Name: isolation and transplantation of different aged murine thymic grafts.
Uploaded: 2015-05-13T14:00:00.000+00:00
Duration: 5 min 47 s
Description: Watch this Scientific Journal Video about Isolation and Transplantation of Different Aged Murine Thymic Grafts. at JoVE.com

This work was supported by funding received from the National Institutes of Health (1R01AI083269).

Los estudios murinos fueron aprobados por el Cuidado y Uso de Animales Comité Institucional (IACUC) de la Universidad de Carolina del Norte en Chapel Hill y todo cuidado de los animales estaba en conformidad con las directrices del IACUC. 1. Preparación del Recién Nacido y del adulto Thymi <ol><li> Preparar todos los reactivos y el equipo antes de la eutanasia de los ratones donantes. </li><li> Esterilizar los instrumentos quirúrgicos de esterilización en autoclave u otros métodos apropiados. Todos los procedimientos quirúrgicos deben realizarse bajo una campana de flujo laminar para mantener las condiciones estériles y evitar la contaminación. Montar las herramientas necesarias para la extracción de timos de ratones donantes. </li><li> Llene un plato de 60 mm con estéril 1x PBS (pH 7,4) y colocar en hielo dentro de la campana. Esto se utilizará para almacenar brevemente timos extirpados de ratones donantes antes del trasplante. </li><li> De acuerdo con las normas éticas, la eutanasia a los ratones donantes recién nacidos por decapitación y los ratones donantes adultos por asfixia de CO 2 seguido por dislocación cervical. </li><li> Para removal del timo: Coloque el ratón en una posición reclinada dorsal sobre una toalla de papel absorbente estéril y rociar con etanol al 70% antes de hacer una incisión. </li><li> Exponer la cavidad abdominal y torácica haciendo una incisión en la línea media a través de la piel. Doble la piel sobre el pecho y extremidades anteriores para revelar la cavidad torácica. </li><li> Haga dos incisiones laterales a través del diafragma y la caja torácica para exponer el mediastino superior y anterior de la cavidad torácica. El timo debe ser visible como dos lóbulos blancos inmediatamente por encima y adyacente al corazón. </li><li> Desmenuzar el tejido conectivo que rodea el timo con unas pinzas finas, asegurándose de no perturbar la cápsula. Mientras mantiene de nuevo la caja torácica con fórceps, utilizar un segundo par de pinzas para extraer los dos lóbulos del timo mediante la colocación de pinzas curvas debajo del órgano y tirando verticalmente. Esto se puede hacer usando un microscopio de disección para extraer timos de ratones recién nacidos. </li><li> Coloque el timo en el 60 mm dish que contiene 1x PBS estéril (pH 7,4) en hielo y los lóbulos tímicos separados por el corte a través del istmo conectivo. Retire cualquier residuo de los lóbulos tímicos asegurándose de no dañar la cápsula, y cortar el timo en el número apropiado de secciones para el trasplante. </li><li> No manipular el timos obtenido a partir de ratones recién nacidos. NOTA: Se utiliza para un máximo de dos ratones receptores (1 lóbulo por destinatario). </li><li> Transplante las timos obtenido a partir de ratones adultos en un máximo de 4-6 ratones receptores. El uso de un par de pinzas, sujete cuidadosamente lóbulo tímico un adulto, como se muestra en la Figura 1, cortar el timo en tres secciones iguales utilizando tijeras quirúrgicas. </li><li> Repita los pasos 1.3 a 1.4 para cada ratón donante. Con el fin de limitar el tiempo timos están expuestos, preparar timo donante 1 a la vez. </li></ol> 2. Implantación timo bajo la cápsula renal <ol><li> Montar el equipo de pre-esterilizados listado en la Tabla 1. Aséptica adecuadotécnica debe ser utilizada durante el curso del procedimiento para prevenir la exposición de trasplante-receptores a las herramientas o reactivos contaminados. </li><li> Antes del trasplante, pesar y etiquetar cada ratón receptor. </li><li> Configure el microscopio de disección y el circuito de la anestesia en la campana de flujo laminar. </li><li> El uso de una máquina de afeitar eléctrica, afeitarse el lado izquierdo de la ratón receptor y asegurar sin pelo permanece alrededor de la zona utilizada para hacer la incisión. </li><li> Encienda vaporizador isofluorano y anestesiar el ratón utilizando una dosis de entre 1-2%. Determinar anestesia adecuada antes de comenzar el procedimiento quirúrgico mediante la comprobación de una ausencia de reflejo siguiente pizca dedo del pie. </li><li> Después de que el ratón está anestesiado adecuadamente, aplicar el ungüento veterinaria a los ojos del ratón para evitar la sequedad y preparar el ratón para el trasplante como sigue: </li><li> Coloque el ratón bajo el microscopio de disección en posición decúbito lateral derecho para que el lado afeitado quede hacia arriba. Starting en el centro de la zona quirúrgica, dispensar en un movimiento circular usando una pipeta de transferencia desechable 70% de etanol, seguido de povidona-yodo (betadine). Repetir el tratamiento con etanol / betadine 3 veces antes de hacer una incisión. </li><li> Usando tijeras de disección hacen una incisión en el flanco 1-2 cm directamente sobre el riñón. </li><li> El uso de pinzas medianas agarrar el tejido conectivo adyacente al riñón y levantar suavemente el riñón de modo que se encuentra encima de la musculatura. Para mantener el riñón expuesto y en su lugar sobre la musculatura, inserte un brazo de fórceps medio por debajo del riñón, prestando especial atención a no perturbar el hueso ilíaco renal. </li><li> Con el fin de evitar la desecación del tejido, irrigar el riñón con 1x PBS estéril (pH 7,4) durante cada paso hasta que la manipulación de la cápsula renal y el riñón es completa. </li><li> Usando unas pinzas finas pellizcan y levantar la cápsula cerca del borde del riñón más distal a las glándulas suprarrenales para separarlo del riñón. </li><li> Utilice un calibre 18aguja para hacer una incisión lo suficientemente grande como para insertar la pieza preparada de timo donante (Figura 2A). Asegúrese de mantener la incisión capsular tan pequeño como sea posible para evitar la dislocación del injerto con el tiempo. </li><li> Mientras se mantiene la cápsula se apartó desde el riñón, utilizar un segundo par de pinzas finas para insertar el injerto debajo de la cápsula y empujar el timo lo más adelante de la incisión capsular como sea posible (Figura 2B). </li><li> Retire las pinzas de debajo de los riñones, y volver suavemente el riñón nuevo en su lugar a través de la incisión. </li><li> Cierre la musculatura suturando la pared peritoneal y aplicar betadine una vez cerrada. Después de sutura es completa, aplicar clips de la herida para cerrar la dermis y aplicar betadine a la zona circundante. </li><li> Devuelva el ratón a una jaula que se ha calentado utilizando una lámpara de calor. </li><li> Monitorear cada ratón postoperatorio hasta que recupera la conciencia plena y la movilidad y no coloque tha ratonest no se han recuperado después de la anestesia en una jaula con otros animales. </li><li> Tratar ratones con fármacos de control del dolor post-operatorio, según sea necesario. Proporcionar ratones con analgésicos post-operatorias tales como el acetaminofeno en el agua de bebida a una concentración de 1,6 mg / ml. </li><li> Repita los pasos 2.3 a 2.9 por cada ratón receptor. </li><li> Asegúrese de que los ratones retorno a la actividad normal dentro de 1 hora después de la cirugía. Monitor de peso post-operatorio, habilidades motoras, así como la actividad de beber y la alimentación de cada ratón para determinar las complicaciones que surgen como resultado del procedimiento de trasplante. NOTA: la pérdida de peso post-operatorio, en comparación con el peso preoperatoria, así como la movilidad alterada o el fracaso para comer o beber puede indicar complicaciones. </li><li> Monitor de reconstitución de células T en la sangre periférica por sangrado ratones a través de la cola nick seguido de separación de linfocitos usando Lympholyte medios de separación de células de acuerdo con las especificaciones del fabricante. Lograr characteriza células Tción por tinción linfocitos con anticuerpos conjugados con fluorescencia específicos para marcadores de células T CD3, CD4, y CD8, así como un discriminador de muertos en vivo, tales como un éster de succinimidilo activado por fluorescencia 8. Análisis como se muestra en la Figura 3 se puede lograr a través de gating en interiores, de células vivas, y las células CD3 positivas. </li></ol>

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	<li>Kappler, J. W., Roehm, N., Marrack, P. <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+tolerance+by+clonal+elimination+in+the+thymus.">T cell tolerance by clonal elimination in the thymus.</a> Cell. 49 (2), 273-280 (1987).</li><li>Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Journey+through+the+thymus:+stromal+guides+for+T-cell+development+and+selection.">Journey through the thymus: stromal guides for T-cell development and selection.</a> Nat. Rev. Immunol. 6, 127-135 (2006).</li><li>Klein, L., Kyewski, B., Allen, P. M., Hogquist, K. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+FOXN1-Deficiency+is+associated+with+ab+double-negative+and+FoxP3++T-cell+expansions+that+are+distinctly+modulated+upon+thymic+transplantation.">Human FOXN1-Deficiency is associated with ab double-negative and FoxP3+ T-cell expansions that are distinctly modulated upon thymic transplantation.</a> PLoS One. 7 (5), e37042 (2012).</li><li>Chinn, I. K., Milner, J. D., Scheinberg, P., Douek, D. C., Markert, M. 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The authors do not have any competing financial interests to disclose.

Events regulating thymic selection are poorly understood. Recent studies have demonstrated ontogenic changes in the efficiency of selection. Coupling this approach with different transgenic thymus donor and recipient mice will further facilitate studies to identify the events and parameters that regulate thymic selection. Notably, thymic transplantation is used for the treatment and management of T cell and thymic disorders seen for instance in athymic infants lacking functional T cells and in patients with DiGeorge synd...

El timo es un órgano en el que los eventos críticos en el desarrollo de células T producen 1. Timocitos Residentes, sobre reordenación del receptor de células T (TCR) α y β genes, se someten a una serie de interacciones con lymphostromal y células presentadoras de antígeno (APC) en las regiones corticales y medulares del timo 2. Selección positiva tímico está mediada por células epiteliales corticales del timo (TEC) para producir los timocitos que reconocen péptidos antigénicos en el contexto de anfitrión complejo mayor de histocompatibilidad (MHC) de clase I y II moléculas 2-3. La posterior selección negativa tímica implica la purga de los timocitos autorreactivos, impulsado por una interacción con TEC medular o células dendríticas (DC) que presentan péptidos derivados de auto-proteínas unidas por MHC de clase I y II moléculas 3. El resultado final de estos procesos es el establecimiento de un grupo de células maduras T CD4 + y CD8 + capaces de responder a un amplio espectrode antígenos extraños mientras que exhiben una reactividad mínima a proteínas propias 4. La eficiencia de eventos de selección tímica está influenciada por una serie de factores, incluyendo la maduración tímica, la frecuencia de TEC medular y cortical, composición subconjunto de tímico DC, y la fuente de los precursores tímicos 3. En particular, la selección tímica aberrante puede resultar en autoinmunes 5 o inmunodeficientes patologías, que surgen de deterioro de la selección negativa o positiva, respectivamente. Los eventos moleculares que regulan la selección tímica, sin embargo, son poco conocidos. In vitro enfoques tales como cultivos de órganos timo reaggregate (RTOC) 6, han demostrado ser útiles para el análisis de los acontecimientos básicos asociados con la selección del timo, pero no para recapitular completamente la dinámica del curso en los eventos in vivo. Como resultado, este enfoque basado en el trasplante de timo se estableció a mejores eventos de selección de timocitos estudio in vivo 7 </sarriba&gt;. Este protocolo describe el trasplante de timos de ratones donantes recién nacidos y adultos en ratones receptores SCID inmunodeficientes. Esta técnica permite el estudio de los mecanismos que regulan la selección tímica positivo y negativo, así como la producción tímica de diversos subconjuntos de células T durante la ontogenia. Más recientemente, este enfoque ha sido utilizado para demostrar que la eficiencia de la selección tímica se limita temprano después del nacimiento en ratones conduce a un mayor desarrollo de las células T autorreactivas, y un reducido repertorio de células T específicas para antígenos extraños 7.

<table><tbody><tr><td>Ethanol&amp;nbsp;</td><td>Decon Laboratories</td><td>2705HC</td><td></td></tr><tr><td>PBS</td><td>GIBCO</td><td>14190-144</td><td>pH 7.4</td></tr><tr><td>Betadine Solution</td><td>Purdue Products</td><td>67618-150-17</td><td></td></tr><tr><td>18 gauge needle</td><td>BD</td><td>305195</td><td></td></tr><tr><td>Sutures (1.0 metric)</td><td>Ethicon</td><td>J493G</td><td>18&quot; (45cm)</td></tr><tr><td>AUTOCLIP Wound Clips (9mm)</td><td>Clay Adams&amp;reg; Brand</td><td>427631</td><td></td></tr><tr><td>Transfer pipette</td><td>Fisher Scientific</td><td>13-711-20</td><td>Sterile, disposable</td></tr><tr><td>Mouse anti-CD3 Ab</td><td>eBioscience</td><td>11-0031-85</td><td>Clone: 1452C11</td></tr><tr><td>Mouse anti-CD4 Ab</td><td>eBioscience</td><td>48-0042-82</td><td>Clone: RM4-5</td></tr><tr><td>Mouse anti-CD8 Ab</td><td>eBioscience</td><td>25-0081-82</td><td>Clone: 53-6.7</td></tr><tr><td>Lancet</td><td>Medipoint</td><td>Goldenrod 4mm</td><td></td></tr><tr><td>Pacific Orange Succinimidyl Ester, Triethylammonium Salt</td><td>Invitrogen</td><td>P30253</td><td></td></tr><tr><td>96-well round botton polypropylene plates</td><td>Corning</td><td>3365</td><td></td></tr><tr><td>1.2 ml polypropylene cluster tubes</td><td>Corning</td><td>4401</td><td></td></tr><tr><td>5 ml polypropylene round-bottom tubes</td><td>BD</td><td>352002</td><td></td></tr><tr><td>40uM Nylon Cell Strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>16% Paraformaldehyde Solution, EM Grade</td><td>Electron Microscopy Services</td><td>15710</td><td>Hazardous</td></tr><tr><td>Puralube Optical Ointment</td><td>Fisher Scientific</td><td>NC0138063</td><td></td></tr><tr><td>Lympholyte</td><td>Cedarlane</td><td>CL5030</td><td></td></tr><tr><td>60 mm cell culture dish</td><td>Corning</td><td>430196</td><td>60 mm x 15 mm, Sterile</td></tr></tbody></table>

isolation and transplantation of different aged murine thymic grafts.

Los mecanismos que regulan la eficacia de la selección tímica permanecen mal definidos. El método presentado aquí permite en los análisis in vivo de el desarrollo y selección de las células T específicas para antígenos propios y extraños. El enfoque implica la implantación de injertos tímicos derivados de diversos ratones de edad en receptores SCID inmunodeficientes. Durante un período relativamente corto de tiempo los destinatarios están completamente reconstituidos con células T derivadas de injerto de timo implantado. Sólo timocitos siembra el timo en el momento de aislamiento se someten a selección y se desarrollan en células T maduras. Como tal, los cambios en la naturaleza y la especificidad de las células T injertadas como una función de los acontecimientos del timo dependientes de la edad pueden ser evaluados. Aunque se requiere experiencia técnica para el trasplante de timo éxito, este método proporciona una estrategia única para estudiar in vivo una amplia gama de patologías que se deben a o un resultado de la función y / o homeostas tímico aberrantees.

El éxito de este procedimiento es dependiente de mínimo trauma quirúrgico, así como el posicionamiento preciso del injerto por debajo de la cápsula renal. El injerto tímico se debe cortar para asegurar apropiadamente dimensionado secciones tímicos para posterior trasplante como se muestra en la Figura 1. Siguiendo el esquema de la Figura 1, los timos de ratones recién nacidos o adulto se puede utilizar para el trasplante subcapsular éxito con resultados injerto de células T co...

Watch this Scientific Journal Video about Isolation and Transplantation of Different Aged Murine Thymic Grafts. at JoVE.com

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

Aislamiento y Trasplante de diferentes Aged murinos tímicos injertos.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

isolation-and-transplantation-of-different-aged-murine-thymic-grafts

Research

JoVE Journal

Immunology and Infection

13.7K vistas.  University of North Carolina at Chapel Hill. This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo. 

Video: Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Caracterización de tímico Instalarse Progenitores en el embrión de ratón Uso<em&gt; En Vivo</em&gt; Y<em&gt; In Vitro</em&gt; Ensayos

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Investigación

Biología del Desarrollo

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imagen intravital del timo de ratón utilizando 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Inmunología e Infección

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

La inducción de la enfermedad de injerto contra huésped y<em&gt; En Vivo</em&gt; T Monitoreo Celular Utilizando un modelo murino MHC de concordancia

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

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 ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

El aislamiento, la identificación y purificación de células epiteliales del timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Murino de grosor completo Trasplante de Piel

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

Un microquirúrgica Modelo Novela de heterotópico, en bloque, pared torácica, el timo y el trasplante cardiaco en ratones

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Medicina

Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation is an important method for investigating thymic epithelial cell function and T cells maturation in vivo. Here we will describe the experimental methods used within our laboratory to transplant 2&#8217;-deoxyguanosine (to deplete donor&#8217;s lymphocytes) treated embryonic thymus into the renal capsule of an athymic nude mouse. This method is both simple and efficient and does not require special skills or devices. The results obtained via this simple method showed that transplanted thymus can effectively support the recipient&#8217;s T cells production. Additionally, several key points with regards to the protocol will be further elucidated.

We provide a simple and efficient method to transplant 2&#8217;-deoxyguanosine treated E18.5 thymus into the renal capsule of a nude mouse. This method should aide in the study of both thymic epithelial cells function and T cells maturation.

Trasplante subcapsular renal de timo embrionario murino tratado con 2'-desoxiguanosina en ratones desnudos

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation ...

Optimización del aislamiento de células estromales del timo y la médula ósea

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

Autor destacado: Avance de la investigación hematopoyética mediante el aislamiento de células estromales para la secuenciación de células individuales

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

Aislamiento de células estromales de órganos hematopoyéticos

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

Aislamiento y Trasplante de diferentes Aged murinos tímicos injertos.

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