Este trabajo es apoyado por una ayuda a la producción de terapias celulares (contrato de NIH-NHLBI (HB-10-03) HHSN26820100000C) (CMR), un Centro Especializado para terapia basada en células subvención del NIH-NHLBI 1 U54 HL081007 (CMR), un ASBMT Premio al Investigador Joven (UG y JV), Miembro Sociedad de Leucemia y Linfoma Especial en el Premio de Investigación Clínica (UG), y una Amy Strelzer Scholar Award Manasevit (AML).

1. DC nucleofection <ol><li> Cosecha de los países en desarrollo derivados de los monocitos, que se han enriquecido con la adhesión de plástico, cultivadas durante 5 días con Cell Genix medios suplementados con IL-4 (1000U/ml), GMCSF (800IU/ml) y además con una maduración de 24 horas de uso de las citocinas IL-4 maduración de las DC (1000U / ml), GMCSF (800IU/ml), IL6100ng/ml, TNF-α 10ng/ml, IL1-β 10ng/ml y PGE2 (1μg/ml) 1, por la resuspensión suave con una pipeta de transferencia de 3 ml. </li><li> Contar con los países en desarrollo viable con azul tripán, la transferencia en tubos de 15 ml 3 veces con no menos de 0.5x10 6 y no más de 2x10 6 células / tubo. </li><li> Centrífuga para los países en desarrollo 10 minutos @ 200g. Durante este tiempo de pre-calentamiento celular Genix medios suplementados con las citoquinas maduración de las DC (DC maduración de los medios de comunicación) - 2ml/well en tres pozos de un cultivo de tejidos de 12 pocillos tratados en una placa de 37 ° C / 5% CO 2 incubadora. </li><li> Una vez que las células han terminado de girar, aspirar el sobrenadante y añadir los plásmidos de ADN correspondientes a cada uno de los tubos en una concentración final de ADN 5μg / tubo. En este caso, añadir el plásmido que codifica IE1-pp65 al tubo # 1, Hexon-Penton al tubo # 2, y EBNA1-LMP2-BZLF1 al tubo # 3. </li><li> Resuspender los países en desarrollo y el ADN con 100μl de solución nucloefection Amaxa, mezclar bien y la transferencia de las cubetas nucleofection. </li><li> Cubetas lugar en el nucleofector 4D, elegir el programa CB150 (Amaxa / Lonza), y pulse Iniciar. </li><li> Inmediatamente después de añadir 500μl nucleofection del 2 ml precalentado celular medios de maduración Genix DC a la cubeta, mezclar suavemente pipeteando arriba y abajo de 3.2 veces, y los países en desarrollo la transferencia de nucleofected el preparado de 12 y placa que contiene los restantes 1,5 ml de precalentado DC medios de maduración. Traslado a los 37 ° C / 5% CO 2 incubadora por un período de 12-18hrs. </li></ol> 2. La estimulación de células T <ol><li> La cosecha y el recuento de los países en desarrollo nucleofected, e irradiar a 30Gy. Lavar una vez con 10 ml de medio de CTL (45% RPMI, 45% EHAA clics, el 10% de SFB, 2 mM Glutamax) y resuspender @ 3 x 10 5 países en desarrollo por ml de los medios de CTL. </li><li> Piscina con un mínimo de 7.5x10 5 (2,5 ml) y un máximo de 15x10 5 (5 ml) de los países en desarrollo que contiene cada uno de los plásmidos y transferencia de los centros de distribución en común para el dispositivo G-Rex que luego serán colocados en la incubadora. </li><li> Para la preparación de células de respuesta utilizar PBMCs previamente congelados o no adherentes células mononucleares que quedan después de la selección de CC (adherencia o la selección de CD14). Descongelar las células, la transferencia al medio de cultivo precalentado, lavar una vez con los medios de comunicación CTL. Resuspender las células en los medios de comunicación CTL, el recuento de las células y llevarlos a una concentración de 2x10 6 células por ml. Tome 15x10 6 células o 7.5ml y complementar con 30000U IL4 (1000U/ml -. Conc final) y 300ng IL7 (10ng/ml - conc final.). </li><li> Transferencia 7.5ml de PBMC (15x10 6 células) a la G-Rex y rellene el biorreactor con los medios de comunicación CTL para un volumen total de 30ml. </li><li> La cultura del G-Rex durante 6-7 días en un 37 ° C / 5% CO 2 humidificado incubadora. </li></ol> 3. La expansión de células T <ol><li> En el día 06/07, 10 ml de aspirado de los medios de comunicación, a continuación, mezclar las células en el resto de los medios de 20ml con una pipeta de 10 ml y el recuento de células viables con azul de tripano. Si hay &lt;50x10 6 llenar con medio fresco + citoquinas. Si hay&gt; 50x10 6 células retirar 10 ml de suspensión celular, la transferencia a un nuevo G-Rex, y luego alimentar el G-Rexs con los medios de comunicación fresca CTL + citoquinas. </li><li> Cultura por un período adicional de 4-6 días. Una vez que suficientes células se han ampliado, lleve a cabo la caracterización fenotípica y funcional de la Franquicia del CTL y criopreservar para uso futuro. </li></ol> 4. Los resultados representativos: Un esquema de nuestra aprobado por la FDA multivirus específico proceso de generación de CTL se muestra en la Figura 1. A diferencia de la convención multivirus CTL protocolos que utilizan los adenovectores y LCL EBV-para estimular la reacción virus de las células T 2 se han sustituido materiales virus infeccioso con plásmidos de ADN que codifican antígenos múltiples derivados de cada uno de los tres virus. Para estimular CTL trivirus hemos diseñado tres multicistronic plásmidos que codifican Hexon y Penton de adenovirus, IE1 y pp65 de CMV, y EBNA1, LMP2, y BZLF1 de EBV. Estos antígenos fueron seleccionados con base en el fomento de los resultados clínicos de los nuestros y de otros grupos que muestran que las células T dirigidas contra las proteínas ADV-hexón y pentón 2,4-6, y CMV-1E1 y CMV-pp65 tienen un efecto protector en vivo 7. Para el VEB, EBNA1 es un inmuno-CD4 + de células T antígeno diana se expresa en todas las neoplasias asociadas al VEB en las células normales y B infectados por EBV 8,9, es inmunogénica LMP2 entre los tipos de HLA múltiples y se expresa en la mayoría de los tumores EBV, 10,11, mientras que BZLF1 codifica una inmunodominantes, inmediata antígeno temprano del ciclo lítico que estimula tanto CD4 + y CD8 + T en la mayoría de las personas y es probable importante para la cooperaciónntrol de reproducción de las células del virus 12. Para optimizar aún más nuestros métodos de fabricación que colaboró ​​con la tecnología de la naturaleza que genera minimizada, sin antibióticos (compatible con la FDA) plásmidos para la estimulación CTL 13,14. Utilizando esta estrategia de forma consistente lograr eficiencias nucleofection de&gt; 35%, mientras que el mantenimiento de la viabilidad celular alta (datos no mostrados) 3. La figura 2 muestra que la frecuencia de células T específicas del virus en respuesta a plásmidos de ADN optimizados medida por IFN ELISPOT, fue mayor que en respuesta a las convencionales plásmidos de expresión pShuttle basado en expresar los mismos antígenos (n = 8 Adenovirus, CMV n = 4 y n = 2 EBV). La relación óptima de DC: PBMC fue importante para el estímulo potente de las células T, como se muestra en la figura 3, donde una proporción de 1:50 produjo debajo del nivel óptimo de activación en comparación con S 01:20: cociente R (n = 2 donantes). La producción de suficientes números de CTL con especificidad de antígeno amplia es un requisito previo para la eficacia clínica contra los tres virus. Esto se logra mediante el cultivo CTL en el G-Rex, que apoya la expansión de células T superiores en comparación con los convencionales de 24 y placas (Figura 4) 15, mientras que la adición de IL-4 y IL7 a los aumentos de las culturas del repertorio y la especificidad, como se muestra en la Figura 4B donde la frecuencia de las células T reactivas contra el CMV-pp65 derivados de HLA-A2 resticted NLV péptido se evaluó en cultivos generados en la presencia o ausencia de IL-4 y / o 16,17 IL7. Para evaluar la capacidad y el fenotipo funcional de las células que amplió el análisis de citometría de flujo, intracelular de citoquinas ELISPOT tinción / IFN, y Cr 51 ensayos de liberación en el producto final para la criopreservación / infusión. Normalmente las células se generan policlonales con una población mixta de células CD4 + y CD8 + T con el antígeno-específico detectable en los dos compartimentos de las células T. Los CTL son capaces de matar a antígenos virales que expresan las células diana, pero no virus parcialmente negativa-HLA objetivos encontrados, lo que indica que no debe inducir de injerto contra huésped (EICH) in vivo (Figura 5). <img src="/files/ftp_upload/2736/2736fig1.jpg" alt="Figura 1"/> Figura 1. Protocolo RCTL generación. En primer lugar, los países en desarrollo son nucleofected con la codificación de antígeno viral plásmidos y luego se mezcla con PBMC autólogo en un R: S de 10 o 20:01. Las células se expanden en el G-Rex durante 10-14 días en presencia de IL-4 y IL7, luego cosechadas, contados, la prueba de la función, la identidad y la esterilidad, y luego criopreservados para su uso clínico. <img src="/files/ftp_upload/2736/2736fig2.jpg" alt="Figura 2"/> Figura 2. Plásmidos de ADN optimizados inducir la activación de células T superiores in vitro. Países en desarrollo se nucleofected con optimizado, compatible con la FDA Hexon plásmidos que codifican y Penton (Avanzado), IE1 y pp65 (CMV), y EBNA1, LMP2, y BZLF1 (EBV) o plásmidos convencionales pShuttle codificación de los mismos antígenos. Estos fueron utilizados para estimular las células T y la especificidad fue analizada por ELISPOT IFN 10 días después de la estimulación. <img src="/files/ftp_upload/2736/2736fig3.jpg" alt="Figura 3"/> Figura 3 DC óptimo:. Ratios de células T para la activación de CTL. Los países en desarrollo a partir de 2 donantes nucleofected con los tres plásmidos optimizados y luego se utiliza para estimular la PBMCs autólogas a las 1:20 o 1:50 DC: relación de PBMC. La frecuencia de las células T reactiva se evaluó en el día 10 de IFN ELISPOT. <img src="/files/ftp_upload/2736/2736fig4.jpg" alt="Figura 4"/> Figura 4. Expansión de células T en el G-Rex con la mejora de las citoquinas. El panel A muestra el dispositivo G-Rex, así como la apariencia CTL en la membrana de gas permeable, evaluadas por microscopía. Una comparación entre la producción de células en cultivo de tejidos logrado convención tratados placas vs G-Rex también se indica. El panel B muestra la frecuencia de CMV CTL pentámero positivos logrados en las culturas se expandió en la presencia de ninguna de citocinas, IL-4 solo, solo y IL7 IL4 IL7 +. <img src="/files/ftp_upload/2736/2736fig5.jpg" alt="Figura 5"/> Figura 5. Fenotipo y la función de CTL ampliado. El panel A muestra un ejemplo representativo del fenotipo de la ampliación del CTL multivirus, que son policlonales con una mezcla de células CD4 + (45% - helper) y CD8 + (42% - citotóxicos), las células T, de los cuales la mayoría (95%) expresó la memoria marcador CD45RO + / CD62L +. El panel B muestra que estas células son específicas para todos los antígenos estimulantes y son polifuncionales, como evaluación de la tinción intracelular de citoquinas para detectar la producción de IFN y TNF después de la estimulación antigénica. Panel C muestra que el CTL ampliado son funcionales medida Cr 51 por ensayo. Autólogo LCL, ya sea solo o transducidas con un vector nulo o un vector adenoviral que expresa CMV-pp65 fueron utilizados como el alquitránse. Alorreactividad se evaluó mediante trasplante alogénico explosiones PHA como un objetivo.

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+cellular+immunity+against+cytomegalovirus+in+recipients+of+allogeneic+bone+marrow+by+transfer+of+T-cell+clones+from+the+donor.">Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.</a> N. Engl. J. Med. 333, 1038-1044 (1995).</li></ol>

Juan F. Vera es un consultor para la fabricación de Wilson Wolf. Los otros autores no se incluyen desgloses pertinentes.

Las infecciones virales representan una importante morbilidad y mortalidad en pacientes inmunodeprimidos por su enfermedad o su tratamiento. Después de TPH, por ejemplo, infecciones causadas por herpesvirus persistentes, tales como EBV y CMV, así como por virus respiratorios como el virus sincitial respiratorio (VSR), son bien conocidos, mientras que la importancia de las infecciones causadas por ADV, virus BK, y los derechos humanos virus del herpes (HHV) -6 más recientemente, han sido apreciados. Mientras que los agentes farmacológicos son la terapia estándar para algunas infecciones, tienen efectos tóxicos importantes, generar variedades resistentes, y con frecuencia ineficaz. En contraste, el virus de las células T específicas derivadas de donantes de células madre han demostrado ser seguros y eficaces para la prevención y el tratamiento de la infección viral o enfermedad en el trasplante de células madre hematopoyéticas (TCPH) configuración 2,5,6,18-21. Sin embargo, la aplicación más amplia de la inmunoterapia de células T es en última instancia, limitada por el costo, la complejidad y el tiempo requerido para la producción de CTL. Nuestro enfoque novedoso y rápido para generar multivirus CTL, descrito en el manuscrito actual, deberían aumentar considerablemente la viabilidad de la terapia de células T citotóxicas para las enfermedades virales, permitiendo que la estrategia para convertirse en un estándar de atención para el huésped inmunocomprometido. El uso de un plásmido DC nucleofected como APC permite la presentación de antígenos en ambos MHC de clase I y II sin la competencia de los vectores virales o de hecho a partir de múltiples antígenos virales se expresan dentro de una célula ya que las diferentes poblaciones de DC se utilizan para cada plásmido 3. El uso de IL-4 / 7 aumenta la supervivencia de las células T y la proliferación, lo que en consecuencia ayuda a aumentar la frecuencia y el repertorio de la respuesta del antígeno específico de células T 16,17. Por último, la cultura en el G-Rex reduce la apoptosis de las células T durante el cultivo. El intercambio de gases (O2 y CO 2) se produce a través de una membrana de gas permeable de silicona en la base del frasco, la prevención de la hipoxia al tiempo que permite una mayor profundidad de medio por encima de las células, proporcionando mayor cantidad de nutrientes y la dilución de los productos de desecho. Esta plataforma también puede ser ampliado a los virus adicionales como antígenos protectores cuando se identifican.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> CellGenix </td><td> CellGenix </td><td> 2005 </td></tr><tr><td> IL4 </td><td> I + D de Sistemas </td><td> 204-IL/CF </td></tr><tr><td> IL7 </td><td> Peprotech </td><td> 200-15 </td></tr><tr><td> Hyclone RPMI 1640 </td><td> Thermo Scientific </td><td> SH30096.01 </td></tr><tr><td> Suero humano AB </td><td> Valle Biomedical Inc. </td><td> HP1022 </td></tr><tr><td> Nucleofector </td><td> Amaxa / Lonza </td><td> AAF-1001B y AAF-1001X </td></tr><tr><td> Nucleofection Kit </td><td> Amaxa / Lonza </td><td> V4XP-3012 </td></tr><tr><td> Plásmidos </td><td> NTC </td><td> n / a </td></tr><tr><td> GM-CSF </td><td> I + D </td><td> 215-GM/CF </td></tr><tr><td> IL1 </td><td> I + D </td><td> 201-LB-025 </td></tr><tr><td> IL6 </td><td> I + D </td><td> 206-IL-CF </td></tr><tr><td> TNF </td><td> I + D </td><td> 210-TA-010 </td></tr><tr><td> PGE2 </td><td> Sigma </td><td> P6532-1mg </td></tr><tr><td> G-Rex </td><td> Wilson Lobo de fabricación </td><td> AY11-00027 </td></tr></tbody></table>

generation of multivirus-specific t cells to prevent/treat viral infections after allogeneic hematopoietic stem cell transplant

Infecciones virales causan morbilidad y mortalidad en trasplante alogénico de células madre hematopoyéticas (HSCT). Nosotros y otros han generado con éxito y se infunden las células T específicas para el virus de Epstein Barr (EBV), citomegalovirus (CMV) y el adenovirus (ADV) con los monocitos y VEB-transformado células linfoblásticas (EBV-LCL) modificadas genéticamente con un vector de adenovirus como células presentadoras de antígeno (APC). Tan sólo 5 2x10 / kg trivirus específicas linfocitos T citotóxicos (CTL) multiplicado por varios registros después de la infusión y apareció para prevenir y tratar las enfermedades virales, incluso graves resistentes a otros tratamientos disponibles. La aplicación más amplia de este enfoque alentador es limitada por altos costos de producción, la complejidad de la fabricación y el tiempo prolongado (4-6 semanas para el VEB-LCL generación, y 4-8 semanas para la fabricación de CTL - total 10 a 14 semanas) para la preparación. Para superar estas limitaciones, hemos desarrollado un nuevo GMP protocolo de producción de CTL. En primer lugar, en lugar de adenovectores para estimular las células T que utilizan las células dendríticas (DC) nucleofected con el ADN de plásmidos que codifican LMP2, EBNA1 y BZLF1 (EBV), Hexon y Penton (Avanzado), y pp65 y IE1 (CMV) como presentadoras de antígeno las células. Estos vehículos blindados reactivar las células T específicas para todos los antígenos estimulantes. Segundo cultivo, de las células T activadas en presencia de IL-4 (1.000 U / ml) e IL-7 (10ng/ml) se incrementa y mantiene el repertorio y la frecuencia de células T específicas en nuestras líneas. En tercer lugar, hemos utilizado un nuevo dispositivo, la cultura de gas permeable (G-Rex), que promueve la expansión y la supervivencia de los números de células grandes después de una estimulación única, eliminando así la necesidad de EBV-LCLs y reducir la intervención del técnico. Mediante la implementación de estos cambios se pueden producir ahora CTL multiespecíficos objetivo EBV, CMV, y Adv a un costo por cada 10 6 células que se reduce&gt; 90%, y en sólo 10 días en lugar de 10 semanas utilizando un enfoque que puede extenderse a otros protección antígenos virales. Nuestro enfoque aprobado por la FDA deben ser de valor para las aplicaciones de profilácticos y de tratamiento para los receptores de alto riesgo TCPH alogénicos.

Watch this Scientific Journal Video about Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant at JoVE.com

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Un protocolo de forma rápida, sencilla y rentable para la generación de donantes de origen multivirus CTL específicos (RCTL) para la perfusión del trasplante alogénico de células madre hematopoyéticas de trasplante (HSCT) con riesgo de desarrollar infecciones por CMV, EBV o Adv. Este proceso de fabricación es conforme a las GMP y debe garantizar la aplicación más amplia de células T inmunoterapia más allá de los centros especializados.

Generación de células T Multivirus específicos para prevenir / tratar las infecciones virales después de trasplante alogénico de células madre hematopoyéticas

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

generation-multivirus-specific-t-cells-to-preventtreat-viral

Research

JoVE Journal

Immunology and Infection

17.1K vistas.  Baylor College of Medicine. Un protocolo de forma rápida, sencilla y rentable para la generación de donantes de origen multivirus CTL específicos (RCTL) para la perfusión del trasplante alogénico de células madre hematopoyéticas de trasplante (HSCT) con riesgo de desarrollar infecciones por CMV, EBV o Adv. Este proceso de fabricación es conforme a las GMP y debe garantizar la aplicación más amplia de células T inmunoterapia más allá de los centros especializados.

Video: Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

A raíz de la celda de destino en<em&gt; E. coli</em&gt; Después de la infección por fagos Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Investigación

Inmunología e Infección

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

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

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#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 ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Aplicación clínica de<em&gt; La Bella Durmiente</em&gt; Artificial y células presentadoras de antígenos para modificar genéticamente las células T de la sangre del cordón umbilical y periférica

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

El establecimiento de un cultivo líquido cubierto de las vías respiratorias humanas epiteliales polarizadas Calu-3 células para estudiar la respuesta del huésped celular a patógenos respiratorios<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

La identificación de mutaciones del ADN en Purificada hematopoyéticas células madre / progenitoras

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

El trasplante de la cola de la piel para el Estudio alogénico de células T CD4 respuestas en ratones

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generación y multi-fenotípicos cribado de alto contenido de<em&gt; Coxiella burnetii</em&gt; transposón mutantes

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

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.

Desarrollo de derivados de células madre Las células T reguladoras específicas de antígeno contra la autoinmunidad

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

La medición de células T Alorreactividad recurriendo a imágenes de Citometría de Flujo

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

Evaluación de respuestas de células T Zika Virus-específica en Immunoprivileged órganos de ratones- / - Ifnar1 infectados

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...