Este trabajo fue apoyado por subvenciones del NIH (5U01CA137070) y la Sociedad Americana del Cáncer (RSG-09-070-01-LIB), un Premio de Investigación del Cáncer del Instituto Investigador (MK), una Asociación Americana del Corazón beca predoctoral (KM) y el Ministerio español de Educación y Ciencia Becas (APG). Nos gustaría agradecer a la Dr. Nicholas Restifo y el Dr. Zhiya Yu (NIH / NCI) por sus útiles sugerencias para el protocolo.

1. Prepare Construir retrovirales <ol><li> Sub-clon del receptor de células T (TCR) de genes de interés en un vector retroviral (Figura 1, los vectores de ejemplo pMSG 7, pMIGII 5,8, pMXs de Cellbiolabs). El TCR α y β gen de la cadena debe estar en el mismo vector bajo el control del mismo promotor para garantizar la igualdad de expresión. Si el TCR de interés humano, los dominios constantes humanos deben ser reemplazados por los dominios del ratón constante 9. Nuestra observación es que los TCR de cuerpo entero humanos no expresan establemente en el ratón de células-T. </li><li> Preparar ADN de alta calidad utilizando el plásmido Qiagen Maxi Kit de Preparación o producto similar. La concentración del plásmido debe estar en alrededor de 1 mg / mL. </li></ol> 2. Transfección y aislamiento de las células T Día 1 (células de la placa de embalaje) <ol><li> Desalojar de platino-E (Plat-E, Cellbiolabs) células retroviral envases con tripsina-EDTA y neutralizar con DMEM los medios de comunicación. </li><li> Centrifugar las células a 1000 xg durante 5 min. Aspirar el sobrenadante y resuspender el botón celular en DMEM los medios de comunicación. </li><li> Determinar el número de células y diluir las células a 0.6x10 6 / mL. Placa 10 ml de suspensión celular en un 10 mm poli-lisina placa recubierta de cultivo de tejidos y crecer durante la noche en una celda de la incubadora a 37 ° C, 5% de CO 2. </li></ol> El día 0 (transfección) <ol start="4"><li> A la mañana siguiente, examinar las células bajo un microscopio de luz. Las células deben ser aproximadamente el 80% confluente. </li><li> Retire con cuidado los medios de comunicación de la placa. Lave las células una vez con PBS 1x y agregar 10 ml previamente calentada, fresco medios DMEM sin penicilina-estreptomicina (penicilina / estreptomicina). (Nota: penicilina / estreptomicina puede interferir con la transfección) </li><li> Preparar complejos de transfección. Prepare la mezcla de A y B de la siguiente manera: <table border="0"><tbody><tr><td> Mezcla A: El ADN del plásmido </td><td> 9 mg </td></tr><tr><td> PCL-Eco plásmido auxiliar </td><td> 6,3 mg </td></tr><tr><td> OptiMEM </td><td> 1,5 ml </td></tr><tr><td colspan="2"></td></tr><tr><td> Mezcla B: Lipopfectime 2000 </td><td> 60 l </td></tr><tr><td> OptiMEM </td><td> 1,5 ml </td></tr></tbody></table> Incubar A y B por separado durante 5 minutos a temperatura ambiente Mezcla de A y B juntos suavemente e incubar durante 20 minutos a temperatura ambiente para formar complejos de transfección. </li><li> Lentamente por goteo la mezcla (un total de 3 ml) en las células Plat-E. Agite suavemente la placa de un lado a otro para distribuir uniformemente la mezcla de transfección. </li><li> Se incuban las células a 37 ° C / 5% CO 2 en un incubador de células. </li><li> Después de 6-8 horas, sustituya medio con 10 ml de medio DMEM fresco (con penicilina / estreptomicina) para la producción viral. </li></ol> Escudo plato con anticuerpos <ol start="10"><li> Ratón las células T necesitan ser activadas antes de transducción viral. Hay varias formas diferentes para activar las células T. Aquí se utiliza la placa de obligado anticuerpos anti-CD3e y anti-CD28. Prepare una mezcla de anticuerpos de 1 mg / ml de anti-CD3e y 2 mg / ml de anti-CD28 en PBS estéril. </li><li> Prescindir de 250 l de la mezcla de anticuerpos a cada pocillo de una placa de tejidos de 24 pocillos. La placa se puede recubrir toda la noche a 4 ° C o 2 horas a 37 ° C. </li></ol> Preparación de una sola célula de suspensión de bazo de ratón <ol start="12"><li> Cosecha de bazo de ratón y traslado al medio RPMI. </li><li> Colocar el bazo en un colador de la célula. Mash el bazo con un émbolo de la jeringa en una placa de cultivo de 5 cm de tejido. Enjuague las células de la células colador con PBS estéril. </li><li> Centrífuga 1000 xg, 5 min. </li><li> Desechar el sobrenadante. Resuspender el sedimento celular en tampón de lisis ACK (2 ml por bazo). Incubar 2 a 3 minutos a temperatura ambiente. Añadir medio RPMI hasta 20 ml y centrifugar 1000 xg durante 5 min. </li><li> Desechar el sobrenadante. Resuspender el sedimento celular en PBS estéril. Determinar el número de células y centrifugar 1000 xg durante 5 min a 4 ° C. Proceder al aislamiento de las células T. </li></ol> El aislamiento y la activación de las células T de ratón <ol start="17"><li> Magnéticamente identificar a las células de acuerdo con el manual del producto (CD8 + T-cell kit de aislamiento de Miltenyi Biotec). </li><li> Coloque una columna LS (Miltenyi Biotec) en el magnetic campo. Solicitar la suspensión de células etiquetadas en la columna. Recoge el flujo a través de (enriquecido ratón CD8 + células T). Lavar la columna tres veces con tampón 3 ml de acuerdo con el manual del producto y se combinan con las anteriores de flujo continuo. </li><li> Tinción de las células con anticuerpos anti-CD3e y anti-CD8a y analizar por citometría de flujo (Figura 2a). Para una separación típica, ~ 8.10% del total de células pueden ser aisladas. ~ 90% de las células aisladas son CD8 + T-células. </li><li> Centrifugar las células a 1000 xg, 5 min. Eliminar el sobrenadante. Resuspender el botón celular en medio RPMI con recombinante humano IL-2 (rhIL2, 20 ng / ml) a 1x10 6 / mL. </li><li> Justo antes de la adición de las células, eliminar la solución de anticuerpos de la placa (preparado previamente en 2.11). Enjuague cada pocillo con PBS estéril y PBS quitar. </li><li> Añadir 1 ml de la suspensión celular a cada pocillo. Poner la placa a 37 ° C / 5% CO 2 en una celda de la incubadora. </li></ol> 3. (Día 2 y Día 3) La infección de células T activadas <ol><li> Después de 2 días de producción de virus, el medio de la placa celular Plat-E debe ser de color amarillo. Transferir el sobrenadante viral a un tubo cónico de 15 ml. Agregar 10 ml de medio DMEM fresco a la plancha para la producción de más virus (Opcional). </li><li> Centrifugar el sobrenadante del virus a 1000 xg durante 5 min para eliminar los restos células. Transferir cuidadosamente el sobrenadante del virus a un nuevo tubo. Deje un poco de líquido en la parte inferior del tubo y no molestar a los restos células. </li><li> Recoger las células T activadas a partir de la placa en un tubo cónico de 50 ml. Determinar el número de células. Ahorra un poco de las células en un tubo separado para ser utilizado como negativos (sin transducidas) control. Centrifugar a 1000 xg durante 5 minutos y desechar el sobrenadante. </li><li> Resuspender el botón celular en virus sobrenadante a 10 6 células por 1 ml con 20 ng / mL rhIL2 y 10 mg / mL de sulfato de protamina. Añadir 1 ml de suspensión celular a cada pocillo de una placa y 24. Resuspender las células en el tubo de control en el medio RPMI a la misma densidad celular con la misma cantidad de sulfato de protamina y rhIL2. Añadir a las células a la misma placa. </li><li> Envuelva el plato con film de plástico. Centrifugar 90 minutos a 2000 xg, a 32 ° C sin freno. </li><li> Después de la centrifugación, quite la película plástica. Añadir 1 ml de medio fresco RPMI con 20 ng / mL rhIL2 a cada pocillo. Colocar la placa de nuevo a la incubadora. </li><li> (Opcional) Mayor nivel de expresión de TCR se puede lograr mediante la recopilación de los virus sobrenadante y repetir el procedimiento de la infección en el día 3. </li></ol> 4. Expansión de las células y la evaluación de la eficiencia de transducción <ol><li> Examinar la transducción de células T al día. División de las células en proporciones 01:03, cuando sea necesario en medio RPMI rhIL2. No deje que un crecimiento excesivo de las células (medio se vuelve amarilla). </li><li> En el día 6 o 7 días, teñir las células con el anticuerpo y tetrámero MHC específicos para el TCR para evaluar la expresión de TCR en las células T a la superficie. Uso de la ONU-transduced las células T como de control (Figura 2c). </li><li> La transducción de células T están preparados para más aplicaciones posteriores. </li></ol> 5. Resultados representante A menudo es ventajoso para purificar subconjuntos de células T antes de la transducción de esplenocitos, aunque pueden ser transducidas sin purificación. De alta pureza subconjuntos de células T se pueden obtener utilizando comerciales bolas magnéticas (Figura 2a). Para evaluar el nivel de expresión de TCR en las células T, anticuerpos específicos para el TCR alfa o beta cadenas se pueden utilizar. Por lo general 30% al 80% de las células transducidas pueden expresar TCR (Figura 2b) en función de los genes de TCR y los títulos de virus. MHC específicos tetrámero para el TCR también se puede utilizar para verificar a fondo la expresión funcional del TCR (Figura 2c). <img src="/files/ftp_upload/2307/2307fig1.jpg" alt="Figura 1" /> Figura 1. Ejemplo de gen del receptor de células T construcción de sub-clonado en un vector retroviral. Alpha de longitud completa y los genes de la cadena beta están unidos por un conector de auto-2A escindibles péptido 8. <img src="/files/ftp_upload/2307/2307fig2.jpg" alt="Figura 2" /> Figura 2. Ejemplo de éxito en el aislamiento y la transducción de las células T de ratón. (A) células T CD8 fueron aislados a partir de esplenocitos con CD8a + T celda de aislamiento Kit (Miltenyi Biotec) y teñidas con anticuerpos anti-CD3e y anti-CD8a. Aisladas células T CD8 fueron transducidas con R6C12 TCR específico para humanos gp100: 209 -217 4 de péptidos y teñidas con anti-anticuerpos humanos vbeta 8 (b) y HLA-A2kb gp100 tetrámero (c).

<ol>
	<li>Krogsgaard, M., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+T+cells+'see'+antigen.">How T cells 'see' antigen.</a> Nat. Immunol. 6, 239-245 (2005).</li><li>Berg, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen/MHC-specific+T+cells+are+preferentially+exported+from+the+thymus+in+the+presence+of+their+MHC+ligand.">Antigen/MHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand.</a> Cell. 58, 1035-1046 (1989).</li><li>Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+antagonist+peptides+induce+positive+selection.">T cell receptor antagonist peptides induce positive selection.</a> Cell. 76, 17-27 (1994).</li><li>Yu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poor+immunogenicity+of+a+self/tumor+antigen+derives+from+peptide-MHC-I+instability+and+is+independent+of+tolerance.">Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance.</a> J. Clin. Invest. 114, 551-559 (2004).</li><li>Holst, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+T-cell+receptor+retrogenic+mice.">Generation of T-cell receptor retrogenic mice.</a> Nat Protoc. 1, 406-417 (2006).</li><li>Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+regression+in+patients+after+transfer+of+genetically+engineered+lymphocytes.">Cancer regression in patients after transfer of genetically engineered lymphocytes.</a> Science. 314, 126-129 (2006).</li><li>Hawley, R. G., Lieu, F. H., Fong, A. Z., Hawley, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+retroviral+vectors+for+potential+use+in+gene+therapy.">Versatile retroviral vectors for potential use in gene therapy.</a> Gene Ther. 1, 136-138 (1994).</li><li>Szymczak, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+multi-gene+deficiency+in+vivo+using+a+single+'self-cleaving'+2A+peptide-based+retroviral+vector.">Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.</a> Nat. Biotechnol. 22, 589-594 (2004).</li><li>Cohen, C. J., Zhao, Y., Zheng, Z., Rosenberg, S. A., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+antitumor+activity+of+murine-human+hybrid+T-cell+receptor+(TCR)+in+human+lymphocytes+is+associated+with+improved+pairing+and+TCR/CD3+stability.">Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability.</a> Cancer Res. 66, 8878-8886 (2006).</li><li>Letourneur, F., Malissen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+a+T+cell+hybridoma+variant+deprived+of+functional+T+cell+receptor+alpha+and+beta+chain+transcripts+reveals+a+nonfunctional+alpha-mRNA+of+BW5147+origin.">Derivation of a T cell hybridoma variant deprived of functional T cell receptor alpha and beta chain transcripts reveals a nonfunctional alpha-mRNA of BW5147 origin.</a> Eur. J. Immunol. 19, 2269-2274 (1989).</li><li>Bendle, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lethal+graft-versus-host+disease+in+mouse+models+of+T+cell+receptor+gene+therapy.">Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy.</a> Nat. Med. 16, 565-570 (2010).</li></ol>

Varios pasos son críticos para lograr resultados óptimos. Plat-E línea celular de empaquetamiento se debe mantener en buenas condiciones. Si es necesario, las células pueden ser pasados ​​un par de veces en medio DMEM con 1 mg / ml puromycin, mg 10 / blasticidin ml para evitar la pérdida de expresión retroviral estructura de proteínas. En el día de la transfección, las células deben ser confluentes ~ 80%. Muy pocas células o demasiadas puede reducir el título del virus. La calidad virus se puede comprobar mediante ensayos con líneas celulares que pueden ser fácilmente transducidas. Desde TCR requiere CD3 complejos que se expresa en la superficie celular, que utilizan de forma habitual 58α-/β- células de hibridoma 10 (una línea celular que no expresa endógeno α TCR o la cadena β, pero no expresa complejo CD3). Se obtiene casi el 100% de eficiencia de transducción de células de hibridoma cuando transducción de las células con 1 ml de sobrenadante de virus. Finalmente, las células T necesitan ser activados y proliferan porque retrovirus sólo puede infectar a las células en división activa. El método que aquí se presenta para la producción de antígenos específicos de células T tiene varias limitaciones. En primer lugar, es difícil alcanzar el 100% de eficiencia de la transducción de principal del ratón las células-T. Una parte de las células no expresan el TCR de interés. En segundo lugar, el TCR α transducidas y las cadenas de β puede mispair con TCR endógeno 9, que puede reducir el nivel de expresión del TCR de interés. Además, el TCR mispaired puede causar autoinmunidad en vivo 11. Finalmente, sólo hay un tiempo limitado de la transducción de células T puede ser utilizado. Después de aproximadamente una semana, las células sufren apoptosis a menos que se re-estimulado. Sin embargo, las células pueden agotarse y perder con funciones repetitivas re-estimulaciones.

Los medios de comunicación: Medio DMEM (calor DMEM +10% FBS inactivado + penicilina / estreptomicina + L-Glu + + piruvato de sodio no aminoácidos esenciales). Medio DMEM sin penicilina / estreptomicina (Igual que el medio DMEM, pero sin la adición de penicilina / estreptomicina). Medio RPMI (calor RPMI 10% FBS inactivado + L-Glu + no aminoácidos esenciales + β-Mercaphoethanol + penicilina / estreptomicina + piruvato sódico).

retroviral transduction of t-cell receptors in mouse t-cells

Receptores de células T (TCR) juegan un papel central en el sistema inmunológico. TCR en la superficie de las células T pueden reconocer específicamente antígenos peptídicos presentados por las células presentadoras de antígeno (APC) 1. Este reconocimiento conduce a la activación de las células T y una serie de resultados funcionales (por ejemplo la producción de citoquinas, causando la muerte de las células diana). La comprensión del papel funcional de la TCR es fundamental para aprovechar el poder del sistema inmunológico para tratar una variedad de enfermedades relacionadas con la inmunología (por ejemplo el cáncer o la autoinmunidad). Es conveniente estudiar TCR en modelos de ratón, que se puede lograr de varias maneras. Hacer TCR transgénicos modelos de ratón es costoso y requiere mucho tiempo y en la actualidad sólo hay un número limitado de ellos dispone de 2-4. Por otra parte, los ratones con antígenos específicos de células T pueden ser generados por la médula ósea quimera. Este método también toma varias semanas y requiere de experiencia 5. Transducción retroviral de TCR en ratón in vitro activa las células T es un método rápido y relativamente fácil de obtener las células T de la deseada péptido-MHC especificidad. De antígenos específicos de células T se pueden generar en una semana y se utiliza en las aplicaciones posteriores. El estudio de las células T transducidas también tiene aplicación directa a la inmunoterapia humanos, como la transferencia adoptiva de células T humanas transducidas con el antígeno específico de TCR es una nueva estrategia para el tratamiento del cáncer 6. Aquí se presenta un protocolo para la transducción retroviral TCR en activa in vitro de las células T de ratón. Humanos y los genes del ratón TCR se puede utilizar. Retrovirus llevar genes específicos TCR se generan y se utilizan para infectar las células T de ratón activados con anticuerpos anti-CD3 y anti-CD28. Después de la expansión in vitro, las células T transducidas son analizadas por citometría de flujo.

Watch this Scientific Journal Video about Retroviral Transduction of T-cell Receptors in Mouse T-cells at JoVE.com

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

Se presenta un protocolo para producir antígenos específicos de células T de ratón usando la transducción retroviral

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 ...

retroviral-transduction-of-t-cell-receptors-in-mouse-t-cells

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

34.8K vistas.  New York University School of Medicine. Se presenta un protocolo para producir antígenos específicos de células T de ratón usando la transducción retroviral

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

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

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

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

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

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

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

Investigación

Inmunología e Infección

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 ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

La transducción adenoviral de células T CD4 + naïve a estudiar la diferenciación Treg

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

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 ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Ratón Naïve CD4<sup&gt; +</sup&gt; Aislamiento de células T y<em&gt; In vitro</em&gt; Diferenciación en subconjuntos de células T

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Retroviral transducción de células progenitoras de médula ósea para generar las células T del receptor Retrogenic ratones

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

Generando<em&gt; De Novo</em&gt; T humanas receptores de células específicas de antígeno por retroviral Transducción de Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Retroviral transducción de células T colaboradoras como un enfoque genético para estudiar mecanismos que controlan su diferenciación y función

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

En Vivo Aumento de la Inducción de Células T Regulatorias Gut-Homing

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

Aislamiento de células CD8 + T residentes en el riñón delratón para el análisis de citometría de flujo

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...