Los autores desean agradecer al Dr. Boris Reizis del Departamento de Microbiología e Inmunología en la Universidad de Columbia útil para los debates. También queremos agradecer a la Dra. Margaret Bynoe, Dr. Rodman Getchell y Mahamed Deequa del Departamento de Microbiología e Inmunología de la Facultad de Medicina Veterinaria de la Universidad de Cornell para la asistencia técnica con la citometría de flujo y Lu Huang para obtener ayuda con la edición de vídeo. Los autores también desean agradecer a los miembros de Duhamel y los laboratorios de Weiss para su revisión crítica de la producción de manuscritos y de vídeo, y Yazinski Stephanie Weiss en el laboratorio de reproducción y genotipo p53 - / - ratones. Los experimentos con animales se realizaron de acuerdo con las directrices y normas establecidas por Cornell Cuidado de Animales Institucional de la Universidad y el empleo. Esta investigación fue financiada en parte con fondos proporcionados por el Departamento de Agricultura de EE.UU., Estado de Investigación Cooperativa, Educación y Servicio de Extensión, Salud Animal y el Programa de Investigación de Enfermedades (para el GED y AMR) y las subvenciones del NIH R03 HD058220 y R01CA108773 (a RSW). El video fue producido utilizando en instalaciones de la casa por el personal de los laboratorios de Duhamel &amp; Weiss.

1. La disección del tumor <ol><li> Para la disección del tumor, una toalla de papel o paño quirúrgico desechable se coloca en la plataforma de la disección y rociado con etanol al 70%. </li><li> Los ratones de este protocolo son habitualmente sacrificados con CO 2. Coloque el ratón sacrificados en la plataforma de la disección y el spray ambos lados del animal con etanol. El ratón se mantiene en su lugar en la almohadilla de la disección a través de cuatro o más alfileres inserta a través de las piernas y los roció abundantemente con etanol al 70%. </li><li> La piel que cubre la línea media se corta desde el pubis hasta la base de la cabeza con unas tijeras estériles, evitando al mismo tiempo los músculos subyacentes y penetrar en las cavidades del cuerpo. </li><li> Abrir la cavidad abdominal con un nuevo par de tijeras y pinzas estériles, teniendo cuidado de no dañar los órganos internos. </li><li> Abrir la cavidad torácica por disección y desviar la caja torácica para revelar el tumor, sin dañar los vasos sanguíneos. </li><li> Separar el tumor de otros órganos, y la transferencia de una pieza de aproximadamente el 40-50% del total del tumor (aproximadamente de 1,5 x 0,8 x 0,8 cm) en PBS frío se mantienen en hielo. </li><li> La parte restante del tumor se pueden guardar para otros ensayos y una necropsia completa puede llevarse a cabo en este momento. </li></ol> 2. El cultivo de células del linfoma <ol><li> Rocíe el tubo que contiene el tumor disecada con un 70% de etanol y la transferencia de un gabinete de bioseguridad. </li><li> Agregar 10 ml de medio de cultivo estéril a un 150 mm 2 placa de Petri. </li><li> La transferencia de la pieza de tumor en la placa de Petri por succión suave con una pipeta. </li><li> Doble dos estéril aguja de calibre 18 frente a la cara biselada hacia fuera. </li><li> Disociar el tumor mediante las agujas dobladas. </li><li> Agite el plato, golpear suavemente y coloque en un ángulo para recoger la suspensión celular a un lado, evitando al mismo tiempo grandes trozos de tumor. </li><li> Aspirar la suspensión celular y transferir a un tubo cónico de 50 ml, evitando grandes trozos de tumor. </li><li> Lavar la placa con 10 ml de medio y de transferencia en el mismo tubo, evitando grandes trozos de tumor. </li><li> Mezcle la suspensión celular compuesto por linfocitos y células del estroma con suavidad y la transferencia de 5 ml en un matraz de tejido T-25 y la cultura de 10 ml en un T-75 frasco de cultivo de tejidos. Hacer girar suavemente cada vial. </li><li> Use la suspensión celular restante para hacer diluciones 2, 4, 10100 y 1000 veces en 2,5 ml de volumen final (el cultivo de células en diferentes densidades, utilizando varios tipos de recipientes de cultivo maximiza la probabilidad de establecer una línea de células T). </li><li> Placa de 2,0 ml de cada dilución en pocillos individuales de una placa de 6 pocillos. Remover con suavidad la placa. </li><li> Se incuban las células durante 48 h en atmósfera de CO 2 humidificado en el aire. </li></ol> 3. La alimentación de las células del linfoma <ol><li> Después de 48 h de incubación, añadir 5 ml de medio en el matraz T-25 y 10 ml de medio en el matraz T-75 a lo largo de la pared con la mínima perturbación de las células. </li><li> Añadir 2 ml de medio en cada pocillo de la placa de 6 pocillos a lo largo de la pared con la mínima perturbación de las células. </li><li> Se incuban las células durante otras 72 h. </li></ol> 4. Pases las células del linfoma <ol><li> Aprobación inicial <ol><li> Al final del quinto día de incubación, las células se pasan en una proporción de 1:1, mientras que conserva los frascos originales y las placas (el mantenimiento de la densidad de las células a una concentración de aproximadamente 3x106 / ml durante los primeros pasos promueve el establecimiento exitoso de una línea celular continua). <ol class="lalpha"><li> Para el frasco T-25, la transferencia de 5 ml de suspensión celular en un nuevo frasco que contenía 5 ml de medio. Añadir 5 ml de medio en el matraz original. </li><li> Para el frasco T-75, la transferencia de 10 ml de suspensión celular en un nuevo frasco contiene 10 ml de medio. Agregar 10 ml de medio en el frasco original. </li><li> Para la placa de 6 pocillos, la transferencia de 2 ml de suspensión celular de cada pozo en los pozos correspondientes de una nueva placa que contiene 2 ml de medio en cada pocillo. Añadir 2 ml de medio en cada pocillo de los pozos original. </li></ol></li><li> Incubar todos los frascos y las placas de un adicional de 72 h. En nuestra experiencia la mayoría de líneas celulares establecidas a partir de estos pasajes una cultura (la presencia de agregados celulares de baja adherencia de suspensión sobre las células del estroma se adjunta una indicación temprana de una línea celular establecida). </li><li> Para los cultivos con menos o ningún agregados suspensión de baja adherencia celular, alimentar a los frascos o placas con cuidado reemplazando aproximadamente el 50% de la media cada 72 h. Para evitar la eliminación de las células de la cultura original, recipientes de cultivo se dejan en pie y en reposo durante al menos 5 minutos a que el sedimento células en la parte inferior por gravedad antes de la aspiración suave del medio sobrenadante. </li></ol></li><li> El mantenimiento de líneas celulares establecidas <ol><li> Las líneas celulares establecidas se pasan en nuevos frascos y las placas. Para T-25 y T-75 frascos, añadir 5 ml del original o depaso de una suspensión en 5 ml de medio. Para placas de 6 pocillos, agregar 3 ml de la suspensión celular original o un paso en 7 ml de medio en un frasco T-25. Alimentar y conservar el original o el paso de una cultura mediante la sustitución del volumen extraído con medio de cultivo fresco. </li><li> Paso las líneas celulares establecidas en los nuevos T-25 frascos. Las líneas de células tienen un tiempo de duplicación de 18 a 24 h. Los cultivos se mantienen rutinariamente a una densidad de 1-3x106 / ml y se pasaron cada 3 días. Añadir el volumen necesario de la cultura pasaje anterior al nuevo medio para hacer hasta 10 ml. Alimentar y mantener la cultura original. </li><li> Después de unos cuantos pasajes, las células no adherentes se auto sostenerse sin las células del estroma adherentes y un número menor de células adherentes se realizan a través de cada paso. Más allá de este punto, las culturas se pueden mantener en cultivo de tejidos no tratados T-25 frascos. </li></ol></li></ol> 5. Congelación y recuperación de líneas celulares de linfoma <ol><li> Las líneas celulares se congelan en el recién preparada RPMI 1640 que contenía FBS 20% y el 10% de DMSO. </li><li> Las células de un cultivo de 48 h de edad en el frasco T-25 (aproximadamente 1.5x10 6 células / ml) se centrifuga a 1500 rpm durante 5 min a temperatura ambiente. Después de descartar el sobrenadante, las células se resuspendieron en 2 ml de medio de un frío. Dos volúmenes de 1,0 ml de suspensión celular de cada frasco se colocan en viales de congelación, inmediatamente congelado a -80 ° C, y se transfieren a nitrógeno líquido al día siguiente. </li><li> Las células se recuperan de almacenamiento de nitrógeno líquido por descongelación rápida, y resuspender el contenido en 10 ml de medio de cultivo fresco. Alternativamente, las células recién descongeladas se lavan una vez en medio de cultivo caliente para eliminar el DMSO antes de resuspender en 10 ml de medio de cultivo fresco. Las células se cultivan durante al menos 72 horas antes de la aprobación posterior. </li></ol> 6. Resultados representante Un cajero automático - / - T-línea celular desarrollada con este protocolo y designado DWJ-ATM-1 se caracterizó por la tinción con anti-CD3-FITC, anti-CD4-APC y anti-CD8-PE y el análisis de citometría de flujo (Figura 1). Más del 90% de las células T se CD3, CD4 y CD8 de tres positivos. <img src="/files/ftp_upload/2598/2598fig1.jpg" alt="Figura 1" /> Figura 1. Caracterización de una línea de células T desarrollado utilizando este protocolo. Los marcadores de superficie celular de un cajero automático establecido - / - línea celular designado DWJ-ATM-1 se caracteriza por la tinción con anti-CD3-FITC, anti-CD4-APC, anti-CD8-PE y el análisis de citometría de flujo. En pocas palabras, 1x10 6 células frescas se lavaron una vez en PBS frío (1500 rpm / 5 min), y se incubaron a 4 ° C durante 10 minutos con L de cóctel de anticuerpos (cada anticuerpo en la dilución 1:200 en PBS con BSA al 1% 200. Las células pueden ser examinados de inmediato mientras que las células fijas fresco o fijado para su posterior análisis. se preparan mediante la adición de formalina neutra tamponada a una concentración del 4% (vol / vol) final y se incubaron a 4 ° C durante 30 min, cosechadas por centrifugación (1500 rpm / 5 min) y se resuspendió en 200 l de PBS con BSA al 1%. Un total de 1 x 10 4 células fueron analizadas mediante un analizador de BD FACS Calibur y CellQuest software (BD Biosciences, San Jose, CA).

<ol>
	<li>Barlow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atm-deficient+mice:+a+paradigm+of+ataxia+telangiectasia.">Atm-deficient mice: a paradigm of ataxia telangiectasia.</a> Cell. 86, 159-171 (1996).</li><li>Elson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+defects+in+ataxia-telangiectasia+protein-deficient+mice.">Pleiotropic defects in ataxia-telangiectasia protein-deficient mice.</a> Proc Natl Acad Sci U S A. 93, 13084-13089 (1996).</li><li>Kuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Atm-/-+thymic+lymphoma+cell+proliferation+in+vitro+and+in+vivo+by+dexamethasone.">Control of Atm-/- thymic lymphoma cell proliferation in vitro and in vivo by dexamethasone.</a> Cancer Chemother Pharmacol. 55, 203-212 (2005).</li><li>Browne, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+a+catalytic+antioxidant+corrects+the+neurobehavioral+defect+in+ataxia-telangiectasia+mice.">Treatment with a catalytic antioxidant corrects the neurobehavioral defect in ataxia-telangiectasia mice.</a> Free Radic Biol Med. 36, 938-942 (2004).</li><li>Jacks, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+spectrum+analysis+in+p53-mutant+mice.">Tumor spectrum analysis in p53-mutant mice.</a> Curr Biol. 4, 1-7 (1994).</li><li>Chervinsky, D. S., Lam, D. H., Zhao, X. F., Melman, M. P., Aplan, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+T+cell+leukemia+cell+lines+established+from+SCL/LMO1+double+transgenic+mice.">Development and characterization of T cell leukemia cell lines established from SCL/LMO1 double transgenic mice.</a> Leukemia. 15, 141-147 (2001).</li><li>Sharma, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1+contributes+to+mouse+T-cell+leukemia+by+directly+inducing+the+expression+of+c-myc.">Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc.</a> Mol Cell Biol. 26, 8022-8031 (2006).</li><li>Maser, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomally+unstable+mouse+tumours+have+genomic+alterations+similar+to+diverse+human+cancers.">Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers.</a> Nature. 447, 966-971 (2007).</li></ol>

En este protocolo, se proporcionan procedimientos detallados para establecer murino líneas de células T a partir de dos genotipos diferentes del ratón, ATM - / - [FVB/N- Atm tm1Led / J] y p53 - / - [129/S6- Trp53 tm1Tyj / J] . Mediante el uso de este protocolo, un total de 6 (de 7 intentos) ATM - / - y tres (de 5 intentos) p53 - / - murino líneas de células T se han establecido en nuestro laborato...

<ul><li> Ramo de hielo </li><li> 70% de etanol </li><li> Disección de plataforma que consta de bloques de espuma cubierta con papel de aluminio y pasadores </li><li> Dos conjuntos de pequeñas tijeras y pinzas estériles </li><li> Estéril de 150 mm 2 placa de Petri (Thermo Fisher Scientific, Waltham, MA;. Cat # 08-757-13) </li><li> Estéril 18 agujas de calibre </li><li> 50 ml tubos cónicos (Corning Inc., Corning NY, cat # 430829) </li><li> 10% de formol </li><li> Frascos de cultivo de tejidos <ul><li> T-25 (TPP, Trasadingen, Suiza, cat # 90025) </li><li> T-75 (TPP, Trasadingen, Suiza, cat # 90075) </li></ul></li><li> Placas de 6 pocillos (Corning Inc., Corning NY, cat # 3506) </li><li> Estéril tamponada con fosfato salino pH 7.2 (PBS) </li><li> Medio de cultivo <ul><li> RPMI 1640 con HEPES 25 mM, 200 mM L-glutamina (Lonza, Walkersville, MD, cat # 12-115F), complementado con lo siguiente: <ul><li> 10% inactivado por calor (56 ° C, 30 min) de suero fetal bovino (SFB; Hyclone, Logan, UT, cat # SH30088.03) </li><li> 1% de penicilina y estreptomicina (Mediatech, Manassas, VA; gato # 30-002-CI) </li><li> 1% aminoácidos no esenciales (Mediatech, cat # 25-025-CI) </li><li> 55 mM 2β-mercaptoetanol (Sigma, St Louis, MO, cat # M752) </li></ul></li></ul></li><li> Dimetilsulfóxido (DMSO; Calbiochem, La Jolla, CA, cat # 317275) </li><li> Anticuerpos (eBioscience, San Diego, CA) <ul><li> anti-CD3-FITC (cat. # 11-0031-85) </li><li> anti-CD4-APC (cat. # 17-0041-83) </li><li> anti-CD8-PE (cat. # 12-081-85) </li></ul></li><li> Albúmina sérica bovina (BSA, Life Technologies, Grand Island, NY;. Cat # 11018-017) </li><li> Concanavalina A (Sigma cat # C5275) </li><li> 1,8 mL viales de congelación (Nunc, Roskilde, Dinamarca, cat # 368632) </li></ul>

derivation of thymic lymphoma t-cell lines from atm-/- and p53-/- mice

Las líneas celulares establecidas son una herramienta de investigación fundamental que puede reducir el uso de animales de laboratorio en la investigación. Ciertas cepas de ratones genéticamente modificados, tales como ATM - / - y p53 - / - consistentemente desarrollar linfoma del timo primeros años de vida 1,2, y por lo tanto, puede servir como una fuente confiable para la derivación de murino líneas de células T. Aquí presentamos un protocolo detallado para el desarrollo de linfomas tímicos murinos establecido líneas de células T, sin necesidad de añadir las interleucinas como se describe en los protocolos anteriores 1,3. Los tumores se cosecharon de los ratones de tres a seis meses, en la primera indicación de tumores visibles sobre la base de la observación de la postura encorvada, dificultad para respirar, la preparación pobre y perder en un 1,4 cepa susceptible. Hemos establecido con éxito varias líneas de células T que utilizan este protocolo y cepas puras de ATM - / - [FVB/N- Atm tm1Led / J] 2 y p53 - / - [129/S6- Trp53 tm1Tyj / J] 5 ratones. Además, demuestran que más del 90% de lo establecido población de células T expresa CD3, CD4 y CD8. De acuerdo con las líneas celulares establecidas de forma estable, las células T generados por el uso del presente Protocolo se han pases para más de un año.

Watch this Scientific Journal Video about Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice at JoVE.com

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

En este video se demuestra un protocolo para establecer ratón timo líneas celulares de linfoma. Siguiendo este protocolo, hemos establecido con éxito varias líneas de células T de la ATM-/ - y p53-/ - ratones con linfoma del timo.

Derivación de linfoma tímico líneas de células T de Atm - / -</sup Y P53 - / -</sup Ratones

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

derivation-of-thymic-lymphoma-t-cell-lines-from-atm-and-p53-mice

Research

JoVE Journal

Immunology and Infection

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

12.2K vistas.  Cornell University. En este video se demuestra un protocolo para establecer ratón timo líneas celulares de linfoma. Siguiendo este protocolo, hemos establecido con éxito varias líneas de células T de la ATM-/ - y p53-/ - ratones con linfoma del timo.

Video: Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Los ratones con Transnuclear predefinidos Especificidades receptor de células T contra la Toxoplasma gondii Obtenidos a través de la SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Investigación

Inmunología e Infección

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

El examen de selección tímica positivo y negativo por citometría de flujo

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

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

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

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

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

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

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&amp;#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 ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

Un método eficiente y Simple para establecer líneas de células T de pacientes con infección crónica activa Virus Epstein - Barr y NK

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

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

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

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

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

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

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

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

Knock-in de genes por CRISPR/Cas9 y clasificación celular en líneas de macrófagos y células T

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and significant role as carriers of biological information make them intriguing study objects, requiring reliable and repetitive protocols for their isolation. However, realizing their full potential is difficult as there are still many technical obstacles related to their research (like proper acquisition). This study presents a protocol for the isolation of small EVs (according to the MISEV 2018 nomenclature) from the culture supernatant of tumor cell lines based on differential centrifugation. The protocol includes guidelines on how to avoid contamination with endotoxins during the isolation of EVs and how to properly evaluate them. Endotoxin contamination of EVs can significantly hinder subsequent experiments or even mask their true biological effects. On the other hand, the overlooked presence of endotoxins may lead to incorrect conclusions. This is of particular importance when referring to cells of the immune system, including monocytes, because monocytes constitute a population that is especially sensitive to endotoxin residues. Therefore, it is highly recommended to screen EVs for endotoxin contamination, especially when working with endotoxin-sensitive cells such as monocytes, macrophages, myeloid-derived suppressor cells, or dendritic cells.

The proposed protocol includes guidelines on how to avoid contamination with endotoxin during the isolation of extracellular vesicles from cell culture supernatants, and how to properly evaluate them.

Derivación de linfoma tímico líneas de células T de Atm - / - Y P53 - / - Ratones

Derivación de linfoma tímico líneas de células T de Atm^{- / - Y P53^{- / - Ratones}}