Este trabajo fue apoyado por becas de los Institutos Nacionales de Salud y DK074656 AI56374 (CEM), así como la Fundación de Investigación de Diabetes Juvenil.

1. Preparación de las células <ol><li> Objetivo el cultivo de células: la cultura de las líneas celulares de ratón beta, NIT-1 y NIT-4, fue de 1,2 se ha descrito anteriormente en DMEM suplementado con FBS al 10%, 0,02% de BSA, no de aminoácidos esenciales, 15 mM HEPES, 16,5 mm Glocose y penicilina / estreptomicina. El 51 Cr etiquetado, las células de las semillas en un fondo plano de 96 pocillos placa de cultivo de 5x10 4 células / pocillo en 200 l medio de cultivo. Para los experimentos de microscopía, las células de las semillas en un 8 y Lab-Tak cubreobjetos II de cámara en cámara de 5x10 4 por 500 en el medio de cultivo l, y dejar crecer durante 48 horas antes de usar en los experimentos de citotoxicidad. </li><li> El control de las células T cultura línea: Utilice la línea celular Jurkat (ATCC, CD3 + línea celular humana de linfocitos) como control negativo. Cultivo de células Jurkat en RPMI1640 complementado con 10% de SFB, 2 mM L-glutamina, 1,5 g / L de bicarbonato de sodio, 10 mM HEPES y 1,0 mM piruvato de sodio. </li></ol> 2. Recolección y la activación de efectores autorreactivos células T CD8 + NOD.Cg-Rag1 tm1Mom Tg (TcraAI4) 1Dvs/DvsJ [NOD-AI4a] y NOD.Cg-Rag1 tm1Mom Tg (TcrbAI4) 1Dvs/DvsJ [NOD-AI4b] fueron adquiridos de El Laboratorio Jackson (Bar Harbor, ME) y criados en nuestras instalaciones de ratón. Progenie híbrida F1 de cruces de NOD-AI4a con NOD-AI4b [NOD-AI4a / b] desarrollar diabetes en 3-5 semanas de edad. Todos los ratones fueron alojados en libres de patógenos específicos y aprobados por las instalaciones de Cuidado de Animales de la institución en cuestión y el empleo. <ol><li> 3-4 semanas de edad, la eutanasia AI4a NOD-/ b ratones utilizando CO 2. </li><li> Recoger el bazo de los ratones. Los ratones sacrificados se preparan con spray de etanol y se abrió bajo el capó quirúrgica. Bazos se quitan y se ponen en helado buffer HBSS inmediatamente. </li><li> Recoger las células del bazo con un homogeneizador de vidrio de 7 ml. De dos a tres bazos cada uno se puso en 5 ml de HBSS frío y homogeneizados con un homogeneizador de 7 ml de vidrio. Homogeneizado se recoge y se centrifuga a 1200rpm durante 5 minutos a 4 ° C utilizando una centrifugadora Sorvall RT7 +. Gránulos de las células se recogen. </li><li> Eliminar las células rojas de la sangre mediante un tratamiento con una solución hipotónica. Los pellets son tratados con 5 ml de solución hipotónica / bazo en hielo durante 5 minutos, luego se neutraliza mediante la adición de un volumen igual de HBSS. Recoger las células por centrifugación, como el anterior, y resuspender en 50 ml de HBSS. Pase la suspensión de células a través de células colador y contar con un hemocitómetro con tinción TrypanBlue. </li><li> Activar las células. Resuspender las células en 5x10 6 / ml y activar mediante un cultivo de 3 días con 0,1 M AI4 mimotopo (YFIENYLEL secuencia de aminoácidos) y 25 U / mL de IL-2 en RPMI 1640 suplementado con 10% de SFB, 2 mM L-glutamina, 1,5 g / L de bicarbonato de sodio, 10 mM HEPES y 1,0 mM piruvato de sodio. </li></ol> 3. Ensayo de liberación de 51 Cr para examinar CML <ol><li> Células etiqueta de destino: Agregar 51 Cr a las células diana en un Ci / pocillo y la cultura durante 3 horas, y lavar 3 veces con medio de cultivo. </li><li> Suspender las células efectoras en medio RPMI 1640 como se mencionó en el paso 2.5 para que el volumen final a cada pocillo es de 200 l. </li><li> Después del último lavado de las células diana marcada, añada activar las células efectoras de las culturas de las células diana a efector deseado al objetivo (E: T) ratios. Por lo general, el uso E: T ratios de 0.4:1, 1:1, 2:1, 5:1, 10:1, 20:1 y 50:1. </li><li> Añadir nuevo modificado RPMI 1640 (véase el paso 2.5) a 6 pozos de las células diana para actuar como un control de lisis espontánea. </li><li> Co-cultivo las células efectoras y diana durante 16 horas. </li><li> Recoger el líquido sobrenadante en tubos de 6x50 mm de vidrio de cal </li><li> Lisis de las células mediante la adición de 100μl 2% de SDS y pipetear varias veces con suavidad. </li><li> Recoger lisado celular en otro juego de tubos de vidrio 6x50 mm de cal. </li><li> Lavar una vez con la limpieza de pozos H 2 O y añadir el lavado de las trompas de lisado celular. </li><li> Contar con los sobrenadantes y lisados ​​de células utilizando un contador gamma. </li><li> Calcular lisis específica de la siguiente manera: <img src="files/ftp_upload/2724/2724eq1.jpg" alt="La ecuación 1" /></li></ol> 4. Tinción de las células de imágenes de células vivas Entre los más utilizados potencial de membrana mitocondrial colorantes, TMRM exhibe la menor toxicidad mitocondrial. 3 Por lo tanto, elegimos TMRM para estos estudios de imágenes de células vivas. <ol><li> Prepare una solución de 40 mM de TMRM en DMSO y almacenar a -20 ° C. Haga un [25 nM] solución de trabajo fresca en rojo fenol libre de DMEM con todos los suplementos de NIT-1 cultivo celular 1. </li><li> Mancha objetivo NIT-1 con células TMRM 25 nM durante 30 minutos a 37 ° C. </li><li> Vuelva a colocar los medios de comunicación con los medios de comunicación fresco que contiene 5 nM TMRM para mantener el equilibrio de distribución del colorante 4. </li><li> Conde activa las células T efectoras AI4 utilizando un hematocitómetro. </li><li> Mancha activa las células T efectoras AI4 con 1 l / mL para Picogreen5 minutos a temperatura ambiente y lavar con rojo fenol libre de medio de cultivo 3 veces. </li></ol> 5. La evaluación de los linfocitos T mediada por la muerte de las células beta usando imágenes de células vivas <ol><li> Monte la cámara cubre objetos con células NIT manchado en el escenario de un Zeiss LSM 510 microscopio confocal. Retire la lengüeta con una dremmel y gas esterilizar el vidrio con óxido de cámaras Etheline. El escenario está equipado con una cámara de imágenes de células vivas, que tiene la temperatura controlada a 37 ° C y 5% de CO 2 con la humedad. </li><li> Añadir activa y se tiñeron las células T a las cámaras designadas en diferentes E: T ratios. </li><li> Añadido el mismo número de células Jurkat manchado a las cámaras de control. </li></ol> 6. La obtención de imágenes de células vivas El microscopio está equipado con una platina motorizada que nos permite adquirir imágenes en repetidas ocasiones en diferentes lugares de las cámaras de iguales o diferentes de forma automática en el transcurso del experimento. <ol><li> El uso de un lente de 63x de aceite objetivo, capturar imágenes a intervalos de cada 3 minutos para un total de 300 cuadros por ubicación. </li><li> Detectar manchas TMRM con una longitud de onda de excitación de 543 nm y el conjunto de filtros para la emisión de 565-619 nm </li><li> Detectar manchas Picogreen con una excitación de 488 nm y un conjunto de filtros para la emisión de 500-630 nm. El video fue creado usando el software Zeiss LSM. </li></ol> 7. De conversión de vídeo para su publicación <ol><li> Usando el software LSM Zeiss, crear un archivo de película sin comprimir en formato AVI. </li><li> Para convertir el vídeo en el formato de publicación, importar el vídeo en el paquete de software FIJI ( <a href="http://pacific.mpi-cbg.de/wiki/index.php/Fiji">http://pacific.mpi-cbg.de/wiki/index.php/Fiji</a> ), un ImageJ ( <a href="http://rsb.info.nih.gov/ij/">http://rsb.info. nih.gov / ij /</a> ) la distribución, usando el plugin Bioformats de loci ( <a href="http://www.loci.wisc.edu/software/bio-formats">http://www.loci.wisc.edu/software/bio-formats</a> ). </li><li> Ajustar la configuración del brillo y el contraste para equilibrar las señales de color verde y rojo y la velocidad de fotogramas se establece en 2 fotogramas / segundo utilizando el menú de opciones de animación. </li><li> Recortar el vídeo a 512 x 512 píxeles y la exportación como un archivo AVI sin comprimir. </li><li> Abrir el archivo AVI en el software Quicktime 7 (ordenador de Apple, Cupertino, CA) y comprimir y exportar en el formato de archivo de vídeo abierto H264/MPEG-4 a 1200 kbs / segundo. </li></ol> 8. Los resultados representativos: <ol><li> Un resultado representativo de 51 Cr ensayo de LMC. La figura 1 muestra un resultado representativo de un ensayo de LMC en la lisis por ciento específica (eje Y) aumenta a medida que el efector: relación de destino (eje X) se incrementa. Las células diana son islotes aislados de primaria inmunodeficientes NOD Rag1-/ -. Ratones y etiquetados con 51 Cr. Esplenocitos de ratones transgénicos NOD.AI4α / β son aislados y se activa como se describe en el paso 2. Las metas y los efectores fueron co-cultivadas durante 16 horas. Lisis por ciento determinado se calcula como se describe en el paso por encima de 3,11. </li><li> Dos representantes (positivo y negativo) los resultados de una mediada por células T disipación de las células beta del potencial de membrana mitocondrial se presentan. Las células se tiñen y las reacciones se registran como se describe en los pasos 4, 5 y 6, los archivos de vídeo se crean como en el paso 7. Video 1: La versión beta del ratón línea celular NIT-1 se tiñó el potencial de membrana mitocondrial tinte TMRM (rojo). Activado, autorreactivos células T CD8 + AI4 (teñido de verde con Picogreen) fueron co-cultivadas con estas NIT-1 en las células en un E: T relación de 50:1. NIT-1 potencial de membrana mitocondrial se disipa poco a poco durante una duración de 400 minutos, pero sólo en los grupos que se interactúa con las células T AI4. Video 2: Como un experimento de control, NIT-1 células se tiñeron con TMRM y co-cultivadas con células Jurkat Picogreen manchada en un E: T relación de 50:1. Células Jurkat es un ser humano línea de células T que es MHC no coincidentes. NIT-1 células mantiene el potencial de membrana mitocondrial durante toda la duración de los 400 minutos de co-período de incubación. Células Jurkat no interactúan con NIT-1 clusters y por lo tanto, no provocar el asesinato. </li></ol><img src="files/ftp_upload/2724/2724fig1.jpg" alt="Figura 1" /> Figura 1 resultado Representante de la LMC utilizando como células isletas objetivo principal aislado de inmunodeficiencia NOD.Rag1-/ -. Ratones y esplenocitos de ratones transgénicos NOD.AI4α / β. Las metas y los efectores fueron co-cultivadas durante 16 horas. Lisis porcentaje específico aumenta a medida que la unidad de efectos: aumenta la proporción de destino. Video 1. Beta ratón línea celular NIT-1 se tiñó el potencial de membrana mitocondrial TMRM colorante (rojo). Activado autorreactivos células T CD8 + AI4 (teñido de verde con Picogreen) fueron co-cultivadas con estas NIT-1 células en E: T relación de 50:1. NIT-1 membrana mitocondrial potencial de disipaciónTed gradualmente durante un período de 400 minutos, pero sólo en los grupos que se interactúa con las células T AI4 verde. <a href="http://www.jove.com/files/ftp_upload/2724/video.mov">Haga clic aquí para ver el video</a> . Video 2. NIT-1 las células se tiñeron mismo que se describe en el video 1 y co-cultivadas con Picogreen manchada Jurkat línea de células T humanas. NIT-1 células fueron capaces de mantener el potencial de membrana mitocondrial pensado la duración de 400 minutos. Células Jurkat no interactúan con NIT-1 clusters y por lo tanto no inducen muerte. <a href="http://www.jove.com/files/ftp_upload/2724/Video2.mp4">Haga clic aquí para ver el video</a> .

<ol>
	<li>Hamaguchi, K., Gaskins, H. R., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIT-1,+a+pancreatic+beta-cell+line+established+from+a+transgenic+NOD/Lt+mouse.">NIT-1, a pancreatic beta-cell line established from a transgenic NOD/Lt mouse.</a> Diabetes. 40, 842-849 (1991).</li><li>Chen, J., Gusdon, A. M., Piganelli, J., Leiter, E. H., Mathews, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mt-Nd2a+modifies+resistance+against+autoimmune+Type+1+diabetes+in+NOD+mice+at+the+level+of+the+pancreatic+beta+cell.">mt-Nd2a modifies resistance against autoimmune Type 1 diabetes in NOD mice at the level of the pancreatic beta cell.</a> Diabetes. , (2010).</li><li>Scaduto, R. C., Grotyohann, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mitochondrial+membrane+potential+using+fluorescent+rhodamine+derivatives.">Measurement of mitochondrial membrane potential using fluorescent rhodamine derivatives.</a> Biophys J. 76, 469-477 (1999).</li><li>Lemasters, J. J., Ramshesh, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+mitochondrial+polarization+and+depolarization+with+cationic+fluorophores.">Imaging of mitochondrial polarization and depolarization with cationic fluorophores.</a> Methods Cell Biol. 80, 283-295 (2007).</li><li>Abel, M., Krokowski, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+immune-mediated+(type+1)+diabetes+mellitus:+potential+for+immunotherapy.">Pathophysiology of immune-mediated (type 1) diabetes mellitus: potential for immunotherapy.</a> BioDrugs. 15, 291-301 (2001).</li><li>Mathews, C. E., Graser, R. T., Savinov, A., Serreze, D. V., Leiter, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unusual+resistance+of+ALR/Lt+mouse+beta+cells+to+autoimmune+destruction:+role+for+beta+cell-expressed+resistance+determinants.">Unusual resistance of ALR/Lt mouse beta cells to autoimmune destruction: role for beta cell-expressed resistance determinants.</a> Proc Natl Acad Sci U S A. , 235-240 (2001).</li><li>Szabadkai, G., Duchen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+mediated+cell+death+in+diabetes.">Mitochondria mediated cell death in diabetes.</a> Apoptosis. 14, 1405-1423 (2009).</li><li>Ly, J. D., Grubb, D. R., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mitochondrial+membrane+potential+(deltapsi(m))+in+apoptosis;+an+update.">The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update.</a> Apoptosis. 8, 115-128 (2003).</li><li>Dudek, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+T-cells+from+T-cell+receptor+transgenic+NOD8.3+mice+destroy+beta-cells+via+the+perforin+and+Fas+pathways.">Cytotoxic T-cells from T-cell receptor transgenic NOD8.3 mice destroy beta-cells via the perforin and Fas pathways.</a> Diabetes. 55, 2412-2418 (2006).</li><li>Pakala, S. V., Chivetta, M., Kelly, C. B., Katz, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+autoimmune+diabetes+the+transition+from+benign+to+pernicious+insulitis+requires+an+islet+cell+response+to+tumor+necrosis+factor+alpha.">In autoimmune diabetes the transition from benign to pernicious insulitis requires an islet cell response to tumor necrosis factor alpha.</a> J Exp Med. 189, 1053-1062 (1999).</li></ol>

Los experimentos con animales se realizaron de acuerdo con las directrices y normas establecidas por la Universidad de Florida Cuidado de Animales institucional y el empleo.

Células T citotóxicas mediadas por muerte de las células beta es la fisiopatología principal de DT1 5. Ensayos de CML con 51 Cr liberación nos permite estudiar el grado de respuesta del efector objetivo 6. Sin embargo, el proceso detallado y las vías de las células T mediada por la muerte de las células beta aún no está totalmente entendido. Dado que las mitocondrias son esenciales para la función celular beta y la muerte de siete, nos hemos centrado en cambios en l...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> CR-51 </td><td> Perkin Elmer </td><td> NEZ030500IMC </td><td></td></tr><tr><td> PBS </td><td> Cellgro </td><td> 21-040-60 </td><td></td></tr><tr><td> Tetrametil rodamina éster metílico (TMRM) </td><td> Invitrogen </td><td> T668 </td><td></td></tr><tr><td> Picogreen </td><td> Invitrogen </td><td> P7581 </td><td></td></tr><tr><td> AI4 mimotopo </td><td> mimotopes.com </td><td> secuencia de aminoácidos: YFIENYLEL </td><td></td></tr><tr><td> IL-2 </td><td> I + D </td><td> 485-MI </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 11885 </td><td></td></tr><tr><td> FBS </td><td> HyClone </td><td> SH30071.03 </td><td></td></tr><tr><td> Albúmina de suero bovino </td><td> Sigma </td><td> A8412 </td><td></td></tr><tr><td> HEPES </td><td> Cellgro </td><td> 25-060-Cl </td><td></td></tr><tr><td> MEM no aminoácidos esenciales </td><td> GIBCO </td><td> 11140 </td><td></td></tr><tr><td> Penicilina / estreptomicina </td><td> Géminis </td><td> 400-109 </td><td></td></tr><tr><td> Rojo fenol libre de DMEM </td><td> Sigma </td><td> D5303 </td><td></td></tr><tr><td> Incubadora de CO2 </td><td> Thermo Fisher </td><td> HeraCell 150i </td><td> Mantenerse estéril para el cultivo celular </td></tr><tr><td> Cabina de seguridad biológica </td><td> Thermo Fisher </td><td> Forma 1440 </td><td></td></tr><tr><td> Tubos desechables cultura, 6x50 mm de vidrio de cal </td><td> Pescador </td><td> 14 a 958-A </td><td></td></tr><tr><td> Gamma Contador </td><td> Perkin Elmer </td><td> Asistente de contador automático de Gamma 1470 </td><td></td></tr><tr><td> Microscopio confocal </td><td> Zeiss </td><td> LSM 510 Meta Confocal </td><td> Con platina motorizada y una cámara de células vivas </td></tr><tr><td> Pipeta (1 ml, 200μl, 20μl, 10μl, 2μl) </td><td> Eppendorf </td><td></td><td></td></tr><tr><td> Tubos (ámbar) </td><td> Pescador </td><td> 50819772 </td><td></td></tr><tr><td> Centrífugo </td><td> Sorvall Legend RT + </td><td> 75004377 </td><td></td></tr><tr><td> Hemocitómetro </td><td> Fisher Scientific </td><td> 267110 </td><td></td></tr><tr><td> Microscopio vertical </td><td> Zeiss </td><td> Axioskop40 </td><td></td></tr><tr><td> NOD.Cg-Rag1 tm1Mom Tg (TcraAI4) 1Dvs/DvsJ ratones </td><td> El Laboratorio Jackson </td><td> 004347 </td><td></td></tr><tr><td> NOD.Cg-Rag1 tm1Mom Tg (TcrbAI4) 1Dvs/DvsJ ratones </td><td> El Laboratorio Jackson </td><td> 004348 </td><td></td></tr><tr><td> NOD-AI4α / ratones F1 β </td><td> N / A </td><td> N / A </td><td> Criados en instalaciones para animales UF </td></tr></tbody></table>

methods to assess beta cell death mediated by cytotoxic t lymphocytes

La diabetes tipo 1 (DT1) es una enfermedad mediada por células T autoinmunes. Durante la patogénesis, los pacientes se vuelven cada vez más insulinopenia como la producción de insulina se pierde, probablemente esto se debe a la destrucción de las células beta del páncreas por las células T. Comprensión de los mecanismos de la muerte de las células beta en el desarrollo de DT1 proporcionará información para generar una cura efectiva para esta enfermedad. Mediada por células linfocitotoxicidad (LMC) los ensayos se han utilizado históricamente el radionúclido cromo 51 (51 Cr) para etiquetar las células diana. Estos objetivos se exponen a las células efectoras y la liberación de 51 Cr de las células diana se lee como una indicación de la muerte celular mediada por linfocitos. Los inhibidores de la consecuencia de muerte celular en menor liberación de 51 Cr. Como células efectoras, se utilizó una población activa autorreactivos clonal de las células CD8 + linfocitos T citotóxicos (CTL) aislado de un ratón transgénico de valores, tanto para las cadenas alfa y beta del receptor de la célula AI4 T (TCR). Activa las células T AI4 fueron co-cultivadas con 51 Cr células diana marcadas NIT durante 16 horas, la liberación de 51 Cr se registró para el cálculo de lisis específica Las mitocondrias participar en importantes eventos fisiológicos, tales como la producción de energía, la regulación de la transducción de señales, y la apoptosis. El estudio de las células beta mitocondrial cambios funcionales durante el desarrollo de DT1 es una nueva área de investigación. Utilizando el potencial de membrana mitocondrial colorante tetrametil rodamina éster metílico (TMRM) y confocal de imágenes microscópicas de células vivas, que supervisó el potencial de membrana mitocondrial en el tiempo en la línea de células beta NIT-1. Para los estudios de imagen, las células T efectoras AI4 fueron marcadas con el colorante fluorescente tinción nuclear Picogreen. NIT-1 en las células y las células T fueron co-cultivadas en cubreobjetos y cámaras montadas en la platina del microscopio equipado con una cámara de células vivas, controlada a 37 ° C, con 5% de CO 2, y húmedo. Durante estos experimentos se tomaron imágenes de cada grupo cada 3 minutos durante 400 minutos. Más de un curso de 400 minutos, se observó la disipación del potencial de membrana mitocondrial en NIT-1, donde grupos de células AI4 las células T se adjunta. En el experimento de control simultáneo donde NIT-1 células fueron co-cultivadas con MHC mal emparejado las células Jurkat de linfocitos, el potencial de membrana mitocondrial se mantuvo intacta. Esta técnica se puede utilizar para observar en tiempo real los cambios en el potencial de membrana mitocondrial en las células bajo el ataque de los linfocitos citotóxicos, citocinas u otros reactivos citotóxicos.

Watch this Scientific Journal Video about Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes at JoVE.com

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Mediadas por células linfocitotoxicidad (LMC) los ensayos se pueden utilizar para evaluar las respuestas autorreactivas y los mecanismos de estudio de la muerte celular in vitro. Sin embargo, utilizando células vivas, las técnicas de microscopía confocal microscópicos con tintes fluorescentes, el tipo y la cinética de muerte celular, así como las rutas utilizadas se pueden estudiar con mayor detalle.

Métodos para evaluar la Beta mediada por la muerte celular por linfocitos T citotóxicos

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

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Immunology and Infection

13.3K vistas.  University of Florida. Mediadas por células linfocitotoxicidad (LMC) los ensayos se pueden utilizar para evaluar las respuestas autorreactivas y los mecanismos de estudio de la muerte celular in vitro. Sin embargo, utilizando células vivas, las técnicas de microscopía confocal microscópicos con tintes fluorescentes, el tipo y la cinética de muerte celular, así como las rutas utilizadas se pueden estudiar con mayor detalle.

Video: Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

La expansión de los linfocitos T citotóxicos sangre del cordón umbilical que Citomegalovirus Target, Virus de Epstein-Barr, adenovirus y

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

Investigación

Inmunología e Infección

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

La determinación óptima de la actividad citotóxica de las células T específicas Her2Neu CD8 Humanos comparando el Ensayo de liberación de Cr51 al Sistema xCELLigence

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Diferenciación dirigida de células madre pluripotentes inducidas hacia los linfocitos T

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Aislamiento y diferenciación de Th17 Naïve linfocitos T CD4

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Radial Mobility and Cytotoxic Function of Retroviral Replicating Vector Transduced, Non-adherent Alloresponsive T Lymphocytes

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on extracellular matrix proteins, for measuring the motility of fluorescently-labeled, non-adherent human or murine effector immune cells. This technique employs a stainless steel manifold and 10-well Teflon slide to focally deposit non-adherent T cells into wells prepared with either confluent tumor cell monolayers or extracellular matrix proteins. Light and/or multi-channel fluorescence microscopy is used to track the movement and behavior of the effector cells over time. Fluorescent dyes and/or viral vectors that code for fluorescent transgenes are used to differentially label the cell types for imaging. This method is distinct from similar-type in vitro assays that track horizontal or vertical migration/invasion utilizing slide chambers, agar or transwell plates. The assay allows detailed imaging data to be collected with different cell types distinguished by specific fluorescent markers; even specific subpopulations of cells (i.e., transduced/nontransduced) can be monitored. Surface intensity fluorescence plots are generated using specific fluorescence channels that correspond to the migrating cell type. This allows for better visualization of the non-adherent immune cell mobility at specific times. It is possible to gather evidence of other effector cell functions, such as cytotoxicity or transfer of viral vectors from effector to target cells, as well. Thus, the method allows researchers to microscopically document cell-to-cell interactions of differentially-labeled, non-adherent with adherent cells of various types. Such information may be especially relevant in the assessment of biologically-manipulated or activated immune cell types, where visual proof of functionality is desired with tumor target cells before their use for cancer therapy.

We describe a protocol to monitor radial mobility of non-adherent immune cells in vitro using a cell sedimentation manifold/slide apparatus. Cell migration is tracked on monolayers of tumor cells or on extracellular matrix proteins. Examination by light and fluorescence microscopy allows for observation of cell mobility and cytotoxic functionality.

Radial Movilidad y citotóxicos Función de Retroviral Replicar Vector transducidas, no adherente Alloresponsive linfocitos T

We report a novel adaptation of the Radial Monolayer Cell Migration assay, first reported to measure the radial migration of adherent tumor cells on ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

Difusión molecular en el plasma membranas de linfocitos primarios medidos por espectroscopia de correlación de fluorescencia

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

La cuantificación rápida de Blastogénesis inducida por mitógeno de linfocitos T para identificar fármacos inmunomoduladores

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

Un método para evaluar las funciones efectoras mediadas por Fc inducidas por los anticuerpos específicos de la hemaglutinina de la gripe

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Using Terminal Transferase-mediated dUTP Nick End-labelling (TUNEL) and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which infects and disrupts frog epidermis; however, pathological changes have not been explicitly characterized. Apoptosis (programmed cell death) can be used by pathogens to damage host tissue, but can also be a host mechanism of disease resistance for pathogen removal. In this study, we quantify epidermal cell death of infected and uninfected animals using two different assays: terminal transferase-mediated dUTP nick end-labelling (TUNEL), and caspase 3/7. Using ventral, dorsal, and thigh skin tissue in the TUNEL assay, we observe cell death in the epidermal cells in situ of clinically infected animals and compare cell death with uninfected animals using fluorescent microscopy. In order to determine how apoptosis levels in the epidermis change over the course of infection we remove toe-tip samples fortnightly over an 8-week period, and use a caspase 3/7 assay with extracted proteins to quantify activity within the samples. We then correlate caspase 3/7 activity with infection load. The TUNEL assay is useful for localization of cell death in situ, but is expensive and time intensive per sample. The caspase 3/7 assay is efficient for large sample sizes and time course experiments. However, because frog toe tip biopsies are small there is limited extract available for sample standardization via protein quantification methods, such as the Bradford assay. Therefore, we suggest estimating skin surface area through photographic analysis of toe biopsies to avoid consuming extracts during sample standardization.

Using Terminal Transferase-mediated dUTP Nick End-labelling TUNEL and Caspase 3/7 Assays to Measure Epidermal Cell Death in Frogs with Chytridiomycosis

We quantify epidermal cell death in frogs with chytridiomycosis using two methods. First, we use terminal transferase-mediated dUTP nick end-labelling (TUNEL) in situ histology to determine differences between clinically infected and uninfected animals. Second, we conduct a time series analysis of apoptosis over infection using a caspase 3/7 protein analysis.

Utilizando mediada por la transferasa Terminal dUTP Nick End-etiquetado (TUNEL) y caspasas 3/7 ensayos a muerte celular epidérmica, medida en ranas con la quitridiomicosis

Amphibians are experiencing a great loss in biodiversity globally and one of the major causes is the infectious disease chytridiomycosis. This disease is ...