Christian Welker is funded by a grant provided by the J&#252;rgen-Manchot-Stiftung.

Declaración de Ética: Todos los procedimientos se llevaron a cabo de acuerdo con la Declaración de Helsinki y fue aprobado por el Comité de Ética Clínica en la Universidad de Tubinga (proyecta 38 / 2009B02 y 470 / 2013B02). 1. Aislamiento de células mononucleares de sangre periférica (PBMCs) <ol><li> Tome 50-100 ml de un voluntario sano a través de la punción venosa utilizando una jeringa de 50 ml que contiene 1.000 UI de heparina sulfato y diluir la sangre 1: 2 con PBS (pH = 7,2). </li><li> La capa cuidadosamente 35 ml de la sangre: solución de PBS en solución separando 15 ml de sangre tales como Biocoll (d = 1,077 g / ml) en un tubo cónico de 50 ml. Centrifugar durante 15 min a 800 xg a TA. NOTA: El freno no debe ser utilizado. </li><li> Recoger las células de la capa linfocítica con una pipeta y se lavan las células dos veces en por lo menos 50 ml de PBS (centrífuga de 12 min; a 400 xg). Eliminar el sobrenadante después de la centrifugación con una pipeta. </li><li> Lyse restante eritrocitos suspendiendo y incubAting la fracción linfocítica con 1-5 ml de una solución tampón hipotónico durante 2-4 min. NOTA: Duración de la exposición y el volumen de tampón de lisis depende del tampón de lisis utilizado y de la cantidad de glóbulos rojos que quedan en la fracción de linfocitos. </li><li> Diluir las células con PBS (01:10) y se centrifuga a 250 xg durante 12 min y, posteriormente, volver a suspender y se lava el sedimento celular una vez en 10 ml de PBS a 300 xg durante 12 min. </li><li> Resuspender los linfocitos en PBS y contar las células en una cámara de recuento Neubauer utilizando solución de azul de tripano. NOTA: Por lo general,&gt; 1 x 10 8 linfocitos se reciben de 100 ml recién extraída de la sangre periférica. </li></ol> 2. Aislamiento de células T Vδ1 <ol><li> Tome &lt;1 x 10 6 linfocitos aislados durante iniciales FACS que determinan la frecuencia de células T CD4 Vδ1. NOTA: Tamaño de la población de esta entidad de células T puede diferir mucho entre individuos y en función de su estado inmunológico. Typicaliado, aproximadamente 1% de las células T son Vδ1 + y aproximadamente 1-5% de las células son Vδ1 + CD4 +. <ol><li> Diluir las células con 1 ml de tampón FACS-, que consiste en PBS que contenía 2% de FCS y EDTA 5 mM. Centrifugar a 660 xg durante 2 minutos y decantar el sobrenadante. El volumen de reflujo de tampón FACS sobre 100 l es suficiente para la posterior tinción. Vórtice brevemente e incubar las células con 5 l de reactivo Fc-bloqueo durante 5 min a RT para aumentar la especificidad de la tinción FACS. </li><li> Añadir anticuerpos monoclonales humanos directamente a las células sin retirar el reactivo Fc-bloqueo. Asegúrese de teñir las células con anticuerpos contra Vδ1 (01:50), CD3 (01:50), CD4 (1: 200), CD8 (1: 200) y TCRαβ (1:25). Preparación del control isotipo con los isotipos de inmunoglobulinas correspondientes. Se incuban las células con anticuerpos durante 20 min a 4 ° C en la oscuridad. </li><li> Lavar las células en 1 ml de tampón FACS (Centrifugar durante 2 min; a 660 xg), se decanta el supernatant y proceder a la adquisición FACS en el volumen de tampón restante. </li></ol></li><li> Peletizar las células (que no se utilizan en el análisis FACS) por centrifugación durante 12 min; a 300 g; 8 ° C. Eliminar el sobrenadante y resuspender el celular pelletin 60 l MACS-buffer por 1 x 10 7 células. Añadir 20 l de reactivo Fc-bloqueo por 1 x 10 7 células e incubar durante 5 min a 4 ° C. </li><li> Añadir 10 l de anticuerpo anti-Vδ1 humana conjugado con FITC por 1 x 10 7 células directamente a las células. Incubar durante 12 min a 4 ° C en la oscuridad. </li><li> Lavar las células utilizando 14 ml MACS-buffer (centrifugar durante 12 min; a 300 g; 8 ° C). Desechar el sobrenadante y resuspender completamente las células en 80 l de tampón MACS por 1x10 7 células. </li><li> Tome 1-2 x 10 5 células y las diluir en tampón FACS 100 l para la verificación del etiquetado por análisis FACS. No se necesitan aditivos adicionales para este paso. Asegúrese de que no es un suficiente diferencia en el brillo para Vδ1 - y las células Vδ1 +. Si este no es el caso, repita los pasos 2.3) y 2.4). </li><li> Añadir 20 l microperlas anti-FITC por 1 x 10 7 células para las células restantes, mezclar bien e incubar durante 15 min en la oscuridad a 4 ° C. A partir de entonces, se lavan las células utilizando al menos 10 ml de tampón MACS (centrifugar durante 12 min, a 300 xg; 8 ° C). </li><li> Durante la centrifugación, equilibrar la columna magnética pre-enfriado colocado en un imán con tampón MACS 500 microlitros. </li><li> Resuspender las células en 500 l de tampón MACS-(para el número de células mayor que 1 x 10 8, ampliar este volumen en consecuencia) y aplicar cuidadosamente las células en la columna. Recoger el flujo a través que contiene el Vδ1 - células. </li><li> Lavar la columna tres veces con l MACS-buffer 500 y recoger el flujo a través de nuevo en el mismo tubo. Tenga en cuenta que el depósito debe estar vacío antes de aplicar tampón en la columna. </li><li> Retire la columna del dispositivo magnético y colocarlo en un tubo cónico de 15 ml. Vaciar rápidamente la columna con tampón MACS 1 ml empujando firmemente el émbolo en la columna. Recoger el eluido en un tubo. NOTA: Para obtener una pureza&gt; 98%, por lo general es necesario repetir los pasos 2,7 a 2,9) con una segunda columna. </li><li> Verificar la pureza de las células mediante análisis FACS 18 con el mismo panel de anticuerpos como antes (ver 2.1.2) y contar las células. </li></ol> 3. Aislamiento de células T + Vδ1CD4 <ol><li> Resuspender 25 l de cuentas de CD4 con un vórtice de&gt; 30 seg. Lavar las perlas (la cantidad mínima tal como se representa por el fabricante) en 1 ml de tampón MACS en un tubo de 1,5 ml de reacción colocando el tubo en un imán durante 1 min. Descartar el sobrenadante cuidadosamente con una pipeta a pequeña escala (200 l) y resuspender las perlas en el volumen original de tampón MACS-. </li><li> Granular Vδ1 + células (centrífuga de 12 minutos; a 300 xg) y aspirar el sobrenadante y resuspender todas las células + Vδ1 en 500 l de tampón MACS-y añadirlos al tubo que contenía las perlas. Mezclar vigorosamente y se incuba durante 20 min a 4 ° C con inclinación constante (por ejemplo, en un rotador). </li><li> Se coloca el tubo que contiene las células en un imán para 2 min. Asegúrese de que no hay restos en la tapa. NOTA: Las células CD4 + se han unido las perlas magnéticas y adjuntar a un lado del tubo frente al imán. </li><li> Abrir con cuidado la tapa mientras se mantiene el tubo en el interior del dispositivo magnético y recoger el sobrenadante que contiene el CD4 - células Vδ1 + usando una pipeta de pequeña escala. Coloque CD4 - las células Vδ1 + en un tubo separado y colocar este tubo en el imán de nuevo para evitar posiblemente restante células CD4 o perlas de la población. <ol><li> Lave el CD4 - las células en 5 ml MACS-buffer (centrifugar durante 12 min; a 300 xg) unnd resuspender en una concentración de 1 x 10 5 células por 100 medios de comunicación mu l. CD4 - las células pueden cultivarse fácilmente en las condiciones dadas a continuación (4.2). </li></ol></li><li> Coloque el tubo con los objetivos de células CD4 + fuera del campo magnético, resuspender las células en 500 l MACS-buffer y ponerlos de nuevo en el dispositivo magnético. Repetir los pasos 3.3 a 3.5 dos veces para obtener una mayor pureza. Aspirar el sobrenadante con la pipeta </li><li> Resuspender las células CD4 + en 100 l de medios de cultivo (RPMI 1640, 10% de FCS, 1% de L-glutamina, 1% de penicilina / estreptomicina) y añadir 10 l de una solución de perlas de desprendimiento. Incubar a TA durante 45 min con inclinación constante (por ejemplo, en un rotador). </li><li> Colocar las células en un imán para 1 min. Recoger cuidadosamente el sobrenadante que contiene células CD4 + + Vδ1-talón gratis con una pipeta de pequeña escala. </li><li> Fuera del imán, resuspender las perlas con medios de cultivo y 100 l de repeticiónpasos 3.6 y 3.7 dos veces para obtener el número de células más altos de células CD4 +. </li><li> Granular las células (centrifugar a 300 xg, durante 12 minutos) y descartar el sobrenadante por completo con la pipeta. Resuspender las células en los medios de comunicación, pre-calentado frescos y contarlos. Examinar la pureza de las células Vδ1 + + CD4 por análisis FACS representados en los pasos 2.1.1-2.1.3. </li></ol> Clonación 4.-célula individual por dilución limitada <ol><li> Aislar las células mononucleares de sangre periférica (PBMCs) de un donante alogénico como se representa en 1/1 a 1/6. </li><li> Irradiar 2,5 x 10 7 PBMC alogénicas con 80 Gy en 25 ml de medio de cultivo utilizando γ-radiación. </li><li> Añadir IL-2 (200 U / ml), IL-7 (20 ng / ml) y PHA (2 mg / ml) a las células alimentadoras irradiadas y distribuirlos en placas de 96 pocillos en forma de U, 5 x 10 4 alimentador células en 50 l por pocillo. </li><li> Diluir las células Vδ1 + CD4 + a una concentración de 0,3 células por 50 l. Pipetate 50 l de la solución de células a cada pocillo que alberga las células irradiadas y citoquinas. NOTA: Por lo tanto, las citoquinas se diluyen a la concentración final de 100 U / ml de IL-2, 10 ng / ml de IL-7 y 1 mg / ml de PHA. Se incuban las placas de 96 pocillos en una incubadora a 37 ° C, 5% de CO 2 humidificado atmósfera. <ol><li> Opcionalmente, cultivar restante purificado células Vδ1 + CD4 + como cultivo en masa en las mismas condiciones de cultivo como los clones. </li></ol></li><li> Suministrar las células cada 3-4 días con citoquinas y medio fresco. Para ello, el intercambio de la mitad del medio en los pocillos con 50 l de medio fresco mediante pipeteo. Añadir concentración 2 veces de citoquinas para cada suplementación. Añadir 5x10 4 células alimentadoras irradiados por pozo cada otra ronda de alimentación. </li><li> Usando un microscopio, observar primeros clones se hacen visibles después de 3-4 semanas, ya que metabolizan y por lo tanto cambiar el color de las colonias del medio de cultivo y la forma. NOTA: Para pielesTher cultivo, resuspender los clones bien y transferirlos para separar las placas de 96 pocillos. Analizar las células a través de FACS como se indica en 2.1.1-2.1.3. Los clones mantenidos en estas condiciones pueden llegar a cambiar su TCR de Vδ1 a αβTCR. El punto de tiempo para esto transdiferenciación varía de clon para clonar. </li></ol>

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The authors have nothing to disclose.

Para estudiar el fenotipo, la biología y la función de una entidad de células escasas (T-), es decir, las células T CD4 + + Vδ1, se utilizaron dos marcadores: Vδ1 y CD4 para su aislamiento celular magnética positiva. Vδ1 es un receptor huérfano, mientras que CD4 se expresa en células T helper, a un nivel inferior en monocitos y células dendríticas, y en un nivel muy bajo en las células progenitoras hematopoyéticas. Las técnicas para el enriquecimiento y la selecció...

En los vertebrados, la inmunidad adaptativa que se estructura en el celular y una parte de la inmunidad humoral desempeña un papel importante en la defensa contra patógenos. El reconocimiento de una amplia gama de antígenos está mediada por T- hyperpolymorphic y receptores de células B (TCR / BCR), que con respecto a las células T se supone que se produce principalmente en el timo 1. A esto, las células madre hematopoyéticas (HSC), derivadas de la médula ósea, el timo semillas y diferenciar a lo largo de las etapas bien definidas, finalmente, que dan lugar a todos los linajes de células T. Thymus progenitores siembra son CD4 - y CD8 - y por lo tanto constituyen la fracción de timocitos inmaduros, doble negación (DN). Señales derivadas del timo entonces inducen su compromiso linaje y la diferenciación en cualquiera de las células T delta gamma o delta gamma. La expresión de genes funcionalmente reordenados cadena TCR-gamma y TCR-delta en DN2 / 3 timocitos conduce a gamma δTCR complejos, que impulsan una proliferación celulard promover la diferenciación en células DT γ 2,3. En contraste, el reordenamiento de una cadena TCR-β funcional, que puede emparejarse con preTα para construir un preTCR pT, induce el silenciamiento transcripcional de la cadena TCR-γ en timocitos DN3 y su transición a CD4 + CD8 + doble positivas timocitos 4 . En esta etapa, la recombinación de la cadena TCR-α se produce, eliminar el locus TCR-δ que está ubicado dentro del locus TCR-α, abrogando así la producción de un γδTCR en estas células irrevocablemente 5-9. ΑβTCRs reordenados se seleccionan posteriormente por su capacidad para unirse a la auto-MHC débilmente (selección positiva), que no podrá exceder de un cierto umbral para evitar la autoinmunidad (selección negativa). De acuerdo con su capacidad de unión a MHC de clase I o II, las células αβT seleccionados se convierten en células CD4 + de un solo positivo o T CD8 +, que salga el timo los linfocitos T como ingenuos. Sin embargo, la involución del timo comienza temprano en la vida que conduce a la producción de forma exponencial reducido de células T ingenuas que es casi extinguido después de la adolescencia 10. Sin embargo, el tamaño del grupo de células T permanece constante durante toda la vida, lo que se explica sólo en parte por la proliferación homeostática post-timo de las células T y la proliferación de larga vida memoria inmunológica 11. En consecuencia, debe ocurrir extrathymic desarrollo de las células T. Investigaciones recientes han ganado atractivo importante que caracterizó progenitores de células αβT, que -al extrathymic sitios dieron origen al delta gamma funcional células T 12-17. Sin embargo, un conocimiento detallado de extrathymic precursores de células αβT que independiente de un timo diferenciarse en células αβT es tan fragmentaria como el fondo que tenemos en la ruta que toman los mismos. Recientemente hemos identificado la pequeña entidad de células T de Vδ1 + </sup&gt; células CD4 + γδT como un extrathymic prognitor celular αβT 18, que cuando se aislaron de sangre periférica de donantes humanos sanos pueden transdiferenciarse en células αβT en un entorno inflamatoria leve. Curiosamente y contrario a la proliferación homeostática de las células T post-tímicos, transdiferenciación de las células Vδ1 CD4 + genera nuevos receptores de células T, ampliando así la diversidad repertorio, por lo que potencialmente nuevos antígenos pueden ser reconocidos y puede protección del huésped contra los patógenos recién adquiridas. Esto se suma a la plasticidad de las células T y añade una nueva vía hasta ahora no apreciado para extrathymic desarrollo de células T. El aislamiento cuantitativa de fuentes linfocítica, la generación de clones de células individuales y su expansión eficiente son esenciales para el objetivo de identificar a los marcadores y moléculas que desencadenan esta célula αβT precursor`s desarrollo extrathymic.

<table border="0" cellpadding="0" cellspacing="0" width="789">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biocoll Solution</td>
			<td style="width:93px;">Biochrom</td>
			<td style="width:119px;">L 6113</td>
			<td style="width:361px;">lymphocyte separating solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lysing Buffer</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>555899</td>
			<td>lysis of erythrocytes&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate-buffered Saline</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td style="width:119px;">D8537&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MACS buffer</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td>supplement with BSA and pre-cool before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BSA</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td>not mandatorily from this supplier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human Vd1 FITC (clone:&#160; TS8.2)</td>
			<td style="width:93px;">Thermo Scientific</td>
			<td>TCR2730</td>
			<td>not mandatorily from this supplier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD3 PerCP (clone: SK7)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>345766</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human TCRab PE (clone: T10B9.1A-31)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>555548</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD4 VioBlue (clone: M-T466)&#160;</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-097-333</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD8 APC-H7 (clone: SK1)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>641400</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Anti-FITC MultiSort Kit</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-058-701</td>
			<td>yields better results than anti-FITC MicroBeads</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MS columns</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-201</td>
			<td>pre-cool before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MiniMACS Separator</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CD4 Positive Isolation Kit</td>
			<td style="width:93px;">life technologies</td>
			<td>11331D</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and ex vivo culture of v&#948;1+cd4+&#947;&#948; t cells, an extrathymic &#945;&#946;t-cell progenitor

El timo, el órgano principal para la generación de células T delta gamma y columna vertebral del sistema inmune adaptativo en los vertebrados, ha sido considerado como la única fuente de células αβT. Sin embargo, la involución del timo comienza temprano en la vida que conduce a una reducción de la producción de manera drástica de las células αβT ingenuos en la periferia. Sin embargo, incluso los centenarios pueden crear inmunidad contra patógenos recién adquiridas. Investigaciones recientes sugieren el desarrollo de células αβT extrathymic, sin embargo, nuestra comprensión de las vías que pueden compensar la pérdida de la función del timo son todavía rudimentario. células γδ T son linfocitos innatas que constituyen la principal subconjunto de células T en los tejidos. Recientemente nos atribuimos una función destacada hasta ahora poco apreciado a un γδ T subconjunto de células al mostrar que la entidad escasa de células CD4 + Vδ1 + γδ células T pueden transdiferenciarse en células αβT en condiciones inflamatorias. En este sentido, provide el protocolo para el aislamiento de este progenitoras de la sangre periférica y su cultivo posterior. Vδ1 células se enriquecen positivamente a partir de PBMCs de donantes humanos sanos, utilizando perlas magnéticas, seguido de una segunda etapa en la que nos dirigimos a la escasa fracción de células CD4 + con una técnica de etiquetado magnético más. La fuerza magnética del segundo etiquetado excede el de la primera etiqueta magnética, y por lo tanto permite el aislamiento positivo eficiente, cuantitativa y específica de la población de interés. Se introduce entonces la condición técnica y la cultura requerida para la clonación y eficientemente la expansión de las células y para la identificación de los clones generados por análisis FACS. Por lo tanto, ofrecemos un protocolo detallado para la purificación, la cultura y la expansión ex vivo de delta gamma células T CD4 + Vδ1 +. Este conocimiento es requisito indispensable para los estudios que se relacionan con este αβT progenitor`s celulares biología y para aquellos que pretenden identify los desencadenantes moleculares que intervienen en su transdiferenciación.

La Figura 1 representa las diferentes etapas y el resultado del aislamiento de células T Vδ1 partir de sangre periférica. La Figura 1A muestra una distribución típica de las células Vδ1 + en los linfocitos CD3 +, así como la expresión co-receptor de la población Vδ1 +. En este donante, la frecuencia de Vδ1 + células (rojo) es 2,3% de total de linfocitos y la expresión de CD4 (verde) de Vδ1 + linfocitos es 2,6%. En tot...

Watch this Scientific Journal Video about Isolation and Ex Vivo Culture of VÎ´1+CD4+Î³Î´ T Cells, an Extrathymic Î±Î²T-cell Progenitor at JoVE.com

Isolation and Ex Vivo Culture of V&amp;#948;1+CD4+&amp;#947;&amp;#948; T Cells, an Extrathymic &amp;#945;&amp;#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 ...

isolation-ex-vivo-culture-v1-cd4-t-cells-an-extrathymic-t-cell

Research

JoVE Journal

Immunology and Infection

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

9.2K vistas.  Children's University Hospital Tübingen. Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral Vδ1+CD4+ T cells that is, as we showed recently, an extrathymic αβ T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

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

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Aislamiento de linfocitos T humanos, cultura y análisis de la migración In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Investigación

Inmunología e Infección

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Expansión ex vivo de células T tumor-reactiva por medio de la 1/Ionomycin briostatina y la formulación de la cadena gamma común de citoquinas

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Ex vivo de imágenes de células T en los ganglios linfáticos murino Rebanadas con campo amplio y microscopios confocal

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

Isolation of Double Negative &amp;#945;&amp;#946; T Cells from the Kidney

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

Aislamiento de doble negación células T αβ del Riñón

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

La evaluación de la innata de detección de VIH-1 CD4 infectados<sup&gt; +</sup&gt; Células T por las células dendríticas plasmocitoides Usando un<em&gt; Ex vivo</em&gt; Co-cultura del sistema.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Una<em&gt; Ex vivo</em&gt; Modelo de un ataque de células T Oligodendrocyte dirigida en Rebanadas cerebral agudo

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

El aislamiento de linfocitos de la piel humana para el Análisis y fenotípica<em&gt; Ex Vivo</em&gt; Cultivo Celular

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

los<em&gt; Ex Vivo</em&gt; Cultura y Reconocimiento de Patrones receptor de estimulación de ratón intestinal organoides

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

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

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

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

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

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

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Un modelo de cultivo primario de células Bursal de pollo Ex Vivo para estudiar la patogenesia de la enfermedad Bursal infecciosa Virus

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...