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.

In questa pubblicazione video e protocollo, sono state condotte tutte le procedure sugli animali conformemente al protocollo animali approvato dall&#39;Autorità cantonale di Basilea-Città, Svizzera. Eseguire tutte le procedure in condizioni sterili, ove possibile. 1. Preparazione di Chirurgia <ol><li> Sterilizzare in autoclave tutti gli strumenti chirurgici e garza prima dell&#39;uso. </li><li> Riscaldare il pad calda e organizzare strumenti chirurgici sulla tabella (tabella dei materiali / metodi). </li><li> Aprire le bende striscia dito. Applicare vaselina sul pad ferita con un batuffolo di cotone (assicurarsi che la garza è completamente coperto). </li><li> Pesare i topi e somministrare analgesia Buprenorfina 0,05 mg / kg di peso corporeo. Nota: questo fornirà analgesia subito dopo il trapianto. </li></ol> 2. Preparazione della coda-pelle per il trapianto <ol><li> Euthanize topi donatori (ad esempio, IA BM12) per CO 2 asfissia e disinfect le code con una soluzione di disinfezione prima escissione. </li><li> Posizionare la coda con la striscia nera verso il basso su una tavola di plastica pulito e tagliato longitudinalmente tutta la linea mediana, facendo una incisione superficiale coinvolge solo lo strato di pelle. </li><li> Estrarre la pelle dalla coda con pinze di presa. </li><li> Mettere la coda pelle in una coltura di cellule piatto di 10 centimetri riempito con 10 ml HBSS. </li></ol> 3. Procedura chirurgica <ol><li> Anestetizzare topi riceventi (ad esempio, C57BL / 6) per inalazione di una soluzione isofluorano 3% in una scatola trasparente induzione. Nota: Ci vogliono 3-5 minuti per il mouse per raggiungere l&#39;anestesia piano chirurgico. Utilizzare veterinario unguento sugli occhi per prevenire la secchezza mentre sotto anestesia. </li><li> Posizionare il mouse sul tappetino tiepida e applicare 1,5% isofluorane tramite una maschera bocca e asini ogni riflesso animale punta pizzicare. </li><li> Shave il sito dorsale dei topi riceventi (ad esempio, C57BL / 6) e rimuovere i peli contaminanti utilizzando una garza sw asciuttoab. </li><li> Disinfettare sito trapianto rasata con una soluzione asettica. </li><li> Pizzicare la pelle con una pinza nel sito centrale / destra del dorso e usare le forbici curve per fare un giro incisione di circa 1 x 0,6 centimetri. </li><li> Se l&#39;emorragia si verifica, quindi pulire la ferita con un tampone di cotone sterile. </li><li> Valutare la dimensione della incisione e tagliare un trapianto della dimensione stimata dalla coda-pelle con il bisturi. </li><li> Tagliare gli angoli per arrotondare il trapianto. </li><li> Caricare il trapianto sul bisturi, eccesso secco di HBSS su un batuffolo di cotone sterile e posizionarlo nel letto innesto del mouse destinatario. </li><li> Evitare la sovrapposizione di donatore e la pelle host e applicare l&#39;adesivo di tessuto sulla zona di contatto in esclusiva. </li><li> Applicare un cerotto, senza pieghe intorno alla vita del mouse. </li><li> Applicare l&#39;adesivo bendaggio non elastico per fissare l&#39;intonaco al mouse (3 mm spostato). </li><li> Incidere con le forbici circa 3 mm nel sito ventrale superiore del bendaggio. Nota: Questo per evitare trANALITICI dei denti del mouse durante la fase di recupero. </li><li> Rimuovere l&#39;anestetico-bocca-maschera e mantenere i topi trapiantati sulla rampa di riscaldamento fino sveglio. </li><li> Ritorno topi trapiantati nelle loro gabbie solo quando completamente recuperato e valutare la loro mobilità per garantire che la fasciatura non sia troppo stretto. </li><li> Sterilizzare tutti gli strumenti chirurgici con etanolo al 70% prima di procedere con un intervento chirurgico successivo. </li></ol> 4. Cure post-operatorie <ol><li> Aggiungere sciroppo contenente paracetamolo per l&#39;acqua potabile (4 mg / ml) e somministrare per 7 giorni. </li><li> Ritorno mouse per stanza degli animali una volta che ha recuperato. </li><li> Monitorare attentamente i topi per segni clinici, tra cui la depressione o altri cambiamenti comportamentali (alimentari o di immobilità), aspetto anomalo (mancanza di grooming) o la postura (pilo-erezione o postura ricurva) per il tempo la benda è a posto (1 settimana). </li></ol> 5. Fasciatura Removal (6-7 giorni post-trapianto) <ol><li> Anesthetize topi per inalazione di una soluzione isofluorano 3% in una scatola trasparente induzione (vedi sopra). </li><li> Tagliare benda e gesso con le forbici arterie e cura rimuoverle. Nota: evitare di allungamento del sito trapianto. </li><li> Monitorare i topi trapiantati di segni clinici (v. 4.3) e il punteggio per il rifiuto di innesti cutanei (Tabella 1). Nota: Il sistema di punteggio innesto sfrutta le diverse texture dei capelli sulla coda-pelle rispetto alla dorsale pelle. L&#39;aspetto del trapianto e il suo rifiuto è classificato in 3 gradi. Pelle allogenico (MHC II non corrispondenti, ad esempio, IA BM12) innesti di C57BL / 6 topi immunocompetenti di solito sono rifiutati entro 12-15 giorni dopo il trapianto (dati propri e 15-20). </li></ol>

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The authors declare that they have no competing financial interests.

Trapianto di pelle è un metodo ampiamente usato per studiare rifiuto e tolleranza dipende cellule T. Poiché il modello di trapianto di pelle è stato istituito, sono stati applicati diversi adattamenti e modifiche. Nella procedura descritta, IA BM12 trapianto coda-pelle viene eseguita utilizzando il gas anestetico (isofluorane). L&#39;uso di anestesia gas diminuisce il tempo di esecuzione e topi di recupero, che diminuisce lo stress sui topi trapiantati. La procedura impiega tessuto adesivo per fissare la c...

Il trapianto di organi solidi come la pelle, cuore e reni è ormai una procedura standard nella pratica medica in tutto il mondo 1. Organi trapiantati con successo possono essere rifiutati dalla attivazione del sistema immunitario ricevente, che riconosce i principali antigeni di istocompatibilità del donatore. Pertanto, i pazienti trapiantati necessitano di un trattamento con farmaci immunosoppressori 2. Il trapianto allogenico pelle nei topi è stata fondata da Medawar e colleghi nel 1955 ed è stato utile per identificare le molecole mirate successivamente descritto come complesso maggiore di istocompatibilità (MHC) di classe I e II. Da allora, il modello di trapianto di pelle è stato costantemente modificato e adattato per studiare il ruolo di sottoinsiemi di cellule T e la rilevanza di prodotti chimici e di intervento biologico nel sopprimere rigetto 2-4. Pelle dall&#39;orecchio e il tronco sono più difficili da preparare e sono più suscettibili di ipossia e necrosi di coda pelle 5; tuttavia,la procedura di trapianto è simile. Inoltre il monitoraggio dei trapianti coda-pelle è facile a causa della caratteristica struttura dei capelli della pelle. Questo articolo fornisce una procedura dettagliata per MHC di classe II trapianto coda della pelle disadattamento che permette lo studio dei diversi aspetti del CD4 + T cellulo-mediata rigetto e la tolleranza nei topi. La mutazione naturale tre punti nella molecola MHC di classe II IA b (chiamato IA BM12) 6-9 è sufficiente per indurre il rigetto di alloinnesti cutanee in topi C57BL / 6 8. Il BM12 molecola IA attiva le cellule T CD4 + con vari recettore delle cellule T αβ-(TCR) catene da C57BL / 6 topi, tra cui cellule T Vα2Vβ8-TcR-specifici sono stati identificati al fine di generare un topo transgenico TcR-10. Il trasferimento adottivo di cellule Vα2Vβ8-TcR specifiche T è stato utilizzato per creare un modello di rigetto nei C57B immunodeficientiL / 6 Rag2 - / - topi trapiantati con la pelle IA BM12. Differenze genetiche tra donatore e ricevente impatto l&#39;esito di trapianto di accettazione e di rifiuto. Ci sono diversi tipi di trapianti: autologhi sono trapianti dal singolo destinatario stesso; syngrafts e allotrapianti sono trapianti da individui geneticamente identici e geneticamente imparentati, rispettivamente. L&#39;accettazione di diversi trapianti di organi allogenici è stata dimostrata dagli interventi chimici e biologici nei pazienti e nei modelli murini 11,3,4. In un approccio di base, anti-CD3 anticorpo trattati C57BL / 6 topi hanno mostrato una sopravvivenza prolungata di IA BM12 tail-pelle (dati non pubblicati). L&#39;esaurimento delle CD4 + e CD8 + T cellule prima del trapianto in topi riceventi provocato l&#39;accettazione delle MHC di classe I e II innesti non corrispondenti (Rev. in 12). È interessante notare che il rigetto di innesti di pelle dipende dalla presenza di CD4 + </sup&gt; Cellule T (Rev. in 12). In questo modello, il targeting interazioni specifiche tra le diverse cellule immunitarie, bloccando le molecole di costimolazione con anticorpi o soppressione di cellule T regolatorie potrebbe indurre tolleranza (dati non pubblicati). Infatti, il blocco sia CD40 e CD28 ha portato a lungo termine di tolleranza allogenico pelle 13,14. Trapianto Tail-pelle è facile da eseguire e facile da controllare rispetto al trapianto di altri organi. Inoltre, trapianti coda-pelle sono facili da preparare e sono meno sensibili a ischemia di altri tessuti cutanei. In contrasto con anestetici iniettati, l&#39;uso di gas anestetico (isofluorano) durante il trapianto accorcia sia la procedura e tempi di recupero destinatario. Arricciamenti del trapianto coda-pelle, che può portare alla guarigione della ferita incompleta e rigetto del trapianto, è impedita dalla applicazione di adesivo tissutale. Inoltre, il modello di trapianto di coda-pelle IA BM12 attiva esclusivamente CD4 <sup&gt; + Cellule T sia immunocompetenti e topi immunodeficienti (dello stesso background genetico) facilitare l&#39;interpretazione dei risultati. Questo protocollo descrive un modello di topo affidabile, riproducibile e facilmente controllato che permette intervento chimico e biologico. Il modello è stato progettato per indagare il rifiuto e la tolleranza induzione di trapianti di coda della pelle.

<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.

In un primo approccio C57BL / 6 topi sono stati trapiantati con IA allotrapianti BM12 e IA b syngrafts. Dopo la rimozione della fasciatura, innesti segni manifesti di guarigione della ferita senza chiusura della zona di contatto in topi C57BL / 6 (Figura 1A). Dopo la rimozione della fasciatura, CD4 + T infiammazione cellulo-mediata portato alla comparsa di zone necrotiche (macchie rosse) e rigetto di alloinnesti IA BM12 in C57BL / 6 topi entro 13 giorni dopo i...

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.

Il trapianto di coda della pelle per studiare allogenico di cellule T CD4 risposte in topi

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

12.5K Views.  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.

Trapianto di pelle 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 ...

Ricerca

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.

Induzione di Graft-versus-host disease e<em&gt; In Vivo</em&gt; T monitoraggio cellulare Utilizzando un MHC-abbinato modello murino

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 ...

Immunologia e infezioni

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.

Trapianto di tumore per valutare la dinamica delle cellule T CD8+ infiltranti il tumore nei topi

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

Ricerca sul cancro

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.

Induzione di Murine infiammazione intestinale per trasferimento adottivo di Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alto</sup&gt; T Cells in topi immunodeficienti

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.

Imaging CD4 T cellule interstiziali migrazione nel derma infiammate

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.

Murine tutto spessore trapianto della pelle

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).

Ortotopico degli arti posteriori del trapianto di 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.

Espressione di citochine esogene in Xenografts derivati ​​da pazienti mediante iniezione con una linea cellulare stromalica trasdotta a citocina

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.

Piattaforma di trapianto di midollo osseo per studiare il ruolo delle cellule dendritiche nella malattia di Graft-versus-Host

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>

Il trapianto di coda della pelle per studiare allogenico di cellule T CD4 risposte in topi

Riepilogo

Esplora Altri Video

Capitoli in questo video

Trapianto di pelle murina

Induzione di Graft-versus-host disease e

Trapianto di tumore per valutare la dinamica delle cellule T CD8⁺ infiltranti il tumore nei topi

Induzione di Murine infiammazione intestinale per trasferimento adottivo di Effector CD4

Imaging CD4 T cellule interstiziali migrazione nel derma infiammate

Murine tutto spessore trapianto della pelle

Ortotopico degli arti posteriori del trapianto di mouse

Espressione di citochine esogene in Xenografts derivati da pazienti mediante iniezione con una linea cellulare stromalica trasdotta a citocina

Piattaforma di trapianto di midollo osseo per studiare il ruolo delle cellule dendritiche nella malattia di Graft-versus-Host

Trasferimento di cellule adottive: introduzione degli splenociti di topo donatore a un topo ospite e valutazione del successo tramite FACS

Il trapianto di coda della pelle per studiare allogenico di cellule T CD4 risposte in topi

Riepilogo

Esplora Altri Video

Capitoli in questo video

Trapianto di pelle murina

Induzione di Graft-versus-host disease e

Trapianto di tumore per valutare la dinamica delle cellule T CD8+ infiltranti il tumore nei topi

Induzione di Murine infiammazione intestinale per trasferimento adottivo di Effector CD4

Imaging CD4 T cellule interstiziali migrazione nel derma infiammate

Murine tutto spessore trapianto della pelle

Ortotopico degli arti posteriori del trapianto di mouse

Espressione di citochine esogene in Xenografts derivati ​​da pazienti mediante iniezione con una linea cellulare stromalica trasdotta a citocina

Piattaforma di trapianto di midollo osseo per studiare il ruolo delle cellule dendritiche nella malattia di Graft-versus-Host

Trasferimento di cellule adottive: introduzione degli splenociti di topo donatore a un topo ospite e valutazione del successo tramite FACS

Trapianto di tumore per valutare la dinamica delle cellule T CD8⁺ infiltranti il tumore nei topi

Espressione di citochine esogene in Xenografts derivati da pazienti mediante iniezione con una linea cellulare stromalica trasdotta a citocina