Name: generation of human alloantigen-specific t cells from peripheral blood
Uploaded: 2014-11-21T15:00:00.000+00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Generation of Human Alloantigen-specific T Cells from Peripheral Blood at JoVE.com

The author would like to thank the NIH Tetramer Core Facility for tetramer production. The author would also like to thank E.D. O&#8217;Connor and K.E. Marquez at the UCSD Human Embryonic Stem Cell Core Facility flow cytometry laboratory for assistance in cell sorting. This work was funded by National Institutes of Health grant K08AI085039 (G.P.M.).

NOTA: Este protocolo requiere el uso de muestras de sangre periférica de voluntarios humanos. Todas las investigaciones con seres humanos debe ser revisado y aprobado por un Estudios Humanos Junta de Revisión Institucional para garantizar el cumplimiento de la Declaración de Helsinki (2013) y la Portabilidad del Seguro de Salud y la Ley de Responsabilidad de 1996. 1. Aislamiento de células T de sangre entera <ol><li> Antes de comenzar, calentar el medio de gradiente de densidad a temperatura ambiente. Alícuota 4 ml de medio de gradiente de densidad en 2-4 estériles de 15 ml tubos de centrífuga cónicos (1 tubo será utilizado por cada 10 ml de volumen total de sangre diluida). </li><li> Obtener 10-20 ml de sangre en aerosol heparina 1-2 de sodio revestido (verde-top) tubos de extracción de sangre venosa. Recoger muestras humanas bajo la supervisión de personas capacitadas y de acuerdo con una Junta de Revisión Institucional protocolo aprobado. </li><li> Limpiar el exterior de los tubos con etanol al 70%. Retire con cuidado la parte superior de la fillenada tubos de recogida y decantar la sangre en un tubo de centrífuga de 50 ml estéril. </li><li> Añadir 10 ml de solución salina tamponada con fosfato estéril (PBS) a cada tubo de recogida de sangre. Recoger el PBS, añadir a la sangre entera se decanta, y mezclar suavemente. </li><li> Añadir 25 mu l T humanas de enriquecimiento celular Cocktail / 2 ml de volumen total. Incubar a temperatura ambiente durante 20 min. </li><li> Usando una pipeta de 10 ml, la capa de hasta 10 ml de la 1: 1 sangre diluida suavemente en la parte superior del medio de gradiente de densidad. Tenga cuidado de no perturbar la superficie del medio de gradiente de densidad. </li><li> Sangre en capas Centrifugar y medio de gradiente de densidad a 1.200 xg durante 20 min a 20 o C. </li><li> Sacar los tubos de la centrífuga, teniendo cuidado de no alterar la interfaz entre el medio de gradiente de densidad y la capa de leucocitos en la interfaz entre el medio de gradiente de densidad y el plasma diluido. Con una pipeta de 5 ml, recoger cuidadosamente la capa leucocitaria y traslado a un nuevo 50 ml bañera cónico estérile. </li><li> Añadir PBS para llevar el volumen de la PBL recogidos a 50 ml y mezclar suavemente. </li><li> Centrifugar a 600 xg durante 5 min a 20 ° C. Decantar. </li><li> Resuspender las células sedimentaron en 10 ml estéril citometría de flujo de la clasificación tampón (PBS filtrada de forma estéril que contiene 1% de BSA). Muestra de 10 l de suspensión celular, se diluye 1:10 (añadir a 90 l) de azul de tripano para contar las células utilizando un hemocitómetro (rendimiento esperado 1-4 x 10 6 células / tubo de sangre total). <ol><li> Eliminar 1 ml alícuota, traslado al tubo de 5 ml, y mantener en hielo para el análisis de las células T no etiquetados por tetrámero. Mantener la suspensión de células en hielo. </li></ol></li></ol> 2. Enriquecimiento magnética de las células T específicas de aloantígeno- <ol><li> Diluir aloantígeno pMHC tetrámero a 1: 100 en tampón de tipo estéril. </li><li> Centrifugar la suspensión celular (9 ml del paso 1,11) a 600 xg durante 5 min a 20 ° C. Decantar. </li><li> Añadir 50 l de diluirse aloantıgeno pMHC tetrámero de peletizadocélulas (hasta 10 7 células). Mezclar por pipeteo suave. Transferir a un tubo de 5 ml estéril. Incubar durante 30 min a temperatura ambiente. </li><li> Añadir 2 ml de tipo buffer. Centrifugar a 600 xg durante 5 min a 20 ° C. Decantar. </li><li> Resuspender las células en 100 l de tampón especie. Añadir 10 l de cóctel de selección de biotina y se incuba durante 15 min a temperatura ambiente. </li><li> Añadir 5 l Magnetic nanopartículas y se incuba durante 10 min a temperatura ambiente. </li><li> Añadir 2,5 ml especie de amortiguamiento y se mezclan suavemente con la pipeta. Quite 100 l alícuota, traslado al tubo de 5 ml, y mantener en hielo para el análisis de las células pre-enriquecimiento. </li><li> Coloque el tubo de 5 ml que contiene la suspensión celular en el imán de separación de células. La tapa debe estar ligeramente encima del tubo. Incubar durante 5 min a temperatura ambiente. </li><li> Retire con cuidado la tapa del tubo. Sosteniendo el tubo y el imán juntos, decantar el contenido del tubo en un tubo de 5 ml fresco. No golpee ni sacuda el contenido del tubo aeliminar las últimas gotas de líquido. </li><li> Retire el tubo del imán. Añadir 2 ml de tampón de tipo frío y mezclar suavemente con la pipeta. Muestra de 10 l de suspensión celular, diluir 1: 2 (añadir a 10 l) de azul de tripano para contar las células utilizando un hemocitómetro. </li></ol> 3. Preparación de las células T de células Individual Citometría de Flujo clasificación de células <ol><li> Añadir tampón de tipo frío a todos los tubos de células (tetrámero no marcado, marcado con tetrámero-un enriquecido, y tetrámero marcado enriquecido) para llevar el volumen a 3 ml. </li><li> Centrifugar a 600 xg durante 5 min a 4 ° C y el tampón de decantación para recuperar las células. </li><li> Resuspender las células sedimentadas en 25 l de tampón especie frío. Añadir 5 l bloque Fc humana. Incubar en hielo durante 20 min. </li><li> Preparar anticuerpos para la clasificación de citometría de flujo. Mezclar 15 l de tampón especie, 15 l anti-CD5, 15 l anti-CD14, y 15 l anti-CD19. </li><li> Añadir 20 l de mezcla de anticuerpos a cada tubo de las células. Incubar en hielo durante 20 min. </li><li> Añadir 2 ml de tampón de tipo frío a cada tubo. </li><li> Centrifugar a 600 xg durante 5 min a 4 ° C y el tampón de decantación para recuperar las células. </li><li> Resuspender las células a una concentración de 1 -2 x 10 6 células / ml en tampón de clasificación. </li><li> Alícuota de 100 l de medio de cultivo de células humanas T (DMEM de Iscove suplementado con 2 mM Glutamax, HEPES 10 mM, 50 mg / ml de gentamicina, 50 mM 2-mercaptoetanol, 10% suero AB humano inactivado por calor, y 2,5 humana recombinante ng / ml IL-2) a cada pocillo de una placa redonda de 96 pocillos de cultivo celular de fondo. Mantener en hielo. </li></ol> 4. Aislamiento de células T tetrámero marcado por celda individual Citometría de Flujo Clasificación <ol><li> Establecer citometría de flujo para identificar los parámetros de compuerta aloantígeno-pMHC tetrámero células T marcados (Figura 2.A). Utilice la fracción tetrámero pre-enriquecimiento etiquetados para establecer gating estrategias para eliminar dobletes, puerta de linfocitos, las células que expresan CD19 excluir B y CD14expresando monocitos, e identificar las células T mediante la expresión CD5. Utilice la muestra no etiquetada con tetrámero como una fluorescencia menos uno de control para establecer la gating para la identificación de las células T tetramer vinculante (con 0% de CD5 + CD14 - CD19 - células de la muestra no etiquetada por tetrámero deben caer dentro de esta puerta) . NOTA: Gating estrategias que no son adecuadamente rigurosas dará como resultado el aislamiento de células no T o células T que no son específicas de antígeno, mientras que las estrategias de compuerta excesivamente restrictivas se reducirá el número de células T aisladas. </li><li> Programar los parámetros de la placa de la célula única especie. Dirigir el clasificador para colocar un CD5 + CD14 - CD19 - tetrámero + células en cada pocillo. Dirigir el plato creado para contener 1 pocillo de control negativo (no hay células ordenadas en el pozo) y 1 control positivo también (100 CD5 + CD14 - CD19 - tetrámero - células) en cada fila (figura 2.B </strong&gt;). </li><li> Uso de la citometría de flujo establecido gating estrategia y la configuración de la placa, aislar células individuales de unión tetrámero-T directamente en la placa de 96 pocillos usando un clasificador de células por citometría de flujo. </li></ol> 5. Cultura y expansión de clones aloantígeno-específicos de células T <ol><li> Después de la finalización de la clasificación de células, la placa se centrifuga a 600 xg durante 5 min a 20 ° C. Lugar placa de cultivo a 37 ° C en un incubador de CO2 al 6%. </li><li> Vortex estimulantes microperlas anti-CD3 / anti-CD28 durante 30 segundos para volver a suspender cuentas. </li><li> Calcular el volumen de perlas de activador necesarios para la estimulación de las células T recogidas (0,5 l de microperlas de activador / pocillo). Traslado volumen calculado de cuentas estimuladoras a un tubo de 5 ml estéril. Añadir 1 ml de medio de cultivo de células T humanas y de vórtice. </li><li> Coloque el tubo con la suspensión de microperlas en imán. La tapa debe estar ligeramente encima del tubo. Incubar durante 2 min a temperatura ambiente. </li><li> Retire con cuidadola tapa del tubo. Sosteniendo el tubo y el imán juntos, contenido del tubo de decantación en un tubo de 5 ml fresco. No golpee ni sacuda el contenido del tubo. </li><li> Retire el tubo del imán. Añadir medio de cultivo de células humanas T (100 medianas l / 0,5 l de microperlas añaden inicialmente). </li><li> Alícuota de 100 l de suspensión de perlas activador a cada pocillo de la placa de 96 pocillos. Incubar placa de cultivo a 37 ° C en un incubador de CO2 al 6%. </li><li> Monitorear el crecimiento celular diariamente por examen microscópico. Pequeños grupos de células proliferantes pueden ser observados microscópicamente después de 5 - 7 días de cultivo (Figura 3.A). NOTA: Visualización de cultivos de células T en este punto puede ser difícil, y la ausencia de grupos de células observables en esta etapa puede no ser indicativo de una falta de células T en crecimiento. </li><li> A los 14 días después del aislamiento de células, cultivos de alimentación retirando cuidadosamente 100 l de los medios de comunicación fuera de la parte superior de la cultura y la reemplazan con 100 μ; L fresco T humano medio de cultivo celular. Continuar la incubación de placa de cultivo a 37 ° C en un incubador de CO2 al 6%. </li><li> Monitorear el crecimiento celular diariamente por examen y evaluación de el color del medio de cultivo microscópico. Grupos de células grandes deben ser visibles al microscopio 2 - 3 días después de que el cambio de medio. Macroscópicamente, los sedimentos celulares deben ser visibles durante los 14 días siguientes al cambio de medio. </li><li> A los 28 días siguientes de aislamiento de células, identificar clones que crecen por el examen microscópico. Transferir el volumen 200 l de los cultivos en placas de 96 pocillos de crecimiento positivo a pocillos individuales de una placa de cultivo de tejidos de 48 pocillos que contenía 200 medio de cultivo de células T humanas l. </li><li> Añadir 100 l que contenían 12,5 l de microperlas anti-CD3 / anti-CD28 estimuladoras (preparada como se describe en las secciones 5.2 a 5.6). Incubar placa de cultivo a 37 ° C en un incubador de CO2 al 6%. </li><li> Monitorear el crecimiento del cultivo a diario por ex microscópicaaminación y evaluación de el color del medio de cultivo. Cuando el cultivo de células T alcanza&gt; 2x10 6 / ml (típicamente 3-5 días después de la re-estimulación, se puede estimar por el día en que los medios de comunicación comienza girando amarillo paja), la cultura transferencia a un pocillo de una placa de cultivo de tejido de 24 pocillos y añadir 500 l medio celular humana T. </li><li> En 10 a 14 días seguir re-estimulación, recoger 200 l de cultivo de células T para evaluar aloantıgeno especificidad por citometría de flujo análisis de pMHC tetrámero de unión (mediante la estrategia de etiquetado y la compuerta se describe en las secciones 3 y 4.1). </li></ol> 6. A largo plazo Re-estimulación y Cultura de clones de células T <ol><li> Continuamente re-estimular y expandir los cultivos de células T clonales con la especificidad deseada puede ser cada 14 días. Recoger cultivos de células T en un tubo de 5 ml estéril a 14 días después de la última estimulación microperlas anti-CD3 / CD28. Combinar múltiples pocillos de la misma clon de células T en un solo tubo, hasta un volumen de 2,5 ml. </li&gt; <li> Añadir medio para llevar el volumen final a 2,5 ml y mezclar suavemente por pipeteado. </li><li> Coloque el tubo de 5 ml que contiene el cultivo de células T en el imán de separación de células para eliminar microperlas de edad del cultivo. La tapa debe estar ligeramente encima del tubo. Incubar durante 5 min a temperatura ambiente. </li><li> Retire con cuidado la tapa del tubo. Sosteniendo el tubo y el imán juntos, decantar la suspensión de células en un tubo de 5 ml fresco. No golpee ni sacuda el contenido del tubo para eliminar las últimas gotas de líquido. </li><li> Añadir medio para llevar el volumen final a 4 ml. Muestra de 10 l para contar las células utilizando un hemocitómetro. </li><li> Recuperar las células T mediante centrifugación a 600 xg durante 5 min a temperatura ambiente. </li><li> Decantar el sobrenadante. Resuspender las células T en 10 6 células / T humanas medio de cultivo celular ml. Transferir 1 ml de alícuotas a cada pocillo de una placa de cultivo tisular de 24 pocillos. </li><li> Vuelva a estimular las células T mediante la adición de 100 medios de comunicación l que contenía 12,5 l de estimulante anti-CD3 / amicroperlas nti-CD28 (como se describe en las secciones 5.2 a 5.6). Se incuba a 37 ° C en un incubador de CO2 al 6%. </li></ol>

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The authors declare no competing financial interests.

T cell alloreactivity is a long-studied and clinically-relevant phenomenon. The robust proliferative and effector responses of T cells to allogeneic stimulation has enabled extensive analyses of human T cell responses in vitro through relatively straightforward mixed lymphocyte reactions of peripheral blood T cells against inactivated allogeneic cells. However, these primary alloreactive T cell responses are oligoclonal, comprised of a large number of individual T cells responding to specific alloantigens. This ...

Los linfocitos T son componentes críticos del sistema inmune adaptativo. Las células T son responsables de mediar no sólo directamente respuestas inmunes protectoras a los agentes patógenos a través de una variedad de mecanismos efectores, sino también mantener activamente la auto-tolerancia inmunológica y dirigir las respuestas de otras células en el sistema inmune. Estas funciones están dirigidas a través de un número de señales integradas, incluyendo receptores de células T (TCR) de la ligación, citocinas y quimiocinas, y los metabolitos 1. De estas señales, el TCR es de particular importancia, ya que proporciona la especificidad característica que define el papel de la célula T en la inmunidad adaptativa. Un TCR interactúa con antígenos peptídicos lineales presentados por MHC (HLA ortólogo humano) moléculas (complejos pMHC) de una manera altamente específica y sensible para proporcionar las señales que inician la función efectora de células T. Los parámetros bioquímicos de las interacciones de TCR con ligandos pMHC proporcionan no sólo la especificidad para Tactivación de las células, sino que también tiene un impacto cualitativo en función de la célula posterior T2. Por lo tanto, el estudio de la función de las células T a menudo requiere el examen de las respuestas de las células T clonales con especificidad antigénica definida. El compartimiento humana de células T, que contiene aproximadamente 10 12 células T αβ, contiene un estimado de julio 10 a agosto 10 αβTCRs distintas 3-4. Este diverso repertorio ofrece oportunidad para el reconocimiento de la gran variedad de péptidos de patógenos potenciales que requeriría una respuesta de células T para la inmunidad protectora. Se estima que la frecuencia de células T en respuesta a un antígeno extraño dado presentado por auto-MHC es del orden de 10 -4 a 10 -7 en ausencia de respuesta inmune antes de que el antígeno 5. El repertorio de células T naive está determinada por la selección tímica para asegurar la capacidad de reconocer auto-MHC presentación de antígenos peptídicos y límite reaccionanividad contra antígenos auto-péptido, la maximización de la utilidad potencial para mediar la inmunidad protectora 2. Sin embargo, en violación de esta diseñado reactividad, una frecuencia relativamente grande, 10 -3-10 -4, de las células T de individuos inmunológicamente ingenuos responder a la estimulación con células alogénicas, reconociendo tanto las moléculas MHC extranjeros así como los péptidos endógenos que presentan 6. El reconocimiento de ligandos pMHC alogénicos es estructuralmente similar al reconocimiento de antígenos extraños presentados por auto-MHC; el TCR hace interacciones bioquímicas críticos tanto con la molécula MHC alogénico, así como el péptido presentado 7. La naturaleza robusta de la respuesta de las células T alogénicas a resultados de la estimulación de la diversidad de los complejos pMHC presente en la superficie de las células alogénicas 8. Se estima que cada MHC presenta aproximadamente 2 x 10 4 diferentes antígenos peptídicos endógenos 9. Este breadth de la respuesta a la estimulación alogénica es un aspecto significativo de la patología clínicamente relevantes, tales como el rechazo de aloinjerto o enfermedad de injerto contra huésped (EICH), como resultado de alorreactividad de células T. Estudio de células T respuestas alorreactivas humanos se ha basado tradicionalmente en el examen de las respuestas policlonales de células T vírgenes después de la estimulación con células alogénicas. La estimulación repetida con la misma línea celular alogénico combinado con análisis de dilución limitante es capaz de generar células T clonales con el reconocimiento alogénico HLA definida de 10. Sin embargo, este enfoque es problemático para el examen de las respuestas a ligandos pMHC alogénicos individuales, como el repertorio amplio y diverso de complejos pMHC endógeno presente durante un determinado alogénico HLA estimula un amplio repertorio de células T. Esta estimulación población mayor y la limitación de la dilución enfoque requeriría de cribado de un gran número de clones para aislar células T con la deseada Reactividad contra un único ligando pMHC. Además, la frecuencia de células T que responden a un ligando pMHC alogénico individual es relativamente baja entre las poblaciones de células T ingenuas, que presenta una barrera para la generación eficiente de clones de células T humanos que responden a un antígeno dado. La identificación y el aislamiento de las células T específicas de antígeno de las poblaciones policlonales se han habilitado por el desarrollo de fluoróforo marcado pMHC multímeros 11. Este enfoque utiliza antígenos peptídicos específicos cargados en moléculas MHC solubles biotiniladas recombinantes, que se etiquetan mediante la unión a un fluoróforo marcado con estreptavidina. La multimerización de pMHC aumenta la avidez, la compensación de la afinidad del TCR intrínsecamente baja (M) para ligandos solubles pMHC. Las células marcadas pueden ser identificados y aislados por citometría de flujo. Sin embargo, este enfoque todavía está limitado por la baja frecuencia de células T específicas de antígeno entre las poblaciones de células T vírgenes, que son típicamenteórdenes de magnitud menor que el límite de la identificación y la cuantificación precisa en la mayoría de los citómetros de flujo. Para abordar esta limitación, un método de etiquetado tetrámero pMHC y posterior enriquecimiento perla magnética para las células tetrámero marcado ha sido desarrollado 12. Este método ha demostrado una detección fiable, enumeración, y el aislamiento de células T específicas de antígeno de baja frecuencia. Este protocolo describe un protocolo eficaz para la generación de clones humanos de células T que responden específicamente a ligandos pMHC alogénicos individuales. El protocolo se aplica pMHC (HLA) etiquetado multímero y enriquecimiento para el aislamiento de células T humanas-aloantígeno específico con citometría de flujo de células de clasificación y un método robusto para el cultivo in vitro de células T humanas para permitir la producción de clones de células T de células clasificadas individuales ( visión general en la Figura 1).

<table><tbody><tr><td>Sodium heparin venous blood collection tube 16 x 100 mm</td><td>Becton, Dickenson and Company</td><td>366480</td><td></td></tr><tr><td>Lymphoprep</td><td>Stemcell Technologies</td><td>7801</td><td></td></tr><tr><td>Rosette Sep Human T Cell Enrichment Kit</td><td>Stemcell Technologies</td><td>15061</td><td></td></tr><tr><td>Dulbecco&#039;s PBS, 1x without Ca or Mg</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6635</td><td></td></tr><tr><td>Fluorophore-labeled pMHC tetramer</td><td>NIH Tetramer Facility</td><td>NA</td><td></td></tr><tr><td>EasySep Biotin Selection Kit</td><td>Stemcell Technologies</td><td>18553</td><td></td></tr><tr><td>EasySep Selection magnet</td><td>Stemcell Technologies</td><td>18000</td><td></td></tr><tr><td>TruStain FcX Human Fc blocking solution</td><td>Biolegend</td><td>422301</td><td></td></tr><tr><td>Anti-CD5 PE-Cy7 (clone UCHT2)</td><td>Biolegend</td><td>300621</td><td></td></tr><tr><td>Anti-CD14 FITC (clone HCD14)</td><td>Biolegend</td><td>325603</td><td></td></tr><tr><td>Anti-CD19 FITC (clone HIB19)</td><td>Biolegend</td><td>302205</td><td></td></tr><tr><td>Iscove&#039;s DMEM, without b-ME or L-glutamine</td><td>Corning</td><td>15-016-CV</td><td></td></tr><tr><td>HEPES</td><td>Corning</td><td>25-060-CI</td><td></td></tr><tr><td>b-Mercaptoethanol&amp;nbsp;</td><td>Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>Gentamicin sulfate (50 mg/ml)</td><td>Omega Scientific</td><td>GT-50</td><td></td></tr><tr><td>Human AB serum, male donor</td><td>Omega Scientific</td><td>HS-30</td><td></td></tr><tr><td>Recombinant human IL-2</td><td>Peprotech</td><td>AF 200-02</td><td></td></tr><tr><td>Dynabeads Human T-Activator CD3/CD28</td><td>Life Technologies</td><td>11131D</td><td></td></tr><tr><td>Media</td><td></td><td></td><td></td></tr><tr><td>Cell sorting buffer</td><td></td><td></td><td></td></tr><tr><td>PBS, pH 7.4</td><td>1 L</td><td></td><td></td></tr><tr><td>BSA</td><td>10 g</td><td></td><td></td></tr><tr><td>EDTA (0.5 M)</td><td>2 ml</td><td></td><td></td></tr><tr><td>Human T Cell Culture Medium</td><td></td><td></td><td></td></tr><tr><td>Iscove&#039;s DMEM</td><td>351.6 ml</td><td></td><td></td></tr><tr><td>Heat-inactivated human AB serum</td><td>40 ml</td><td></td><td></td></tr><tr><td>HEPES (1 M)</td><td>4 ml</td><td></td><td></td></tr><tr><td>Glutamax (100x)</td><td>4 ml</td><td></td><td></td></tr><tr><td>Gentamicin (50 mg/ml)</td><td>0.4 ml</td><td></td><td></td></tr><tr><td>b-mercaptoethanol (14.3 M)</td><td>1.4 ml</td><td></td><td></td></tr><tr><td>Recombinant human IL-2 (1 mg/ml)</td><td>1 ml</td><td></td><td></td></tr></tbody></table>

generation of human alloantigen-specific t cells from peripheral blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

Este protocolo describe la generación de cultivos de células humanas T clonal con aloantígeno especificidad definida a través de un enriquecimiento de perla magnética y flujo de una sola célula de clasificación de citometría de estrategia. La Figura 1 proporciona un esquema del proceso. <img alt="Figura 1" src="/files/ftp_upload/52257/52257fig1highres.jpg" /> Figura 1: Visión general de Protocolo El protoco...

Watch this Scientific Journal Video about Generation of Human Alloantigen-specific T Cells from Peripheral Blood at JoVE.com

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Generación de células T humanas aloantígeno-específicos de la sangre periférica

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

generation-of-human-alloantigen-specific-t-cells-from-peripheral-blood

Research

JoVE Journal

Immunology and Infection

12.9K vistas.  University of California, San Diego. This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Video: Generation of Human Alloantigen-specific T Cells from Peripheral Blood

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

La inducción de anergia aloantígenos específicos en las células mononucleares de sangre periférica mediante estimulación aloantígeno con bloqueo de señales co-estimuladoras

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Investigación

Inmunología e Infección

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&#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 ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Generación de Integración libres de células madre pluripotentes inducidas a partir de células mononucleares de sangre periférica humana Utilizando vectores episomales

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Biología del Desarrollo

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

Uso de células madre pluripotentes inducidas por el hombre para la generación de células T específicas de antígenos tumorales

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Investigación sobre el cáncer

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Generación de humanos CD40 activado por células B

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Biología

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

Aislamiento y<em&gt; Ex Vivo</em&gt; Cultura de Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; Las células γδ T, una célula αβT Progenitor Extrathymic

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

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

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

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

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

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

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

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Generación de células dendríticas derivadas de monocitos humanos de sangre entera

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Generación de anticuerpos monoclonales humanos discriminativo de células B antígeno específicas raras circulan en sangre

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Generation of Human Regulatory T Cell Clones

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as &#8220;Treg&#8221; therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4+CD25+ T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based ...

Generación de células T humanas aloantígeno-específicos de la sangre periférica

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