Name: transplantation of tail skin to study allogeneic cd4 t cell responses in mice
Uploaded: 2014-07-25T13:00:00.000+00:00
Duration: 6 min 48 s
Description: Watch this Scientific Journal Video about Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice at JoVE.com

This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R. We thank E. Palmer and B.T.H. Hausmann for mice and technical expertise.

En esta publicación, el vídeo y el protocolo, se llevaron a cabo todos los procedimientos con animales de acuerdo con el protocolo de los animales aprobado por la autoridad cantonal de Basilea-Ciudad, Suiza. Realice todos los procedimientos en condiciones de esterilidad, cuando sea posible. 1. Preparación de Cirugía <ol><li> Autoclave todos los instrumentos quirúrgicos y gasas antes de su uso. </li><li> Calentar la almohadilla caliente y organizar los instrumentos quirúrgicos en la tabla (Tabla de Materiales / Métodos). </li><li> Abra las vendas de la tira dedo. Aplique vaselina en la plataforma de la herida con un algodón (asegúrese de que la gasa se cubre totalmente). </li><li> Pesar los ratones y administrar analgesia buprenorfina 0,05 mg / kg de peso corporal. Nota: esto proporcionará analgesia inmediatamente después del trasplante. </li></ol> 2. Preparación de la cola de la piel para el Trasplante <ol><li> La eutanasia a los ratones donantes (por ejemplo, IA) BM12, los cuales por asfixia de CO 2 y disinfect las colas con solución de desinfección antes de la escisión. </li><li> Coloque la cola con la franja negro sobre una placa de plástico limpio y cortado longitudinalmente a lo largo de la línea media, haciendo una incisión superficial que afecta sólo la capa de piel. </li><li> Retirar la piel de la cola utilizando pinzas de agarre. </li><li> Ponga la cola de la piel en una placa de cultivo de 10 cm celda llena con 10 ml de HBSS. </li></ol> 3. Procedimiento Quirúrgico <ol><li> Anestesie ratones receptores (por ejemplo, ratones C57BL / 6) por inhalación de una solución de isofluorano 3% en una caja de inducción transparente. Nota: Se necesita 3-5 minutos para el ratón para llegar a la anestesia plano quirúrgico. Use ungüento veterinario en los ojos para evitar la sequedad, mientras que bajo anestesia. </li><li> Coloca el ratón sobre la almohadilla tibia y aplicar el 1,5% isofluorane través de una mascarilla boca y el culo cada reflejo animal toe pellizcos. </li><li> Afeitar el sitio dorsal de los ratones receptores (por ejemplo, C57BL / 6) y quitar los pelos contaminantes utilizando un sw gasa secaab. </li><li> Desinfectar sitio trasplante afeitado con solución aséptica. </li><li> Apriete la piel con unas pinzas en el sitio central / derecha del dorso y el uso de tijeras curvas para hacer una incisión alrededor de alrededor de 1 x 0,6 cm. </li><li> Si se produce una hemorragia, luego limpiar la herida con un hisopo de algodón estéril. </li><li> Evaluar el tamaño de la incisión y cortar un trasplante de el tamaño estimado de la cola-piel utilizando el bisturí. </li><li> Recorte esquinas para redondear el trasplante. </li><li> Cargue el trasplante en el bisturí, el exceso seca de HBSS en un hisopo de algodón estéril y lo coloca en el lecho del injerto del ratón receptor. </li><li> Evitar la superposición de los donantes y de la piel de acogida y aplicar adhesivo tisular en la zona de contacto de forma exclusiva. </li><li> Aplicar un yeso sin arrugas alrededor de la cintura del ratón. </li><li> Aplicar el adhesivo vendaje no elástico para fijar el yeso para el ratón (3 mm desplazado). </li><li> Una incisión con las tijeras alrededor de 3 mm en el sitio ventral superior del vendaje. Nota: Esto es para evitar trapping de los dientes del ratón durante la fase de recuperación. </li><li> Retire la anestesia boca máscara y mantener a los ratones trasplantados en la almohadilla caliente hasta que despierto. </li><li> Volver ratones trasplantados a sus jaulas sólo cuando esté completamente recuperado y evaluar su movilidad para asegurarse de que el vendaje no está demasiado ajustado. </li><li> Esterilizar todos los instrumentos quirúrgicos con etanol al 70% antes de proceder con la cirugía siguiente. </li></ol> 4. Cuidado post-operatorio <ol><li> Añadir jarabe que contiene paracetamol al agua de bebida (4 mg / ml) y administrarlo durante 7 días. </li><li> Ratón Regresar a la sala de los animales una vez que se haya recuperado. </li><li> Seguir de cerca los ratones en busca de signos clínicos como la depresión u otros cambios de comportamiento (comer o inmovilidad), aspecto anormal (falta de aseo personal) o la postura (pilo-erección o postura encorvada) durante el tiempo que el vendaje está en su lugar (1 semana). </li></ol> 5. Eliminación del vendaje (6-7 días post-trasplante) <ol><li> Anesthetize los ratones por inhalación de una solución de isofluorano 3% en una caja de inducción transparente (véase más arriba). </li><li> Cortar el vendaje y el yeso con unas tijeras de la arteria y cuidadosamente eliminarlos. Nota: evitar el estiramiento del sitio de trasplante. </li><li> Monitorear los ratones trasplantados en busca de signos clínicos (ver 4.3) y la puntuación para el rechazo de los injertos de piel (Tabla 1). Nota: El sistema de puntuación de injerto se aprovecha de las diferentes texturas de pelo en la cola de la piel en comparación con el dorsal de la piel. La aparición del trasplante y su rechazo se clasifica en 3 grados. Piel alogénico (MHC II no coinciden, por ejemplo, IA BM12) injertos de C57BL / 6 ratones inmunocompetentes son generalmente rechazados dentro de 12 a 15 días después del trasplante (datos propios y 15-20). </li></ol>

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The authors declare that they have no competing financial interests.

Trasplante de la piel es un método ampliamente utilizado para el estudio de rechazo y dependiente de las células T de la tolerancia. Desde que se estableció el modelo de trasplante de piel, se han aplicado varias adaptaciones y cambios. En el procedimiento descrito, IA BM12 trasplante de cola de piel se realiza con gas anestésico (isofluorano). El uso de la anestesia con gas reduce el tiempo de ejecución y la recuperación de los ratones, lo que disminuye la presión sobre los ratones trasplantados. El p...

El trasplante de órganos sólidos, como la piel, el corazón y los riñones es ahora un procedimiento estándar en la práctica médica en todo el mundo 1. Órganos trasplantados con éxito pueden ser rechazadas por la activación del sistema inmune receptor, que reconoce los antígenos principales de histocompatibilidad del donante. Por lo tanto los pacientes transplantados necesitan tratamiento con medicamentos inmunosupresores 2. El trasplante alogénico de piel en ratones fue establecido por Medawar y sus colegas en 1955 y ha sido útil para la identificación de las moléculas específicas que se describen más adelante como complejo mayor de histocompatibilidad (MHC) de clase I y II. Desde entonces, el modelo de trasplante de piel ha sido modificado y adaptado para estudiar el papel de los subconjuntos de células T y la relevancia de la química y la intervención biológica en la supresión de rechazo de injertos 2-4 continuamente. La piel de la oreja y el tronco son más difíciles de preparar y son más susceptibles a la hipoxia y la necrosis de la piel de la cola-5; Sin embargo,el procedimiento de trasplante es similar. Además, el seguimiento de trasplantes de cola de la piel es fácil debido a la textura del cabello característico de la piel. Este artículo proporciona un procedimiento detallado de MHC de clase II desajuste trasplante de cola de la piel que permite el estudio de los diferentes aspectos de la T CD4 + rechazo de aloinjertos mediada por células y la tolerancia en ratones. La mutación natural de tres puntos en la molécula de MHC clase II IA B (llamado IA BM12) 6-9 es suficiente para inducir el rechazo de aloinjertos de piel en ratones C57BL / 6 8. La molécula de BM12 IA activa las células T CD4 + con diferentes receptor de células αβ-T (TCR) cadenas de ratones C57BL / 6, entre las que se identificaron las células T Vα2Vβ8-TCR-específicas con el fin de generar un ratón transgénico TCR-10. La transferencia adoptiva de células-TCR-específicos Vα2Vβ8 T se ha usado para establecer un modelo de rechazo en C57B inmunodeficienteL / 6 Rag2 - / - ratones transplantados con la piel IA BM12. Las diferencias genéticas entre el donante y el receptor afectan el resultado de la aceptación y el rechazo de trasplante. Hay diferentes tipos de trasplantes: autoinjertos son los trasplantes de la persona beneficiaria misma; syngrafts y aloinjertos son los trasplantes de individuos genéticamente idénticos y no relacionados genéticamente, respectivamente. Aceptación de diferentes trasplantes de órganos alogénicos se ha demostrado por la intervención química y biológica en pacientes y modelos de ratón 11,3,4. En un enfoque básico, anti-CD3 tratados C57BL / 6 ratones mostraron una supervivencia prolongada de IA BM12 cola-piel (datos no publicados). El agotamiento de los CD4 + y CD8 + T las células antes del trasplante en ratones receptores resultó en la aceptación de MHC de clase I y II injertos no coincidentes (Apocalipsis en el 12). Curiosamente, el rechazo de injertos de piel depende de la presencia de células CD4 + </sup&gt; Células T (rev. en 12). En este modelo, la orientación interacciones específicas entre diferentes células inmunes mediante el bloqueo de las moléculas coestimuladoras con anticuerpos o supresión con células T reguladoras podría inducir tolerancia (datos no publicados). De hecho, el bloqueo de ambos CD40 y CD28 dio lugar a la tolerancia 13,14 aloinjerto de piel a largo plazo. Trasplante de Cola-piel es fácil de realizar y fácil de controlar en comparación con el trasplante de otros órganos. Además, los trasplantes de cola de la piel son fáciles de preparar y son menos susceptibles a la isquemia de otros tejidos de la piel. En contraste a los anestésicos inyectados, el uso de gas anestésico (isofluorano) durante el trasplante acorta tanto el procedimiento y el tiempo de recuperación destinatario. Curling del trasplante de cola de la piel, que puede conducir a la cicatrización de heridas incompleta y el rechazo del injerto, se evita mediante la aplicación de adhesivo de tejido. Además, el modelo de trasplante de piel de la cola-IA BM12 activa exclusivamente CD4 <sup&gt; + Células T, tanto inmunocompetentes como ratones inmunodeficientes (de los mismos antecedentes genéticos) facilitar la interpretación de los resultados. Este protocolo describe un modelo de ratón fiable, reproducible y fácilmente controlables que permite la intervención química y biológica. El modelo está diseñado para investigar el rechazo y la tolerancia a la inducción de los trasplantes de cola de la piel.

<table><tbody><tr><td>Betadine standard solution</td><td>Mundipharma</td><td></td><td></td></tr><tr><td>Cotton swab</td><td>Carl Roth GmbH</td><td>31025060</td><td></td></tr><tr><td>Dafalgan , UPSA</td><td>Bristol Myers Squibb UPSA</td><td></td><td></td></tr><tr><td>Hansaplast finger strips</td><td>Beierdorf AG</td><td>REF.76861</td><td></td></tr><tr><td>Histoacryl tissue adhesive</td><td>Braun</td><td>REF.1050052</td><td></td></tr><tr><td>Leukotape classic , 2 cm x 10 m</td><td>BSN Medical SAS</td><td>REF.02204-00</td><td></td></tr><tr><td>PBS, Phosphate Buffered Saline, pH 7.4</td><td>Invitrogen</td><td>10010015</td><td>GIBCO</td></tr><tr><td>Sterile gauze, 5 x 5 cm, 8 ply</td><td>MaiMed GmbH</td><td>21010</td><td></td></tr><tr><td>Narrow pattern forceps</td><td>FST</td><td>11003-12</td><td></td></tr><tr><td>Fine iris scissor curved</td><td>FST</td><td>14095-11</td><td></td></tr><tr><td>Fine iris scissor</td><td>FST</td><td>14094-11</td><td></td></tr><tr><td>Mayo scissors</td><td>FST</td><td>14010-15</td><td></td></tr><tr><td>Artery scissors ball tip 11.5 cm</td><td>FST</td><td>14080-11</td><td></td></tr><tr><td>Tissue forceps</td><td>FST</td><td>11021-14</td><td></td></tr><tr><td>Surgical blade No. 20</td><td>Swann-Morton LTD</td><td>3006</td><td>Carbon steel</td></tr><tr><td>Surgical blade handles</td><td>Swann-Morton LTD</td><td></td><td></td></tr><tr><td>Syringe, 1 ml</td><td>ARTSANA</td><td></td><td>Disposable</td></tr><tr><td>Temgesic, buprenorphine</td><td>ESSEX Chemie AG</td><td></td><td>0.3 mg/ml</td></tr><tr><td>Tissue Culture dishes 10 cm, 60.1 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>TPP</td><td></td><td></td></tr><tr><td>Vaseline</td><td>Vifor SA</td><td></td><td></td></tr><tr><td>Warm pad</td><td>Solis</td><td></td><td>Type 223</td></tr></tbody></table>

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.

En una primera aproximación C57BL / 6 ratones fueron trasplantados con aloinjertos BM12, los cuales IA y b syngrafts IA. Después de retirar el vendaje, injertos signos manifiestos de la cicatrización de heridas y sin cierre de la zona de contacto en C57BL / 6 ratones (Figura 1A). Después de la retirada del vendaje, CD4 + T inflamación mediada por células condujo a la aparición de áreas necróticas (manchas rojas) y el rechazo de aloinjertos BM12, los cuales<...

Watch this Scientific Journal Video about Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice at JoVE.com

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

transplantation-tail-skin-to-study-allogeneic-cd4-t-cell-responses

Research

JoVE Journal

Immunology and Infection

12.4K vistas.  University of Basel and University Hospital Basel. 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.

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

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

Trasplante de piel murina

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

Investigación

Biología

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 ...

Inmunología e Infección

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

Trasplante de tumores para evaluar la dinámica de las células T CD8+ infiltrantes de tumores en ratones

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Investigación sobre el cáncer

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

La inducción de la inflamación intestinal murino por transferencia adoptiva de Efector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; T células en ratones inmunodeficientes

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral tissues through an as-yet undefined mechanism. The application of multiphoton microscopy to the study of the immune system provides a tool to measure the dynamics of immune responses within intact tissues. Here we present a protocol for non-invasive intravital multiphoton imaging of CD4 T cells in the inflamed mouse ear dermis. Use of a custom imaging platform and a venous catheter allows for the visualization of CD4 T cell dynamics in the dermal interstitium, with the ability to interrogate these cells in real-time via the addition of blocking antibodies to key molecular components involved in motility. This system provides advantages over both in vitro models and surgically invasive imaging procedures. Understanding the pathways used by CD4 T cells for motility may ultimately provide insight into the basic function of CD4 T cells as well as the pathogenesis of both autoimmune diseases and pathology from chronic infections.

The mechanisms that govern the interstitial motility of CD4 effector T cells at sites of inflammation are relatively unknown. We present a non-invasive approach to visualize and manipulate in vitro-primed CD4 T cells in the inflamed ear dermis, allowing for study of the dynamic behavior of these cells in situ.

La migración de células T CD4 obtener imágenes intersticial en la dermis inflamadas

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

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 ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

Ortotópico de la extremidad posterior de trasplante en el ratón

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

Medicina

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying the mechanisms of normal and malignant hematopoiesis and are the gold standard for identifying effective chemotherapies for many malignancies. PDX models are possible because many of the mouse cytokines also act on human cells. However, this is not the case for all cytokines, including many that are critical for studying normal and malignant hematopoiesis in human cells. Techniques that engineer mice to produce human cytokines (transgenic and knock-in models) require significant expense before the usefulness of the model has been demonstrated. Other techniques are labor intensive (injection of recombinant cytokine or lentivirus) and in some cases require high levels of technical expertise (hydrodynamic injection of DNA). This report describes a simple method for generating PDX mice that have exogenous human cytokine (TSLP, thymic stromal lymphopoietin) via weekly intraperitoneal injection of stroma that have been transduced to overexpress this cytokine. Use of this method provides an in vivo source of continuous cytokine production that achieves physiological levels of circulating human cytokine in the mouse. Plasma levels of human cytokine can be varied based on the number of stromal cells injected, and cytokine production can be initiated at any point in the experiment. This method also includes cytokine-negative control mice that are similarly produced, but through intraperitoneal injection of stroma transduced with a control vector. We have previously demonstrated that leukemia cells harvested from TSLP-expressing PDX, as compared to control PDX, exhibit a gene expression pattern more like the original patient sample. Together the cytokine-producing and cytokine-negative PDX mice produced by this method provide a model system that we have used successfully to study the role of TSLP in normal and malignant hematopoiesis.

Described here is a method for producing exogenous cytokine in patient-derived xenograft (PDX) mice via weekly intraperitoneal injection of a cytokine-transduced stromal cell line. This method broadens the utility of PDX and provides the option for transient or sustained exogenous cytokine delivery in a multitude of PDX models.

Expresión de citoquina exógena en los xenoinjertos derivados del paciente mediante inyección con una línea de células estromales transducidas por citoquinas

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying ...

Bone Marrow Transplantation Platform to Investigate the Role of Dendritic Cells in Graft-versus-Host Disease

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to eradicate tumors. However, its application is limited by the development of graft-versus-host disease (GVHD), a major complication of BMT. GVHD is evoked when T-cells in the donor grafts recognizealloantigen expressed by recipient cells and mount unwanted immunological attacks against recipient healthy tissues. Thus, traditional therapies are designed to suppress donor T-cell alloreactivity. However, these approaches substantially impair the GVL effect so that the recipient&#39;s survival is not improved. Understanding the effects of therapeutic approaches on BMT, GVL, and GVHD, is thus essential. Due to the antigen-presenting and cytokine-secreting capacities to stimulate donor T-cells, recipient dendritic cells (DCs) play a significant role in the induction of GVHD. Therefore, targeting recipient DCs becomes a potential approach for controlling GVHD. This work provides a description of a novel BMT platform to investigate how host DCs regulate GVH and GVL responses after transplantation. Also presented is an effective BMT model to study the biology of GVHD and GVL after transplantation.

Graft-versus-host disease is a major complication after allogeneic bone marrow transplantation. Dendritic cells play a critical role in the pathogenesis of graft-versus-host disease. The current article describes a novel bone marrow transplantation platform to investigate the role of dendritic cells in the development of graft-versus-host disease and the graft-versus-leukemia effect.

Plataforma de trasplante de médula ósea para investigar el papel de las células dendríticas en la enfermedad de injerto contra huésped

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematological malignancies due to the graft-versus-leukemia (GVL) effect to ...

Adoptive Cell Transfer: Introducing Donor Mouse Splenocytes to a Host Mouse and Assessing Success via FACS

Source: Meunier Sylvain1,2,3, Perchet Thibaut1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphopoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
Adoptive cell transfer is a method for introducing cells into a patient or study organism in order to treat a disease or study a biological process, such as haematopoiesis. Aims of adoptive transfer are various; it can be used in fundamental biology as well as in medical sciences (1, 2). In mouse models, migration and distribution of transferred cells can be studied and followed by a tracking system (cell surface marker, staining by CFSE, etc.). In cancer studies on mouse models, transfer of specific cell populations can be used as experimental treatment against tumors. Another example for this technique is creation of chimeric mice by transfer of bone marrow cells to irradiated mice or mice with a severe immunodeficiency phenotype. This mouse model can be used to assess the impact of gene deletion on a specific cell population for instance. Transfer of bone borrow cells is also used in human medical treatment. When patients are irradiated in case of cancer therapy, adoptive transfer of bone marrow allows immune system reconstitution.
The first step in this technique is to obtain the cell population of interest. The technique chosen to isolate this population depends on the level of specificity of the targeted population. The largest level of selection is the whole organ, in which all cell populations present in the organ are taken. A more precise method is selection of a target cell population, often selected by one cell surface marker. The ideal method to sort cells in this case is by magnetic sorting. Finally, the most stringent level is the selection of cells by several cell surface markers to sort very specific cell populations. Flow cytometry sorting is the most popular method for this level of selection. Once population of interest is obtained, it can be transferred to the host. Before adoptive transfer it is essential to ensure compatibility between host and donor. Indeed, regardless of transfer goal, compatibility is crucial to assure cells adoption by the host without cells rejection.
In this lab exercise, we demonstrate the adoptive cell transfer technique by transferring splenocytes from a CD45.2 mouse into a CD45.1 Rag&#947;c mouse (lacking lymphocytes) and four days later confirm the splenocytes transfer using flow cytometry (see Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10504/10504fig1.jpg" /> 
Figure 1: Schematic representation of adoptive transfer. (1) Splenocytes are isolated from CD45.2 mice and (2) transferred in CD45.1 Rag&#947;c mouse, control mouse is injected with PBS only. (3) 4 days after adoptive transfer, splenocytes are recovered from mice and (4) analyzed by flow cytometry. <a href="https://www.jove.com/files/ftp_upload/10504/10504fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Resumen

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