Los autores desean dar las gracias a Andre Han y Yen Lawrence para la asistencia técnica, y los miembros del laboratorio de Jenkins en busca de ayuda en el desarrollo de este protocolo.

1. Aislamiento de células del tejido linfoide <ol><li> Añadir 1 ml de hielo frío cEHAA (EHAA + 10% de FBS, penicilina / estreptomicina, gentamicina, 2 mM de L-glutamina, 55 mM de 2-mercaptoetanol) u otro medio equivalente de las células T, a una placa de cultivo de 60 mm que contiene un pequeño cuadrado de 100 micras de malla de nylon y colocar en hielo. </li><li> Eutanasiar ratón. </li><li> Retire el bazo y como muchos nodos linfáticos fácilmente accesibles como sea posible. Estos deben incluir al menos las inguinales, axilares, ganglios linfáticos cervicales, braquial, y mesentérica. Colocarlos en la parte superior de la malla de nylon en la placa de cultivo. </li><li> Usando la parte superior plana cerrada de un tubo de microcentrífuga de 1,5 ml, suavemente mezcla el tejido linfoide sobre la malla de nylon para liberar a los linfocitos. Añadir otra ml de 1 cEHAA y pipetear hacia arriba y hacia abajo para trabajar las células en una suspensión. Transferir las células a través de otra pieza de malla de nylon coloca sobre la parte superior de un tubo de centrífuga de 15 ml de polipropileno. Enjuagar la cápsula y de malla con otro 1 ml de hielo frío cEHAA, poOling los volúmenes en el mismo tubo. Repetir para conseguir un volumen de suspensión celular final de 4 ml. </li><li> Añadir tampón clasificador de frío (PBS + 2% de FBS, 0,05% de azida) hasta un volumen final de 15 ml y centrifugar el tubo durante 5 min a 300 xg, 4 ° C. </li><li> Aspirar con cuidado el sobrenadante, asegurándose de que no haya gotas de líquido se quedan en los lados del tubo. Resuspender el sedimento celular en Fc bloque (clasificador de tampón + 2.4G2 anticuerpo) a un volumen final igual a aproximadamente el doble de la del propio gránulo. Por ejemplo, el bazo y nódulos linfáticos de un ratón naive por lo general produce un sedimento celular de aproximadamente 100 l. En este caso, añadir 100 ml de bloque Fc para llevar el volumen a 200 l. Si un alto grado de aglutinación de células se ha producido, retirar cuidadosamente el agregado celular en este punto con una punta de pipeta. </li></ol> 2. Tetramer tinción <ol><li> Añadir PE-o APC marcada con pMHC tetrámero a una concentración final de 10 nM (o concentración empíricamente optimizado). </li><li> Mix and incubaciónTE durante 1 hora a temperatura ambiente (o tiempo empíricamente optimizado y temperatura). </li><li> Añadir tampón clasificador de frío hasta un volumen de 15 ml y centrifugar el tubo durante 5 min a 300 xg, 4 ° C. Mantener las muestras en hielo oa 4 ° C a partir de ahora. </li><li> Aspirar con cuidado el sobrenadante, asegurándose de que no haya gotas de líquido se quedan en los lados del tubo. Resuspender el sedimento celular en tampón clasificador para un volumen final de 200 l. Para el enriquecimiento tetrámero doble, resuspender hasta un volumen final de 150 l. </li></ol> 3. Enriquecimiento magnética <ol><li> Añadir 50 l de microperlas Miltenyi anti-PE o anti-APC. Para el enriquecimiento tetrámero doble, se añaden 50 l de ambos. </li><li> Mezclar e incubar durante 20 min a 4 ° C. </li><li> Añadir tampón clasificador de frío hasta un volumen de 15 ml y centrifugar el tubo durante 5 min a 300 xg, 4 ° C. </li><li> Mientras se espera, configure una columna Miltenyi LS magnético en un imán midiMACS o QuadroMACS. Posicionar un abierto 15 ml de polipropileno en un tubo de centrífugaacumular directamente debajo de la columna. </li><li> Añadir 3 ml de tampón clasificador a la parte superior de la columna, lo que permite que se drene en el tubo de 15 ml. </li><li> Colocar un nylon 100 micras de malla cuadrada en la parte superior de la columna. </li><li> Cuando las células han terminado de hilado, aspirar con cuidado el sobrenadante y resuspender el sedimento en 3 ml de tampón clasificador. </li><li> Transferir la suspensión celular a través de la malla de nylon en la parte superior de la columna. </li><li> Cuando la suspensión de células se haya vaciado completamente en la columna, enjuagar el tubo original con otro ml de tampón 3 clasificador y transferir el tampón a través de la malla de nylon en la columna, lavar la malla en el proceso. Deseche la malla de nylon. </li><li> Cuando la memoria intermedia se haya vaciado completamente en la columna, añadir otro 3 ml de tampón clasificador a la columna. </li><li> Repetir el paso 10 para un total de 3 x 3 lavados ml. </li><li> Retirar la columna del imán y colocada en un nuevo tubo de centrífuga de 15 ml de polipropileno. </li><li> Añadir 5 ml de tampón clasificador para tél columna. </li><li> Inmediatamente inserte el émbolo en la parte superior de la columna y en un movimiento continuo, empuje el émbolo hacia abajo, forzando el tampón de la columna en el tubo. </li><li> Centrifugar los tubos que contenían la fracción eluida unida y la fracción no unida de flujo a través durante 5 min a 300 xg, 4 ° C. </li><li> Aspirar con cuidado el sobrenadante de la fracción unida, asegurándose de que no haya gotas de líquido se quedan en los lados del tubo. Resuspender el sedimento celular en tampón clasificador para un volumen final de 95 l exactamente. Aspirar y volver a suspender la fracción no unida a un volumen final de 2 ml. </li></ol> 4. Citometría de Flujo <ol><li> Para cada fracción, quitar 5 l y añadir a 200 l de perlas de conteo (ajustado a una concentración de 200.000 / ml en tampón clasificador) en un tubo de 5 ml FACS. De lado a 4 ° C para su posterior análisis. Para ahorrar tiempo, los granos pueden ser pre-alícuotas a tubos etiquetados antes del comienzo del experimento. </li><li> Preparar amaster mezcla de anticuerpos para teñir los marcadores de superficie en las células (Tabla 1). Para ahorrar tiempo, esto se puede hacer antes del comienzo del experimento. </li><li> Para la fracción unida, añadir una dosis de cóctel de anticuerpos directamente al ~ 90 l de células en el tubo. Si el análisis de la fracción no unida se desea, transferir 90 l de células a un tubo de 5 ml FACS y añadir una dosis de cóctel de anticuerpos. </li><li> Crear un panel de controles de compensación de un solo color para el citómetro de flujo. Para cada fluorocromo a utilizar, mezclar 50 l de células sobrantes fracción no unida en un tubo de 5 ml FACS con 1 l de anticuerpo anti-CD4 conjugado con el fluorocromo correspondiente. Recuerde que debe establecer un control sin mancha también. </li><li> Agitar e incubar las muestras durante 30 min a 4 ° C. </li><li> Añadir 5 ml de tampón clasificador a cada tubo y centrifugar durante 5 min a 300 xg, 4 ° C. </li><li> Para la fracción unida, aspirar con cuidado el sobrenadante y resuspender las células en 200 l de buf clasificadorfer. Transferir las células a un microtubo de 1,2 ml FACS. Enjuague el tubo con otro 200 l de tampón clasificador y la piscina en el mismo microtubo. Si los agregados celulares son evidentes, pasar las células a través de un filtro de 50 micras. </li><li> Para la fracción no unida y controles de compensación, decantar o aspirar el sobrenadante y se resuspende en 2 ml de tampón clasificador. </li><li> Configurar el citómetro de flujo utilizando los controles de compensación de un solo color. Seleccione el lado de dispersión de ancho (SSC-W) como un parámetro adicional que desee grabar. </li><li> Analizar las muestras teñidas utilizando una secuencia de puertas de inclusión sucesivas para identificar CD4 + o CD8 + células T como se ilustra en las Figuras 1 y 2. Para las fracciones consolidados, recoger las células tanto como sea posible hasta un máximo de 2.000.000 de eventos totales. Para las fracciones no unidas, recoger 1.000.000 eventos totales. Mantener la velocidad de adquisición en o debajo de 3.000 eventos por segundo. </li><li> Uso de los ajustes de la máquina misma, analizar las muestras de conteo de cuentas. Recoge un total de 10.000 eventos. &lt;/ Li&gt; <li> Guarde todos los datos como archivos de FCS. </li></ol> 5. Análisis de Datos <ol><li> Analizar FCS archivos de datos para las muestras de recuento de talón utilizando software FlowJo. Parcela dispersión frontal por FITC y configurar una puerta aroung las perlas de recuento (véanse las Figuras 1 y 2). Determinar el número total de recuento de granos detectados en cada muestra. Restar de la cantidad total de eventos recogidos para determinar el número de eventos celulares recogidas. </li><li> Calcule el número total de todas las células en cada muestra utilizando las ecuaciones descritas en el Cuadro 1. </li><li> Analizar FCS archivos de datos de las muestras teñidas fracción unida y no unida. Configurar una secuencia de puertas de inclusión sucesivas para identificar + linfoide, lo lado de ancho de dispersión, vertimiento, CD3 +, CD4 + o CD8 +, tetrámero + células T en cada muestra como se ilustra en las Figuras 1 y 2. </li><li> Multiplicar el número total de células en la muestra por las frecuencias de cada una de las puertas de inclusión utilizados para definir CD4 + tetrámero+ O CD8 + tetrámero + células para calcular el número total de epítopos de células T específicas (Cuadro 1). </li></ol>

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No hay conflictos de interés declarado.

El pMHC tetrámero basado en el método de enriquecimiento de células presentada por este protocolo es una poderosa herramienta para el estudio de epítopo de células T específicas de repertorios de células T endógenas. El uso de tetrámeros de pMHC permite la detección de células T específicos para el epítopo basado directamente en la capacidad de sus TCR para unir ligandos cognados pMHC. El enriquecimiento proporciona un nivel de sensibilidad de tal manera que las poblaciones extremadamente raros de antígeno...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Reactivo</strong</td&gt;<td<strong&gt; Vendedor</strong</td&gt;<td<strong&gt; El número de catálogo</strong</td&gt;</tr&gt;<tr&gt;<td&gt; PE o APC conjugado tetrámero pMHC (o multímero)</td&gt;<td&gt; Fabricado por investigador, obtenido a partir del núcleo NIH tetrámero, o adquiridos de fuentes comerciales</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-PE microbolas magnéticas conjugadas</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-048-801</td&gt;</tr&gt;<tr&gt;<td&gt; Anti-APC microesferas magnéticas conjugadas</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-090-855</td&gt;</tr&gt;<tr&gt;<td&gt; Ls columnas magnéticas</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-042-401</td&gt;</tr&gt;<tr&gt;<td&gt; MidiMACS o imán QuadroMACS</td&gt;<td&gt; Miltenyi</td&gt;<td&gt; 130-042-302 o 130-090-976</td&gt;</tr&gt;<tr&gt;<td&gt; Cuentas de recuento celular</td&gt;<td&gt; Life Technologies</td&gt;<td&gt; PCB-100</td&gt;</tr&gt;</tbody&gt;</table

peptide:mhc tetramer-based enrichment of epitope-specific t cells

Una necesidad básica para los investigadores que estudian la inmunidad adaptativa con modelos experimentales in vivo es la capacidad de identificar las células T en función de su receptor de antígeno de células T (TCR) especificidad. Muchos métodos indirectos están disponibles en el que una población mayor de células T se estimularon in vitro con un antígeno específico y el epítopo de células T específicas se identifican a través de la medición de una respuesta funcional, tal como la proliferación, producción de citoquinas, o la expresión de marcadores de activación 1. Sin embargo, estos métodos sólo identificar epítopos de células T específicas que presentan una de las muchas posibles funciones, y que no son lo suficientemente sensibles como para detectar epítopos de células T específicas en las frecuencias precursoras ingenuas. Una alternativa popular es el modelo de transferencia adoptiva TCR transgénico, en la que las células T monoclonales de un ratón TCR transgénico se siembran en hosts histocompatibles para crear una población grande precursor de células T específicos para el epítopo que pueden ser easiLy seguido con el uso de un anticuerpo marcador congenic 2,3. Mientras potente, este método adolece de artefactos experimentales asociados con la frecuencia no fisiológica de las células T con especificidad para un epítope 4,5 sola. Además, este sistema no se puede utilizar para investigar la heterogeneidad funcional del epítopo específicos de clones de células T dentro de una población policlonal. La forma ideal para estudiar la inmunidad adaptativa debe implicar la detección directa de epítopo de células T específicas del repertorio de células T endógeno utilizando un método que distingue la especificidad TCR únicamente por su unión a péptido cognado: complejos de MHC (pMHC). El uso de tetrámeros y citometría de flujo pMHC logra esto 6, pero se limita a la detección de poblaciones de alta frecuencia de células T específicos para el epítopo que sólo se encuentran siguiente inducida por antígeno expansión clonal. En este protocolo, se describe un método que coordina el uso de tetrámeros pMHC y magnetic tecnología de enriquecimiento de células para permitir la detección de células de frecuencia extremadamente baja T específicos para el epítopo de los tejidos linfoides de ratón 3,7. Con esta técnica, se puede realizar un seguimiento integral enteros específicos de epítopo poblaciones endógenas de las células T en ratones en todas las etapas de la respuesta inmune.

La Figura 1 muestra representativas parcelas de citometría de flujo de pMHCII tetrámero bazo enriquecidos y muestras de ganglios linfáticos de ratones sin tratamiento previo, mientras que la Figura 2 muestra los datos representativos de ratones previamente inmunizados con el péptido relevante + CFA. Gating serie elimina eventos no deseados autofluorescentes y otro a partir del análisis de las células CD4 + T poblaciones celulares. El CD8 + población de células T sirve como un co...

Watch this Scientific Journal Video about Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells at JoVE.com

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

Este protocolo describe el uso de péptido: MHC tetrámeros y microperlas magnéticas para aislar poblaciones de baja frecuencia de células T específicos para el epítopo y analizarlas por citometría de flujo. Este método permite el estudio directo de sus propias poblaciones de células T de interés de<em&gt; In vivo</em&gt; Los sistemas experimentales.

Péptido: MHC tetrámero basado en el enriquecimiento de células T específicos para el epítopo

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

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17.8K vistas.  Massachusetts General Hospital and Harvard Medical School. Este protocolo describe el uso de péptido: MHC tetrámeros y microperlas magnéticas para aislar poblaciones de baja frecuencia de células T específicos para el epítopo y analizarlas por citometría de flujo. Este método permite el estudio directo de sus propias poblaciones de células T de interés de In vivo Los sistemas experimentales.

Video: Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Transducción retroviral de receptores de células T en ratones las células T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

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&#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 ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

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

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

Diferenciación dirigida de células madre pluripotentes inducidas hacia los linfocitos T

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

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac Transposón modificación del sistema de células T humanas primarias

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

Aislamiento de doble negación células T αβ del Riñón

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

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 ...

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

Enriquecimiento de la célula automatizada de citomegalovirus células T específicas para aplicaciones clínicas utilizando el Sistema de citoquinas de captura

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

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 ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

Detección y enriquecimiento de Rare células B específicas de antígeno para el análisis del fenotipo y la función

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...