Apoyado en parte por el NIH R01 CA121258, CA125244 R01, R01 CA154256, NIH / NCATS UCLA CTSI la subvención Número UL1TR000124, USAMRMC W81XWH-08-1-0734, y el Fondo de Investigación Joan S. Holmes Memorial. MJH y GCO recibieron el apoyo de la Joan S. Holmes Memorial beca posdoctoral en la UCLA. El dispositivo CSM se obtuvo de métodos científicos creativos: www.creative-sci.com. El vector lentiviral se recibió de la UCLA Vector Core, que es apoyado por DK041301 CURE / P30.

1. Preparación de los portaobjetos <ol><li> Insertar diapositivas colector de sedimentación celular en bolsas de esterilización y selle con cinta de autoclave. Oriente el lado recubierto de teflón de la corredera del lado del papel de la bolsa para evitar los depósitos de plástico en los pozos. </li><li> Bolsas de autoclave durante 15 min a 121 ° C. </li><li> Saque los portaobjetos de la bolsa de esterilización dentro de una cabina de bioseguridad y colocar en un 150 x 15 mm placa de Petri estéril estéril. Hasta cuatro diapositivas puede caber por plato. Colocar una placa de 35 x 10 mm Petri junto a las diapositivas. Añadir 2-3 ml de H2O estéril para proporcionar humidificación. </li><li> Diapositivas pre-capa con poli-D-lisina en 100 g / ml mediante el uso de un volumen suficiente para cubrir la totalidad de bien. Después de una hora a RT, aspirar la solución y enjuague la superficie del pozo dos veces pipeteando 1x PBS sobre la superficie de los pocillos. </li><li> Opcionalmente, añadir fibronectina u otros componentes de ECM para asegurar más fuerte adherencia de las células del tumor. Añadir fibronectina a 5 g / ml en un volumen suficiente That los pozos no se secará dentro de un período corto de tiempo a temperatura ambiente. <ol><li> Después de 1 hora, aspirar la solución sobrante y lavar dos veces con la pipeta hacia arriba y abajo con 1x PBS. </li></ol></li><li> Mantenga PBS en los pozos hasta que el tumor está listo para la siembra. Uso desliza el mismo día. </li><li> Cosecha de un matraz confluente de el tipo de célula tumoral adherente deseado. Contar las células viables mediante exclusión con colorante azul de tripano por microscopía de luz y resuspender en 5 x 10 6 células / ml en el ejemplo medio para el crecimiento adecuadamente tamponada con, medio esencial mínimo de Dulbecco (DMEM) con suero bovino fetal al 10% (FBS), L- glutamina y piruvato de sodio. </li><li> Si se desea imágenes fluorescentes de la población de células adherentes, etiquetar la población celular adherente con un colorante fluorescente vital o un colorante de la proliferación de células siguiendo los protocolos proporcionados por el fabricante. </li><li> Suavemente pipeta de 10 l de medio completo en cada pocillo de la atención de diapositivas tomar para evitar la creación de burbujas en el wells, ya que estos pueden prevenir uniforme adherencia de las células del tumor. </li><li> Añadir 2,5-5,0 x 10 4 células (5-10 l de suspensión celular) al medio en cada pocillo (Figura 1A). Ajuste el número óptimo dependiendo del tipo de célula tumoral y el número de días de crecimiento deseados. Las células tumorales más grandes o células de crecimiento rápido pueden requerir un menor número de células inicialmente. Aumentar el volumen así hasta 40 l mediante la adición de medio y pipeta de arriba y abajo para asegurar una distribución uniforme de las células. </li><li> Después de todos los pozos se siembran, permitir que las células tumorales se adhieran a temperatura ambiente, a continuación, coloque la tapa en el 150 x 15 mm placa de Petri y se mueven con cuidado el plato a un incubador humidificado 37 ° C / 5% de CO 2 durante al menos 24 horas. </li><li> Cambie el medio de cultivo a diario. Pipetear con cuidado una porción del medio gastado de la monocapa y reemplazar con medio completo fresco. NOTA: Debido a la evaporación, será necesario añadir que fue tomada más volumen. Wells estará listo para el ensayo cuando un par, confluente capa de células está presente. Esto es típicamente 1-3 días después de la siembra inicial. </li><li> Lave suavemente monocapa una vez con PBS como se describe en el paso 1.4 y, o bien continúe con el paso 2 si se desean monocapas, o al paso por debajo de 1,13 para extraer proteínas ECM de la monocapa. </li><li> Hacer una Triton-X (v / v) solución al 0,5% en medio completo y añadir 30 l a cada pocillo. Deje monocapa digerir en campana durante 2 min, luego lavar dos veces con medio completo mediante pipeteo como se describe anteriormente. NOTA: La capa de ECM puede ser visible al microscopio óptico (Figura 2). Utilice diapositivas de inmediato, o mantener en medio completo en el humidificado incubadora de CO 2 durante 1-2 días. No permita que los pocillos se sequen. </li></ol> 2. La sedimentación de las células T no adherentes en un portaobjetos <ol><li> Si se desea una imagen fluorescente de la población de células no adherentes, etiquetar las poblaciones de células no adherentes con un tinte fluorescente de vital importancia, como la CFSE, Cell Rastreador Red CMPTX o eFluor670 siguiendo los protocolos proporcionados por el fabricante al menos 1 hr antes de la prueba. Alternativamente, manipular las células para expresar proteínas fluorescentes codificadas por los vectores virales. </li><li> Autoclave el colector de sedimentación celular en una bolsa de autoclave como se describe en 1.2 anteriormente. </li><li> Retire la cámara de humidificado contiene diapositivas recubiertos de teflón de la incubadora y el lugar en el gabinete de seguridad biológica. </li><li> Lavar los pocillos con PBS 1x pipeteando arriba y abajo, a continuación, añadir 45 l de medio completo que contiene al menos 10% de suero. </li><li> Retire el colector de la envoltura de esterilización y deslice cuidadosamente en más de los pocillos hasta que el &quot;gancho&quot; en el extremo del colector toca la parte inferior de la corredera (Figura 1B). </li><li> Asegúrese de que el medio de cultivo es visible en cada uno de los canales. Si ninguno se ve, quitarse el colector y añadir más medio al pocillo correspondiente, luego vuelva a colocar y puedes volver a intentarlo. </li><li> Repita el paso 2.5 para cada channel del colector, o para tantos pozos como se desee. </li><li> Contar las células no adherentes y volver a suspender a no menos de 1,0 a 2,0 x 10 5 / l. Elaborar 1 l de células y lentamente la pipeta ellos en el canal (Figura 1C). Haga esto para cada bien deseado. </li><li> Deja todo el aparato cargado célula, es decir, del colector y de diapositivas, dentro de la bio-campana durante 20-30 min para permitir que las células en el canal se asienten. </li><li> Retire el colector al tocar ambos extremos y levantando suavemente de él hacia arriba de la diapositiva con el fin de no molestar a las células focalmente sembradas en el centro de los pozos (Figura 1D). </li><li> Inspeccionar cada pocillo para asegurar que las células no adherentes se han sembrado con éxito en el centro de la así permanecer dentro de la circunferencia del canal colector. Las células no adherentes se adhieran idealmente ligeramente a la monocapa o ECM y entre sí, por lo tanto, moviendo suavemente el portaobjetos alrededor no causará la pelle celulart para dispersar. No empujar la diapositiva. </li></ol> 3. Microscopía de Fluorescencia <ol><li> Con el equipo experimental apropiado, realizar la proyección de imagen después de que se confirmó la sedimentación. Lo ideal es utilizar un microscopio que está equipado con una cámara de CO 2 y la humedad. Imagen a baja potencia, es decir, 4X o 10X, para permitir múltiples imágenes para ser tomado de un área más grande para que la totalidad del bien puede ser visto. </li><li> Adquirir imágenes digitales usando el software de captura de imágenes. Realizar campo claro y / o imágenes de fluorescencia multi-canal. </li><li> Si lo desea, configure una de lapso de tiempo para grabar imágenes en diferentes canales sobre un área determinada, o mantener la diapositiva humidifica y en un / 5% de CO 2 incubadora a 37 ° C y manualmente sacar a la imagen en los puntos de tiempo deseados (Figura 1E y F, se recomienda para los puntos de tiempo más largos). </li></ol> 4. Análisis y Visualización <ol><li> El uso de software de edición de imagen, combinar la luz y la flimágenes fluorescentes en una sola capa para múltiples tipos de células y los marcadores se pueden ver en la misma imagen. Arrastre y suelte los archivos de imagen para cada color individual en el programa y, cuando se le solicite, seleccione la opción &quot;Añadir Capa&quot; para crear un archivo de imagen con múltiples capas. <ol><li> A mayor aumento, tomar, alinear y unir juntos las imágenes de todo el bien digitalmente. Alinear archivos mirando a regiones conservadas entre dos imágenes y la superposición de una imagen en la parte superior de la otra hasta que una imagen completa se genera. </li></ol></li><li> Añadir bares micras a las imágenes durante la adquisición usando el software de captura para determinar la distancia células individuales han migrado. </li><li> Para determinar la migración de células en diversos momentos, hacer mapas de intensidad de fluorescencia superficie con la imagen del software, es decir, ImageJ, para cuantificar la agregación de células T fluorescente o extendido en el tiempo. <ol><li> Para generar representaciones gráficas de superficie, tomar una imagen con capas generada en el paso 4.1 y bajo la ventana de capas,desactive todas las capas excepto las correspondientes al canal deseado. Por ejemplo, si se desea un gráfico de superficie para visualizar la expresión de EMD, sólo las capas &quot;verdes&quot; que corresponde al filtro utilizado para este marcador debe ser visible. </li><li> Aplanar la imagen y guardarla en un formato de archivo reconocido (por ejemplo, .png) y cargar la imagen en el programa. Para generar el gráfico de superficie, cambiar el tipo de imagen de 8 bits, cambiar el brillo / contraste, según sea necesario para reducir el fondo, y seleccione la opción &quot;Analizar / Superficie Terreno». Las imágenes de la Figura 5 se generaron mediante la comprobación de la &#39;sombra&#39;, &#39;Dibuja Axis&#39;, &#39;Un polígono por línea &quot;, y casillas de verificación&quot; suave &quot;. </li><li> Otra opción es tomar mediciones utilizando el radio o el diámetro del depósito de células focal en el tiempo cero y comparar a las células en el punto más exterior en diferentes momentos. El diámetro de los pozos de teflón es 6,0 mm. </li></ol></li></ol>

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The authors declare they have no competing financial interests.

Las células tumorales en una suspensión de células se pipetearon en los pocillos de un portaobjetos de Teflon-enmascarado. Las células se dejaron adherir y luego formaron monocapas en un humidificado 5% de CO 2, 37 ° C incubadora (Figura 1A). Monocapas establecidas o proteínas ECM derivados de la monocapa pueden ser cosechadas para estos ensayos (Figura 1B). Linfocitos T efectores marcados con colorantes fluorescentes vitales o transducidas con vectores que codifican pa...

El ensayo de migración de células Radial monocapa se desarrolló originalmente para medir las propiedades infiltrantes de células tumorales adherentes 1-4 en portaobjetos recubiertos con matriz extracelular (ECM), las proteínas 5-7 o con componentes de ECM individuales, tales como fibronectina o laminina 1,2. La técnica involucrada sembrar una suspensión de células individuales de células tumorales en el centro de pozos utilizando un colector de células de sedimentación de acero inoxidable (CSM). Después de la sedimentación, las células tumorales se adherirá a la parte inferior del pozo y el cambio en el diámetro de la población celular inicial en el tiempo se utilizó para establecer una tasa de movilidad horizontal. El ensayo de migración de células Radial Monocapa proporciona una ventaja visual sobre otros métodos existentes que emplean transwell placas de ensayo de las capacidades migratorias in vitro de células; estos ensayos no son propicias para la imagen 8. Además, también proporciona una gran cantidad de libertad en la elección del punto de tiempos cuando se evalúa la migración, sin límite en el número de puntos de tiempo un investigador podría elegir a la imagen después de la sedimentación. Debido a que la capacidad de migrar es una funcionalidad importante para las células no adherentes, especialmente en el área de la inmunoterapia o donde pueden ser utilizados como vehículos de administración para los vectores virales, se adaptó el uso de la CSM para evaluar la migración de células no adherentes tipos en monocapas de células tumorales, además de las proteínas de ECM. El beneficio añadido de microscópicamente la visualización de la migración de las células no adherentes en monocapas de células tumorales viables, en ECM complejo aislado del tumor, o en los componentes de ECM individuales hace que este ensayo versátil. Los ensayos que emplean pocillos recubiertos con una sola proteína extracelular no reflejan con exactitud el sustrato tejido o tumor ECM las células se migrar a través in vivo. Aquí, utilizamos alorreactivas linfocitos T citotóxicos (alloCTL), sensibilizados a gran histocompcomplejo atibility (MHC) de proteínas utilizando reacciones unidireccional de células tumorales de linfocitos mixtos (MLTR) o reacciones de linfocitos mixtos (MLR) 9, como nuestro representante tipo de células no adherentes. Hemos probado células tanto de origen humano y murino. Cuando la migración se midió en monocapas tumorales, las células tumorales empleadas eran objetivos, ya sea parcial pertinentes, se presentan algunas de las mismas proteínas MHC se encuentran en la población de células utilizado para sensibilizar a los efectores, o metas plenamente pertinentes, con un conjunto completo de moléculas del MHC que el efectores habían sensibilizado hacia. En algunos experimentos, se utilizó CellTracker fluorescente de color rojo CMPTX o tinte proliferación celular eFluor 670 para diferenciar entre las células efectoras y diana. También utilizamos la transducción con codificación de vectores virales para proteínas fluorescentes como una forma adicional de visualizar las células. Para ciertos ensayos, transduced la alloCTL con vectores retrovirales de replicación (RRV) que codifican para el verde esmeralda (EMD) de la proteína fluorescente 10,11; para othres, las células tumorales fueron transducidas con vectores lentivirales que codifican mStrawberry. El alloCTL se sembraron a través de un canal del colector en el centro de cualquiera de las monocapas de células tumorales o ECM cosechadas a partir de monocapas de células tumorales. Interacciones de las células adherentes y no adherentes se visualizaron por la luz y / o por microscopía de fluorescencia con el tiempo. Interrupción en la monocapa de células tumorales a baja potencia, o tumorales células con núcleos fragmentados a alta potencia fueron los indicadores de lesión celular por lisis y la apoptosis, respectivamente. Hemos creado digitalmente mapas fluorescentes intensidad superficie que muestran la migración de las células fluorescentes T no adherentes en los cultivos en monocapa. También notamos la citotoxicidad engendró a la monocapa de células de glioma adherente después de la formación de agrupaciones de la alloCTL no adherente superpuesto. Además, se observó la transducción horizontal de RRV-EMD desde el alloCTL a la monocapa de glioma.

<table border="0" cellpadding="0" cellspacing="0" width="920">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Name of Reagent/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:239px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:312px;">Teflon-masked microscope slides</td>
			<td style="width:203px;">Creative Scientific Methods</td>
			<td style="width:239px;">CSM002</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:312px;">Cell Sedimentation Manifold</td>
			<td style="width:203px;">Creative Scientific Methods</td>
			<td style="width:239px;">CSM001</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Petri Dish, 150mm</td>
			<td style="width:203px;">Corning</td>
			<td style="width:239px;">430597</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Petri Dish, 35mm</td>
			<td style="width:203px;">Corning</td>
			<td style="width:239px;">430588</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Phosphate-Buffered Saline (PBS)</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">21-040</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">20 &#956;L Pipetman</td>
			<td style="width:203px;">Gilson</td>
			<td style="width:239px;">F123600</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">200 &#956;L Pipetman</td>
			<td style="width:203px;">Gilson</td>
			<td style="width:239px;">F123601</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">200 &#956;L pipette tips</td>
			<td style="width:203px;">VWR</td>
			<td style="width:239px;">89003-060</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Distilled, deionized water (sterile)</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">25-055</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Trypsin</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">25-054-CL</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Celltracker Red CMPTX</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:239px;">C34552</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">TrypLE Express (optional)</td>
			<td style="width:203px;">Gibco</td>
			<td style="width:239px;">12604</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Tumor cell culture media (e.g. DMEM)</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">10-013</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">AIM-V serum-free media</td>
			<td style="width:203px;">Invitrogen</td>
			<td style="width:239px;">12055-083</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Fetal Bovine Serum</td>
			<td style="width:203px;">Omega Scientific</td>
			<td style="width:239px;">FB-02</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Inverted microscope</td>
			<td style="width:203px;"></td>
			<td style="width:239px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">SPOT Advanced</td>
			<td style="width:203px;">Diagnostic Instruments</td>
			<td style="width:239px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Poly-D-Lysine</td>
			<td style="width:203px;">Millipore</td>
			<td style="width:239px;">A-003-E</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Fibronectin</td>
			<td style="width:203px;">BD</td>
			<td style="width:239px;">354008</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">31/2 x 51/4 Autoclave Pouches</td>
			<td style="width:203px;">Crosstex</td>
			<td style="width:239px;">SCXS2</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Trypan Blue</td>
			<td style="width:203px;">Mediatech</td>
			<td style="width:239px;">25-900-CI</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">Cell Proliferation eFluor 670</td>
			<td style="width:203px;">eBioscience</td>
			<td style="width:239px;">65-0840-85</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:312px;">ImageJ</td>
			<td style="width:203px;">NIH</td>
			<td style="width:239px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

radial mobility and cytotoxic function of retroviral replicating vector transduced, non-adherent alloresponsive t lymphocytes

Presentamos una adaptación de la novela del ensayo de migración celular Radial monocapa, informó por primera vez para medir la migración radial de las células tumorales adherentes en las proteínas de la matriz extracelular, para la medición de la motilidad de las células marcadas con fluorescencia, humanos no adherente o efectoras inmunes murino. Esta técnica emplea un colector de acero inoxidable y 10 pocillos deslizante de Teflon para depositar focalmente células T no adherentes en los pocillos preparadas con monocapas confluentes de células tumorales o proteínas de la matriz extracelular. Microscopía de fluorescencia de luz y / o multi-canal se utiliza para seguir el movimiento y comportamiento de las células efectoras con el tiempo. Los colorantes fluorescentes y / o vectores virales que codifican para transgenes fluorescentes se utilizan para etiquetar diferencialmente los tipos de células para la imagen. Este método es distinto del de tipo similar en ensayos in vitro que el seguimiento de la migración / invasión horizontal o vertical utilizando cámaras de diapositivas, agar o transwell placas. El ensayo permite que los datos detallados de imagen para Be recogido con diferentes tipos de células que se distinguen por marcadores fluorescentes específicos; incluso subpoblaciones específicas de células (es decir, transducidas / nontransduced) pueden ser monitoreados. Parcelas de superficie intensidad de fluorescencia se generan utilizando canales específicos de fluorescencia que se corresponden con el tipo de célula que migra. Esto permite una mejor visualización de la movilidad de las células inmunes no adherente en momentos específicos. Es posible reunir pruebas de otras funciones de células efectoras, tales como la citotoxicidad o la transferencia de vectores virales de efector a células diana, también. Por lo tanto, el método permite a los investigadores documentan microscópicamente-célula a célula interacciones de diferencialmente marcado con, no adherente con células adherentes de diferentes tipos. Dicha información puede ser especialmente relevante en la evaluación de tipos de células inmunes biológicamente manipuladas o activados, cuando se desea una prueba visual de la funcionalidad con las células diana tumorales antes de su uso para la terapia del cáncer.

Los vectores virales que codifican para las proteínas fluorescentes se pueden utilizar además de, o en lugar de, tinte fluorescente. Transducción viral se debe realizar antes de el ensayo de motilidad. Ambos tipos de células adherentes y no adherentes pueden ser marcados de forma diferencial. El protocolo para la transducción dependerá del tipo de vector utilizado. Aquí, transduced la alloCTL en las figuras 3, 5 y 6 con RRV-EMD al menos dos días de anticipación de la prueba uti...

Watch this Scientific Journal Video about Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes at JoVE.com

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

Radial Movilidad y citotóxicos Función de Retroviral Replicar Vector transducidas, no adherente Alloresponsive linfocitos T

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...

radial-mobility-cytotoxic-function-retroviral-replicating-vector

Research

JoVE Journal

Immunology and Infection

7.7K vistas.  UCLA David Geffen School of Medicine. We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality. 

Video: Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Transducción retroviral de receptores de células T en ratones las células T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Investigación

Inmunología e Infección

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

Preparación y uso de VIH-1 en células CD4 infectadas Primaria + T-células como células diana en las células asesinas naturales ensayos citotóxicos

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Métodos para evaluar la Beta mediada por la muerte celular por linfocitos T citotóxicos

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

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 ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Diferenciación dirigida de células madre pluripotentes inducidas hacia los linfocitos T

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Aislamiento y diferenciación de Th17 Naïve linfocitos T CD4

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits &#945;L and &#946;2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in&#160;vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g.&#160;VLA-4 - fibronectin).

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

Ensayo de adherencia estática para el Estudio de la integrina activación en linfocitos T

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Protocolo Aislamiento eficiente para B y linfocitos T a partir Humanos Palatine amígdalas

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Retroviral transducción de células progenitoras de médula ósea para generar las células T del receptor Retrogenic ratones

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

Generando<em&gt; De Novo</em&gt; T humanas receptores de células específicas de antígeno por retroviral Transducción de Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...