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.

Nesta publicação de vídeo e protocolo, todos os procedimentos com animais foram conduzidos de acordo com o protocolo de animais aprovados pela Autoridade cantão Basileia-Cidade, Suíça. Execute todos os procedimentos em condições estéreis, sempre que possível. 1. Preparação de Cirurgia <ol><li> Autoclave todos os instrumentos cirúrgicos e gaze antes de usar. </li><li> Aqueça a almofada quente e organizar instrumentos cirúrgicos sobre a mesa (Tabela de Materiais / Métodos). </li><li> Abra as ataduras tira dedo. Aplique vaselina na almofada ferida com um cotonete (certifique-se a gaze é completamente coberto). </li><li> Pesar os ratinhos e administrar analgesia buprenorfina 0,05 mg / kg de peso corporal. Nota: este vai proporcionar analgesia imediatamente após o transplante. </li></ol> 2. Preparação do Cauda-pele para Transplante <ol><li> Eutanásia camundongos doadores (por exemplo, BM12 IA) por asfixia CO 2 e disinfect as caudas com solução desinfetante antes da excisão. </li><li> Posicione o rabo com a tarja preta para baixo em uma placa de plástico limpo e corte-o longitudinalmente ao longo da linha do meio, fazendo uma incisão superficial envolvendo apenas a camada de pele. </li><li> Retire a pele da cauda usando uma pinça agarrando. </li><li> Coloque o rabo-de pele em uma placa de cultura de 10 centímetros célula cheia com 10 ml de HBSS. </li></ol> 3. Procedimento Cirúrgico <ol><li> Anestesiar ratos receptores (por exemplo, ratinhos C57BL / 6) por inalação de uma solução a 3% de isofluorano em uma caixa de indução transparente. Nota: Demora 3-5 min para o mouse para chegar a anestesia plano cirúrgico. Use veterinário pomada nos olhos para evitar a secura sob anestesia. </li><li> Coloque o mouse sobre a almofada morna e aplique 1,5% isofluorano através de uma máscara boca e bundas cada reflexo animais por toe beliscar. </li><li> Raspar o site dorsal dos ratos destinatário (por exemplo, C57BL / 6) e remover os pêlos contaminantes usando um sw gaze secaab. </li><li> Desinfetar local transplante raspada com uma solução asséptica. </li><li> Aperte a pele com uma pinça no site meio / direita do dorso e usar tesoura curva para fazer uma incisão em torno de cerca de 1 x 0.6 cm. </li><li> Se ocorrer sangramento, em seguida, limpar a ferida com um cotonete estéril. </li><li> Avaliar o tamanho da incisão e corta um transplante do tamanho estimado a partir da cauda para a pele usando o bisturi. </li><li> Apare os cantos para arredondar o transplante. </li><li> Carregue o transplante no bisturi, o excesso de seca de HBSS em um cotonete estéril e colocá-lo na cama enxerto do mouse destinatário. </li><li> Evitar a sobreposição de doador e pele de acolhimento e aplicar adesivo de tecido na zona de contato com exclusividade. </li><li> Aplicar um emplastro sem vincos ao redor da cintura do mouse. </li><li> Aplique o adesivo curativo não-elástico para fixar o gesso para o mouse (três milímetros deslocado). </li><li> Inciso com a tesoura cerca de 3 mm no site ventral superior do curativo. Nota: Isto é para evitar trapping dos dentes do rato durante a fase de recuperação. </li><li> Remova a boca-máscara anestésica e manter os ratos transplantados com a almofada de aquecimento até acordado. </li><li> Retornar ratinhos transplantados para as suas gaiolas apenas quando totalmente recuperado e avaliar a sua mobilidade para assegurar que a ligadura não seja demasiado apertado. </li><li> Esterilizar todos os instrumentos cirúrgicos com etanol 70%, antes de prosseguir com a próxima cirurgia. </li></ol> 4. Cuidados pós-operatórios <ol><li> Adicionar contendo paracetamol xarope para a água de beber (4 mg / ml), e administrá-lo por 7 dias. </li><li> Retorno do mouse para a sala de animais, uma vez que se recuperou. </li><li> Acompanhar de perto os ratos para sinais clínicos, incluindo depressão ou outras alterações comportamentais (comer ou imobilidade), a aparência anormal (falta de higiene) ou postura (pilo-ereção ou postura arqueada) para o tempo o curativo está em vigor (1 semana). </li></ol> 5. Remoção Bandage (6-7 dias pós-transplante) <ol><li> Anesthetize ratos por inalação de uma solução Isofluorano 3% em uma caixa de indução transparente (ver acima). </li><li> Corte curativo e gesso com uma tesoura da artéria e, cuidadosamente, remova-os. Nota: evitar o estiramento do local do transplante. </li><li> Monitorar os ratos transplantados para os sinais clínicos (ver 4.3) e marcar para a rejeição de enxertos de pele (Tabela 1). Nota: O sistema de pontuação do enxerto aproveita as diferentes texturas de cabelo na cauda-pele em comparação com o dorsal pele. A aparência do transplante e sua rejeição é classificado em 3 classes. Pele alogênico (MHC II incompatíveis, por exemplo, IA BM12) enxertos de camundongos C57BL / 6 imunocompetentes geralmente são rejeitados dentro de 12-15 dias pós transplante (próprios dados e 15-20). </li></ol>

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The authors declare that they have no competing financial interests.

O transplante de pele é um método amplamente utilizado para estudar a rejeição e de tolerância em células T dependentes. Desde foi criado o modelo de transplante de pele, várias adaptações e mudanças foram aplicadas. No procedimento descrito, IA BM12 transplante de cauda-de pele é realizada usando gás anestésico (isoflurano). O uso de anestesia gás diminui o tempo de execução e recuperação de ratos, o que diminui o stress em ratos transplantados. O procedimento utiliza tecido adesivo para fi...

O transplante de órgãos sólidos, tais como a pele, o coração e os rins já é um procedimento padrão na prática médica mundial 1. Órgãos transplantados com sucesso pode ser rejeitado pela activação do sistema imune receptor, o qual reconhece os principais antigénios de histocompatibilidade dos dadores. Pacientes transplantados Portanto precisam de tratamento com drogas imunossupressoras 2. O transplante alogênico de pele em ratos foi estabelecida por Medawar e seus colegas em 1955 e foi útil para identificar as moléculas-alvo mais tarde descrito como complexo principal de histocompatibilidade (MHC) de classe I e II. Desde então, o modelo de transplante de pele tem sido continuamente modificado e adaptado para estudar o papel das subpopulações de células T e a relevância da química e biológica de intervenção na supressão da rejeição do enxerto 2-4. Pele da orelha e tronco são mais difíceis de preparar e são mais suscetíveis à hipóxia e necrose de pele tail-5; no entanto,o procedimento de transplante é semelhante. Além disso, o monitoramento de transplantes de cauda de pele é fácil, devido à textura do cabelo característico da pele. Este artigo fornece um procedimento detalhado para a classe II do MHC transplante incompatibilidade cauda-de pele que permite que para o estudo de diferentes aspectos dos linfócitos T CD4 + mediada por células T e tolerância rejeição do enxerto em murganhos. A mutação de três pontos natural na molécula de MHC de classe II IA b (chamado IA BM12) 6-9 é suficiente para induzir a rejeição de enxertos de pele em ratos C57BL / 6 8. A molécula BM12 IA activa as células T CD4 + com vários receptores de células αβ-T (TCR) cadeias de ratinhos C57BL / 6, entre os quais as células T Vα2Vβ8-específicos de TCR foram identificados, a fim de gerar um ratinho transgénico Tc-10. A transferência adoptiva de células T específicos de TCR Vα2Vβ8 tem sido usado para estabelecer um modelo de rejeição em C57B imunodeficienteL / 6 RAG2 - / - ratos transplantados com IA BM12 pele. As diferenças genéticas entre doador e receptor influência sobre o resultado de aceitação de transplante e rejeição. Existem diferentes tipos de transplantes: auto-enxertos são transplantados a partir do próprio indivíduo receptor; syngrafts e enxertos são transplantes de indivíduos geneticamente idênticos e geneticamente não relacionadas, respectivamente. A aceitação de transplante de órgãos alogénicos foi demonstrada por intervenção química e biológica de pacientes e de modelos de rato 11,3,4. Numa abordagem básica, anti-CD3 tratadas com anticorpo C57BL / 6 ratos mostraram sobrevivência prolongada de IA BM12 cauda-de pele (dados não publicados). A depleção de células T CD4 + e CD8 +, células T antes do transplante em ratinhos receptores resultou na aceitação do MHC de classe I e II enxertos incompatíveis (rev. em 12). Curiosamente, a rejeição de enxertos de pele depende da presença de células T CD4 + </sup&gt; As células T (Rev. em 12). Neste modelo, tendo como alvo as interacções específicas entre as células imunes diferentes, bloqueando as moléculas de co-estimulação com anticorpos ou com supressão de células T reguladoras, pode induzir tolerância (dados não publicados). Na verdade, bloqueando tanto CD40 e CD28 levou a longo prazo a tolerância do enxerto de pele 13,14. O transplante de pele da cauda-é fácil de realizar e é fácil de monitorizar em comparação com o transplante de outros órgãos. Além disso, os transplantes de pele de cauda são fáceis de preparar e são menos susceptíveis à isquemia do que os outros tecidos da pele. Em contraste com os anestésicos injectáveis, o uso de gás anestésico (isoflurano) durante o transplante de ambos encurta o processo e o tempo de recuperação do destinatário. Curling do transplante de cauda-de pele, o que pode conduzir a cicatrização incompleta e rejeição de enxerto, é evitada por aplicação de adesivo de tecido. Além disso, o modelo de transplante de pele da cauda-IA BM12 ativa exclusivamente CD4 <sup&gt; + Células T, tanto em imunocompetentes e camundongos imunodeficientes (do mesmo fundo genético) facilitando a interpretação dos resultados. Este protocolo descreve um modelo fiável, reprodutível e facilmente monitorado rato que permite a intervenção química e biológica. O modelo destina-se a investigar a rejeição ea tolerância indução de transplantes de rabo-de pele.

<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.

Numa primeira abordagem C57BL / 6 foram transplantadas com IA allografts BM12 e IA b syngrafts. Após a remoção curativo, enxertos sinais manifestos de cicatrização de feridas, sem encerramento da zona de contato em camundongos C57BL / 6 (Figura 1A). Após a remoção do penso, a inflamação mediada por células T CD4 +, conduziu ao aparecimento de áreas necróticas (manchas vermelhas) e rejeição de aloenxertos IA BM12 em ratinhos C57BL / 6 no espaço ...

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.

O transplante de pele da cauda para estudar as respostas das células T CD4 alogênico em camundongos

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

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JoVE Journal

Immunology and Infection

12.4K visualizações.  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.

Vídeo: 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.

Transplante de pele murinos

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

Biologia

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.

Indução de enxerto-versus-hospedeiro de Doenças e<em&gt; Em Vivo</em&gt; Monitoramento celular T Usando um modelo murino de MHC de correspondência

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 ...

Imunologia e Infecção

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.

Transplante de tumor para avaliar a dinâmica das células CD8+ T que se infiltram em tumores em camundongos

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

Pesquisa do câncer

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.

Indução de Murino inflamação intestinal por transferência adotiva de Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; Células T em imunodeficientes Mice

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.

Migração intersticial imagens de células CD4 T na derme 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 Transplante de pele de espessura total

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 Hind Limb Transplantation no mouse

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.

Expressão de citocina exógena em xenotransplantes derivados de pacientes via injeção com uma linha de células estromáticas transduzidas por citocinas

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 transplante de medula óssea para investigar o papel das células dendríticas na doença enxerto-versus-hospedeiro

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>

O transplante de pele da cauda para estudar as respostas das células T CD4 alogênico em camundongos

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