Este trabajo fue apoyado por un Dan L. Duncan beca de investigación colaborativa (CMB y EJS), el Instituto Nacional del Corazón Pulmón, y Sangre (US4HL081007), una Sociedad de Leucemia y Linfoma Investigación Clínica premio Scholar (CMB), y el Instituto Nacional del Cáncer (RO1 CA06150816; EJS).

1. Aislamiento de células mononucleares (día 0) <ol><li> Insertar espiga de adaptador luer hembra en el puerto de salida de la fracción de 20% de la unidad de sangre del cordón umbilical, conecte la jeringa y retirar la sangre. Transferir sangre descongeladas a 20 ml de RPMI calentó en tubo de centrífuga 50 mL. Enjuague la bolsa de sangre de cordón con 5 ml de RPMI y transferir al tubo de centrífuga mismo. </li><li> Centrifugar las células durante 10 minutos a 400 x g. Aspirar el sobrenadante. </li><li> Resuspender las células en 20 ml de RPMI caliente. Células de capa en 15 ml de Lymphoprep en 50 ml tubo de centrífuga. Centrifugar 40 minutos a 400 x g. </li><li> Cosechar la interfaz que contiene las células mononucleares y se lava en 20 ml de RPMI. Centrifugar durante 10 minutos @ 450 x g. </li><li> Aspirar el sobrenadante y lavar con 20 ml de RPMI. Contar las células y centrifugar durante 5 minutos a 400 x g. </li><li> Aspirar el sobrenadante y volver a suspender las células a 5 x 10 6 células mononucleares / mL de CellGenix DC medios de comunicación (sin suero). Retirar 1 mL para EBV-LCL generación y puesta en 15 ml centrifuge tubo. </li></ol> 2. Generación de células dendríticas (comenzando en el día 0) <ol><li> Placa de 2 ml de células (ya se encuentran en 5 x 10 6 células / ml de medios de comunicación DC) por pocillo en una placa de 6-así. Dejar en incubación durante 1-2 horas. </li><li> Después de 1-2 horas, se lavan las células no adherentes por lavado 3-4 veces con PBS. Recoger las células no adherentes y congelar. Añadir 2 ml / pocillo de medio de DC que contienen 1000 U / ml de IL-4 y 800 U / ml de GM-CSF. </li><li> 3-4 días después de la iniciación DC, reponer la IL-4 y GM-CSF mediante la adición de 100 l / pocillo de medio que contenía una concentración final de 1000U/mL IL-4 y 800 U / ml de GM-CSF. </li><li> En el día 5-6, la cosecha DC raspando el pocillo con una pipeta de transferencia. Contar las células grandes y centrifugar durante 5 minutos @ 400 x g. Aspirar los medios de comunicación y DC resuspender en 2x10 6 células / ml de los medios de comunicación de CC. Añadir 0,5 ml / pocillo de una placa de 24 pocillos. </li><li> Transducir la DC usando el vector Ad5f35pp65 en una celda por MOI de 10 unidades infecciosas. Añadir vector a cada pocillo en una placa de 24 pocillos. Se incuban las células durante 1,5 horas. Después de 1,5 horas, agregar 1,5 ml / pocillo de medio que contiene DC: 1000 U / ml de IL-4, 800 U / ml de GM-CSF, 10 ng / ml de TNF-alfa, 1 mg / ml de PGE-1, 100 ng / mL IL-6, y 10 ng / ml de IL-1beta. Estas citoquinas madurar los países en desarrollo. </li></ol> 3. Generación de EBV-LCL (a partir del día 0) <ol><li> Centrifugar el tubo que contiene las 5 x 10 6 células mononucleares. Añadir 200 l de concentrado EBV B95.8 sobrenadante y 1,8 ml de medio completo (RPMI + 10% de FBS + 2 mM L-glutamina) que contiene ciclosporina (1 ug / ml). </li><li> Alícuota de 100 l de células en 10 pocillos de una placa de 96 pocillos y 200 mu l en 5 pozos. Añadir 100 l de medios de comunicación completa ciclosporina + a los pocillos que contienen sólo 100 l de células. Llenar los pocillos restantes con agua estéril. </li><li> Alimente el semanario LCL y ampliar de una placa de 96 pocillos a una placa de 24 pocillos después de 2 semanas. Después de otra semana, transferir a un matraz T25 y en la transferencia de semana posteriorun matraz T75. En esta etapa se recomienda congelar las alícuotas de LCL. Por lo general se toma 1 mes para generar un número suficiente de LCL. </li></ol> 4. CTL Iniciación - 7 días después del inicio de los DC <ol><li> Los países en desarrollo Harvest e irradiar a los 30 Gy. Lave 4 x y el recuento. Resuspender DC en los medios de células T que contienen suero humano (45% de RPMI, 45% CLICK, 10% de suero humano, 2 mM de L-glutamina) @ 1 x 10 5 DCS / ml. </li><li> Descongelar las células no adherentes de la etapa 2.2. Lavar las células, y contar. Resuspender en 2 x 10 6 células no adherentes / mL en medio de células T que contienen suero humano. Añadir 10 ng / ml de IL-7 e IL-12-, y 5 ng / ml de IL-15. Añadir 1 ml de células por pocillo en una placa de 24 pocillos. Añadir 1 ml de DC por pocillo. Llenar los pocillos vacíos de 24-así placa con agua estéril. </li><li> 7 días después de la iniciación de células T, la alimentación y / o dividir las células con medio de células T que contienen suero humano. </li><li> 9-12 días después de la iniciación de células T, las células T congelar hasta que la LCL están listos. </li><li> ONCE LCL se han expandido y se pasan en matraces T75, la transducción de la LCL por centrifugación durante 5 minutos a 400 x g. Añadir el vector al sedimento de células en una MOI de 100 unidades infecciosas por célula. Incubar durante 1,5 horas. Después de 1,5 horas, resuspender en medio completo a 5 x 10 5 LCL / ml y añadir 2 ml por pocillo en una placa de 24 pocillos. 12 </li><li> Después de 2 días, la cosecha de la LCL e irradiar a los 40 Gy. Lave 4 x y el recuento. Resuspender el LCL en los medios de comunicación de células T que contienen suero humano a 2,5 x 10 5 células / ml. Añadir 1 ml de LCL por pocillo de una placa de 24 pocillos. Además, añadir 5 x 10 6 LCL a un dispositivo de cultivo Grex40 así como 5 ng / ml de IL-15. 13 </li><li> Recoger las células T. Centrifugar durante 5 minutos a 400 x g, aspirar el sobrenadante y se resuspenden en medios de células T que contienen suero humano a 1 x 10 6 células T / mL. Añadir 5 ng / ml de IL-15 y se añade 1 ml / pocillo de células T (un total de 1 x 10 6 células T) para el LCL que ya están en la placa de 24 pocillos. En el Grex40, llevarel volumen total de medios de hasta 30 ml. </li><li> En días 3-4 después de la estimulación, si las células son confluentes después dividir (en la placa) 1:1 y añadir medio fresco que contenía 50 U / ml de IL-2. Si ellos no son confluentes después simplemente aspirar ½ los medios de comunicación y sustituir con medio que contenía 50 U / ml de IL-2. En la Grex40, aspirar el medio de los medios de comunicación, reemplazar con medio fresco, y se añaden 50 U / ml de IL-2. </li><li> En el día 7, repetir como en el paso 4,6 pero el uso de IL-2 en el día de la estimulación no, IL-15. </li></ol> 5. Los resultados representativos Un esquema del protocolo de fabricación GMP aprobado por la FDA se representa en la figura 1. El proceso dura unos 50 días. Los métodos típicos de la generación de virus específicos de las células T expandir pre-existentes de células T de memoria, sin embargo, carece de experiencia en la sangre del cordón virus de las células T y por lo tanto necesitamos vivo prime ingenuo células T ex. Para ello, se utilizan células dendríticas, así como las citoquinas IL-7, IL-12 e IL-15,necesario para la generación de especificidad viral. Después de 3 estimulaciones, el rendimiento debería ser más de 100x10 6 células. Si las células no hay suficientes, estimulaciones adicionales se puede realizar hasta que el número deseado de CTLs están disponibles. La mayoría de estas células debe ser CD3 + con una mezcla de CD4 + y CD8 + células T. No debe ser menos de 15% de células NK (CD3-/CD56 +) y menos de 1% células B CD19 + (Figura 2). El CTL expandido debe reconocer el antígeno pp65 de CMV, hexón y pentón de adenovirus, así como numerosos antígenos de EBV que se expresan en EBV-LCL. Cuando se probó en un ensayo ELISPOT, CTL debería secretar más IFN-? en respuesta a estos antígenos que los antígenos irrelevantes (Figura 3). El CTL también debe lisar virales pulsadas con péptidos tales objetivos blastos PHA. En un ensayo de liberación de 51Cr, CTL lyse debe LCL, CMVpp65 de pulso y Adenovirus hexón y /o objetivos pentón de pulso pero no objetivos pulsadas con péptidos irrelevantes (Figura 4). <img src="/files/ftp_upload/3627/3627fig1.jpg" alt="Figura 1."/> Figura 1. Generación de múltiples virus CTL específicos de sangre del cordón umbilical. Esquemática que muestra todo el proceso de fabricación de CTL a partir de sangre del cordón umbilical. En primer lugar las células mononucleares de sangre de cordón umbilical se aíslan de la fracción de 20% de la unidad de sangre de cordón umbilical fraccionado. Con la excepción de 5x10 6 células que se guardan para la generación de LCL, todas las células se colocaron en placas en medio de células dendríticas durante 1-2 horas, momento en el cual las células no adherentes se recogieron y congelaron. La DC se alimenta entonces DC de medios que contiene IL-4 y GM-CSF. Después de 5 días de cultivo, la DC se maduran y transducidas con un vector adenoviral que contiene el antígeno inmunodominante CMV pp65. En la iniciación, estas DC se combinan con las células no adherentes, así como las citoquinas IL-7, IL-12,y IL-15. En estimulaciones posteriores, el vector adenoviral se utiliza el mismo para transducir EBV-LCL, que se utilizan como células presentadoras de antígeno. IL-15 se utiliza en la segunda estimulación y la IL-2 a partir de entonces. <img src="/files/ftp_upload/3627/3627fig2.jpg" alt="Figura 2."/> Figura 2. Fenotipo de las células T resultante. Se muestra el porcentaje de células vivas incluidas en la puerta de linfocitos. CTL son CD3 + y CD4 + o CD8 +, pero en gran parte CD3-/CD56- y CD19-. <img src="/files/ftp_upload/3627/3627fig3.jpg" alt="Figura 3."/> Figura 3. Funcionalidad de células T. Especificidad de las células T se ensayó por el IFN-γELISPOT. Las células T fueron pulsadas con péptidos solapantes que abarcan toda la proteína de hexón, pentón, pp65, y el antígeno irrelevante 1-IE. CTL solo indica los medios de comunicación solo. LCL autólogas fueron irradiadas y se añadió a 1x10 5 / pocillo. Se muestra el punto medio de la formación de células count de pocillos por triplicado. <img src="/files/ftp_upload/3627/3627fig4.jpg" alt="Figura 4."/> Figura 4. Actividad citolítica de CTL. La capacidad de los CTL resultante para lisar dianas que expresan antígenos virales fue probado en un ensayo de 51 Cr. 51 Cr-autólogas marcadas blastos PHA fueron pulsadas con péptidos solapantes que abarcan el antígeno completo o 51 Cr-LCL autólogas marcadas se cocultivaron con CTL. Después de 4 horas, la liberación gamma se contaron en un contador gamma.

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<html> Las actuales estrategias encaminadas a controlar las infecciones virales después de la TCC puede ser eficaz, pero están asociados con toxicidades significativas, son caros, y no confieren protección a largo plazo contra la infección posterior. De hecho, el uso de algunos fármacos antivirales pueden limitar la expansión de las células T específicas de virus que de otro modo serían de protección. 14 Otra opción es la infusión de las células derivadas del donante T específicos para el virus. ...

<table border="1"><tbody><tr><td> Nombre de reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 21870-076 </td></tr><tr><td> DC medios de comunicación </td><td> CellGenix </td><td> 20801-0500 </td></tr><tr><td> EHAA (Medio de Click) </td><td> Irvine Scientific </td><td> 9195 </td></tr><tr><td> Suero Humano </td><td> Gemini Bio Products </td><td> 100-110 </td></tr><tr><td> Gas Permeable Cultureware 18 </td><td> Wilson-Wolf </td><td> 80040S </td></tr><tr><td> IL-2 </td><td> Chiron (TCH Farmacia) </td><td></td></tr><tr><td> IL-12 </td><td> NCI / CTEP </td><td></td></tr><tr><td> IL-15 </td><td> CellGenix </td><td> 1013-050 </td></tr><tr><td> IL-7 </td><td> De I + D </td><td> AFL207 </td></tr><tr><td> IL-1 beta </td><td> De I + D </td><td> AFL201 </td></tr><tr><td> IL-6 </td><td> CélulaGenix </td><td> 1004-050 </td></tr><tr><td> GM-CSF </td><td> TCH Farmacia </td><td></td></tr><tr><td> IL-4 </td><td> De I + D </td><td> <a href="http://www.rndsystems.com/pdf/AFL204.pdf" target="_blank">AFL204</a> </td></tr><tr><td> TNF-alfa </td><td> De I + D </td><td> AFL210 </td></tr><tr><td> Ad5f35pp65 </td><td> BCM Vector CAGT Instalaciones de Producción </td><td></td></tr><tr><td> Transferencia de plasma con adaptador luer hembra </td><td> Carta Médica </td><td> 89-550-66J </td></tr><tr><td> Lymphoprep </td><td> Nycomed </td><td> 1114550 </td></tr></tbody></table>

expanding cytotoxic t lymphocytes from umbilical cord blood that target cytomegalovirus, epstein-barr virus, and adenovirus

Las infecciones de virus después de un trasplante de células madre se encuentran entre las causas más comunes de muerte, sobre todo después de la sangre del cordón (CB) trasplante (CBT), donde el CB no contiene cantidades apreciables de células T del virus con experiencia que pueden proteger al destinatario de la infección 1. - 4 Nosotros y otros han demostrado que el virus de CTL específico generado a partir de donantes seropositivos y se infunde al destinatario son seguros y protectores. 5-8 Sin embargo, hasta hace poco, virus específicos de las células T no podría ser generado a partir de sangre de cordón, probablemente debido a la ausencia de virus específicos de las células T de memoria. En un esfuerzo para imitar mejor las condiciones en el cebado in vivo de las células T vírgenes, se estableció un método que utiliza CB-derivados de las células dendríticas (DC) transducidas con un vector adenoviral (Ad5f35pp65) que contiene el antígeno inmunodominante CMV pp65, por lo tanto, conducir especificidad de las células T hacia CMV y adenovirus. 9 Al inicio, utilizamos estas máquinasDCs estructurados, así como CB derivados de células T en la presencia de las citoquinas IL-7, IL-12 e IL-15. 10 En la segunda estimulación se utilizó el EBV de células B transformadas, o EBV-LCL, que expresan tanto latente y antígenos para el VEB lítico. Ad5f35pp65-EBV-LCL transducidas se utilizan para estimular las células T en presencia de IL-15 en la segunda estimulación. Estimulaciones posteriores utilizar Ad5f35pp65-EBV-LCL transducidas y IL-2. De 50x10 6 células mononucleares CB somos capaces de generar más de 150 x 10 6 virus específicos de las células T que lisan los impulsos de antígeno-objetivos y liberación de citoquinas en respuesta a la estimulación antigénica. 11 Estas células se fabrican de una manera GMP usando sólo la fracción de 20% de una unidad de sangre de cordón se fraccionó y se han traducido para el uso clínico.

Watch this Scientific Journal Video about Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus at JoVE.com

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

Aquí se describe la primera práctica de fabricación (GMP)-cumple método de producción de virus específicos de los linfocitos T citotóxicos (CTL) de la sangre del cordón umbilical, una fuente de células T predominantemente ingenuo.

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

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

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Immunology and Infection

12.7K vistas.  Baylor College of Medicine. Aquí se describe la primera práctica de fabricación (GMP)-cumple método de producción de virus específicos de los linfocitos T citotóxicos (CTL) de la sangre del cordón umbilical, una fuente de células T predominantemente ingenuo.

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

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

Preparación y uso de VIH-1 en células CD4 infectadas Primaria + T-células como células diana en las células asesinas naturales ensayos citotóxicos

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Investigación

Inmunología e Infección

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

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

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

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

La introducción de la tensión de cizallamiento en el Estudio de la adhesión bacteriana

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Establecimiento de Epstein-Barr transformado al crecimiento líneas celulares linfoblastoides

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

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 ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Aislamiento de los precursores de células B subconjuntos de Sangre del Cordón Umbilical

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

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

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

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

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

Aislamiento y diferenciación de Th17 Naïve linfocitos T CD4

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

Development of an IFN-&#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, antiherpetic prophylaxis is administrated systematically to pediatric UCBT recipients to prevent complications associated with VZV infection, but there is no strong, evidence based consensus that defines its optimal duration. Because T cell mediated immunity is responsible for the control of VZV infection, assessing the reconstitution of VZV specific T cell responses following UCBT could provide indications as to whether prophylaxis should be maintained or can be discontinued. To this end, a VZV specific gamma interferon (IFN-&#947;) enzyme-linked immunospot (ELISpot) assay was developed to characterize IFN-&#947; production by T lymphocytes in response to in vitro stimulation with irradiated live attenuated VZV vaccine. This assay provides a rapid, reproducible and sensitive measurement of VZV specific cell mediated immunity suitable for monitoring the reconstitution of VZV specific immunity in a clinical setting and assessing immune responsiveness to VZV antigens. &#160;

Development of an IFN-&amp;#947; ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

Novel generations of functional assays such as gamma interferon (IFN-&#947;) ELISpot, which detect cytokine production at the single cell level and provide both quantitative and qualitative characterization of T cell responses can be used to assess cell-mediated immune responses directed against varicella zoster virus (VZV).

Desarrollo de un Varicela-Zoster inmunidad mediada por células específicas del virus de IFN-γ ELISPOT ensayo para evaluar la raíz del cordón umbilical de Trasplante de Sangre

Varicella zoster virus (VZV) is a significant cause of morbidity and mortality following umbilical cord blood transplantation (UCBT). For this reason, ...

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 ...

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

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

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

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

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