Este trabajo fue apoyado por la Asociación Americana de Gastroenterología (AGA) Investigación estudiosos Premio y la enfermedad de Crohn y Colitis Foundation of America (CCFA) Premio Desarrollo Profesional (a ENS), NIH NIDDK F30 DK089692 (ECS), y la Universidad de Carolina del Norte Centro de Biología Gastrointestinal y Grant Enfermedades P30 DK34987 (Histología Core). El Fondo para el flujo de Citometría de UNC es apoyado en parte por una subvención del NCI Core Support Center (P30CA016086) al Centro Integral del Cáncer de la UNC Lineberger. Damos las gracias a Lucas B. Borst de North Carolina State University College de Medicina Veterinaria por su ayuda con el análisis histopatológico e inmunohistoquímica.

NOTA: Asegúrese de que todos los animales protocolos son aprobados por y en cumplimiento con el empleo Comisión (IACUC) regulaciones Institucional Cuidado de Animales y el y el Manual del Consejo Nacional de Investigación para el Cuidado y Uso de Animales de Laboratorio. Los ratones donantes pueden ser ya sea hombre o mujer, pero los ratones receptores deben ser hombres. Si hembras receptoras se van a utilizar, los ratones donantes deben ser mujeres 5. Mantener colonias utilizando, ropa de cama no estéril y agua regulares no acidificado, porque éstos pueden afectar la microbiota intestinal y, por lo tanto, el fenotipo colitis de los ratones 5,6. 1. Preparación Experimental <ol><li> Utilice los medios y tampones heladas. Mantener células en hielo durante todo el experimento. </li><li> Lleve a cabo el experimento en campana de bioseguridad estéril utilizando una técnica estéril. </li></ol> 2. Aislamiento de células T esplénica <ol><li> La eutanasia de ratón donante / ratones en CO 2 cámara seguidos por dislocatio cervicaln. Pulverizar abdomen con etanol al 70%. </li><li> Haga una incisión horizontal en el abdomen y pelar la piel para exponer peritoneo. Mantenga peritoneo fuera de los órganos internos con los fórceps y hacer una incisión en el peritoneo abdominal izquierda para exponer y extirpar el bazo. </li><li> Coloque el bazo en 10 ml de medios completos en un plato de Petri. Retire y deseche el exceso de tejido de bazo. </li><li> Utilice 2 portaobjetos de vidrio esterilizados para aplastar y desmenuzar el bazo en la suspensión de una sola célula. Suspensión de células Filtrar a través de un colador de 70 micras en un tubo cónico de 50 ml y enjuague filtro con 5 ml de medios completos. Coloque un máximo de 5 bazos en un tubo cónico de 50 ml. </li><li> Centrifugar las células a 450 xg durante 7 minutos. Desechar el sobrenadante vertiendo o por succión al vacío en un contenedor de residuos. </li><li> Resuspender suavemente las células en 5 ml por bazo de tampón de lisis para lisar las células rojas de la sangre durante 10 min a temperatura ambiente. Añadir un volumen igual de completa Media (5 ml por bazo) en el tubo. </li><li&gt; centrifugar las células a 450 xg durante 7 minutos. Desechar el sobrenadante vertiendo o por succión al vacío en un contenedor de residuos. </li><li> Resuspender las células en 10 ml de Etiquetado Buffer. </li><li> Contar las células por exclusión de azul tripán. <ol><li> Retire 20 l de suspensión celular y añadir 180 l de azul de tripano a un tubo de microcentrífuga y mezclar bien. Después de 5 min, añadir 10 l de células hemocitómetro y contar las células no azules bajo el microscopio etiquetados. Determinar el número total de células viables. Deseche celular / azul tripán mezcla. </li></ol></li><li> Centrifugar las células en 10 ml de Etiquetado Buffer a 450 xg durante 7 minutos. Desechar el sobrenadante vertiendo o por succión al vacío en un contenedor de residuos. </li></ol> 3. Enriquecimiento de las células T CD4 + NOTA: Siga las instrucciones del fabricante para los productos específicos que se utilizan en esta sección. <ol><li> Resuspender suavemente las células a una suspensión de una sola célula de 20 x 10 6 células / ml en frío Labeling Buffer. </li><li> Añadir 5 l por 1 x 10 6 células T CD4 anticuerpos enriquecimiento celular con biotina. Se incuban las células en hielo durante 15 min. </li><li> Añadir 10x volumen de Etiquetado Buffer. Centrifugar las células a 450 xg durante 7 minutos. Aspirar con cuidado todo el sobrenadante mediante aspiración al vacío en un contenedor de residuos. </li><li> Completamente vórtice partículas magnéticas conjugadas con estreptavidina. Añadir 5 l de partículas por 1 x 10 6 células. </li><li> Mezclar bien pero con suavidad. Mantener la mezcla a 6-12 ° C durante 30 min. </li><li> Añadir Labeling Buffer a una concentración de 20-80 x 10 6 células / ml. Transferencia de hasta 1,0 ml células marcadas por tubo de ensayo de fondo redondo de 12 x 75 mm (en adelante, el &quot;tubo-fracción positiva&quot;). </li><li> Colocar cada tubo de fracción positiva en el imán durante 6-8 min. </li><li> Con los tubos de fracción positiva fijas en el imán, transferir cuidadosamente el sobrenadante de tubo de fracción positiva con pipeta Pasteur de vidrio a un nuevo tubo cónico de 50 ml estériles (referred como la &quot;fracción enriquecida&quot;). Esta fracción enriquecida contiene células T CD4 +. Tenga cuidado de no romper las células marcadas atraídos por el imán. </li><li> Resuspender las células dejan en los tubos de la fracción positiva en el mismo volumen de Etiquetado Buffer como en el paso 3.6 pipeta hacia arriba y hacia abajo con fuerza. Coloque el tubo-fracción positiva de vuelta en el imán de 6-8 min. </li><li> Con tubos de fracción positiva fijas en el imán, transferir cuidadosamente el sobrenadante (fracción enriquecida, CD4 +) de tubo de fracción positiva con vidrio pipeta Pasteur estéril de 50 ml tubo cónico de paso 3.8 sin afectar las células unidas al imán. </li><li> Repetir los pasos 3.9 a 3.10 para aumentar el rendimiento de las células T CD4 + obtenidos. Continuar utilizando protocolo fracción enriquecida (células CD4 +). </li></ol> 4. Etiquetado y ordenar celdas 7 <ol><li> Centrifugar las células enriquecido a 450 xg durante 7 min. Desechar el sobrenadante vertiendo o SUCT vacíoiones en un contenedor de residuos. </li><li> Resuspender las células en 1 ml de Etiquetado Buffer. Eliminar alícuota de células para contar y para evaluar la viabilidad celular por exclusión con azul de tripano como en el paso 2.9. </li><li> Añadir un volumen de Etiquetado Buffer de 10 x 10 6 células / ml; si las células ya están &lt;10 x 10 6, centrifugar a 450 xg durante 7 min, desechar el sobrenadante vertiendo o succión de vacío en el contenedor de residuos, y aumentar el volumen del etiquetado Buffer de 10 x 10 6 células / ml. <ol><li> Establecer alícuotas separadas de aproximadamente 5 a 10 × 10 5 células cada una de las células de control sin teñir, isotipo manchados, y de un solo manchado en tubos de microcentrífuga. </li></ol></li><li> Añadir 5 g / ml-CD4 FITC y 1 mg / ml CD45RB-PE a las células. Añadir manchas de control de isotipo y las manchas individuales a la misma concentración a partes alícuotas en tubos de microcentrífuga apropiarse. Mezclar bien pero suavemente y se incuba en hielo protegidas de la luz durante 30 min. </li><li> Añadir 10x volumen de Etiquetado Buffer a las células y el controls. Centrífuga 450 xg durante 7 minutos. Desechar el sobrenadante por succión al vacío en un contenedor de residuos. </li><li> Resuspender en Labeling Buffer de volumen en el paso 4.5. Centrífuga 450 xg durante 7 minutos. Desechar el sobrenadante por succión al vacío en un contenedor de residuos. </li><li> Resuspender en Labeling Buffer de 10 x 10 6 células / ml. Pase células a través de 70 micras filtro en el tubo de FACS. Mantener en hielo protegido de la luz hasta el momento de FACS. </li><li> Establecer y determinar la indemnización correspondiente en el clasificador de células con células sin teñir y controles individuales de colores. </li><li> Excluir células no viables con gating a plazo y de dispersión lateral (Figura 1). Configure gating CD4 + y células CD45RB + con controles de isotipo manchados. Células Gate sobre la población CD4 +. </li><li> Ordenar CD4 + CD45RB células en alto y CD45RB poblaciones de bajos utilizando un histograma simple para células PE-manchado en tubos con 2 ml de medios completos. El CD45RB alta </sup&gt; población representa el más alto 40% de CD4 + CD45RB + células (CD45RB alta), y la baja población CD45RB es el 20% más bajo de células CD4 + CD45RB + (CD45RB baja; Figura 1B). </li></ol><img alt="Figura 1" src="/files/ftp_upload/52533/52533fig1.jpg" /> Figura 1: Flujo de Representante citometría de parcelas de poblaciones de células CD4 + CD45RB T durante el análisis FACS (A - C) FITC y CD4 PE esplenocitos CD45RB manchada-de donantes C57BL / 6 ratones fueron ordenados por FACS en CD4 + CD45RB alta y CD4 +. CD45RB poblaciones de células T baja. Eventos (A) Doublet fueron excluidos del gráfico de dispersión hacia adelante. (B) Los linfocitos fueron cerrada en la dispersión frontal y lateral trama. (C) CD4 + </sup&gt; células T fueron cerrada, y las células T (D) CD4 + CD45RB + se representaron en un PE frente Eventos histograma. Bajo las células CD4 + CD45RB se consideraron el 20% más bajo de células CD45RB +. Pilas de gran CD4 + CD45RB se definieron como la más alta 40% de las células CD45RB +. <ol start="11"><li> Ejecutar una alícuota de cada población celular en la máquina de FACS para evaluar la pureza de las poblaciones. </li><li> Centrífuga ordenadas células a 450 xg durante 7 minutos. Resuspender en 1 ml de PBS. Eliminar alícuota de células para contar y para evaluar la viabilidad celular por exclusión con azul de tripano como en el paso 2.9. </li></ol> 5. La inyección de células en Destinatarios <ol><li> Resuspender las células ordenadas a 4 x 10 6 células / ml (CD45RB alta) o 2 x 10 6 células / ml (CD45RB baja) en PBS. </li><li> Transfiera 100 l de células de alta CD45RB por destinatario al nuevo tubo estéril. Añadir 100 l de PBS pordestinatario a este tubo. Por lo tanto, el volumen total de inyección por animal es de 200 l, y la cantidad total de células por beneficiario es de 4 altos células T ingenuas x 10 5 células CD4 + CD45RB. </li><li> Si se desea grupo experimental que recibe las células T reguladoras, transferir 100 l de células de alta CD45RB por destinatario al nuevo tubo estéril. Añadir 100 l de células de baja CD45RB por destinatario al mismo tubo. Volumen total de inyección por animal es de 200 l; relación de alta CD45RB: células de baja CD45RB es 2: 1. </li><li> Inyectar 100 l de CD45RB alta o CD45RB células de alta CD45RB bajos CD4 / + por vía intraperitoneal en el lado derecho e izquierdo del abdomen (un total de 200 l) de cada destinatario. </li></ol> 6. Seguimiento de la progresión de la enfermedad en los animales receptores <ol><li> Para evaluar el estado clínico de los animales receptores, asignar puntuaciones clínicas agregadas para los siguientes Parameters 8 en el día de la inyección, después semanalmente, y en el momento del sacrificio: <ol><li> Determinar perder midiendo la pérdida de peso: 0 - sin pérdida de peso; Pérdida de 0,1 a 10% del peso corporal inicial - 1; 2 - la pérdida de más de 10% del peso corporal inicial (Figura 2A). </li></ol></li></ol><img alt="Figura 2" src="/files/ftp_upload/52533/52533fig2highres.jpg" width="650" /> Figura 2: signos patológicos y clínicos graves de inflamación se producen después de la transferencia de tipo salvaje células de alta T CD4 + CD45RB en Rag1 - / - y ratones receptores NRDKO 11 (A) los receptores NRDKO perdieron en promedio el 10% de su peso corporal inicial en un 5. semanas después de la transferencia, mientras que Rag1- / - Receptores no mostraron signos clínicos de la enfermedad en este momento. Cada punto representa la media del porcentaje de peso corporal inicial para la cohorte ± SEM. **, P &lt;0,005. (B) algunas ratones desarrollaron inflamación intestinal grave, como se demuestra por la presencia de prolapso rectal. Esta es una imagen representativa de prolapso rectal en un ratón receptor NRDKO. (C) Grueso, dos puntos, tanto de Rag1 - / - y ratones receptores NRDKO se espesan y acortan en comparación con los dos puntos de Rag1 - / - y ratones NRDKO sin transferencia adoptiva de células T. Los dos puntos de los ratones receptores NRDKO muestran inflamación severa y aumento del peso de los dos puntos (datos no mostrados). <a href="https://www.jove.com/files/ftp_upload/52533/52533fig2highres.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a> <ol><li><ol start="2"><li> Determinar la calidad de las heces mediante la colocación de los animales en un recipiente limpio hasta que haya pasadotaburete: 0 - ninguno; 1 - heces blandas; 2 - heces acuosas y / o con sangre. </li><li> Determinar la salud general de los animales por la presencia de los siguientes signos de enfermedad: 0 - sin postura encorvada, erizado la piel, o la piel lesiones; 1 - cualquiera de los siguientes presente: encorvado postura, cerdas de piel o lesiones en la piel. </li><li> Determinar la presencia de prolapso rectal: 0 - ausente; 1 - presente (Figura 2B). </li></ol></li><li> Sacrificar animales por inhalación de CO 2 seguido por dislocación cervical cuando han perdido el 20% de su peso corporal inicial o en el punto de tiempo deseado, lo que ocurra primero. La enfermedad clínica suele ser evidente a partir de las 5 semanas después de la reposición. </li><li> Valorar la gravedad de la enfermedad a lo descrito previamente 5,7. <ol><li> Asignar puntuaciones clínicas como en el paso 6.1 8. </li><li> Medir la longitud del colon y el peso (Figura 2C). </li><li> Realizar el análisis histológico de la inflamación5. </li><li> Determinar la expresión de citoquinas espontánea en cultivos de explantes de tejido intestinal 9, nódulos linfáticos mesentéricos 10, y / o suero 11. <ol><li> En pocas palabras, para cultivos de explantes 9, quitar dos puntos después del sacrificio, abierta longitudinalmente, y limpia con PBS. Incubar dos puntos en un agitador orbital fijado en 250 rpm en medios completos de 30 min a temperatura ambiente. </li><li> Picar el tejido en trozos pequeños y se incubaron a 37 ° C en medios completos para 24 hr. Recoger el sobrenadante y usar para la cuantificación de citoquinas por 100 mg de tejido por ELISA. </li></ol></li><li> Realizar ex vivo caracterización de fenotipos de células T y / o la función 10. </li></ol></li></ol>

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J Immunol. 175 (8), 5412-5422 (2005).</li><li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN-gamma-induced+TNFR2+expression+is+required+for+TNF-dependent+intestinal+epithelial+barrier+dysfunction.">IFN-gamma-induced TNFR2 expression is required for TNF-dependent intestinal epithelial barrier dysfunction.</a> Gastroenterology. 131 (4), 1153-1163 (2006).</li></ol>

The authors declare that they have no competing financial interests.

Aquí se describe un protocolo paso a paso la inducción de la inflamación del colon en ratones mediante la transferencia adoptiva de células T CD4 + CD45RB + en ratones inmunodeficientes. Se utilizó bazo C57BL / 6 donantes y Rag1 singénico - / - ratones receptores, aunque otras cepas (por ejemplo, BALB / c, 129S6 / SvEv, diabético no obeso (NOD)) modelos y genéticos de la inmunodeficiencia (por ejemplo, SCID, Rag2 - / -) también se p...

The inflammatory bowel diseases (IBD) Crohn&#8217;s disease and ulcerative colitis result from an incompletely defined and complex interaction between host immune responses, genetic susceptibility, environmental factors, and the enteric luminal contents1. Recent genome-wide association studies report associations between immune cell regulatory genes and IBD susceptibility2,3. Both innate and adaptive immune cell intrinsic genes are represented in these studies, indicating a central role for these cell populations in IBD pathogenesis.
There currently exist more than 50 animal models of human IBD. While no one model perfectly phenocopies human IBD, many are useful for studying various aspects of human disease, including disease onset and progression and the wound-healing response. In the method described here, intestinal inflammation is initiated with syngeneic splenic CD4+CD45RBhigh T cell adoptive transfer into T and B cell deficient recipient mice4. The CD4+CD45RBhigh T cell population contains mainly na&#239;ve T cells primed for activation that are capable of inducing chronic small bowel and colonic inflammation. This method allows the researcher to modify key experimental variables, including both innate and adaptive immune cell populations, to answer biologically relevant questions relating to disease pathogenesis. Additionally, this method provides precise initiation of disease onset and a well-characterized experimental time course. This permits the kinetic study of clinical features of disease progression in mice. Intestinal inflammation induced by this method shares many features with human IBD, including chronic large and small bowel transmural inflammation, pathogenesis driven by cytokines such as TNF and IL-12, and systemic symptoms such as wasting5. Thus, it is an ideal model system for studying the pathogenesis of human IBD.
The method here describes in detail the protocol for inducing experimental colitis by adoptive transfer of CD4+CD45RBhigh T cells into Rag1-/- mice. We discuss key technical steps, expected results, optimization, and trouble-shooting. We address the required elements for the successful development of this murine model of intestinal inflammation for research purposes.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Name of Reagent/ Equipment</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments/Description</td>
		</tr>
		<tr >
			<td >10x PBS</td>
			<td >Gibco</td>
			<td >14200075</td>
			<td></td>
		</tr>
		<tr >
			<td >12x75mm round-bottom tube</td>
			<td >Falcon</td>
			<td >352052</td>
			<td></td>
		</tr>
		<tr >
			<td >15 ml conical</td>
			<td >Corning</td>
			<td >430790</td>
			<td></td>
		</tr>
		<tr >
			<td >26g x 3/8 Needle</td>
			<td >BD Biosciences</td>
			<td >305110</td>
			<td></td>
		</tr>
		<tr >
			<td >50 ml conical</td>
			<td >Corning</td>
			<td >430828</td>
			<td></td>
		</tr>
		<tr >
			<td >70 um Cell Strainer</td>
			<td >Fisherbrand</td>
			<td >22363548</td>
			<td></td>
		</tr>
		<tr >
			<td >BD IMagnet</td>
			<td >BD Biosciences</td>
			<td >552311</td>
			<td></td>
		</tr>
		<tr >
			<td >&#946;-mercaptoethanol</td>
			<td >Thermo Scientific</td>
			<td >35602</td>
			<td></td>
		</tr>
		<tr >
			<td >CD4-FITC IgG2b</td>
			<td >eBioscience</td>
			<td >11-0041</td>
			<td></td>
		</tr>
		<tr >
			<td >CD45RB-PE IgG2a</td>
			<td >BD Pharminogen</td>
			<td >553101</td>
			<td></td>
		</tr>
		<tr >
			<td >Complete Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 10% FBS, 0.0004% &#946;-ME</td>
		</tr>
		<tr >
			<td >FACS tube + strainer</td>
			<td >BD Falcon</td>
			<td >352235</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Microscope Slides</td>
			<td >Fisherbrand</td>
			<td >12550A3</td>
			<td></td>
		</tr>
		<tr >
			<td >Heat-inactivated FBS</td>
			<td >Gemini</td>
			<td >100-106</td>
			<td></td>
		</tr>
		<tr >
			<td >Labeling Buffer</td>
			<td ></td>
			<td ></td>
			<td >1x PBS, 0.5% BSA, 2 mM EDTA</td>
		</tr>
		<tr >
			<td >Lysis Buffer</td>
			<td ></td>
			<td ></td>
			<td >0.08% NH4Cl, 0.1% KHCO3, 1 mM EDTA</td>
		</tr>
		<tr >
			<td >MoFlo XDP</td>
			<td >Beckman Coulter</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse CD4 T lymphocyte Enrichment Set - DM</td>
			<td >BD Biosciences</td>
			<td >558131</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2a-PE</td>
			<td >BD Pharminogen</td>
			<td >553457</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2b-FITC</td>
			<td >eBioscience</td>
			<td >11-4732</td>
			<td></td>
		</tr>
		<tr >
			<td >Pasteur pipet</td>
			<td >Fisherbrand</td>
			<td >13-678-20D</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin Solution, 100X</td>
			<td >Corning Cellgro</td>
			<td >30-002-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Petri Dish</td>
			<td >Fisherbrand</td>
			<td >875713</td>
			<td></td>
		</tr>
		<tr >
			<td >Pure Ethanol 200 Proof</td>
			<td >Decon Labs</td>
			<td >2705-HC</td>
			<td></td>
		</tr>
		<tr >
			<td >RPMI-1640</td>
			<td >Gibco</td>
			<td >11-875-093</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringe</td>
			<td >BD Biosciences</td>
			<td >309597</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypan blue</td>
			<td >Corning Cellgro</td>
			<td >25-900-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Wash Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 0.0004% &#946;-ME</td>
		</tr>
	</tbody>
</table>

induction of murine intestinal inflammation by adoptive transfer of effector cd4+cd45rbhigh t cells into immunodeficient mice

Hay muchos modelos diferentes de animales disponibles para el estudio de la patogénesis de las enfermedades humanas inflamatorias del intestino (IBD), cada uno con sus propias ventajas y desventajas. Se describe aquí un modelo de colitis experimental que se inicia por la transferencia adoptiva de células de alta T CD4 + CD45RB singeneicos bazo en ratones receptores deficientes de células T y B. La población de células de alta T CD4 + CD45RB que consiste en gran parte de las células efectoras no tratados previamente es capaz de inducir la inflamación intestinal crónica, muy parecidas a aspectos clave de la EII humana. Este método puede ser manipulado para estudiar aspectos de la aparición de la enfermedad y la progresión. Además, puede ser utilizado para estudiar la función de innato y adaptativo, y las poblaciones de células inmunes reguladoras, y el papel de la exposición ambiental, es decir, la microbiota, en la inflamación intestinal. En este artículo se expone la metodología para la inducción de la colitis con un protocolo paso a paso. Esta incLudes un video de demostración de los aspectos técnicos clave necesarios para desarrollar con éxito este modelo murino de colitis experimental con fines de investigación.

Aproximadamente 10 x 10 6 células de alta T CD4 + CD45RB de 10 bazos de adultos C57BL / 6 ratones donantes son confiablemente aislado. Este número variará dependiendo de la edad y la cepa del ratón donante y el dominio del investigador. Cuando 4 x 10 5 C57BL / 6 celdas de alta T CD4 + CD45RB se transfieren a C57BL / 6 Rag1 - / - ratones receptores, los signos clínicos de la enfermedad surgen alrededor de la semana 5 después de la...

Watch this Scientific Journal Video about Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice at JoVE.com

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

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

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

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

induction-murine-intestinal-inflammation-adoptive-transfer-effector

Research

JoVE Journal

Immunology and Infection

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

16.2K vistas.  University of North Carolina at Chapel Hill. Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

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

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

La metástasis experimentales y CTL Modelo inmunoterapia adoptiva de transferencia de ratón

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Investigación

Inmunología e Infección

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Análisis por citometría de flujo de células inmunes dentro de aortas murino

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

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

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

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

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

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

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

Evaluar el desarrollo de las células murino plasmocitoide dendríticas en las placas de Peyer Usando la transferencia adoptiva de Progenitores Hematopoyéticas

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

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

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

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

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

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

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Análisis de<em&gt; Yersinia enterocolitica</em&gt; Efector translocación en las células huésped Usando beta-lactamasa Efector fusiones

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

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

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

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

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

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

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Un método no invasivo y técnicamente no intensivos para la inducción y Fenotipificación Experimental de neumonía bacteriana en ratones

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

Inducción de parálisis y lesión del sistema Visual en ratones por T las células específicas para el Optica del Neuromyelitis Autoantigen Acuaporina-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

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

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

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

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

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