The authors would like to acknowledge Dr. Laura Johnson and Dr. Richard Morgan for providing the CAR retroviral construct. The authors also thank Giao Ngyuen for her assistance with dosimetry for whole brain irradiation. This work was supported by an NIH NCI grant 1R01CA177476-01.

Este protocolo se basa en un diseño experimental donde 10 los ratones se tratan con 10 7 CAR células T cada uno. Esto significa que se necesitarán 10 8 células CAR T; el rendimiento debe ser sobrestimada por 5 x 10 7 -1 x 10 8 a la cuenta de la pérdida de viabilidad. El siguiente protocolo se escala para generar aproximadamente 200 x 10 6 células. Las células se administran por vía intravenosa a la hembra C57BL / 6 ratones con 9 días tumores intracraneales EGFRvIII positivos singénicos establecidos, desarrollado a partir de los astrocitoma KR158B o líneas celulares de melanoma B16 existentes. Concomitantemente con CAR generación de células T, los ratones portadores de tumores se administran dosis clínicamente relevantes de TMZ lymphodepletive (60 mg / kg) y WBI (16,5 Gy). Los ratones fueron mantenidos y criados en condiciones libres de patógenos en Duke University Medical Center (DUMC). Todos los experimentos con animales se realizaron de acuerdo con los protocolos aprobados por la Institución Universidad de Dukeal Cuidado de Animales y el empleo Comisión (IACUC). 1. Administración de temozolomida a ratones portadores de tumores Días 0 a 4: <ol><li> Calcular el número total de ratones que será inyectada y multiplicar este por 0,5 para determinar el volumen de solución TMZ sea necesario (por ejemplo, 30 ratones x 0,5 ml = 15 ml TMZ) y añadir 1,5 ml a este volumen para dar cuenta de desbordamiento (16,5 ml TMZ ). </li><li> Multiplique este volumen total de 3 mg / ml para determinar el peso de liofilizado TMZ necesario (por ejemplo, 16,5 x 3 = 49,5 mg TMZ). Pesar esto en un Erlenmeyer de 50 ml. PRECAUCIÓN: TMZ es tóxico por inhalación, ingestión y contacto con los ojos, la piel y las membranas mucosas. Consulte con la seguridad de la institución en el trabajo y / o la salud del medio ambiente y el departamento de salud para obtener recomendaciones sobre la reducción de riesgo de exposición. </li><li> Multiplique el volumen total de 0,15 para determinar el volumen de dimetilsulfóxido (DMSO) necesario (por ejemplo, 16,5 x0,15 = 2,475 ml de DMSO). Filtrar esterilizar este volumen de DMSO (más un adicional de 2 ml para dar cuenta de desbordamiento) a través de un filtro de 0,2 micras en un tubo estéril. Añadir 2,475 ml de DMSO estéril al polvo TMZ. </li><li> Coloque la solución TMZ / DMSO en un vaso de agua sobre una placa caliente a 65 ° C durante 10-15 minutos. Una vez TMZ se haya disuelto completamente, la solución debería ser de color amarillo pálido; Si la solución se vuelve de color rosa, a continuación, la TMZ se degrada, y una nueva solución debe ser preparada con un montón fresco de TMZ. </li><li> Calcular el volumen apropiado de solución salina estéril necesaria multiplicando 0,85 por el volumen total (0,85 x 16,5 ml = 14,025 ml de solución salina). Añadir 15 ml de solución salina estéril a una cónica 50 ml. Mientras TMZ se está disolviendo en el DMSO, solución salina de calor a 65 ° C. </li><li> Vortex la solución / DMSO TMZ para asegurar que el polvo de TMZ se haya disuelto completamente y lentamente añadir solución salina calentada a la solución / DMSO TMZ. </li><li> Inmediatamente después de la preparación, cargue completamente 15 x 1 ml de tuberculina syringí con solución TMZ para la administración a 4 días establecidos ratones con tumor. </li><li> Repita este procedimiento en los días 1 al 4 de un total de cinco administraciones de TMZ. </li></ol> 2. Todo el Cerebro La irradiación de ratones portadores de tumores Días 2 al 4: <ol><li> Encienda el irradiador de rayos X y deje que se caliente hasta el voltaje completo. Asegúrese de que el filtro adecuado está en su lugar y la entrada de la tensión y corriente apropiados (Figura 2A). NOTA: La dosimetría del irradiador debe determinarse con antelación para determinar los valores de voltaje y diseño de la red (Figura 2A, B) correspondiente. </li><li> Calcular la longitud de tiempo que es necesario para dar lugar a 5,5 Gy de irradiación de rayos X. Por ejemplo, si los rayos X se entregan a una velocidad de 2 Gy / min, a continuación, 2,75 min es necesario para entregar un total de 5,5 Gy. Entrada de la duración de tiempo apropiado. NOTA: La Tabla 2 proporciona ratón WBI dosifica unaND sus equivalentes clínicos en seres humanos. En el contexto de los gliomas de alto grado, 60 Gy fraccionó WBI (fracciones de 2 Gy, 5 días / semana durante 6 semanas) es el estándar de la atención clínica, y el equivalente murino utilizado aquí es 16,5 Gy (5,5 Gy x 3). </li><li> Preparar una solución fresca de ketamina / xilazina para la anestesia sistémica mediante la adición de 2 ml de ketamina y xilazina 1 ml a 17 ml de solución salina de tal manera que la solución final es de 10 mg / ml de ketamina y 1 mg / ml de xilazina. </li><li> Pesar los ratones y administrar 10 l de ketamina / xilazina por vía intraperitoneal por gramo de peso corporal de tal manera que los animales reciben una dosis de ketamina a 100 mg / kg y 10 mg / kg de xilazina. Ratones debe estar completamente sedado y visiblemente respirar menos de 2 minutos de la ketamina administración / xilazina. </li><li> Para mantener la humedad oftálmica en animales sedados, frote suavemente una pequeña cantidad de lágrimas artificiales pomada en cada ojo. </li><li> Coloque los ratones sedados en la parrilla de tal manera que las cabezas se colocan en el área que recibe la más alta de rayos X enintensidad (Figura 2B, C). Cuerpos Escudo de cuello para abajo con el tamaño apropiado de tubos de plomo para bloquear la entrega de rayos X sistémica (Figura 2D). </li><li> Coloque los ratones colocados bajo el haz de rayos X de tal manera que el láser que denota el punto focal del haz de rayos X está en la coordenada (0,0) en el diseño de cuadrícula. Comience la entrega de rayos X. </li><li> Cuando la entrega de rayos X, retire los animales del irradiador y colóquela sobre una almohadilla térmica. No albergar animales sedados con animales conscientes hasta que los animales han recuperado el conocimiento suficiente para mantener la posición decúbito esternal. Una vez que los animales pueden mantener decúbito esternal, coloque animales conscientes de vuelta en sus jaulas apropiadas. </li><li> Repita este procedimiento en los días 3 y 4 de un total de tres dosis fraccionadas de radiación. </li></ol> 3. La transfección Día -1: <ol><li> Preparar los medios de comunicación D10 mediante la adición de 50 ml de suero bovino fetal (FBS) a 500 ml de Dulbecco Modified medio Eagle (DMEM). </li><li> Preparar los medios de comunicación de células T (TCM) mediante la adición de 5,5 ml de L-glutamina, piruvato de sodio 5,5 ml, 5,5 ml aminoácidos no esenciales, y 5,5 ml penicilina / estreptomicina (pen / strep) a 500 ml de medio RPMI-1640. A continuación, añadir 550 l de 2-mercaptoetanol y 550 l de gentamicina. Por último, añadir 50 ml de FBS. </li><li> Harvest in vitro células HEK293T cultivadas y llevar a una concentración de 7,5 x 10 6 células / ml en medio D10. </li><li> Placa de 10 ml de medio D10 y añadir 1 ml de suspensión celular HEK293T en cada uno de 16 x 10 cm poli-D-lisina (PDL) placas recubiertas. </li><li> Incubar durante la noche a 37 ° C con 5% de CO 2. </li></ol> Día 0: <ol start="6"><li> Reemplace los medios con 10 ml D10 fresco al menos 30 minutos antes de la transfección de las células. </li><li> Determinar la cantidad de plásmido vector, PCL-Eco vector, y el reactivo de transfección liposomal requerido para la transfección multiplicando el número total de placas + 1 (16 + 1 = 17) por 14,1 gplásmido vector (14,1 x 17 = 239,7 g), 9,9 g PCL-Eco vector (9.9 x 17 = 168,3 g), y 60 l de reactivo de transfección liposomal (60 x 17 = 1020 l). Todos los reactivos deben estar a temperatura ambiente antes de la preparación de soluciones. </li><li> Etiquetar dos tubos A y B. En el tubo A, añadir 239,7 g plásmido vector y 168,3 g PCL-Eco vector a (1,5 x 17) ml de suero reducido medio de Eagle modificado (RS-MEM). En el tubo B, añadir 1.020 l de reactivo de transfección liposomal a (1,5 x 17) ml RS-MEM. </li><li> Incubar A y B por separado durante 5 min a temperatura ambiente. </li><li> Mezclar A y B juntos suavemente (vórtex durante 1-2 seg o invertir varias veces) y se incuba durante 20 min a temperatura ambiente para formar lípido / complejo de ADN. </li><li> Añadir lípido / ADN gota complejo gota a células HEK293T en placa de 10 cm. </li><li> Se incuba a 37 ° C durante 6-8 horas o durante la noche (no exceda de 24 horas). </li><li> Después de 6-8 horas de incubación, reemplace medio con 12 ml de TCM fresco para la producción viral. Este medio plateadose utilizará en el Día 2 como sobrenadante viral para la transducción de células T. </li></ol> 4. La esplenectomía y T Preparación de la célula Día 0: <ol><li> Verter 10 ml de TCM en un cónico de 50 ml y el lugar en el hielo para la recolección de bazo. </li><li> Sacrificar el número apropiado de animales por asfixia de CO 2 y la decapitación secundario: Coloque los animales en una jaula recibir CO 2 a una velocidad de flujo de 10 a 30% jaula volumen / minuto, según las directrices de la Asociación Americana de Medicina Veterinaria (no más de 5 animales pueden ser sacrificado al mismo tiempo) hasta que termina la respiración y durante dos minutos después. Retire los animales de la cámara de CO 2 y decapitar. NOTA: Una bazo de un 6-12 semanas de edad hembra C57BL / 6 mouse producirá aproximadamente 4,5-5 x 10 7 esplenocitos. Aquí, 4 bazos se cosecharán durante aproximadamente 200 x 10 6 células. </li><li> Coloque el ratón de modo que su lado derecho quede hacia arriba, yrocíe con etanol al 70%. Con las pinzas, tomar un pliegue delgado de piel debajo de la caja torácica izquierda y cortar una ligera incisión con las tijeras. Pele la piel, tomar cuidadosamente un pliegue delgado del peritoneo con una pinza y se corta una pequeña cavidad. </li><li> El bazo es un órgano pequeño alargado de color rojo, y oscuro que se parece a un grano de aplanado; delicadamente agarrar el bazo con las pinzas y especial cortando el tejido conectivo circundante. Coloca los bazos extirpados en la cónica que contiene 10 ml de TCM en el hielo. </li><li> Verter bazos más de un 70 micras filtro de células de malla y desagregar por maceración con el extremo romo de la parte interior de una jeringa de 5 ml para generar una suspensión de una sola célula. Un máximo de dos bazos debe desglosarse por colador de malla. </li><li> Use un pequeño volumen de TCM para lavar cuidadosamente el filtro siguiente desagregación, a percibir los esplenocitos restantes. Piscina todo bazos desglosados ​​en una suspensión de una sola célula. Aumentar el volumen final a 50 ml con TCM para un lavado und girar a 300 xg durante 10 min. </li><li> Preparar una solución de tampón de lisis 1x mediante la adición de 5 ml de 10x tampón de lisis a 45 ml de agua estéril. Para eliminar las células rojas de la sangre, pellet se resuspende en 5 ml de 1x tampón de lisis por bazo en un Erlenmeyer de 50 ml, mezclando bien pipeteando suavemente hacia arriba y hacia abajo. Si superior a 5 bazos, utilice un tubo de centrífuga de 250 ml. Coloque cónico o tubo de centrífuga en un baño de 37 ° C el agua durante 5 min. </li><li> Retire la reacción de lisis del baño de agua y añadir TCM en una relación 1: 1 con tampón de lisis para neutralizar la reacción. Lavar centrifugando a 300 xg durante 10 min. </li><li> Aspirar el sobrenadante y pellet totalmente resuspender en TCM (2 ml / bazo, 8 ml total de 4 bazos) pipeteando arriba y abajo. </li><li> Contar las células mediante la adición de 10 l de suspensión celular a 190 l de azul de tripano (1:20 dilución). Multiplicar el número obtenido en uno de los cuatro cuadrados reticulados por 20 x 10 4 x volumen total (8 ml) para obtener el número total de células (por ejemplo, células 125 x 20 x 10 4 </sarriba&gt; x 8 ml = 200 x 10 6 esplenocitos). </li><li> Diluir las células a una concentración de 2 x 10 6 células / ml en TCM, suplementado con 2 mg / ml de concanavalina A (ConA) y 50 UI / ml recombinante humana interleucina-2 (rhIL-2). Por lo tanto, para 200 x 10 6 esplenocitos, añadir 92 ml de medio a 8 ml células, 200 mg ConA y 5.000 UI rhIL-2. </li><li> Añadir 2 ml de células a cada pocillo de placas tratadas de cultivo de tejidos de 24 pocillos, de tal manera que 4 x 10 6 células están en cada pocillo (por ejemplo, 100 ml de células requerirán aproximadamente 4 placas). </li><li> Incubar durante la noche a 37 ° C con 5% de CO 2. </li></ol> 5. Transducción Día 1: <ol><li> Calcular el número de cultura no tejido tratado placas de 24 pocillos necesarios para la transducción de multiplicar el número de bazos cosechados por 2 (se necesitan 8 platos para 4 bazos). </li><li> A continuación, calcular el volumen requerido de fragmento de fibronectina humana recombinante solución (RHFF)necesaria para placas capa multiplicando (número total de pozos + 3) x 0,5 ml (8 placas x 24 pozos = 192 + 3 = 195) x 0,5 ml = 97,5 ml. </li><li> Preparar 97,5 ml de solución salina tamponada con fosfato (PBS) que contiene RHFF a una concentración de 25 g / ml multiplicando el volumen total por 25 g (97,5 x 25 = 2437,5 g). Añadir esta cantidad de RHFF a 97,5 ml de PBS. </li><li> Escudo cultura no tejido tratado placas de 24 pocillos añadiendo 0,5 ml de solución de PBS / RHFF por pocillo. </li><li> Incubar toda la noche a 4 ° C. </li></ol> Día 2: <ol start="6"><li> Volcado de solución de PBS / RHFF de cultura no tejido tratado placas de 24 pocillos. </li><li> Añadir 1 ml / pocillo de albúmina de suero bovino al 2% (BSA) en PBS y se incuba a temperatura ambiente durante 30 min. </li><li> Retire BSA por volcar firmemente plato. Lave añadiendo 2 ml de PBS. </li><li> Recoger el líquido sobrenadante viral mediante la transferencia de los medios de comunicación de cada placa PDL HEK293T en un tubo de 250 ml de centrífuga y centrifugar 10 min a 500 x g. </li><li> Transferir cuidadosamente su viralpernatant en un tubo de centrífuga de 250 ml fresca, asegurándose de no perturbar el sedimento de células que puedan haber formado. </li><li> Añadir TCM fresco al sobrenadante viral tal que el volumen final es de 3 ml más de la cantidad necesaria para pocillos recubiertos con RHFF. Por ejemplo, para 192 pocillos recubiertos con RHFF, llevar el volumen de sobrenadante viral a 192 + 3 ml = 195 ml. </li><li> Eliminar esplenocitos cultivados de la incubadora y resuspender pipeteando suavemente arriba y abajo 2 - 3 veces en cada pocillo. Pasar a un Erlenmeyer de 250 ml. Si se utiliza una pipeta multicanal, utilizando un depósito estéril antes de la transferencia ayudará a agilizar este paso. Count como se describe anteriormente y centrifugado a 300 xg durante 10 min. </li><li> Añadir rhIL-2 de sobrenadante viral a una concentración de 50 UI / ml. Por ejemplo, añadir 50 x 195 = 9750 UI rhIL-2 a 195 ml de sobrenadante viral. </li><li> Resuspender esplenocitos en sobrenadante viral a 1 x 10 6 células / ml </li><li> Añadir 1 ml / pocillo de suspensión de esplenocitos a placas de 24 pocillos recubiertas con RHFF. </li><li>Centrifugado durante 90 minutos de acuerdo a los siguientes valores: 770 × g, aceleración = 4, freno / deceleración = 0, 32 ° C. </li><li> Preparar una solución de TCM con 50 UI / ml de rhIL-2. Por ejemplo, preparar una solución de 200 TCM mediante la adición de 10.000 IU rhIL-2. Añadir 1 ml de rhIL-2 / TCM a cada pocillo después de la centrifugación. </li><li> Cultura durante la noche a 37 ° C en 5% de CO 2. </li></ol> 6. CAR células T cultura y la Cosecha Días 3 y 4: <ol><li> Si las células T a alcanzar&gt; 80% de confluencia, las células se pueden dividir (generalmente, al día 3 o el día 4). Para dividir celdas, pipetear suavemente hacia arriba y hacia abajo en cada pocillo 2-3 veces, y pasar 1 ml de cada pocillo en nuevos pozos de una placa de 24 pocillos frescos de cultivo de tejidos tratados. A continuación, añadir 1 ml de TCM fresco con 50 UI / ml de IL-2 a cada pocillo de tal manera que el volumen final es de 2 ml en todos los pocillos. </li><li> Si las células no llegan a&gt; 80% de confluencia, realizar un cambio de medio de comunicación por pipeteando lentamente fuera de 1 ml de medios de comunicación desde la parte superior de cada pocillo. Evite disturbing células asentadas en la parte inferior, mientras que la eliminación de los medios de comunicación. Añadir 1 ml de TCM fresco que contiene 50 IU / ml de rhIL-2. </li></ol> Día 5: <ol start="3"><li> Resuspender las células T CAR pipeteando suavemente hacia arriba y hacia abajo 3 veces en cada pozo y la transferencia a un tubo de centrífuga de 250 ml. Girar las células a 300 xg durante 10 min. </li><li> Completamente sobrenadante aspirado sin molestar a pellet. Lavar una vez con PBS, contar las células como se describió anteriormente, y se lava con PBS una segunda vez. Un rendimiento celular CAR T típica es de aproximadamente 1 x 10 6 células por pocillo (8 placas células x 24 pozos = 192 x 10 6 CAR T). </li><li> Resuspender las células en PBS a una concentración de 5 x 10 7 / ml para una inyección de 1 x 10 7 en un volumen de 200 g (por ejemplo, para 192 x 10 6 células T CAR, resuspender lavaron sedimento en 3,84 ml PBS). </li></ol> 7. CAR Administración de células T a ratones portadores de tumores Día 5: <ol><li> Transpocélulas rt sobre hielo para animalario para inyección intravenosa. Cargar 500-1.000 l en jeringa de insulina con 27-31 G aguja, lo que garantiza que todas las burbujas son expulsadas. </li><li> Coge un ratón en la base de la cola y colocar en la traba de tubo. </li><li> Tirar de la cola tensa con la vena hacia arriba, y se deslizan la aguja aproximadamente 1-2 mm debajo de la piel en la vena mediante la inserción paralela a la vena de la cola. Lentamente expulsar a 200 l en la vena. Si la aguja está correctamente insertado en la vena, el volumen fluirá fácilmente en; de lo contrario habrá resistencia y necesitarán la aguja para ser removido de la cola y se vuelven a insertar correctamente. </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+radiation+on+the+immune+response+to+cancers.">The effect of radiation on the immune response to cancers.</a> Int J Mol Sci. 15 (1), 927-943 (2014).</li><li>Fritzell, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+temozolomide+synergizes+with+immunotherapy+in+a+T+cell-dependent+fashion.">Intratumoral temozolomide synergizes with immunotherapy in a T cell-dependent fashion.</a> Cancer Immunol Immunother. 62 (9), 1463-1474 (2013).</li><li>Park, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-priming+by+temozolomide+enhances+antitumor+immunity+of+dendritic+cell+vaccination+in+murine+brain+tumor+model.">Cross-priming by temozolomide enhances antitumor immunity of dendritic cell vaccination in murine brain tumor model.</a> Vaccine. 25 (17), 3485-3491 (2007).</li><li>Emens, L. A., Jaffee, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leveraging+the+activity+of+tumor+vaccines+with+cytotoxic+chemotherapy.">Leveraging the activity of tumor vaccines with cytotoxic chemotherapy.</a> Cancer Res. 65 (18), 8059-8064 (2005).</li><li>Murphy, K. 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The authors have no conflicts of interests to declare.

La línea de tiempo de tratamiento descrito aquí fue diseñado para modelar estándar clínico de atención y potenciar sus efectos para la terapia adoptiva CAR. CAR dosis de células T, los regímenes de TMZ, y la administración de la radioterapia pueden ser modificados para mejorar la actividad in vivo de células T, lymphodepletion, y el asesinato del tumor en. Regímenes de TMZ se puede aumentar para producir mielosupresión de host y una mayor expansión de las células transferidas adoptivament...

El glioblastoma (GBM) es el tumor cerebral maligno primario más común y es invariablemente fatal. La resección quirúrgica junto con estándar no específica de la quimioterapia y la radioterapia cuidado de no eliminar completamente las células malignas, que resulta en un pronóstico sombrío de menos de 15 meses en los pacientes con esta enfermedad 1. En contraste, la inmunoterapia ofrece un enfoque preciso para apuntar específicamente a las células tumorales, y por lo tanto tiene el potencial para servir como una plataforma de tratamiento altamente eficaz con menor riesgo de toxicidad colateral 2-4. Células T diseñadas ex vivo para expresar receptores de antígenos quiméricos (CAR) ofrecen una estrategia versátil para la inmunoterapia tumoral. CARs se generan mediante la fusión de la región variable extracelular de un anticuerpo con la molécula de señalización de una o más células T intracelular (s), en lugar de una de longitud completa complejo mayor de histocompatibilidad (MHC) -restringido receptor de la célula T 5. Este modo de recono antígeno-anticuerpo-comoOn permite para las células T específicas de antígeno reactivos para reconocer y responder a los antígenos tumorales en ausencia de MHC y se pueden adaptar para un repertorio de antígeno virtualmente infinito. Células T CAR ingeniería contra una variedad de antígenos tumorales han demostrado eficacia preclínica y la promesa excepcional en la clínica 6-9. En concreto, en el contexto de GBM, una variante de crecimiento epidérmico receptor del factor de CAR plataforma de células T focalización III (EGFRvIII), una mutación específica del tumor expresa en la superficie de la célula 10, se ha demostrado prolongar la supervivencia en los ratones portadores de glioma 11. A pesar de su versatilidad, sin embargo, el beneficio clínico de la terapia adoptiva CAR no se ha realizado plenamente, en parte debido a la inmunosupresión asociada al tumor y la evasión inmune 12-16, así como los desafíos en el establecimiento y mantenimiento de las células T específicas de antígeno in vivo. Aprovechando estándar de cuidado (SOC) con inmunoterapia potencialmente puede superar varios de estos limitaciones, resulta en una mayor eficacia tanto en la configuración preclínica y clínica. SOC para el post-resección GBM consiste en altas dosis de temozolomida (TMZ), un agente alquilante de ADN 17, y la irradiación de todo el cerebro (WBI) 1. Estos tratamientos se presume que en sinergia con vacunas tumorales a través de la regulación positiva de la expresión de MHC tumor 18-20 y el derramamiento de antígenos por las células tumorales muertas 17,19,21,22. De hecho, la adición de TMZ 20,23 o 18,24 WBI conduce a una mayor eficacia antitumoral de los tratamientos de base inmunitaria en el entorno preclínico. Además, como muchos agentes quimioterapéuticos citotóxicos no específicos, TMZ se sabe que causa 25,26 linfopenia sistémica, que puede ser aprovechada como medio de host-acondicionado para las plataformas de terapia adoptiva 27-29. Lymphodepletion mediada por TMZ se ha demostrado para mejorar la frecuencia y la función de las células T específicas de antígeno, lo que aumenta la eficacia de un ADOPplataforma de terapia tiva contra los tumores intracraneales 30. En el contexto de la terapia CAR, lymphodepletion sirve como un medio de host-acondicionado por tanto reducir el número de células T supresoras endógena 31, y la inducción de la proliferación homeostática 32 a través de la competencia reducido para citoquinas 33, mejorando así la actividad antitumoral 11,34. Dada la relación sinérgica entre GBM SOC e inmunoterapia plataformas, evaluar nuevas terapias adoptivas y plataformas de vacunas en el contexto del SOC es crítico para sacar conclusiones significativas respecto a la eficacia. En este protocolo, se describe un método para la generación y administración intravenosa de células T CAR-EGFRvIII murino específico junto a TMZ y WBI en ratones con tumores intracraneales EGFRvIII-positivos (ver Figura 1 para la línea de tiempo de tratamiento). En pocas palabras, se producen las células CAR T ex vivo por transducción retroviral. Riñón embrionario humano (HEK) 293T células se transfectaron usando un complejo de ADN / lípido (que contiene el vector y los plásmidos CAR PCL-Eco) para producir virus, que luego se utiliza para transducir esplenocitos murinos activados que se cosechan y se cultivaron en paralelo. Durante el curso de la generación de CAR, los anfitriones murinos con tumores intracraneales EGFRvIII positivos se administran fraccionado de todo el cerebro de la irradiación de rayos X y el tratamiento sistémico TMZ a dosis comparables a SOC clínica. Las células T CAR se entregan por vía intravenosa a los anfitriones lymphodepleted. El siguiente procedimiento se describe en siete fases separadas: (1) La administración de temozolomida a (3) Transfección Ratones portadores de tumores, (2) la irradiación de todo el cerebro de ratones portadores de tumores,, (4) La esplenectomía y células T Preparación, (5 ) Transducción, (6) Cultura de células T CAR y cosecha, y (7) la administración de células T CAR a ratones portadores de tumores. Estas fases consisten en varias etapas que abarcan 6-7 días y se realizan simultáneamente.

<table border="0" cellpadding="0" cellspacing="0" width="1130">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:384px;">Name of Material</td>
			<td style="width:172px;">Company</td>
			<td style="width:125px;">Catalog Number</td>
			<td style="width:449px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pCL-Eco Retrovirus Packaging Vector</td>
			<td>Imgenex</td>
			<td>10045P</td>
			<td>Helper vector for generating CAR retrovirus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Concanavalin A</td>
			<td>Sigma Aldrich</td>
			<td>C2010</td>
			<td>Non-specific mitogen to induce T cell proliferation and viral transduction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Retronectin</td>
			<td>ClonTech/Takara</td>
			<td>T100B</td>
			<td>Facilitates retroviral transduction of T cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine 2000</td>
			<td>Life Technologies</td>
			<td>11668-019</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, high glucose, pyruvate</td>
			<td>Life technologies</td>
			<td>11995-065</td>
			<td>HEK293 culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>T cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM I Reduced Serum Medium</td>
			<td>Life technologies</td>
			<td>11058-021</td>
			<td>Transfection media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 mM L-Glutamine&#160;</td>
			<td>Life technologies</td>
			<td>25030-081</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mM Sodium Pyruvate</td>
			<td>Life technologies</td>
			<td>11360-070</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100X MEM Non-Essential Amino Acids Solution&#160;</td>
			<td>Life technologies</td>
			<td>11140-050</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">55 mM 2-Mercaptoethanol&#160;</td>
			<td>Life technologies</td>
			<td>21985-023</td>
			<td>Reducing agent to remove free radicals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin (50 mg/mL)&#160;</td>
			<td>Life technologies</td>
			<td>15750-060</td>
			<td>T cell culture media supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GemCell U.S. Origin Fetal Bovine Serum</td>
			<td>Gemini Bio Products</td>
			<td>100-500</td>
			<td>Provides growth factors and nutrients for in vitro cell growth&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin (BSA), Fraction V&#8212;Standard&#160;Grade&#160;</td>
			<td>Gemini Bio Products</td>
			<td>700-100P</td>
			<td>Blocks non-specific binding of retrovirus to retronectin-coated plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pharm Lyse (10X concentrate)&#160;</td>
			<td>BD Biosciences</td>
			<td>555899</td>
			<td>Lyses red blood cells during splenocyte processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 &#181;m Sterile Cell Strainers</td>
			<td>Corning</td>
			<td>352350</td>
			<td>Filters away large tissue particles during splenocyte processing&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm BioCoat Culture Dishes with Poly-D-Lysine&#160;</td>
			<td>Corning</td>
			<td>356469</td>
			<td>Promotes HEK293 cell adhesion to maximize proliferation after transfection&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temozolomide</td>
			<td>Best Pharmatech</td>
			<td>N/A</td>
			<td>Lyophilized powder prepared on the day of administration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl Sulfoxide</td>
			<td>Sigma Life Sciences</td>
			<td>D2650</td>
			<td>Necessary for complete dissolution of temozolomide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline</td>
			<td>Hospira</td>
			<td>IM 0132 (5/04)</td>
			<td>Solvent for temozolomide and ketamine/xylazine&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketathesia HCl</td>
			<td>Henry Schein Animal Health</td>
			<td>11695-0701-1</td>
			<td>Ketamine solution&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AnaSed</td>
			<td>Lloyd Inc</td>
			<td>N/A</td>
			<td>Xylazine sterile solution 100 mg/mL&#160;</td>
		</tr>
	</tbody>
</table>

generation of car t cells for adoptive therapy in the context of glioblastoma standard of care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

Las células T CAR son generados por la transducción con el vector retroviral EGFRvIII CAR 11. Este vector, MSGV1, se desarrolló a partir del vector SFGtcLuc_ITE4 35, que contiene el virus de células madre murinas (MSCV) repeticiones terminales largas, la extendida región gag y sitio de empalme sobre (donador de empalme, sd, y aceptor de empalme, sa), y viral señal de empaquetamiento (ψ). El CAR EGFRvIII que contiene el fragmento humano anti-EGFRvIII de una sola cadena variable (scFv) 139, en...

Watch this Scientific Journal Video about Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care at JoVE.com

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Generación de células T CAR para realizar la terapia adoptiva en el contexto de Glioblastoma estándar de cuidado

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

generation-car-t-cells-for-adoptive-therapy-context-glioblastoma

Research

JoVE Journal

Immunology and Infection

21.2K vistas.  Duke University. The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care. 

Video: Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

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

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Generación de células T Multivirus específicos para prevenir / tratar las infecciones virales después de trasplante alogénico de células madre hematopoyéticas

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

La generación de células T reguladoras inducidas de células humanas primarias T vírgenes y de memoria

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac Transposón modificación del sistema de células T humanas primarias

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

Péptido: MHC tetrámero basado en el enriquecimiento de células T específicos para el epítopo

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

Tipo 1 Diabetes inducción acelerada en ratones por transferencia adoptiva de células CD4 + células T diabetogénico

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

La inducción de la inflamación intestinal murino por transferencia adoptiva de Efector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; T células en ratones inmunodeficientes

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

Enriquecimiento de la célula automatizada de citomegalovirus células T específicas para aplicaciones clínicas utilizando el Sistema de citoquinas de captura

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Estereotáctica transferencia adoptiva de células inmunes citotóxicas en modelos murinos de xenoinjertos de Glioblastoma Multiforme de Orthotopic humanos

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.