This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1A6A3A01020610 to HJP) and a grant from the Korean Health Technology R&amp;D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea (HI15C0493 to SJH).

En este estudio, los ratones se mantuvieron en una instalación específica libre de patógenos del Centro de Investigación de Animales de Laboratorio de la Universidad de Yonsei de Yonsei. Todos los experimentos con animales se realizaron de acuerdo con las directrices de Corea Administración de Alimentos y Drogas utilizando los protocolos aprobados por el Comité Internacional de Cuidado y Uso de Animales del Centro de Investigación de Animales de Laboratorio de Yonsei en la Universidad de Yonsei. 1. Preparación de soluciones <ol><li> Preparar 2% de medio RPMI diluyendo suero bovino fetal (FBS) al 2% y penicilina-estreptomicina al 1% en RPMI </li><li> Preparar medio RPMI completo. Para RPMI noticias agregar 10% de FBS, 1% de penicilina-estreptomicina, 1% de L-glutamina, y 50 mM 2-mercaptoetanol. </li><li> Prepare el buffer de fluorescencia de clasificación de células activadas (FACS). Para ello la solución salina es así, buffer fosfato suplemento (PBS) con 2% de FBS. </li><li> Preparar tampón de aislamiento de células T. Para ello, complementar PBS con 2% de SFB y 2 mM ethylenediaminetetraaácido ascético. </li></ol> 2. Aislamiento de linfocitos del bazo <ol><li> Retire los bazos de los ratones no tratados previamente o infectados por el CL13 LCMV como se ha descrito previamente 19 y colocarlos en 60 mm x 15 mm placas de Petri que contenían 10 ml de 2% medio RPMI. NOTA: En estos experimentos de estudio, de 5 a 6 semanas de edad C57B1L / 6J hembra recibió 2 x 10 6 unidades formadoras de placa (pfu) de LCMV CL13 a través de una inyección intravenosa a través de la vena de la cola. Sacrificar los ratones en el día 16 después de la infección (16 d pi) 25. ratones ingenuos analizar emparejados-ge estaban en el mismo día. </li><li> Colocar un filtro de células en un tubo de 50 ml y enjuague con 2 ml de 2% de medio RPMI. Coloque el bazo en el filtro de células 70 micras y se muelen usando el émbolo de una jeringa. Enjuague el filtro de células con 2 ml de 2% medio RPMI y sacarlo de tubo de 50 ml. </li><li> Llenar el tubo con un 2% de medio RPMI y se centrifuga a 300 g durante 5 min a 4 ° C. Eliminar el sobrenadante y resuspender elsedimento celular en 1 ml de tampón de lisis ACK. Se incuban las muestras a temperatura ambiente (RT) durante 5 min. Nota: El volumen de tampón de lisis ACK indicado arriba es para un bazo. Ampliar el volumen en consecuencia para las muestras de bazo agrupadas. </li><li> Llenar el tubo con un 2% de medio RPMI y se centrifuga a 300 xg durante 10 min a 4 ° C. Eliminar el sobrenadante y volver a suspender las células a una densidad de 1 x 10 7 células / ml en medio RPMI completo. NOTA: Para el recuento de células vivas, células de tinción con azul de tripano y cuenta sólo las células vivas que no están teñidas utilizando hemocitómetro. </li></ol> 3. Fenotipificación de esplénica convencional T (T conv) Las células y las células T reg NOTA: Antes de aislamiento de células T reg, examinar el fenotipo de linfocitos esplénicos aislados de los ratones no tratados previamente o infectados por tinción de las células con varios anticuerpos y análisis de los mismos por citometría de flujo. <ol><li> Transferencia de 50 l de las células(5 x 10 5 células) entre los linfocitos esplénicos totales en cada pocillo de una nueva placa de 96 pocillos de fondo en U y añadir 150 l de tampón FACS por pocillo. Centrifugar a 300 xg durante 2 minutos a 4 ° C. </li><li> Descartar el sobrenadante. Agitar el sedimento de células y añadir 200 l de tampón de FACS por pocillo. Centrifugar a 300 xg durante 2 minutos a 4 ° C (2 veces). </li><li> Preparar el cóctel de anticuerpos para marcadores de superficie celular de tinción en 50 l de tampón FACS por pocillo con los siguientes anticuerpos y reactivos. colorante Anti-CD4 violeta, colorante verde Anti-CD25, anti-PD-1 de colorante violeta, Anti-CD8-PerCP Cy5.5 reactivo de detección de tinte y la viabilidad celular en el infrarrojo cercano (IR) colorante reactivo fluorescente. NOTA: A fin de analizar el fenotipo de las células T reg activados, anti-CD103 23,25 se puede añadir para la tinción de la superficie celular marcador. </li><li> Resuspender el sedimento celular con 50 l de cóctel de anticuerpo por pocillo. Incubar durante 20 minutos en la oscuridad a 4 ° C. Se lavan las células dos veces contampón FACS mediante centrifugación a 300 xg durante 2 minutos a 4 ° C. </li><li> Después de la etapa de lavado final, se decanta el sobrenadante y fijar las células durante 20 min en la oscuridad a 4 ° C con 100 l de tampón de fijación preparados de acuerdo con el protocolo del fabricante. </li><li> Lavar las células dos veces con el tampón de lavado de permeabilización (preparado de acuerdo con el protocolo del fabricante) por centrifugación a 300 xg durante 2 minutos a 4 ° C. </li><li> Preparar la solución de anticuerpos para la tinción intracelular Foxp3, y volver a suspender el sedimento celular con 50 l de solución de anticuerpo Anti-Foxp3. NOTA: Para analizar más a fondo los fenotipos de conv T y las células T reg, anti-CTLA se puede agregar en este paso. </li><li> Incubar durante 20 minutos en la oscuridad a 4 ° C y repetir el paso 3.6. Después de la etapa de lavado final, resuspender el sedimento celular en 200 l de tampón de FACS y examinar el fenotipo de las células por citometría de flujo 25. </li><li> Puerta de la población de células vivasción. Para el análisis de los fenotipos de las células T durante la infección conv LCMV CL13, examinar la frecuencia de Foxp3 - PD-1 + células entre las células CD4 + o T CD8 +. NOTA: En base a los resultados experimentales, el porcentaje de Foxp3 - PD-1 + es más del 50% en las poblaciones de células CD4 + y CD8 + a los 16 d pi con LCMV CL13. <ol><li> Para analizar la frecuencia y fenotipos de las células T reg, examinar las frecuencias de Foxp3 + o Foxp3 + PD-1 + células entre las células T CD4 +. NOTA: En base a los resultados experimentales, el porcentaje de Foxp3 + o células Foxp3 + PD-1 + es más de 20% en la población de células T CD4 +, respectivamente. </li></ol></li></ol> 4. Aislamiento de células Treg CD4 + CD25 + T NOTA: Los volúmenes de todos los reactivos indicados a continuación es para unaa partir del número de células de 1 x 10 7 esplenocitos totales. <ol><li> Preparar las células como se describe en la sección 2, utilizando ratones infectados crónicamente ingenuos y LCMV. Se lavan las células por adición de 10 ml de tampón de aislamiento de células T. Centrifugar a 300 xg durante 10 min a 4 ° C. Eliminar el sobrenadante por completo y volver a suspender el sedimento celular en 40 l de tampón. </li><li> Para el enriquecimiento de células T CD4 + utilizando el sistema de separación celular magnética, añadir 10 l de cóctel de biotina-anticuerpo y mezclar bien. Incubar durante 10 minutos a 4 ° C. </li><li> Añadir 30 l de tampón, 20 l de anti-biotina micro-perlas para el etiquetado de las células no CD4 + T y 10 l de anticuerpo CD25-PE para el etiquetado fluorescente de células CD25 +. Mezclar bien e incubar durante 15 minutos en la oscuridad. </li><li> Añadir 2 ml de tampón y pasar las células a través de un filtro de 40 micras de células en un nuevo tubo de 50 ml para eliminar los restos celulares. Se lavan las células por centrifugación a 300 xg durante 10 min a 4 ° C.Eliminar el sobrenadante por completo y volver a suspender el sedimento celular en 500 l de tampón a una densidad de hasta 1,25 x 10 8 células. </li><li> Aplicar las células en la columna y recoger las células no marcadas que pasan a través de la columna. Lavar la columna mediante la adición de 2 ml de tampón y se centrifuga a 300 xg durante 10 min a 4 ° C. Eliminar el sobrenadante por completo y volver a suspender las células T CD4 + aisladas en 90 l de tampón. </li><li> Para el marcaje magnético de células CD25 +, añadir 10 l de microperlas anti-PE, mezclar bien e incubar durante 15 minutos en la oscuridad a 4 ° C. </li><li> Añadir 2 ml de tampón y se lavan las células por centrifugación a 300 xg durante 10 min a 4 ° C. Eliminar el sobrenadante por completo y volver a suspender el sedimento celular en 500 l de tampón a una densidad de células de hasta 1 x 10 8. </li><li> Para enriquecer los reg células T CD4 + CD25 +, aplique las células en la columna de selección positiva, y se lava la column mediante la adición de 2 ml de tampón. Repita el lavado tres veces. </li><li> Cuando la columna está vacía después de la última etapa de lavado, añadir 1 ml de tampón en la columna, y expulsar el marcado magnéticamente CD4 + CD25 + células utilizando el émbolo. </li><li> Repita los pasos 4.8 a 4.9 para mejorar la pureza de los aislados reg células CD4 + CD25 + T. Se lavan las células con tampón FACS mediante centrifugación a 300 xg durante 10 min a 4 ° C. Eliminar el sobrenadante y resuspender las células aisladas reg CD4 + CD25 + T en una concentración de 2 x 10 6 células / ml en medio RPMI completo. </li><li> Para comprobar la pureza de las células T reg + aisladas-CD4 + CD25, utilizar 2 x 10 5 células de los totales reg células aisladas-CD4 + CD25 + T. Teñir las células con 50 l de tampón FACS que contiene anti-CD4 FITC y la viabilidad celular reactivo de detección de cerca-IR colorante reactivo fluorescente. NOTA: CD25 ya se marcó con PE durante el aislamiento. </li><li> Incubar durante 20 minutos en la oscuridad a 4 ° C. Se lavan las células con tampón FACS mediante centrifugación a 300 xg durante 2 minutos a 4 ° C. Después del lavado, resuspender el sedimento celular en 100 l de tampón de FACS y comprobar la pureza por citometría de flujo. </li><li> Puerta de la población de células vivas. Para analizar la pureza de las células T reg, confirmar el porcentaje de células CD4 + CD25 +. NOTA: En base a los resultados experimentales, el porcentaje de células CD4 + CD25 + entre las células purificadas es por encima de aproximadamente 80%. </li></ol> 5. Aislamiento de células T CD8 + y etiquetado de las células T CD8 + NOTA: Los volúmenes de todos los reactivos se indican a continuación son para un número de células a partir de 1 x 10 7 esplenocitos totales. <ol><li> Preparar las células como se describe en la sección 2 utilizando ratones no tratados previamente. Se lavan las células mediante la adición de 10 ml de piel de ante de aislamiento de células Ter. Centrifugar a 300 xg durante 10 min a 4 ° C. Eliminar el sobrenadante por completo, y volver a suspender el sedimento celular en 40 l de tampón. </li><li> Para el aislamiento de células T CD8 + usando el sistema de separación celular magnética, añadir 10 l de cóctel de biotina-anticuerpo para el etiquetado + T cell no CD8 y mezclar bien. Incubar durante 5 min a 4 ° C. </li><li> Añadir 30 l de tampón y 20 l de microperlas anti-biotina. Mezclar bien e incubar durante 10 minutos a 4 ° C. </li><li> Aplicar las células en la columna y recoger las células no marcadas que pasan a través de la columna. Lavar la columna mediante la adición de 2 ml de tampón tres veces. </li><li> Centrifugar a 300 xg durante 10 min a 4 ° C. Eliminar el sobrenadante por completo y volver a suspender las células T CD8 + aisladas en 2 ml de tampón. </li><li> Para comprobar la pureza de las células aisladas, preparar 50 l de solución de anticuerpo en tampón FACS. Volver a suspender las 2 x 10 5 células aisladas entre T CD8 + cells en 50 l de solución de anticuerpo. NOTA: Para preparar la solución de anticuerpo, añadir anti-CD8 FITC, y el reactivo de detección de la viabilidad celular (fluorescente cerca-IR colorante reactivo) en tampón de FACS. </li><li> Incubar durante 20 minutos en la oscuridad a 4 ° C. Se lavan las células con tampón FACS mediante centrifugación a 300 xg durante 2 minutos a 4 ° C. Después del lavado, resuspender el sedimento celular en 100 l de tampón FACS, y comprobar la pureza de las células mediante citometría de flujo. </li><li> Puerta de la población de células vivas. Para comprobar la pureza de las células T CD8 +, confirmar el porcentaje de células T CD8 +. NOTA: En base a los resultados experimentales, el porcentaje de células CD8 + entre las células purificadas es por encima de aproximadamente 90%. </li><li> Añadir PBS a las células T CD8 + aisladas. Centrifugar a 300 xg durante 10 min a 4 ° C. Descartar el sobrenadante. Resuspender las células T CD8 + aisladas a una concentración de 1 x 10 7 células / ml en PBS. </li><li> Para etiquetar la ce T CD8 +LLS para el ensayo de supresión in vitro, diluir la proliferación de células de colorante de seguimiento violeta en PBS para obtener una concentración de 5 mM a temperatura ambiente. NOTA: Los excitación y de emisión aproximados picos de la proliferación celular colorante guía violeta utilizado en el estudio son 405 y 450 nm, respectivamente. </li><li> Mezclar volúmenes iguales de bien la proliferación de células de colorante de seguimiento violeta (5 M) y la suspensión de células (1 x 10 7 células / ml de células T CD8 +) en un tubo de 15 ml, y se incuba a 20 min a 37 ° C. Vortex el tubo cada 10 min. </li><li> Llenar el tubo con medio RPMI completo frío, y dejar el tubo durante 10 min a RT. Centrifugar a 300 xg durante 10 min a TA. Eliminar el sobrenadante por completo, y volver a suspender las células a una concentración de 2 x 10 6 células / ml con medio RPMI precalentado completas. Se incuban las células durante 15 min a TA. </li></ol> 6. Configuración del ensayo de supresión in vitro utilizando células CD4 + CD25 + T &lt;sub&gt; Reg y células T CD8 + <ol><li> Para preparar anti-CD3 / CD28 perlas recubiertas de, transferir el volumen apropiado de perlas magnéticas a una 15 ml de tubo (2,5 l / 1 x 10 5 células). Añadir un volumen igual de PBS y mezclar. Lavar por centrifugación a 300 xg durante 2 min a 4 ° C y descartar el sobrenadante. Diluir las perlas magnéticas en medio completo (50 l / pocillo). </li><li> Alícuota de 50 l de reg células + T CD4 + CD25 por pocillo de placa de 96 pocillos de fondo en U (1 x 10 5 células / pocillo). Añadir 50 l de células T CD8 + como de respuesta T (T resp) células por pocillo (1 x 10 5 células / pocillo). Añadir 50 l de perlas de anti-CD3 / CD28 recubierto diluido en por pocillo. NOTA: En este paso, la etiqueta y configurar los pocillos de control de la siguiente manera: &quot;Sólo no estimulada de células T CD8 +&quot; sin perlas anti-CD3 / CD28-revestido; &quot;Sólo las células T CD8 +&quot; con anti-CD3 / CD28 perlas recubiertas con; Sólo &quot;células T CD8 +&quot;Con perlas de CD28 con recubrimiento anti-CD3 /;&quot;. Células T reg única &quot;con-anti CD3 perlas / CD28 revestido con células T reg pueden diluirse por medio completo y co-cultivadas con células T resp en una proporción diferente de T resp células: las células T reg (1: 0,25-1: 1). </li><li> Añadir 50 l o volumen apropiado de medios de comunicación en todos los pocillos hasta un volumen total de 200 l. Cubrir la placa con papel de aluminio y se incuba en un incubador de CO2 a 37 ° C durante 72 horas. </li></ol> 7. Análisis de las células CD8 + T de proliferación celular y la producción de citoquinas de las células T CD8 + <ol><li> Para el análisis de la producción de citoquinas, después de 3 días de cultivo, separar el sobrenadante de cada pocillo a otra placa y realizar ensayo de inmunoabsorción ligado a enzimas (ELISA). NOTA: El sobrenadante puede alícuotas y se almacenó a -70 ° C. En este experimento, se utilizó la placa recubierta con anticuerpo anti-ratón de IFN-γ para detectar IFN-γ acuerdo produccióning el protocolo del fabricante s. Para determinar la producción de IFN-γ de la proliferación de células T CD8 + en un solo nivel celular, tinción intracelular de citoquinas se puede realizar. </li><li> Después de separar el sobrenadante de cada pocillo, se lava la placa que contiene las células con tampón FACS y se centrifuga a 300 xg durante 2 minutos a 4 ° C (3 veces). </li><li> Después del lavado, descartar el sobrenadante. Resuspender el sedimento celular con 50 l de cóctel de anticuerpos para la tinción de las células T CD8 + proliferado. Incubar durante 20 minutos en la oscuridad a 4 ° C. NOTA: Para preparar cóctel de anticuerpos, añadir anti-CD4 FITC, anti-CD8-PerCP Cy5.5, y el reactivo de detección de la viabilidad celular (fluorescente cerca-IR colorante reactivo) en tampón de FACS. NOTA: Los anticuerpos contra diversos marcadores tales como CD44 o CD69 se pueden combinar con otros anticuerpos para confirmar la activación de las células T CD8 +. Recuerde que las células T CD8 + que ya han sido etiquetados con la proliferación celular de seguimiento violet tinte en el Paso 5.10. </li><li> Lavar dos veces por centrifugación a 300 xg durante 2 min a 4 ° C. Después de la etapa de lavado final, descartar el sobrenadante, y fijar las células durante 20 min en la oscuridad a 4 ° C con 100 l de tampón de fijación. </li><li> Lavar dos veces por centrifugación a 300 xg durante 2 min a 4 ° C. Resuspender las células con 200 l de tampón de FACS y se mide la proliferación de las células T CD8 + marcados con colorante de seguimiento de violeta de proliferación celular por citometría de flujo. <ol><li> Puerta de la población de células T CD8 + entre las células vivas. Medir los porcentajes de células divididas y no divididas de acuerdo con la dilución de la proliferación de las células de colorante de seguimiento de violeta y la dilución de células T CD8 + de acuerdo con la siguiente ecuación. % De inhibición = [(% de células proliferadas T CD8 + en la ausencia de células T reg -% de las células T CD8 + proliferado en la presencia de células T reg) / (% de células T CD8 + proliferado enla ausencia de células T reg)] x 100. analizar más a fondo los datos mediante el uso de la citometría de flujo del software 25. </li></ol></li></ol>

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J., 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=Specific+recruitment+of+regulatory+T+cells+in+ovarian+carcinoma+fosters+immune+privilege+and+predicts+reduced+survival.">Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival.</a> Nat Med. 10 (9), 942-949 (2004).</li><li>Dittmer, U., 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=Functional+impairment+of+CD8(+)+T+cells+by+regulatory+T+cells+during+persistent+retroviral+infection.">Functional impairment of CD8(+) T cells by regulatory T cells during persistent retroviral infection.</a> Immunity. 20 (3), 293-303 (2004).</li><li>Robertson, S. J., Messer, R. J., Carmody, A. B., Hasenkrug, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+suppression+of+CD8++T+cell+function+by+Friend+virus-induced+regulatory+T+cells.">In vitro suppression of CD8+ T cell function by Friend virus-induced regulatory T cells.</a> J Immunol. 176 (6), 3342-3349 (2006).</li><li>Iwashiro, M., 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=Immunosuppression+by+CD4++regulatory+T+cells+induced+by+chronic+retroviral+infection.">Immunosuppression by CD4+ regulatory T cells induced by chronic retroviral infection.</a> Proc Natl Acad Sci U S A. 98 (16), 9226-9230 (2001).</li><li>Suvas, S., Kumaraguru, U., Pack, C. D., Lee, S., Rouse, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4+CD25++T+cells+regulate+virus-specific+primary+and+memory+CD8++T+cell+responses.">CD4+CD25+ T cells regulate virus-specific primary and memory CD8+ T cell responses.</a> J Exp Med. 198 (6), 889-901 (2003).</li><li>Suvas, S., Azkur, A. K., Kim, B. S., Kumaraguru, U., Rouse, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4+CD25++regulatory+T+cells+control+the+severity+of+viral+immunoinflammatory+lesions.">CD4+CD25+ regulatory T cells control the severity of viral immunoinflammatory lesions.</a> J Immunol. 172 (7), 4123-4132 (2004).</li><li>Veiga-Parga, T., 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=On+the+role+of+regulatory+T+cells+during+viral-induced+inflammatory+lesions.">On the role of regulatory T cells during viral-induced inflammatory lesions.</a> J Immunol. 189 (12), 5924-5933 (2012).</li><li>Wherry, E. J., 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=Molecular+signature+of+CD8++T+cell+exhaustion+during+chronic+viral+infection.">Molecular signature of CD8+ T cell exhaustion during chronic viral infection.</a> Immunity. 27 (4), 670-684 (2007).</li><li>Jin, H. T., 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=Cooperation+of+Tim-3+and+PD-1+in+CD8+T-cell+exhaustion+during+chronic+viral+infection.">Cooperation of Tim-3 and PD-1 in CD8 T-cell exhaustion during chronic viral infection.</a> Proc Natl Acad Sci U S A. 107 (33), 14733-14738 (2010).</li><li>Punkosdy, G. A., 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=Regulatory+T-cell+expansion+during+chronic+viral+infection+is+dependent+on+endogenous+retroviral+superantigens.">Regulatory T-cell expansion during chronic viral infection is dependent on endogenous retroviral superantigens.</a> Proc Natl Acad Sci U S A. 108 (9), 3677-3682 (2011).</li><li>Blackburn, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coregulation+of+CD8++T+cell+exhaustion+by+multiple+inhibitory+receptors+during+chronic+viral+infection.">Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection.</a> Nat Immunol. 10 (1), 29-37 (2009).</li><li>Park, H. J., 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=PD-1+upregulated+on+regulatory+T+cells+during+chronic+virus+infection+enhances+the+suppression+of+CD8++T+cell+immune+response+via+the+interaction+with+PD-L1+expressed+on+CD8++T+cells.">PD-1 upregulated on regulatory T cells during chronic virus infection enhances the suppression of CD8+ T cell immune response via the interaction with PD-L1 expressed on CD8+ T cells.</a> J Immunol. 194 (12), 5801-5811 (2015).</li><li>Virgin, H. W., Wherry, E. J., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+chronic+viral+infection.">Redefining chronic viral infection.</a> Cell. 138 (1), 30-50 (2009).</li><li>Penaloza-MacMaster, P., 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=Interplay+between+regulatory+T+cells+and+PD-1+in+modulating+T+cell+exhaustion+and+viral+control+during+chronic+LCMV+infection.">Interplay between regulatory T cells and PD-1 in modulating T cell exhaustion and viral control during chronic LCMV infection.</a> J Exp Med. 211 (9), 1905-1918 (2014).</li><li>Chang, M., 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=The+ubiquitin+ligase+Peli1+negatively+regulates+T+cell+activation+and+prevents+autoimmunity.">The ubiquitin ligase Peli1 negatively regulates T cell activation and prevents autoimmunity.</a> Nat Immunol. 12 (10), 1002-1009 (2011).</li><li>Krishnamoorthy, N., 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=Early+infection+with+respiratory+syncytial+virus+impairs+regulatory+T+cell+function+and+increases+susceptibility+to+allergic+asthma.">Early infection with respiratory syncytial virus impairs regulatory T cell function and increases susceptibility to allergic asthma.</a> Nat Med. 18 (10), 1525-1530 (2012).</li><li>Yadav, M., 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=Neuropilin-1+distinguishes+natural+and+inducible+regulatory+T+cells+among+regulatory+T+cell+subsets+in+vivo.">Neuropilin-1 distinguishes natural and inducible regulatory T cells among regulatory T cell subsets in vivo.</a> J Exp Med. 209 (10), 1713-1722 (2012).</li><li>Tai, X., 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=Basis+of+CTLA-4+function+in+regulatory+and+conventional+CD4(+)+T+cells.">Basis of CTLA-4 function in regulatory and conventional CD4(+) T cells.</a> Blood. 119 (22), 5155-5163 (2012).</li><li>Rushbrook, S. M., 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=Regulatory+T+cells+suppress+in+vitro+proliferation+of+virus-specific+CD8++T+cells+during+persistent+hepatitis+C+virus+infection.">Regulatory T cells suppress in vitro proliferation of virus-specific CD8+ T cells during persistent hepatitis C virus infection.</a> J Virol. 79 (12), 7852-7859 (2005).</li><li>Sekiya, T., 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=The+nuclear+orphan+receptor+Nr4a2+induces+Foxp3+and+regulates+differentiation+of+CD4.">The nuclear orphan receptor Nr4a2 induces Foxp3 and regulates differentiation of CD4.</a> T cells. Nat Commun. 2 (269), (2011).</li><li>Merianos, D. J., 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=Maternal+alloantibodies+induce+a+postnatal+immune+response+that+limits+engraftment+following+in+utero+hematopoietic+cell+transplantation+in+mice.">Maternal alloantibodies induce a postnatal immune response that limits engraftment following in utero hematopoietic cell transplantation in mice.</a> J Clin Invest. 119 (9), 2590-2600 (2009).</li><li>Allakhverdi, Z., 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=Expression+of+CD103+identifies+human+regulatory+T-cell+subsets.">Expression of CD103 identifies human regulatory T-cell subsets.</a> J Allergy Clin Immunol. 118 (6), 1342-1349 (2006).</li><li>Camisaschi, C., 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=LAG-3+expression+defines+a+subset+of+CD4(+)CD25(high)Foxp3(+)+regulatory+T+cells+that+are+expanded+at+tumor+sites.">LAG-3 expression defines a subset of CD4(+)CD25(high)Foxp3(+) regulatory T cells that are expanded at tumor sites.</a> J Immunol. 184 (11), 6545-6551 (2010).</li><li>Wang, R., 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=Expression+of+GARP+selectively+identifies+activated+human+FOXP3++regulatory+T+cells.">Expression of GARP selectively identifies activated human FOXP3+ regulatory T cells.</a> Proc Natl Acad Sci U S A. 106 (32), 13439-13444 (2009).</li><li>Myers, L., 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=IL-2-independent+and+TNF-alpha-dependent+expansion+of+Vbeta5++natural+regulatory+T+cells+during+retrovirus+infection.">IL-2-independent and TNF-alpha-dependent expansion of Vbeta5+ natural regulatory T cells during retrovirus infection.</a> J Immunol. 190 (11), 5485-5495 (2013).</li></ol>

S.-J.H. has a patent and receives patent royalties related to the PD-1 pathway. The other authors have no financial conflicts of interest.

Aunque existe sólo un pequeño número de células T reg en ratones y seres humanos, es importante para entender su función ya que desempeñan un papel crucial en la regulación de la respuesta inmune y el mantenimiento de la tolerancia inmune. El número y supresores funciones de T reg células aumenta durante una infección por el virus crónica 15-20, así como la progresión del cáncer 13,14. Esto es probablemente debido a la estimulación de antígeno continuado. Para ...

Células T reguladoras (Treg) expresan caja forkhead P3 (Foxp3) como un factor de transcripción para su desarrollo y función 1. Además, las células T reg expresan varias otras moléculas tales como CD25 2, el gen de activación de linfocitos 3 (LAG-3) 3, inducida por glucocorticoides factor de necrosis tumoral receptor 4, y linfocitos T citotóxicos asociada a la proteína 4 (CTLA-4) 5 en su superficie o región intracelular. Durante la infección crónica con diversos tipos de agentes patógenos tales como virus, bacterias 6,7 8,9, y parásitos 10-12, o en el curso del desarrollo del cáncer de 13,14, las células T reg se diferencian en células activadas, se presentan función de supresión mejorada células de orientación efectora CD4 + y T CD8 +. Varios trabajos han sugerido que las células T reg expandidas y activadas contribuyen a los respons a alteraciones celulares T CD8 +e durante amigo retrovirus (FV), la infección 15-17. Células T reg inducidas-FV inhiben IFN-γ o expresión de granzima B y reactividad citotóxica de las células T CD8 + 15-17. Por otra parte, en un modelo de infección por el virus herpes simplex, se informó de que el agotamiento de células T CD4 + reg + CD25 resultó en la expansión de células T CD8 + específicos del virus y daños graves en los tejidos por la infiltración de células T CD4 + inmunopatogénicos 18-20. Los ratones infectados crónicamente con la cepa clon 13 del virus de la coriomeningitis linfocítica (LCMV CL13) 21 a 24 han sido ampliamente utilizados para caracterizar el fenotipo y la función de las células T efectoras (T eff) y células T reg durante la infección crónica por virus. Durante la infección por LCMV persistente, las células T eff específico del virus pierden progresivamente su función efectora y se convierten en células T agotados (T) exh. Por otra parte, Treg células refuerzan su capacidad para suprimir la respuesta de células T específica para el virus 25. La disminución de la capacidad de funcionamiento de las células T eff puede explicarse por varios factores tales como la regulación positiva de los receptores inhibidores sobre las células T eff, función alterada de las células presentadoras de antígeno, la producción de citocinas inmunorreguladoras, y aumento de la frecuencia o el incremento de la función de T reg 26 células. Entre los factores implicados en la supresión de células T, la muerte celular programada proteína-1 (PD-1) que expresan las células EXH T y las células T reg han sido ampliamente considerado como las características de persistencia de antígeno y el medio ambiente de supresión. Recientemente, se informó de que el bloqueo de la vía de PD-1 y la ablación de células T reg conducen a la función de células T mejorado y la disminución de la carga viral durante la infección crónica por LCMV 27. Además, las células T reg se activan durante la infección crónica de ratones con LCMV 23,25 </sarriba&gt; y su función supresora se fortalece 25. PD-1 es altamente expresado en las células T reg así como las células T EXH, y el nivel de PD-1 expresada por las células T reg se correlaciona con la fuerza de su función supresora para inhibir la proliferación de células T 25. A continuación, se describe un método para comparar las características de las células T reg activados aislados de ratones infectados con LCMV CL13 y reg células T en reposo aislados de ratones no tratados previamente. Por otra parte, explicamos una serie de procesos para separar las reg células T activadas y examinar su fenotipo ex vivo, así como medir su actividad supresora in vitro.

<table border="0" cellpadding="0" cellspacing="0" width="976">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">FITC Rat Anti-Mouse CD4</td>
			<td style="width:67px;">RM4-5</td>
			<td style="width:173px;">BD Biosciences</td>
			<td style="width:104px;">553047</td>
			<td style="width:249px;">Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytofix/Cytoperm</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td>Use this reagent for cell surface staining.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">U-Bottom Tissue Culture Plates</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>353077</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation buffer</td>
			<td></td>
			<td>BD Biosciences</td>
			<td align="right">554655</td>
			<td>Use this reagent for cell surface staining.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC Rat Anti-Mouse CD25</td>
			<td>7D4</td>
			<td>BD Biosciences</td>
			<td>553072</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer, 70mm</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>352350</td>
			<td>Use this strainer for grinding the whole spleen.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell strainer, 40mm</td>
			<td></td>
			<td>BD Biosciences</td>
			<td>352340</td>
			<td>Use this strainer for filtering the cells before column enrichment.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brilliant Violet 421 Anti-mouse CD279 (PD-1)</td>
			<td>29F.1A12</td>
			<td>BioLegend</td>
			<td>135217</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brilliant Violet 605 Anti-Mouse CD4</td>
			<td>RM4-5</td>
			<td>Biolegend</td>
			<td align="right">100547</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">APC Anti-Mouse/Rat Foxp3&#160;</td>
			<td>FJK-16s</td>
			<td>eBioscience</td>
			<td>17-5773</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Foxp3 / Transcription Factor Staining Buffer Set</td>
			<td></td>
			<td>eBioscience</td>
			<td>00-5223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PerCP-Cyanine5.5 Anti-Mouse CD8a</td>
			<td>53-6.7</td>
			<td>eBiosicence</td>
			<td>45-0081</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 100X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse IFN-gamma Platinum ELISA</td>
			<td></td>
			<td>eBiosicence</td>
			<td>BMS606</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640</td>
			<td></td>
			<td>GE Life Sciences</td>
			<td>SH30027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS (1X)</td>
			<td></td>
			<td>GE Life Sciences</td>
			<td>SH30256</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ACK Lysing Buffer</td>
			<td></td>
			<td>Gibco</td>
			<td>A10492-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamine, 200mM solution</td>
			<td></td>
			<td>Gibco&#160;</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin, 10,000U/mL</td>
			<td></td>
			<td>Gibco&#160;</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit</td>
			<td></td>
			<td>Life technologies</td>
			<td>L-34975</td>
			<td>Please determine appropriate concentration. In this protocol, this reagent was diluted 500X in FACS buffer.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD8a+ T Cell Isolation Kit, mouse</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-104-075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD4+CD25+ Regulatory T Cell Isolation Kit, mouse</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-091-041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS Separation Columns, LD columns</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-042-901</td>
			<td>Use this column for Treg cell isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACS Separation Columns, LS columns</td>
			<td></td>
			<td>Miltenyibiotec</td>
			<td>130-042-401</td>
			<td>Use this column for CD8+ T cell and Treg cell isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA, 0.5M (pH 8.0)</td>
			<td></td>
			<td>Promega</td>
			<td>V4231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Mercaptoethanol</td>
			<td></td>
			<td>Sigma Life Science</td>
			<td>M7522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td></td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30919.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTrace Violet Cell Proliferation Kit</td>
			<td></td>
			<td>Thermo Fisher Scientific</td>
			<td>C34557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Canto II flowcytometer</td>
			<td></td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer*</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flowjo</td>
			<td></td>
			<td>TreeStar</td>
			<td></td>
			<td>Flow cytometry software&#8224;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematocytomer</td>
			<td></td>
			<td>Marienfeld superior</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

phenotypic and functional analysis of activated regulatory t cells isolated from chronic lymphocytic choriomeningitis virus-infected mice

Células reguladoras T (T reg), que expresan Foxp3 como un factor de transcripción, son subconjuntos de células T CD4 +. Células T reg desempeñan papeles cruciales en la tolerancia inmune y el mantenimiento de la homeostasis mediante la regulación de la respuesta inmune. La función principal de las células T reg es la de suprimir la proliferación de células T efectoras T (FEP) y la producción de citoquinas tales como IFN-γ, TNF-α e IL-2. Se ha demostrado que la capacidad de las células T reg &#39;para inhibir la función de las células T eff es mayor durante la infección por patógenos persistente y el desarrollo del cáncer. Para aclarar la función de las células T reg en condiciones de reposo o inflamadas, una variedad de ensayos de supresión in vitro utilizando células de ratón o de reg T humanas se han ideado. El objetivo principal de este estudio es desarrollar un método para comparar las diferencias en el fenotipo y la función supresora de descanso entre y reg T activadasCélulas. Para aislar reg células T activadas, los ratones fueron infectados con el virus de la coriomeningitis linfocítica (LCMV) clon 13 (CL13), una cepa crónica de LCMV. Células T reg aisladas del bazo de ratones infectados por LCMV CL13 exhiben tanto el fenotipo activado y actividad supresora mejorada en comparación con las células en reposo T reg aisladas de ratones no tratados previamente. A continuación, se describe el protocolo básico para el análisis ex vivo fenotipo de distinguir reg células T activadas de descansar células T reg. Además, se describe un protocolo para la medición de la actividad supresora de las células T reg totalmente activadas.

Hemos generado ratones con infección por el virus persistente mediante la inyección de ellos con 2 x 10 6 pfu de LCMV CL13 por vía intravenosa. Para investigar los cambios fenotípicos en las células T reg y las células T durante la infección conv virus crónica, linfocitos esplénicos obtenidos de ratones no tratados previamente y infectadas se tiñeron con diversos anticuerpos y se analizaron por citometría de flujo. A los 16 d PI, PD-...

Watch this Scientific Journal Video about Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice at JoVE.com

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

El análisis fenotípico y funcional de las células reguladoras T activadas Aislado de la coriomeningitis linfocítica crónica por el virus ratones infectados

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

phenotypic-functional-analysis-activated-regulatory-t-cells-isolated

Research

JoVE Journal

Immunology and Infection

9.7K vistas.  Yonsei University. Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from naïve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Video: Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

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

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

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

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

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

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

Investigación

Inmunología e Infección

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

Nuevas herramientas para expandir células T reguladoras de las personas VIH-1-infected

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Systemic Injection of Neural Stem/Progenitor Cells in Mice with Chronic EAE

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative neurology. This directive has arisen from the extensive evidence that brain repair is achieved after focal or systemic NPC transplantation in several preclinical models of neurological diseases.
These experimental data have identified the cell delivery route as one of the main hurdles of restorative stem cell therapies for brain diseases that requires urgent assessment. Intraparenchymal stem cell grafting represents a logical approach to those pathologies characterized by isolated and accessible brain lesions such as spinal cord injuries and Parkinson's disease. Unfortunately, this principle is poorly applicable to conditions characterized by a multifocal, inflammatory and disseminated (both in time and space) nature, including multiple sclerosis (MS). As such, brain targeting by systemic NPC delivery has become a low invasive and therapeutically efficacious protocol to deliver cells to the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system (CNS).
This alternative method of cell delivery relies on the NPC pathotropism, specifically their innate capacity to (i) sense the environment via functional cell adhesion molecules and inflammatory cytokine and chemokine receptors; (ii) cross the leaking anatomical barriers after intravenous (i.v.) or intracerebroventricular (i.c.v.) injection; (iii) accumulate at the level of multiple perivascular site(s) of inflammatory brain and spinal cord damage; and (i.v.) exert remarkable tissue trophic and immune regulatory effects onto different host target cells in vivo.
Here we describe the methods that we have developed for the i.v. and i.c.v. delivery of syngeneic NPCs in mice with experimental autoimmune encephalomyelitis (EAE), as model of chronic CNS inflammatory demyelination, and envisage the systemic stem cell delivery as a valuable technique for the selective targeting of the inflamed brain in regenerative neurology.

The transplantation of neural stem/progenitor cells (NPCs) holds great promises in regenerative neurology. The systemic delivery of NPCs has turned into effective, low invasive, and therapeutically very efficacious protocol to deliver stem cells in the brain and spinal cord of rodents and nonhuman primates affected by experimental chronic inflammatory damage of the central nervous system.

Inyección sistémica de Neuronales células madre / progenitoras en ratones con EAE crónica

Neural stem/precursor cells (NPCs) are a promising stem cell source for transplantation approaches aiming at brain repair or restoration in regenerative ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Derivación de células T<em&gt; In Vitro</em&gt; De células madre embrionarias de ratón

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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

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

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

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

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

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

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

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

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

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

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Células T Captura bacterias por transinfection de células dendríticas

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

Determinación de Biofilm Iniciación, el virus de las células infectadas por bacterias y hongos

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

Caracterización funcional de macrófagos reguladores que inhiben la inmunidad reactiva al injerto

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

El análisis fenotípico y funcional de las células reguladoras T activadas Aislado de la coriomeningitis linfocítica crónica por el virus ratones infectados

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