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 diesem Video-Veröffentlichung und Protokoll wurden alle Tier Verfahren in Übereinstimmung mit der von der Kantonsbehörde Basel-Stadt, Schweiz zugelassenen Tier Protokoll durchgeführt. Führen Sie alle in sterilen Bedingungen, soweit möglich. 1. Vorbereitung der Chirurgie <ol><li> Autoklav alle chirurgischen Instrumenten und Gaze vor dem Gebrauch. </li><li> Erhitzen Sie die warmen Kissen und organisieren chirurgische Instrumente auf dem Tisch (Table of Materialien / Methoden). </li><li> Öffnen Sie die Fingerleiste Bandagen. Vaseline auf die Wundauflage mit einem Wattestäbchen (sicherstellen, dass die Gaze vollständig bedeckt ist). </li><li> Wiegen Sie die Mäuse und verwalten Analgesie Buprenorphin 0,05 mg / kg Körpergewicht. Hinweis: dies wird Analgesie unmittelbar nach der Transplantation bieten. </li></ol> 2. Vorbereitung der Schwanzhaut für Transplantation <ol><li> Euthanize Spendermäusen (zB IA BM12) pro CO 2-Erstickung und disinfect die Schwänze mit Desinfektionslösung vor Exzision. </li><li> Positionieren Sie den Schwanz mit dem schwarzen Streifen unten auf eine saubere Plastikbrett und schneiden Sie es in Längsrichtung über die Mittellinie, so dass eine oberflächliche Inzision, die nur die Hautschicht. </li><li> Ziehen Sie die Haut vom Schwanz mit Greifzange. </li><li> Setzen Sie den Schwanz-Haut in einer 10 cm Zellkulturschale mit 10 ml HBSS gefüllt. </li></ol> 3. Chirurgisches Vorgehen <ol><li> Betäuben Empfängermäuse (beispielsweise C57BL / 6) durch Inhalation einer 3% Isofluran Lösung in einem transparenten Induktionsfeld. Hinweis: Es dauert 3-5 min für die Maus, um chirurgische Anästhesie-Ebene zu erreichen. Verwenden Tierarzt Salbe auf die Augen zu Trockenheit während der Narkose zu vermeiden. </li><li> Legen Sie die Maus auf den warmen Pad und gelten 1,5% Isofluran über eine Mundmaske und Esel jedes Tier Reflex Zehen Kneifen. </li><li> Rasieren Sie die dorsale Seite der Empfängermäuse (zB C57BL / 6) und entfernen verunreinigende Haare mit einem trockenen Gaze swab. </li><li> Desinfizieren Sie rasiert Transplantation Website mit aseptischen Lösung. </li><li> Klemmen Sie die Haut mit einer Pinzette in der Mitte / rechts Seite des Rückens und einer gebogenen Schere verwenden, um eine Runde Schnitt von etwa 1 x 0,6 cm zu machen. </li><li> Wenn Blutungen auftreten, dann reinigen Sie die Wunde mit einem sterilen Wattestäbchen. </li><li> Bewerten Sie die Größe des Einschnitts und schneiden eine Transplantation von der geschätzten Größe von der Schwanzhaut mit dem Skalpell. </li><li> Schneiden Sie die Ecke der Transplantation runden. </li><li> Laden Sie die Transplantation auf dem Skalpell, trockenen Überschuss von HBSS auf einem sterilen Wattestäbchen und legen Sie sie in das Transplantat Bett der Empfängermaus. </li><li> Vermeiden Sie Überlagerung von Spender und Empfänger Haut und wenden Gewebekleber auf die Kontaktzone ausschließlich. </li><li> Tragen Sie eine Putz ohne Falten rund um die Maus Taille. </li><li> Kleber nicht-elastische Binde, das Pflaster auf die Maus zu beheben (3 mm verschoben). </li><li> Einzuschneiden mit der Schere etwa 3 mm im oberen ventralen Stelle der Bandage. Hinweis: Dies ist zu vermeiden, trapping von der Maus die Zähne während der Erholungsphase. </li><li> Entfernen Sie die Narkose-Mund-Maske und halten die transplantierten Mäusen auf dem Heizkissen bis wach. </li><li> Zurück transplantierten Mäusen in ihre Käfige nur bei vollständig erholt und beurteilen ihre Mobilität zu gewährleisten, dass der Verband nicht zu eng. </li><li> Sterilisieren Sie alle chirurgischen Instrumente mit 70% Ethanol, bevor mit der nächsten Operation fortfahren. </li></ol> 4. Post-operative Betreuung <ol><li> Hinzufügen Paracetamol enthaltenden Sirup zum Trinkwasser (4 mg / ml) und zu verwalten, für 7 Tage. </li><li> Zurück Maus an den Tierraum, sobald sie sich erholt hat. </li><li> Mäuse auf klinische Anzeichen wie Depressionen oder andere Verhaltensänderungen (Essen oder Immobilität), abnorme Erscheinung (mangelnde Pflege) oder Körperhaltung (Piloerektion oder gekrümmte Haltung) für die Zeit, die Binde ist vorhanden (1 Woche) genau beobachten. </li></ol> 5. Bandage Removal (6-7 Tage nach der Transplantation) <ol><li> Anesthetize Mäuse durch Inhalation einer 3% Isofluoran Lösung in einem transparenten Induktionsfeld (siehe oben). </li><li> Schneiden Verband und Gips mit Arterie Schere und sorgfältig zu entfernen. Hinweis: Vermeiden Sie Strecken des Transplantations Ort. </li><li> Überwachen Sie den transplantierten Mäusen für die klinische Anzeichen (siehe 4.3) und Punkte für die Ablehnung von Hauttransplantaten (Tabelle 1). Hinweis: Das Transplantat Score-System nutzt die Vorteile der verschiedenen Haartypen auf den Schwanz-Haut im Vergleich zu der Rückenhaut. In drei Typen wird das Aussehen des Transplantates und seiner Ablehnung kategorisiert. Die allogene Haut (MHC II nicht übereinstimm, z. B., IA BM12) Transplantate von C57BL / 6 immunkompetenten Mäusen in der Regel innerhalb von 12 bis 15 Tage nach der Transplantation (eigene Daten und 15-20) abgelehnt. </li></ol>

<ol>
	<li>Mahillo, B., Carmona, M., &#193;lvarez, M., White, S., Noel, L., Matesanz, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Data+in+Organ+Donation+and+Transplantation.">Global Data in Organ Donation and Transplantation.</a> transplantation. 92 (10), 1069-1074 (2011).</li><li>Halleck, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+perspectives+of+immunosuppression.">New perspectives of immunosuppression.</a> Transplantation proceedings. 45 (3), 1224-1231 (2013).</li><li>Wood, K. J., Bushell, A., Jones, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+unresponsiveness+to+alloantigen+in+vivo.+a+role+for+regulatory+T+cells.">Immunologic unresponsiveness to alloantigen in vivo. a role for regulatory T cells.</a> Immunological reviews. 241 (1), 119-132 (2011).</li><li>Sykes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+tolerance:+mechanisms+and+application+in+clinical+transplantation.">Immune tolerance: mechanisms and application in clinical transplantation.</a> Journal of Internal Medicine. 262 (3), 288-310 (2007).</li><li>McFarland, H. I., Rosenberg, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+allograft+rejection..">Skin allograft rejection..</a> Current protocols in immunology. , (2009).</li><li>McKenzie, I. F., Morgan, G. M., Sandrin, M. S., Michaelides, M. M., Melvold, R. W., Kohn, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B6.C-H-2bm12.+A+new+H-2+mutation+in+the+I+region+in+the+mouse.">B6.C-H-2bm12. A new H-2 mutation in the I region in the mouse.</a> The Journal of experimental medicine. 150 (6), 1323-1338 (1979).</li><li>McIntyre, K. R., Seidman, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+sequence+of+mutant+I-A+beta+bm12+gene+is+evidence+for+genetic+exchange+between+mouse+immune+response+genes.">Nucleotide sequence of mutant I-A beta bm12 gene is evidence for genetic exchange between mouse immune response genes.</a> Nature. 308 (5959), 551-553 (1984).</li><li>Stuart, P. M., Beck-Maier, B., Melvold, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Provocation+of+skin+graft+rejection+across+murine+class+II+differences+by+non--bone-marrow-derived+cells.">Provocation of skin graft rejection across murine class II differences by non--bone-marrow-derived cells.</a> Transplantation. 37 (4), 393-396 (1984).</li><li>Hausmann, B., Palmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+selection+through+a+motif+in+the+alphabeta+T+cell+receptor.">Positive selection through a motif in the alphabeta T cell receptor.</a> Science. 281 (5378), 835-838 (1998).</li><li>Bill, J., Ronchese, F., Germain, R. N., Palmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+mutant+amino+acids+to+alloantigenicity.">The contribution of mutant amino acids to alloantigenicity.</a> The Journal of experimental medicine. 170 (3), (1989).</li><li>Monaco, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosuppression+and+tolerance+for+clinical+organ+allografts.">Immunosuppression and tolerance for clinical organ allografts.</a> Current Opinion in Immunology. 1 (6), 1174-1177 (1989).</li><li>Rosenberg, A. S., Singer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+basis+of+skin+allograft+rejection:+an+in+vivo+model+of+immune-mediated+tissue+destruction.">Cellular basis of skin allograft rejection: an in vivo model of immune-mediated tissue destruction.</a> Annual Review of Immunology. 10, 333-358 (1992).</li><li>Kingsley, C. I., Nadig, S. N., Wood, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+tolerance:+lessons+from+experimental+rodent+models.">Transplantation tolerance: lessons from experimental rodent models.</a> Transplant international : official journal of the European Society for Organ Transplantation. 20 (10), 828-841 (2007).</li><li>Larsen, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+acceptance+of+skin+and+cardiac+allografts+after+blocking+CD40+and+CD28+pathways.">Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways.</a> Nature. 381 (6581), 434-438 (1996).</li><li>Tomura, M., Nakatani, I., Murachi, M., Tai, X. G., Toyo-oka, K., Fujiwara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+allograft+responses+induced+by+interleukin-6,+which+selectively+modulates+interferon-gamma+but+not+interleukin-2+production.">Suppression of allograft responses induced by interleukin-6, which selectively modulates interferon-gamma but not interleukin-2 production.</a> Transplantation. 64 (5), 757-763 (1997).</li><li>Ring, G. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-gamma+is+necessary+for+initiating+the+acute+rejection+of+major+histocompatibility+complex+class+II-disparate+skin+allografts.">Interferon-gamma is necessary for initiating the acute rejection of major histocompatibility complex class II-disparate skin allografts.</a> Transplantation. 67 (10), 1362-1365 (1999).</li><li>Rosenberg, A. S., Finbloom, D. S., Maniero, T. G., Vander Meide, P. H., Singer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+prolongation+of+MHC+class+II+disparate+skin+allografts+by+in+vivo+administration+of+anti-IFN-gamma+monoclonal+antibody.">Specific prolongation of MHC class II disparate skin allografts by in vivo administration of anti-IFN-gamma monoclonal antibody.</a> Journal of immunology. 144 (12), 4648-4650 (1950).</li><li>Goes, N., Sims, T., Urmson, J., Vincent, D., Ramassar, V., Halloran, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disturbed+MHC+regulation+in+the+IFN-gamma+knockout+mouse.+Evidence+for+three+states+of+MHC+expression+with+distinct+roles+for+IFN-gamma.">Disturbed MHC regulation in the IFN-gamma knockout mouse. Evidence for three states of MHC expression with distinct roles for IFN-gamma.</a> Journal of immunology. 155 (10), 4559-4566 (1995).</li><li>Surquin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-4+deficiency+prevents+eosinophilic+rejection+and+uncovers+a+role+for+neutrophils+in+the+rejection+of+MHC+class+II+disparate+skin+grafts.">IL-4 deficiency prevents eosinophilic rejection and uncovers a role for neutrophils in the rejection of MHC class II disparate skin grafts.</a> Transplantation. 80 (10), 1485-1492 (2005).</li><li>Gaylo, A. E., Laux, K. S., Batzel, E. J., Berg, M. E., Field, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+rejection+of+MHC+class+II-disparate+skin+allografts+in+mice+treated+with+farnesyltransferase+inhibitors.">Delayed rejection of MHC class II-disparate skin allografts in mice treated with farnesyltransferase inhibitors.</a> Transplant immunology. 20 (3), 163-170 (2009).</li><li>Bose, A., Inoue, Y., Kokko, K. E., Lakkis, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+perforin+down-regulates+CD4+and+CD8+T+cell-mediated+immune+responses+to+a+transplanted+organ.">Cutting edge: perforin down-regulates CD4 and CD8 T cell-mediated immune responses to a transplanted organ.</a> Journal of immunology. 170 (4), 1611-1614 (2003).</li></ol>

The authors declare that they have no competing financial interests.

Hauttransplantation ist ein weit verbreitetes Verfahren zur Untersuchung Ablehnung und Toleranz abhängig von T-Zellen. Da die Haut Transplantationsmodell gegründet wurde, wurden mehrere Anpassungen und Änderungen angewandt. Bei dem beschriebenen Verfahren wird IA-BM12 Schwanz Hauttransplantation durchgeführt unter Verwendung von Narkosegas (Isofluran). Die Verwendung von Gas Narkose verringert die Zeit der Ausführung und Mäuse Erholung, die die Belastung der transplantierten Mäusen abnimmt. Das Verfahr...

Transplantation von soliden Organen wie Haut, Herz und Nieren ist inzwischen ein Standardverfahren in der medizinischen Praxis weltweit ein. Erfolgreich transplantierten Organen kann durch Aktivierung des Immunsystems des Empfängers, der die Haupt-Histokompatibilitäts-Antigene des Spenders erkennt zurückzuweisen. Daher transplantierten Patienten müssen die Behandlung mit Immunsuppressiva 2. Die allogene Hauttransplantation bei Mäusen wurde durch Medawar und Kollegen im Jahr 1955 gegründet und war hilfreich für die Identifizierung der Zielmoleküle später als Major Histocompatibility Complex (MHC) Klasse I und II beschrieben. Seitdem hat sich die Haut Transplantationsmodell kontinuierlich modifiziert und angepasst ist, um die Rolle der T-Zelluntergruppen und die Bedeutung von chemischen und biologischen Intervention bei der Unterdrückung der Abstoßung 2-4 studieren. Haut aus dem Ohr und Rumpf sind schwieriger herzustellen und sind anfälliger für Hypoxie und Nekrose als Schwanzhaut 5; jedochdas Transplantationsverfahren ist ähnlich. Neben der Überwachung der Schwanzhauttransplantaten ist einfach aufgrund der charakteristischen Haarstruktur der Haut. Dieser Artikel enthält eine detaillierte Prozedur für die MHC-Klasse-II-Fehlanpassung Schwanzhauttransplantation, die für die Untersuchung verschiedener Aspekte der CD4 + T-Zell-vermittelten Abstoßung und Toleranz bei Mäusen ermöglicht. Die natürliche Dreipunktmutation in der MHC-Klasse-II-Molekül IA b (genannt IA BM12) 6-9 ist ausreichend, um eine Abstoßung von Hauttransplantate in C57BL / 6 Mäusen 8 induzieren. Die IA BM12 Molekül aktiviert CD4 + T-Zellen mit verschiedenen αβ-T-Zellrezeptor (TCR)-Ketten von C57BL / 6 Mäusen, unter denen Vα2Vβ8-TCR-spezifische T-Zellen, um eine TcR-transgenen Maus 10 erzeugt identifiziert. Der adoptive Transfer von Vα2Vβ8-TCR-spezifische T-Zellen wurde verwendet, um eine Abstoßungsmodell in immundefizienten C57B etablierenL / 6 Rag2 - / - Mäuse mit IA BM12 Haut transplantiert. Genetische Unterschiede zwischen Spender und Empfänger beeinflussen das Ergebnis der Transplantat-Akzeptanz und Zurückweisung. Es gibt verschiedene Arten von Transplantationen: autologen Transplantationen sind vom Empfänger Individuum selbst; syngrafts und Allografts sind Transplantationen von genetisch identisch und genetisch nicht verwandten Individuen sind. Akzeptanz unterschiedlicher allogenen Organtransplantationen durch chemische und biologische Intervention bei Patienten und Mausmodellen 11,3,4 nachgewiesen. In einem grundlegenden Ansatz zeigte anti-CD3-Antikörper-behandelten C57BL / 6 Mäuse verlängerten Überlebenszeit von IA BM12 tail-Haut (unveröffentlichte Daten). Depletion von CD4 + und CD8 + T-Zellen vor der Transplantation in Empfängermäuse führte die Annahme der MHC-Klasse I und II nicht übereinstimm Transplantate (rev. 12). Interessanterweise Abstoßung von Hauttransplantaten, von der Anwesenheit von CD4 + </sup&gt; T-Zellen (rev. 12). In diesem Modell, auf bestimmte Interaktionen zwischen den verschiedenen Immunzellen durch Blockierung kostimulatorische Moleküle mit Antikörpern oder Unterdrückung der regulatorischen T-Zellen könnte Toleranz (unveröffentlichte Daten) zu induzieren. In der Tat, Sperrung sowohl CD40 und CD28 führte zu langfristigen Hauttransplantattoleranz 13,14. Schwanz-Hauttransplantation ist einfach durchzuführen und einfach zu überwachen, im Vergleich zur Transplantation anderer Organe. Darüber hinaus sind Schwanzhauttransplantate leicht herzustellen und sind weniger anfällig für Ischämie als andere Hautgewebe. Im Gegensatz zu injizierenden Anästhetika, die Verwendung von Narkosegas (Isofluran) während der Transplantation verkürzt sowohl die Verfahren und Empfänger Erholungszeit. Curling der Schwanzhaut-Transplantation, die unvollständig Wundheilung und Transplantat-Abstoßung führen kann, wird durch die Anwendung von Gewebekleber verhindert. Darüber hinaus wird die IA-BM12 Schwanz Hauttransplantation Modell aktiviert CD4 ausschließlich <sup&gt; + T-Zellen sowohl in der immunkompetenten und immungeschwächten Mäusen (aus dem gleichen genetischen Hintergrund) die Erleichterung der Interpretation der Ergebnisse. Dieses Protokoll beschreibt einen zuverlässigen, reproduzierbaren und leicht überwacht Maus-Modell, das für die chemische und biologische Eingriffe ermöglicht. Das Modell ist für die Untersuchung von Ablehnung und Toleranzinduktion von tail-Hauttransplantationen vorgesehen.

<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 einem ersten Ansatz wurden C57BL / 6 Mäuse mit IA BM12 Allografts und IA b syngrafts transplantiert. Bandage nach Entfernung Transplantate offensichtlichen Anzeichen der Wundheilung ohne Schließung der Kontaktzone in C57BL / 6 Mäusen (Fig. 1A). Bandage nach Entfernung führte CD4 + T-Zell-vermittelte Entzündung zu dem Auftreten von Nekrosen (rote Punkte) und Ablehnung IA BM12 Allotransplantaten in C57BL / 6 Mäusen innerhalb von 13 Tagen nach der Transp...

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.

Transplantation von Haut-Schwanz zu allogene CD4-T-Zellantworten in Mäusen untersucht

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 Aufrufe.  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.

Serie: 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.

Murine Hauttransplantation

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

Forschung

Biologie

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.

Induktion der Graft-versus-Host-Krankheit und<em&gt; In Vivo</em&gt; T-Zell-Überwachung mit einem MHC-matched Mausmodell

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 ...

Immunologie und Infektion

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.

Tumortransplantation zur Beurteilung der Dynamik tumorinfiltrierender CD8+ T-Zellen in Mäusen

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

Krebsforschung

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.

Die Induktion von Murine Darmentzündungen durch adoptiven Transfer von Effektor CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Hoch</sup&gt; T-Zellen in immundefizienten Mäusen

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-Zell-Interstitial Migration in den entzündeten 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 ...

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 Vollhauttransplantation

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).

Orthotopische Hind Gliedmaßen Transplantation in der Maus

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

Medizin

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.

Expression von exogenem Cytokin in Patient-abgeleiteten Xenotransplantaten über Injektion mit einer Cytokin-transduzierten Stromzelllinie

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.

Knochenmarktransplantationsplattform zur Untersuchung der Rolle dendritischer Zellen bei Graft-versus-Host-Krankheit

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>

Transplantation von Haut-Schwanz zu allogene CD4-T-Zellantworten in Mäusen untersucht

Zusammenfassung

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Imaging CD4 T-Zell-Interstitial Migration in den entzündeten Dermis

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Orthotopische Hind Gliedmaßen Transplantation in der Maus

Expression von exogenem Cytokin in Patient-abgeleiteten Xenotransplantaten über Injektion mit einer Cytokin-transduzierten Stromzelllinie

Knochenmarktransplantationsplattform zur Untersuchung der Rolle dendritischer Zellen bei Graft-versus-Host-Krankheit

Adoptivzelltransfer: Spendermaus-Splenozyten einer Host-Maus einführen und Erfolg über FACS bewerten