D.E. and M.B.D. are members of a multidisciplinary research platform (MRP) of Ghent University, and the Ghent Researchers on Unfolded Proteins in Inflammatory Disease (GROUP-ID) consortium.

En este estudio, se utilizaron adultos (6-8 semanas) hembra C57Bl / 6 ratones. Los animales fueron alojados y criados de acuerdo con las directrices del vivero Universidad de Gante. Todos los animales procedimientos fueron aprobados por el Comité de Cuidado y Ética Animal Institucional. 1. Preparación de células mononucleares (CMN) de ratón Bazo <ol><li> Sacrificar el ratón por dislocación cervical. </li><li> Coloque el mouse hacia abajo en el tablero de disección y fijar la cierva y patas delanteras con alfileres. </li><li> Esterilizar la superficie de la piel con 70% (vol / vol) de etanol / H 2 O. </li><li> Cortar la piel a lo largo de la línea media abdominal al tórax y exponer el bazo usando instrumentos quirúrgicos estériles. </li><li> Después de recortar los tejidos grasos circundantes, colocar el bazo en una placa de 24 pocillos que contenía 1 ml de 1x tampón fosfato salino (PBS) a TA. </li><li> Diseccionar el bazo utilizando un bisturí estéril y transferir las piezas en un filtro de nylon de 70 micras colocado dentro de una tina 50 mle. </li><li> El uso de un émbolo de la jeringa 2 ml, puré suavemente las piezas de bazo a través de la filtro de células y lavar con 5 ml de PBS 1x. </li><li> Añadir 3 ml de Ficoll-Paque a un tubo de 15 ml y superponer el medio de gradiente de densidad con el 5 ml de suspensión celular. </li><li> Centrifugar a 400 xg durante 20 min a TA sin freno. </li><li> La transferencia de la interfase a un tubo de 15 ml fresca, añadir 10 ml de PBS frío 1x y centrifugar a 300 xg durante 10 min a 4 ° C. </li><li> Resuspender las células en 2 ml de 1x PBS que contenía 0,5% de albúmina de suero bovino (BSA) y 1 mM de ácido etilendiaminotetraacético (EDTA) (tampón FACS). Transferencia de 10 l de la suspensión celular a un tubo pequeño, se mezcla con 10 l de 0,4% </li></ol> 2. Enriquecimiento, detección y purificación de células de ratón iNKT <ol><li> Enriquecimiento de esplénicas de ratón CD5positive (CD5 +) linfocitos. <ol><li> (Opcional) Manchas 10 5 células con FITC-conjugado anti-CD5 (4 l de 1:30 dilución / 10 6 células)durante 30 min a 4 ° C en la oscuridad, se lava con 200 l de tampón FACS y resuspender en 500 l de tampón FACS. Preparar una solución de trabajo 20 mM de 4 &#39;, 6-diamidino-2-fenilindol (DAPI) en PBS 1x, añadir 1 l del mismo a 10 5 células en tampón FACS y se incuba a temperatura ambiente durante 5 min. Adquirir 10 4 células pre-enriquecido en el citómetro de flujo. <ol><li> Usando FCS-H y FCS-W, electrónicamente puerta en las células del lo FSC-W para excluir dobletes de células y agregados (Figura 1a). Entonces electrónicamente puerta hacia fuera + células (muertos) DAPI dentro de la W-FSC lo población (Figura 1B), y utilizan SSC-A y FCS-A para seleccionar electrónicamente la población de linfocitos por tamaño (Figura 1C). </li><li> El uso de células dentro de la puerta de linfocitos, trazar SSC-A versus CD5 y electrónicamente puerta CD5 + linfocitos como se muestra en la Figura 1D. Adquirir el resto de la muestra pre-enriquecido en el citómetro de flujo. </li&gt; </ol></li><li> Ajustar los recuentos de células a 10 7 células por 90 l de tampón FACS y añadir 10 l microperlas anti-CD5. </li><li> Se incuba a 4 ºC durante 15 min, se lava una vez con 4 ml de tampón FACS y resuspender en 500 l de tampón FACS frío. Enriquecer para CD5 + linfocitos utilizando separación celular magnética (MACS) columnas de acuerdo con las recomendaciones del fabricante. </li><li> Opcional) Stain 10 5 células con FITC-conjugado anti-CD5 (4 l de dilución 1:30 / 10 6 células) y DAPI como en 2.1.1. Adquirir 10 4 linfocitos enriquecidos en el citómetro de flujo y verifique que las puertas electrónicas creadas en 2.1.1. seleccionar CD5 + linfocitos en la fracción celular enriquecida. Adquirir 10 5 células en el citómetro de flujo y analizar el porcentaje CD5 + linfocitos presentes en la fracción enriquecida como se muestra en la Figura 1E - H. </li></ol></li><li> Detección y aislamiento de ratón iNKT ceLLS por FACS. <ol><li> Resuspender CD5 + linfocitos enriquecidos en una concentración de aproximadamente 10 6 células por 20 l en tampón FACS que contiene anti-CD16 / CD32 (4 l de dilución 1:50 / 10 6 células) y αGalCer PE-conjugado / tetrámero CD1d (1 g / 10 6 células). </li><li> Incubar durante 30 minutos a temperatura ambiente en la oscuridad. </li><li> Diluir los anticuerpos de ratón (mAb) anti-CD3ε V500 (4 l de dilución 1:20 / 10 6 células), anti-CD19 E450 (4 l de dilución 1:50 / 10 6 células) y anti-CD8a E450 (4 l de dilución 1:50 / 10 6 células) en tampón FACS, añadir a CD5 + linfocitos e incubar durante 30 minutos a 4 ° C en la oscuridad. </li><li> Lavar las células con 1 ml de tampón FACS mediante centrifugación a 300 xg durante 10 min a 4 ° C y resuspender en 4 ml de tampón de FACS a una concentración final de 10 7 células / ml. </li><li> Filtrar las células a través de un filtro de células de 30 micras y colocar las células en hielo. </li><li> Calibrar el citómetro de flujo para la clasificación de células de acuerdo con las recomendaciones del fabricante 14,15. Nota: La calibración de un citómetro de flujo para la clasificación de células requiere entrenamiento y puede ser realizada por un operador con experiencia, o un técnico capacitado. </li><li> Añadir 10 l de una solución de trabajo 20 mM de DAPI por cada 10 6 células y se incuba a temperatura ambiente durante 5 min. Adquirir aproximadamente 10 4 CD5 + linfocitos enriquecidos en el citómetro de flujo. Puerta electrónicamente en las células del lo FSC-W para excluir dobletes de células y agregados (Figura 2a). Entonces electrónicamente puerta a cabo DAPI + CD8 + CD19 + células (canal volcado) dentro de la FSC-W lo población (Figura 2B), y el uso de SSC-A y FCS-A para seleccionar electrónicamente linfocitos (Figura 2C) como se describe en 2.1.1.1 . <ol><li> Terreno αGalCer / CD1d tetrámero por CD3ε y electrónicamente puerta αGalCer / CD1dcélulas tetrámero + CD3ε + iNKT como se muestra en la Figura 2D. Células presentes en la fracción celular enriquecida Adquirir no más de 2 x 10 5 CD5 + linfocitos enriquecidos para determinar el porcentaje αGalCer / CD1d tetrámero + CD3ε + iNKT. </li></ol></li><li> Utilizando las puertas electrónicas creadas en 2.2.7., Células FACS tipo αGalCer / CD1d tetrámero + CD3ε + iNKT en el citómetro de flujo a 70 psi utilizando una boquilla de 70 micras, con un caudal de &#39;1.5&#39;. Recoger las células iNKT ordenados en un tubo de FACS que contiene 1 ml de 100% de FCS. Posteriormente enjuague células iNKT ordenadas por el lado del tubo de FACS utilizando 10 ml RMPI 1640 y centrifugar las células a 300 xg durante 10 min a 4 ° C. </li><li> Resuspender las células ordenadas iNKT en 1 ml de medio RPMI 1640 y adquirir 40 l del mismo sobre un citómetro de flujo para evaluar la pureza de las células iNKT ordenados. Seleccione FSC-W lo DAPI - linfocitos como se describe en 2.2.7. (Figura 2E - G) y electrónicamente puerta αGalCer / células tetrámero + CD3ε + iNKT CD1d, como se muestra en la figura 2H. </li></ol></li></ol> 3. Cultura y expansión de células de ratón iNKT Día 0 <ol><li> Escudo una placa de pocillos de fondo plano con 96 anti-CD3ε purificada (3 mg / ml) por pocillo durante 1 hora a RT. Lavar dos veces con 200 l de 1x PBS y una vez con 200 l de medio RPMI 1640 completo (10% FCS vol / vol, 100 U / ml de penicilina-estreptomicina, 5,5 mM β-mercaptoetanol). </li><li> Contar el número de células viables según iNKT (paso 2.2.8) utilizando 0,4% como en un hemocitómetro 1.11., Centrifugar a 300 xg durante 10 min a 4 ° C y resuspender en 5 x 10 5 células / ml en RPMI 1640 medio completo que contiene IL-2 murina (10 ng / ml) recombinante, IL-12 murina (1 ng / ml) recombinante soluble y anti-CD28 de ratón (5 mg / ml). Platosorted células iNKT a 10 5 células / pocillo en placas revestidas anti-CD3ε y se incuba a 37 ° C en una incubadora de CO 2 durante 2 días. </li></ol> Dia 2 <ol start="3"><li> Transferir las células a una placa fresca sin recubrimiento de fondo plano de 96 pocillos y la cultura de las células bajo condiciones de reposo en medio completo RPMI 1640 a 37 ° C en una incubadora de CO 2 durante 2 días. </li></ol> Día 4 <ol start="4"><li> Desalojar a los células de la placa por pipeteado suave y transferir las células a un tubo de 15 ml fresco. Centrifugar a 300 xg durante 10 min a 4 ° C, se resuspenden a 5 x 10 5 células / ml en RPMI completo 1640 que contienen IL-7 murina (5 ng / ml) recombinante, y la placa 10 5 células / pocillo en una Flat- fondo placa de 96 pocillos durante 4 días a 37 ° C en una incubadora de CO 2. </li></ol> Día 8 <ol start="5"><li> Recoger las células en un tubo nuevo, centrifugar a 300 xg durante 10 min a 4 ° C y resuspender en 5 x 10 5 células / ml en medio completo RPMI 1640 que contenía soluble anti-CD28 (5 g / ml). </li><li> Escudo una placa de fondo plano 96 con anti-CD3ε purificada (3 mg / ml) por pocillo y lavar como en 3.1. Placa 10 5 células / pocillo en CD3ε anti-placas recubiertas y se incuba a 37 ° C en una incubadora de CO 2 hasta 11 días. </li></ol> Día 11-18 <ol start="7"><li> Repita los pasos 3,4 a 3,6. </li></ol> Día 19-20 <ol start="8"><li> Transferir las células a un tubo de 15 ml fresca, centrifugar a 300 xg y resuspender en 5 x 10 5 células / ml en medio RPMI 1640 completo. </li><li> Células de la placa en 10 5 células / pocillo en una placa de pocillos de fondo plano 96 y permitir que las células en reposo durante 2 días a 37 ° C en una incubadora de CO 2 antes de su uso en un experimento. </li></ol> 4. Producción de citoquinas por el ratón células iNKT Utilizando un CD1d PlatEnsayo de e-unido <ol><li> Escudo una placa de pocillos de fondo plano 96 con 100 l de murina recombinante CD1d (10 g / ml en PBS 1x) por pocillo e incubar durante 1 hora a 37 ° C. </li><li> Lavar cuatro veces con 200 l de 1x PBS, añadir 200 l de 2% de FCS en 1x PBS (solución de bloqueo) por pocillo y se incuba durante 2 horas a 37 ° C. </li><li> Eliminar la solución de bloqueo, añadir 200 l de αGalCer (5 ng / l) por pocillo, y se incuba la placa a 37 ° C durante 16 horas. [Para la preparación de la solución αGalCer, añadir 1,085 μ 1x PBS a 55 g de αGalCer disuelto en vehículo que contiene 96 mg / ml de sacarosa, 10 mg / ml y desoxicolato de sodio 5 mg / ml tween 20]. </li><li> Después de la incubación, desechar la solución αGalCer y lavar el pozo dos veces con 200 l 1x PBS seguido de 200 l completa medianas RPMI 1640. </li><li> Recoger las células iNKT expandidas in vitro en el día 21, de centrifugación las células a 300 xg durante 10 min a 4 ° C y resuspender en completa RPMI 1640 a una concentración de 0,5 x 10 6 células / ml. </li><li> Añadir 200 l de la resuspensión de células iNKT por pocillo y se incuba durante 72 horas a 37 ° C en una incubadora de CO 2. </li><li> Recoger el sobrenadante y la medición de las citoquinas de interés por el ensayo de inmunoabsorción ligado a enzimas (ELISA) de acuerdo con el protocolo del fabricante. </li></ol>

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The authors have no competing interests to disclose.

Los pasos críticos en el actual protocolo incluyen el aislamiento y posterior enriquecimiento de CD5 + linfocitos (Sección 1 y 2), FACS clasificación (Sección 3) y el recubrimiento inicial de células iNKT (Sección 4). De los pasos realizados en la Sección 1, recuerda a la capa cuidadosamente la suspensión celular esplénica sobre el medio de gradiente de densidad tal que una interfase celular distinta se genera después de la centrifugación. El enriquecimiento posterior para CD5 + linfocitos</su...

Murine células asesinas naturales invariantes T (iNKT) son una población distinta de los linfocitos T innatas seleccionados en el timo por timocitos corticales que expresan CD1d 1,2. iNKT células expresan un receptor de células T (TCR) compuesto de una cadena invariante Vα14-Jα18 TCR se combina con cualquiera de Vß8, Vβ7 o Vβ2 TCR 3, que es capaz de reconocer endógena, así como antígenos lipídicos extranjeros en el contexto de CD1d. Por ejemplo, las células murinas iNKT reconocen y se activan por una llamada isoglobotrihexosylceramide antígeno de lípidos endógenos (iGb3) 4, así como α-galactosylceramide (αGalCer) 5,6, un glicolípido aislado de esponjas marinas. TCR dependiente de la activación de células iNKT promueve el cebado de la respuesta inmune adaptativa, y como resultado, las células iNKT han demostrado ser funcionalmente implicado en la mejora o desarrollo de una gama de patologías incluyendo la enfermedad reumática 7 y el cáncer &lt;sup&gt; 8. Actualmente, los ligandos de células iNKT sintéticos constituyen prometedores nuevos adyuvantes de vacunas que pueden ser capaces de regular una serie de condiciones inmunopatológicas. Previamente se ha demostrado que las células iNKT pueden ser generados in vitro tras el aislamiento a partir de tejido de ratón sin embargo; muchos de estos estudios emplean el uso de células primarias presentadoras de antígeno (APC) y / o líneas celulares 9, Vα14 TCR transgénicos (Tg) 10 ratones, timomas o para la generación de hibridomas de células derivadas de iNKT 11,12. Por otra parte, un gran número de ratones, los altos volúmenes de reactivos tales como dímeros CD1d cargados-αGalCer y largos tiempos de cultivo hacen algunos protocolos publicados menos ética y económicamente atractiva 9,13. En este informe se describe un método adaptado para el aislamiento y en la expansión in vitro de células iNKT de bazo de ratón. Más específicamente, el protocolo describe un métodopara enriquecer células iNKT de bazo de ratón que reduce los ratones, los reactivos y el tiempo necesario para la clasificación de células FACS, y propone un enfoque optimizado para la expansión de las células iNKT esplénica ordenados in vitro.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-2</td>
			<td>eBiosciences</td>
			<td>14-8021</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-12</td>
			<td>eBiosciences</td>
			<td>39-8122-65</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant murine IL-7</td>
			<td>eBiosciences</td>
			<td>14-8071</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD3e (145-2C11)</td>
			<td>eBiosciences</td>
			<td>16-0032-86</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD28 (37.51)</td>
			<td>eBiosciences</td>
			<td>16-0281-85</td>
			<td></td>
		</tr>
		<tr>
			<td>e450-conjugated anti-mouse CD19 (eBio1D3)</td>
			<td>eBiosciences</td>
			<td>48-0193-82</td>
			<td></td>
		</tr>
		<tr>
			<td>e450-conjugated anti-mouse CD8&#945; (53-6.7)</td>
			<td>eBiosciences</td>
			<td>48-0081-82</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-conjugated anti-mouse CD5 (53-7.3)</td>
			<td>eBiosciences</td>
			<td>11-0051-81</td>
			<td></td>
		</tr>
		<tr>
			<td>V500-conjugated anti-mouse CD3&#949; (500A2)</td>
			<td>BD biosciences</td>
			<td>560771</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD5 (Ly-1) microbeads</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-049-301</td>
			<td></td>
		</tr>
		<tr>
			<td>purified anti-mouse CD16/32 (2.4G2)</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-092-575</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MS Columns</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue, 0.4% (wt/vol)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Gibco</td>
			<td>12633-020</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Gibco</td>
			<td>10010-056</td>
			<td>[Ca2+/Mg2+ - free]</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Gibco</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-123</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4458-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>EDS-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque plus</td>
			<td>GE Healthcare</td>
			<td>71-7167-00 AF</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate F-bottom</td>
			<td>Greiner-bio one</td>
			<td>657-160</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Greiner-bio one</td>
			<td>665-180</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Greiner-bio one</td>
			<td>655-180</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml falcon tube</td>
			<td>Greiner-bio one</td>
			<td>188-271</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml falcon tube</td>
			<td>Greiner-bio one</td>
			<td>227-261</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m filter</td>
			<td>Greiner-bio one</td>
			<td>542-070</td>
			<td></td>
		</tr>
		<tr>
			<td>30 &#181;m filter</td>
			<td>Millipore</td>
			<td>SVGP01050</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS separator</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Macs Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Water-jacketed CO2 incubator</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection Kit</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSAria III</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an optimized method for isolating and expanding invariant natural killer t cells from mouse spleen

La capacidad de secretar citocinas rápidamente tras la estimulación es una característica funcional de la invariante natural killer T (iNKT) linaje de células. Por lo tanto, las células iNKT se caracterizan como una población innata de las células T capaz de activar y adaptativa de dirección respuestas inmunes. El desarrollo de técnicas mejoradas para la cultura y la expansión de las células iNKT murinos facilita el estudio de la biología de las células iNKT en in vitro y en sistemas modelo in vivo. Aquí se describe un procedimiento optimizado para el aislamiento y expansión de las células esplénicas murinas iNKT. Se eliminan los bazos de ratones C57BL / 6 ratones, diseccionaron y tensas y la suspensión celular resultante se acoda sobre medios de gradiente de densidad. Después de la centrifugación, las células mononucleares del bazo (CMN) se recogen y (CD5 +) linfocitos CD5 positivas son enriquecidos por usar perlas magnéticas. células iNKT dentro de la fracción CD5 + son posteriormente teñidas con ^5; GalCer-cargado tetrámero CD1d y se purificó mediante clasificación de células activadas por fluorescencia (FACS). FACS ordenadas células iNKT son entonces cultivaron inicialmente in vitro usando una combinación de citocinas murinas recombinantes y receptor de células T unido a placa (TCR) estímulos antes de ser expandido en presencia de IL-7 murina recombinante. Usando esta técnica, aproximadamente 10 8 células iNKT se pueden generar dentro de 18-20 días de cultivo, después de lo cual se pueden utilizar para los ensayos funcionales in vitro, o in vivo experimentos de transferencia en ratones.

Aislamiento de células mononucleares del bazo utilizando un gradiente de densidad tarda aproximadamente 1 hora y se elimina el uso de reactivos necesarios para lisar los glóbulos rojos (GR). Se obtiene un alto rendimiento de células viables utilizando este método y los escombros generados durante el esfuerzo del órgano es eliminado. Típicamente, la frecuencia de células iNKT dentro de la piscina de linfocitos del bazo oscila entre 1 y 5% del total de linfocitos T sin embargo, esto puede variar dependiendo del est...

Watch this Scientific Journal Video about An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen at JoVE.com

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

Un método optimizado para aislar y expandir Invariante Natural Killer células T de ratón Bazo

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

an-optimized-method-for-isolating-expanding-invariant-natural-killer

Research

JoVE Journal

Immunology and Infection

11.9K vistas.  Ghent University Hospital. Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents. 

Video: An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

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 ...

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 ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Antígeno Artificial célula presentadora (AAPC) La activación mediada y expansión de linfocitos T citolíticos naturales

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Killer Artificial células presentadoras de antígenos (KaAPC) para la utilización eficiente<em&gt; In Vitro</em&gt; El agotamiento de células T humanas específicas de antígeno

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Derivación de células T<em&gt; In Vitro</em&gt; De células madre embrionarias de ratón

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

Análisis de citometría de flujo de las células asesinas naturales lítico Actividad en sangre entera humana

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Un novedoso sistema de alimentador-libre para la producción masiva de células de asesino naturales murinos In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Aislar los linfocitos desde el sistema de inmune Intestinal pequeño ratón

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

Cuantificación de basados en citometría de flujo multicolor de las mitocondrias y los lisosomas en las células T

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...