentes necesarios lentiviral gagpol, rev y el sistema tet-off. En primer lugar, la transducción retroviral se utiliza para generar una línea celular de empaquetamiento GPRS que codifica una glicoproteína SVGmu tet-dependiente. Entonces, concatemer array transfección se utilizó para transfectar la línea celular GPRS con un vector lentiviral transgén como GFP. Esta línea celular productora estable, designado como LV-MGFP, se puede probar in vitro e in vivo por su capacidad para producir una vacuna CC-LV frente a GFP. 1. Generación de una línea celular SVGmu Tet-dependiente El plásmido PRX-SVGmu es un constructo en el que SVGmu glicoproteína DC-específica es clonado corriente abajo del promotor TTA-avanzada de plásmido retroviral pRetroX-Tet-Off 1 (Figura 1C). Cultura células 293T en D10 (medio de Eagle modificado de Dulbecco con10% de suero bovino fetal, 2 mM de L-glutamina). Cultura GPR línea celular de empaquetamiento en D10 con doxiciclina (1 ng / ml) y puromicina (2 g / ml). Todas las células se cultivan en un humidificado 37 ° C incubadora con 5% de CO 2. <ol><li> 16-18 h antes de la transfección transitoria, placa de 2 x 10 6 células 293T en 4 ml de D10 en una placa de cultivo tisular de 6 cm de tal manera que las células se acercan a 90% de confluencia en el momento de la transfección. </li><li> La transfección de plásmidos retrovirales: mezcla 100 l 1,25 M CaCl2 solución, agua Milli-Q estéril y los siguientes plásmidos: 5 mg PRX-SVGmu, 2,5 g PGAG-Pol, 2,5 g pVSV-G. El volumen final es de 500 l. Para un tubo de poliestireno ml de fondo redondo de 5, añadir 500 l de 2X HBS (50 mM HEPES, 10 mM de KCl, 12 mM de Dextrosa, 280 mM NaCl, 1,5 mM Na 2 HPO 4 * 7H 2 O, pH 7,05). Añadir gota a gota la mezcla de plásmido en el tampón HBS 2X mientras se burbujeaba el tampón vigorosamente con una pipeta Pasteur de vidrio. Después de añadir el mixture, continúe burbujeando durante otros 30 segundos. A continuación, añadir toda la mezcla sobre las células 293T en la placa de cultivo de 6 cm, y se incuba a 37 ° C. </li><li> 4 horas después de la transfección, vuelva a colocar cuidadosamente el medio en la placa de cultivo con 4 ml de D10 precalentado. </li><li> Girar la infección: 48 h después de la transfección, la cosecha SVGmu-que codifica partículas retrovirales en el sobrenadante por el que pasa el sobrenadante a través de un filtro de 0,45 micras-. Placa de las células empaquetadoras GPR en una placa de 24 pocillos a 2 x 10 4 células / pocillo. Añadir filtró el sobrenadante a células de empaquetamiento GPR en el plato (2 ml / pocillo) y las células de centrifugación durante 90 min a 1050 x g y 25 º C. Cambio de medio en D10 fresco con doxiciclina (1 ng / ml) después de spin-infección (2 ml / pocillo). </li><li> 72 horas después de la transfección, expandir el cultivo de las células transducidas de embalaje en D10 con doxiciclina (1 ng / ml) y puromicina (2 ng / ml). </li><li> Para confirmar la expresión de SVGmu, cultivar las células sin doxiciclina durante 48 horas. Mida ex superficiesión de SVGmu con citometría de flujo utilizando anticuerpos anti-Sindbis suero. Estas células son designadas como línea celular de empaquetamiento GPRS. </li></ol> 2. Construir DC-LV células productoras de matriz concatemer Transfección Lentiviral transferencia de plásmido TL20-GFP es un auto-inactivación lentiviral vector de transferencia de plásmido basado en pCL20c-MSCV-GFP con un sistema de Dox-regulable genoma viral ARN expresión y la sustitución del citomegalovirus (CMV), potenciador con 7 operadores tet (Figura 1C) 11 , 12,15. Plásmido PGK-ble es un resistente casete bleomicina (BLE), impulsados ​​por la debilidad del promotor PGK 2. <ol><li> Resumen 20 g TL20-GFP plásmido con la enzima de restricción SfiI en 50 ° C. Resumen 20 g plásmido PGK-ble con PflMI a 37 ° C. </li><li> Purificar los fragmentos de ADN (el casete PGK-ble de 1011 pb y el vector TL20-GFP es 6861 pb) mediante electroforesis en gel de agarosa. </li><li> Ligar el vector TL20-GFP y PGK-casete ble en una relación molar de 25:1 (NEB ADN ligasa de T4). Incubar toda la noche a temperatura ambiente. </li><li> Después de la ligación durante la noche, purificar ADN a partir de la mezcla de ligación (Qiagen DNeasy Tissue Kit y sangre). Asegúrese de que la cantidad de ADN purificado es de alrededor de 5 g. </li><li> 16-18 h antes de la transfección, las células de la placa de embalaje GPRS en una placa de cultivo tisular de 6 cm de tal manera que la confluencia será de aproximadamente 90% en el momento de la transfección. </li><li> Usar el método de transfección con fosfato de calcio descrito en el paso 1.2 para transfectar 5 ug del ADN concatemeric purificada en las células de empaquetamiento GPRS en la 6 cm de placa de cultivo tisular. 4 horas después de la transfección, quitar cuidadosamente el medio y reemplazar con 4 ml de D10 precalentado con doxiciclina (1 ng / ml). </li><li> 48 h después de la transfección, cambiar el medio en D10 con doxiciclina (1 ng / ml) y puromicina (2 g / ml). Seleccionar para clones transfectados mediante la adición de zeocina (50 g / ml) en el medio de cultivo. Cultivar las células durante aproximadamente 2 semanas hasta celular colonizaciónES se puede ver en la parte inferior de los platos. </li><li> Etiquetar las colonias de células en la parte inferior de los platos. Tome placas de cultivo de tejidos de 24 pocillos, el número de los pocillos y añadir a cada pocillo 2 ml de D10 que contiene zeocina (50 g / ml), doxiciclina (1 ng / ml), y puromicina (2 g / ml). Aspirar el medio de las células transducidas, a continuación, añadir una gota de tripsina en cada una de las colonias por menos de un minuto. A continuación, añadir una o más gotas de D10 en las mismas colonias. Recoger las colonias una a una con una pipeta y transferirlos en pocillos separados de la placa de cultivo de tejidos de 24 pocillos. </li><li> Cultura y expandir todos los clones de células en D10 que contiene zeocina (50 g / ml), doxiciclina (1 ng / ml) y puromicina (2 g / ml) para la evaluación de la capacidad productora de viral. </li></ol> 3. Evaluar Viral producción de cada clon celular <ol><li> Tripsinizar las células productoras y la placa de alrededor de 4x10 6 células en 6 cm de placa de cultivo tisular de D10 sin doxiciclina tal que la confluenciaexcede 90%. Reemplazar el medio con D10 precalentado fresco todos los días y se recoge el medio para el ensayo de título de 72 horas después de la eliminación de DOX. </li><li> Se recoge el sobrenadante viral por filtración el medio con un filtro de 0,45 micras-. </li><li> Las células diana que expresan el receptor de placas DC-SIGN (por ejemplo, 293T.hDC-SIGN o de médula ósea de ratón derivados de células dendríticas 16) y 293T como control negativo en placas de cultivo de 96 pocillos, 1 x 10 4 células / pocillo. Añadir diluciones seriadas de 2 veces del sobrenadante viral a los pocillos (100 l / pocillo). Por lo general, 6-8 diluciones son suficientes para alcanzar el rango en el que las células transducidas GFP-positivas están en una relación lineal con la cantidad de virus añadido. </li><li> Centrifugar las células y reemplazar el medio como se ha descrito en el paso 1.4. BMDCs deben ser cultivadas en medio RPMI con 10% de FBS y GM-CSF (01:20 J558L medio acondicionado). </li><li> Medir la expresión de GFP en células transducidas por citometría de flujo de 4-6 días después de la transducción. El vecto lentiviralr título se calcula en el intervalo de dilución del vector cuando el porcentaje de células positivas para GFP y la cantidad de vectores están en una relación lineal. Elige el clon celular con la producción viral más alta para aplicaciones posteriores. </li></ol> 4. Producir y Concentrado vectores lentiviral <ol><li> Cultura de la línea celular productora (LV-MGFP) en 15 cm de placas de cultivo de tejidos. </li><li> Tripsinizar las células productoras y las células de la placa en placas de cultivo tisular de 15 cm a más de 90% de confluencia en D10 fresco sin doxiciclina. Cambio medio con frescos D10, precalentado a diario. </li><li> En el momento de la producción viral pico (según lo determinado por el usuario), cosechar el sobrenadante viral y filtrarla utilizando un filtro de 0,45 micras-. Cargar el sobrenadante filtrado de pared en tubos de ultracentrífuga 32,5 ml gruesas, sellar con parafilm y se centrifuga a 50.000 xg, 4 º C durante 90 min. A fondo resuspender el precipitado en 50 l o un volumen apropiado de PBS o HBSS dependiendo de la aplicación. &lt;/ Li&gt; </ol> 5. Inmunizar ratones in vivo y analizar la respuesta inmune específica de antígeno <ol><li> Producir virus de alto título como se describe en la sección 4. </li><li> Inyectar ratones BALB / c (6-8 semanas de edad, hembra) a través de una ruta almohadilla de la pata con la suspensión de vector (25 l por almohadilla de la pata). </li><li> 2 semanas después de la inmunización, aislar esplenocitos del bazo y la cosecha. Esplenocitos de cultivo con GFP epítopo péptidos y analizar la presencia de GFP-específica linfocitos T CD8 utilizando tinción intracelular de citoquinas +, como se ha descrito anteriormente 17 (Figura 3B). </li></ol>

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<p class="jove_content"&gt; Los autores declaran que no tienen intereses financieros en competencia.</p

Aquí, hemos esbozado un método para producir grandes cantidades de vectores lentiviral utilizando células 293T transducidas establemente con todos los componentes de lentivirus bajo el tet fuera del sistema regulatorio. Hasta la fecha, la mayoría de los protocolos para producir vectores lentivirales se basan en fosfato de calcio transfección transitoria estándar (ver 18, por ejemplo). Este enfoque ha tenido éxito a nivel clínico, pero puede sufrir de algunas limitaciones que no puedan estar presentes ...

Muchos sistemas de vectores se han desarrollado para la entrega de genes. Los vectores basados ​​en lentivirus han estado entre el sistema viral más comúnmente estudiada. Estos vectores son ventajosas, ya que pueden transducir eficientemente tanto que se dividen y no se dividen las células 1, conseguir la expresión a largo plazo debido a la integración en el genoma huésped, exhibir una baja inmunidad anti-vector natural en la mayoría de las poblaciones humanas 2, y tienen un bajo potencial de genotoxicidad de mutagénesis de inserción 3,4. Producción de lentivirus siempre ha sido coloreado por las preocupaciones de seguridad. Los vectores lentivirales se derivan generalmente de VIH-1, el agente etiológico del SIDA. La transfección transitoria de los componentes individuales de la lentivirus (transferencia, sobre, y los plásmidos de embalaje) es un medio común y flexible de la entrega de material genético en el laboratorio. Sin embargo, la ampliación de las transfecciones transitorias para la aplicación clínica es engorroso y may conducir al desarrollo de lentivirus con capacidad de replicación 5,6. Para superar estos obstáculos, varios envases estable y líneas celulares productoras se han desarrollado 6-11. Una de estas líneas, la línea de envasado GPR 11, tiene la ventaja de ser atractivo regulado por tetraciclina. En este trabajo se demuestra cómo adaptar este sistema para producir auto-inactivación de vectores lentiviral que se dirigen específicamente a las células dendríticas (DC-LV) 12. Las células dendríticas (DC) son las más robustas células presentadoras de antígenos del sistema inmune. Han sido objeto de gran interés en el desarrollo de vacuna contra el cáncer, ya que inician directamente, programa, y regulan las respuestas inmunes específicas de tumor 13. La incorporación de un protocolo de vacunación para incluir a los DC tiene el potencial de provocar una respuesta inmune antitumoral más fuerte que las vacunas de péptidos o ADN. Recientemente, se ha desarrollado un vector lentiviral que specifically dirige a las células dendríticas a través de una glicoproteína del virus de Sindbis modificado, SVGmu 14. Estos vectores son únicos en cuanto a que muestran una alta especificidad para células dendríticas y generan respuestas inmunes más fuertes que los vectores no específicos, VSVG-pseudotyped. Aquí, se presenta un método para la producción de grandes cantidades de estos vectores lentiviral DC-dirigidos. Se demuestra que estos DC-LV puede infectar a los DC y generar fuertes T CD8 + respuestas inmunes celulares. Todos los procedimientos con animales se llevaron a cabo con humanidad, en virtud de la aprobación de la USC Intitutional Cuidado de Animales y el empleo Comisión. Para llevar a cabo experimentos in vivo y clínica, es fundamental para crear una línea de células que pueden producir virus en una alta concentración. Realización de las etapas de transducción y transfección exactamente como se describe será maximizar las posibilidades de la generación de dicho clon.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td>Mediatech Inc.</td>
			<td>10-013-CV</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Glutamine</td>
			<td>Mediatech Inc.</td>
			<td>25-005-Cl</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Doxycycline</td>
			<td>Clontech Laboratories, Inc.</td>
			<td>631311</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Zeocin</td>
			<td>Invitrogen</td>
			<td>R250-01</td>
			<td>Toxic</td>
		 </tr>
		 <tr>
			<td>Puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td></td>
		 </tr>
		 <tr>
			<td>T4 DNA Ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DNeasy Blood &amp; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a tetracycline-regulated cell line produces high-titer lentiviral vectors that specifically target dendritic cells

Los vectores lentivirales (LVs) son un poderoso medio de la entrega de material genético a muchos tipos de células. Debido a los problemas de seguridad asociados a estas VIH-1 vectores derivados, produciendo grandes cantidades de LV es un reto. En este trabajo se presenta un método para la producción de títulos elevados de LV libre inactivar. Nos retroviral transducir la Tet-Off productor estable la línea celular GPR para generar una línea celular, GPRS, que pueden expresar todos los componentes virales, incluyendo una glicoproteína específica de célula dendrítica, SVGmu. A continuación, utilizamos la transfección de ADN concatemeric para transfectar el plásmido que codifica la transferencia de LV un gen reportero GFP en combinación con un marcador seleccionable. Varios de los clones resultantes pueden producir LV a un título 10 veces mayor que lo que logramos con transfección transitoria. Además, estos virus transducen eficazmente células dendríticas in vitro y generan una fuerte respuesta inmune de células T a nuestra antígeno reportero. Este método puede ser una buena opción FOr produciendo fuertes LV vacunas basadas en los estudios clínicos de cáncer o enfermedades infecciosas.

La línea celular estable descrito en este método puede producir grandes cantidades de vectores lentivirales que están dirigidos específicamente a las células dendríticas. Como se muestra en la Figura 1B, el aislamiento de clones individuales produjo líneas celulares estables de calidad variable 12. Entre 26 clones ensayados, 8 produce partículas lentivirales a un título mayor de 10 6 unidades de transducción por ml (UT / ml), que es un punto de referencia típica para los...

Watch this Scientific Journal Video about A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells at JoVE.com

A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Aquí, nosotros usamos la transducción retroviral y transfección concatemeric para crear una línea de células que pueden expresar las componentes de un vector lentiviral (VI) en ausencia de tetraciclina. Esta LV codifica GFP y se pseudotyped con una glicoproteína, SVGmu, que es específico para un receptor en las células dendríticas.

Una línea celular regulada por tetraciclina Produce lentiviral vectores de alta titulación que se dirigen específicamente a las células dendríticas

Lentiviral  vectors (LVs) are a powerful means of delivering genetic material to many types  of cells. Because of safety concerns associated with these ...

a-tetracycline-regulated-cell-line-produces-high-titer-lentiviral

Research

JoVE Journal

Immunology and Infection

14.4K vistas.  University of Southern California, Los Angeles. Aquí, nosotros usamos la transducción retroviral y transfección concatemeric para crear una línea de células que pueden expresar las componentes de un vector lentiviral (VI) en ausencia de tetraciclina. Esta LV codifica GFP y se pseudotyped con una glicoproteína, SVGmu, que es específico para un receptor en las células dendríticas.

Video: A Tetracycline-regulated Cell Line Produces High-titer Lentiviral Vectors that Specifically Target Dendritic Cells

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by na&iuml;ve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems. 
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFN&beta;. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.

We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.

Protocolo optimizado para la transfección eficiente de células dendríticas, sin maduración de las células

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Investigación

Inmunología e Infección

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

La introducción de la tensión de cizallamiento en el Estudio de la adhesión bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Aislamiento de células dendríticas del pulmón de ratón

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

La expansión de los linfocitos T citotóxicos sangre del cordón umbilical que Citomegalovirus Target, Virus de Epstein-Barr, adenovirus y

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

De las Migraciones células dendríticas mediante resonancia 19F / 1H Magnetic

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

Generación de una nueva vacuna de células dendríticas usando melanoma y células madre escamosas del cáncer

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c+), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

Aislamiento de células dendríticas mieloides y células epiteliales de timo humano

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Generación de inmaduras, maduras y tolerogénico células dendríticas con diferentes fenotipos metabólico

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

Un protocolo fácil de generar proteínas Site-Specifically acetiladas en Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&amp;#945;

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

Desarrollo y validación de una sola molécula ultrasensible matriz Digital análisis de enzima-ligado del inmunosorbente para interferón-α humano

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...