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

Die Maus-Studien wurden von der Institutional Animal Care und Verwenden Committee (IACUC) der University of North Carolina in Chapel Hill und alle genehmigten Tierpflege wurde in Übereinstimmung mit den Richtlinien IACUC. 1. Herstellung von Neugeborenen und Erwachsene Thymi <ol><li> Bereiten Sie alle Reagenzien und Geräte vor euthanizing Spendermäusen. </li><li> Sterilisieren chirurgischer Instrumente durch Autoklavierung oder andere geeignete Verfahren. Alle chirurgischen Verfahren müssen unter einer Haube mit laminarer Strömung, um sterile Bedingungen aufrecht zu erhalten und eine Verunreinigung zu vermeiden, durchgeführt werden. Montieren Sie die Werkzeuge für die Gewinnung von Thymi von Spendermäusen benötigt. </li><li> Füllen Sie einen 60-mm-Schale mit steriler 1x PBS (pH 7,4) und auf Eis in der Kapuze. Dies wird zu kurz store Thymi von Spendermäusen vor der Transplantation entnommen werden. </li><li> Nach ethischen Richtlinien, einschläfern Neugeborenen Spendermäusen durch Enthauptung und erwachsene Spendermäusen durch CO 2 Ersticken durch Genickbruch folgte. </li><li> Für removal des Thymus: Legen Sie die Maus in einer dorsalen Rückenlage auf einem sterilen saugfähigen Papiertuch und sprühen mit 70% Ethanol vor der einen Einschnitt. </li><li> Setzen Sie die Bauch- und Brusthöhle, indem Sie einen Mittelschnitt durch die Haut. Falten Sie die Haut über der Brust und Vorderbeine, um die Brusthöhle zu offenbaren. </li><li> Machen Sie zwei seitliche Schnitte durch das Zwerchfell und Brustkorb, den oberen Mediastinum aussetzen und vorderen Brusthöhle. Der Thymus ist als zwei weißen Lappen unmittelbar über sichtbare und neben dem Herzen sein. </li><li> Necken neben der rund um die Thymusdrüse mit feinen Pinzette Bindegewebe, so dass bestimmte nicht, um die Kapsel zu stören. Während den Brustkorb mit einer Pinzette zurückhalten, verwenden Sie eine zweite Zange, um die zwei Lappen des Thymus durch die Positionierung einer gebogenen Pinzette unter der Orgel und vertikal ziehen zu extrahieren. Dies kann unter Verwendung eines Binokular zur Extraktion Thymi von neugeborenen Mäusen durchgeführt werden. </li><li> Setzen Sie den Thymus in der 60 mm dish, die steriles 1x PBS (pH 7,4) auf Eis und separater Thymus Lappen durch Durchtrennen des Bindelandenge. Entfernen Sie alle Fremdkörper aus dem Thymus Lappen mit dem bestimmte nicht, um die Kapsel beschädigt werden, und schneiden Sie die Thymus in die entsprechende Anzahl von Abschnitten für die Transplantation. </li><li> Manipulieren Sie nicht den von neugeborenen Mäusen erhalten Thymi. HINWEIS: Wird für maximal zwei Empfängermäuse (1 Lappen pro Empfänger). </li><li> Transplantation die aus erwachsenen Mäusen erhalten Thymi in maximal 4-6 Empfängermäuse. Mit Hilfe einer Pinzette vorsichtig greifen ein Erwachsener Thymus Lappen, wie in Abbildung 1 dargestellt ist, schneiden Sie den Thymus in drei gleiche Abschnitte mit chirurgische Scheren. </li><li> Wiederholen Sie die Schritte 1,3-1,4 für jede Spendermaus. Um die Zeit zu begrenzen Thymi ausgesetzt sind, bereiten 1 Spender Thymus zu einem Zeitpunkt. </li></ol> 2. Thymus Implantation unter die Nierenkapsel <ol><li> Montieren Sie den pre-sterilisiert Ausrüstung in Tabelle 1 aufgeführt. AseptischeTechnik sollte im Verlauf des Verfahrens Belichten Transplantatrezipienten kontaminierte Werkzeuge oder Reagenzien zu verhindern, verwendet werden. </li><li> Vor der Transplantation wiegen und etikettieren jeden Empfänger der Maus. </li><li> Einrichten des Binokular und Anästhesie-Schaltung in der Sterilbank. </li><li> Mit einem elektrischen Rasierer, rasieren Sie die linke Seite der Empfängermaus und sicherzustellen, dass keine Haare rund um die verwendet werden, um den Schnitt zu machen Bereich bleibt. </li><li> Schalten Sie Isofluoran Verdampfer und betäuben die Maus mit einer Dosis von zwischen 1-2%. Ermitteln ordnungsgemäße Betäubung vor Beginn des chirurgischen Eingriffs durch Prüfen auf die Abwesenheit von reflex folgenden toe Prise. </li><li> Nachdem die Maus ordnungsgemäß betäubt wird, gelten veterinär Salbe auf den Augen der Maus, um Trockenheit zu verhindern und bereiten Sie die Maus für die Transplantation, wie folgt: </li><li> Bewegen Sie die Maus unter dem Binokular in einer rechten Seitenlage, so dass die rasierte Seite nach oben zeigt. Starting in der Mitte der OP-Bereich, verzichten in einer kreisförmigen Bewegung mit einem Einmaltransferpipette 70% Ethanol, gefolgt von PVP-Jod (Betadine). Wiederholen Sie die Ethanol / betadine Behandlung 3 mal vor einen Einschnitt. </li><li> Verwendung Präparierscheren einen 1-2 cm Flankenschnitt direkt über die Niere. </li><li> Verwendung Medium Pinzette fassen Bindegewebe neben der Niere und heben die Nieren, so dass es oben auf der Muskulatur liegt. Um die Niere freigelegt und an Ort und Stelle über die Muskulatur zu halten, legen Sie einen Arm des Mediums Zange unterhalb der Niere, wobei besonderes Augenmerk nicht auf die Nieren ilium stören. </li><li> Um Gewebeaustrocknung zu verhindern, zu bewässern die Niere mit sterilem 1x PBS (pH 7,4) bei jedem Schritt, bis Manipulation der Nieren und Nierenkapsel ist abgeschlossen. </li><li> Verwenden feinen Pinzette kneifen und heben Sie die Kapsel in der Nähe der Kante des Nieren meisten distal zu den Nebennieren, um es von der Niere zu trennen. </li><li> Verwenden Sie eine 18-Gauge-Nadel, um einen Einschnitt groß genug, um das vorbereitete Stück Spender Thymus (2A) einfügen zu machen. Stellen Sie sicher, um die Kapselinzision so klein wie möglich zu Dislokation der Prothese im Laufe der Zeit zu verhindern. </li><li> Während die Kapsel löste sich von der Niere, verwenden Sie ein zweites Paar von feinen Pinzette, um das Transplantat unter der Kapsel einlegen und drücken Sie den Thymus so weit nach vorne aus dem Kapselinzision wie möglich (2B). </li><li> Entfernen Sie die Zange von unterhalb der Niere, und sanft die Niere wieder zurück an seinen Platz durch den Einschnitt. </li><li> Schließen Sie die Muskulatur durch Annähen der Bauchwand und die Anwendung betadine einmal geschlossen. Nach der vollständigen Naht ist, gelten Wundklammern, um die Dermis zu schließen und betadine in die Umgebung. </li><li> Gibt die Maus in einen Käfig mit einer Wärmelampe, die aufgewärmt hat. </li><li> Überwachen sich postoperative Maus, bis es volle Bewusstsein und Mobilität gewinnt und stellen Sie keine Mäuse that wurden nicht nach Betäubung in einen Käfig mit anderen Tieren gewonnen. </li><li> Behandlung von Mäusen mit postoperativen Schmerztherapie Medikamente nach Bedarf. Stellen Mäuse mit postoperativen Schmerzmittel wie Paracetamol im Trinkwasser in einer Konzentration von 1,6 mg / ml. </li><li> Wiederholen Sie die Schritte 2,3-2,9 für jeden Empfänger der Maus. </li><li> Stellen Sie sicher, dass Mäuse, den normalen Aktivitäten innerhalb 1 Stunde nach der Operation. Überwachen Sie die postoperative Gewicht, Motorik, sowie das Tränken und Futter Aktivität jeder Maus, um Komplikationen, die sich als Folge der Transplantation zu bestimmen. HINWEIS: Post-operative Gewichtsverlust im Vergleich zu präoperativen Gewicht sowie veränderte Mobilität oder Misserfolg zu essen oder trinken können Komplikationen hinweisen. </li><li> Überwachen T Zellrekonstitution im peripheren Blut durch Ausbluten Mäusen über die Schwanzvene nick gefolgt von der Abtrennung der Lymphozyten mit Lympholyte Zelltrennungsmedien nach den Angaben des Herstellers. Erreichen T-Zell-characterization durch Anfärben Lymphozyten mit fluoreszenz konjugierte Antikörper spezifisch für T-Zellen-Marker CD3, CD4, CD8, sowie eine Live toten Diskriminator wie einem fluoreszenz aktivierte Succinimidylester 8. Analyse, wie in Figur 3 gezeigt ist über Gating Singuletts, lebenden Zellen und mit CD3-positiven Zellen erreicht werden. </li></ol>

<ol>
	<li>Kappler, J. W., Roehm, N., Marrack, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+tolerance+by+clonal+elimination+in+the+thymus.">T cell tolerance by clonal elimination in the thymus.</a> Cell. 49 (2), 273-280 (1987).</li><li>Takahama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Journey+through+the+thymus:+stromal+guides+for+T-cell+development+and+selection.">Journey through the thymus: stromal guides for T-cell development and selection.</a> Nat. Rev. Immunol. 6, 127-135 (2006).</li><li>Klein, L., Kyewski, B., Allen, P. M., Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+and+negative+selection+of+the+T+cell+repertoire:+what+thymocytes+see+(and+don&#8217;t+see).">Positive and negative selection of the T cell repertoire: what thymocytes see (and don&#8217;t see).</a> Nat. Rev. Immunol. 14 (6), 377-391 (2014).</li><li>Schwartz, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acquisition+of+immunologic+self-tolerance.">Acquisition of immunologic self-tolerance.</a> Cell. 57 (7), 1073-1081 (1989).</li><li>Kishimoto, H., Sprent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+defect+in+central+tolerance+in+NOD+mice.">A defect in central tolerance in NOD mice.</a> Nat. Immunol. 2 (11), 1025-1031 (2001).</li><li>Anderson, G., Jenkinson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+central+tolerance+with+reaggregate+thymus+organ+cultures.">Investigating central tolerance with reaggregate thymus organ cultures.</a> Methods. Mol. Biol. 380, 185-196 (2007).</li><li>He, Q., 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=Thymic+development+of+autoreactive+T+cells+in+NOD+mice+is+regulated+in+an+age-dependent+manner.">Thymic development of autoreactive T cells in NOD mice is regulated in an age-dependent manner.</a> J. Immunol. 191 (12), 5858-5866 (2013).</li><li>Stewart, C. C., Stewart, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+6.+Immunophenotyping.">Chapter 6. Immunophenotyping.</a> Current Protocols in Cytometry. , (2001).</li><li>Rice, H. E., 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=Thymic+transplantation+for+complete+DiGeorge+syndrome:+medical+and+surgical+considerations.">Thymic transplantation for complete DiGeorge syndrome: medical and surgical considerations.</a> J. Pediatr. Surg. 39 (11), 1607-1615 (2004).</li><li>Markert, M. L., 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=Transplantation+of+thymus+tissue+in+complete+DiGeorge+Syndrome.">Transplantation of thymus tissue in complete DiGeorge Syndrome.</a> N. Engl. J. Med. 341, 1180-1189 (1999).</li><li>Market, M. L., Devlin, B. H., McCarthy, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymus+transplantation.">Thymus transplantation.</a> Clin. Immunol. 135 (2), 236-246 (2010).</li><li>Wells, A. D., Li, X., Strom, T. B., Turka, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+peripheral+T-cell+depletion+in+transplantation+tolerance.">The role of peripheral T-cell depletion in transplantation tolerance.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 356 (1409), 617-623 (2001).</li><li>Albuquerque, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+FOXN1-Deficiency+is+associated+with+ab+double-negative+and+FoxP3++T-cell+expansions+that+are+distinctly+modulated+upon+thymic+transplantation.">Human FOXN1-Deficiency is associated with ab double-negative and FoxP3+ T-cell expansions that are distinctly modulated upon thymic transplantation.</a> PLoS One. 7 (5), e37042 (2012).</li><li>Chinn, I. K., Milner, J. D., Scheinberg, P., Douek, D. C., Markert, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymus+transplantation+restores+the+repertoires+of+forkhead+box+protein+3+(FoxP3)++and+FoxP3-+T+cells+in+complete+DiGeorge+anomaly.">Thymus transplantation restores the repertoires of forkhead box protein 3 (FoxP3)+ and FoxP3- T cells in complete DiGeorge anomaly.</a> Clin Exp Immunol. 173 (1), 140-149 (2013).</li><li>Savino, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+thymus+is+a+common+target+organ+in+infectious+diseases.">The thymus is a common target organ in infectious diseases.</a> PLoS Pathog. 2 (6), e62 (2006).</li></ol>

The authors do not have any competing financial interests to disclose.

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

Der Thymus ist ein Organ, in dem kritische Ereignisse in T-Zell-Entwicklung entstehen 1. Resident Thymozyten auf Umlagerung des T-Zell-Rezeptor (TCR) α und β-Gene, durchlaufen eine Reihe von Interaktionen mit lymphostromal und Antigen-präsentierenden Zellen (APC) in der Rinde und Mark Regionen des Thymus 2. Thymus-positive Selektion durch kortikale Thymusepithelzellen (TEC) vermittelte, um Thymozyten, die antigene Peptide im Kontext der Host Major Histocompatibility Complex (MHC) Klasse I und II-Moleküle erkennen 2-3 herzustellen. Nachfolgende Thymus negative Selektion bringt Spülen von autoreaktiven Thymozyten, durch eine Wechselwirkung mit Mark TEC oder dendritischen Zellen (DC), die vorhanden Peptide aus Selbstproteine ​​durch MHC-Klasse-I und II-Moleküle gebunden 3 abgeleitet angetrieben. Das Ergebnis dieser Prozesse ist die Einrichtung eines Pools von reifen CD4 + und CD8 + T-Zellen in der Lage, ein breites Spektrum reagierenFremdgene während sie eine minimale Reaktivität mit Eigenproteine ​​4. Die Effizienz des Thymus Auswahlereignisse durch eine Vielzahl von Faktoren, einschließlich Thymus-Reifung, die Häufigkeit der Mark und kortikaler TEC Teilzusammensetzung Thymus DC, und die Source des Thymus Vorstufen 3 beeinflusst. Bemerkenswert ist, können abweichende Thymus-Auswahl bei Autoimmun 5 oder immundefizienten Pathologien, die von eingeschränkter negative oder positive Selektion entstehen beziehungsweise führen. Die molekularen Ereignisse regulieren Thymus Auswahl, sind jedoch kaum verstanden. In vitro-Ansätze wie reaggregieren Thymus-Organkulturen (RTOC) 6, haben sich als nützlich für die Analyse von Basisereignissen mit Thymus Auswahl zugeordnet ist, aber nicht, um die Dynamik der laufenden vollständig rekapitulieren In-vivo-Veranstaltungen. Als Folge wurde dieses Thymus-Transplantation-basierten Ansatz für eine bessere Studien thymocyte Auswahl Veranstaltungen in vivo 7 fest </sup&gt;. Dieses Protokoll beschreibt die Transplantation Thymi von Neugeborenen und erwachsenen Spendermäusen in immundefiziente SCID-Empfängermäuse. Diese Technik erlaubt die Untersuchung der Mechanismen, die positive und negative Selektion Thymus sowie Thymusfunktion verschiedener T-Zell-Teilmengen während der Ontogenese regulieren. In jüngster Zeit wurde dieser Ansatz verwendet, um zu zeigen, dass die Effizienz des Thymus Auswahl früh nach der Geburt in Mäusen führt zu einer erhöhten Entwicklung von autoreaktiven T-Zellen, und für die Fremdantigene 7 eine reduzierte T-Zell-Repertoire spezifisch.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Decon Laboratories</td>
			<td>2705HC</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>GIBCO</td>
			<td>14190-144</td>
			<td>pH 7.2</td>
		</tr>
		<tr>
			<td>Betadine Solution</td>
			<td>Purdue Products</td>
			<td>67618-150-17</td>
			<td></td>
		</tr>
		<tr>
			<td>18 gauge needle</td>
			<td>BD</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>Sutures (1.0 metric)</td>
			<td>Ethicon</td>
			<td>J493G</td>
			<td>18&#34; (45cm)</td>
		</tr>
		<tr>
			<td>AUTOCLIP Wound Clips (9mm)</td>
			<td>Clay Adams&#174; Brand</td>
			<td>427631</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Fisher Scientific</td>
			<td>13-711-20</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr>
			<td>Mouse anti-CD3 Ab</td>
			<td>eBioscience</td>
			<td>11-0031-85</td>
			<td>Clone: 1452C11</td>
		</tr>
		<tr>
			<td>Mouse anti-CD4 Ab</td>
			<td>eBioscience</td>
			<td>48-0042-82</td>
			<td>Clone: RM4-5</td>
		</tr>
		<tr>
			<td>Mouse anti-CD8 Ab</td>
			<td>eBioscience</td>
			<td>25-0081-82</td>
			<td>Clone: 53-6.7</td>
		</tr>
		<tr>
			<td>Lancet</td>
			<td>Medipoint</td>
			<td>Goldenrod 4mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Pacific Orange Succinimidyl Ester, Triethylammonium Salt</td>
			<td>Invitrogen</td>
			<td>P30253</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well round botton polypropylene plates</td>
			<td>Corning</td>
			<td>3365</td>
			<td></td>
		</tr>
		<tr>
			<td>1.2mL polypropylene cluster tubes</td>
			<td>Corning</td>
			<td>4401</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL polypropylene round-bottom tubes</td>
			<td>BD</td>
			<td>352002</td>
			<td></td>
		</tr>
		<tr>
			<td>40uM Nylon Cell Strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde Solution, EM Grade</td>
			<td>Electron Microscopy Services</td>
			<td>15710</td>
			<td>Hazardous</td>
		</tr>
		<tr>
			<td>Puralube Optical Ointment</td>
			<td>Fisher Scientific</td>
			<td>NC0138063</td>
			<td></td>
		</tr>
		<tr>
			<td>Lympholyte</td>
			<td>Cedarlane</td>
			<td>CL5030</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm cell culture dish</td>
			<td>Corning</td>
			<td>430196</td>
			<td>60mm x 15mm, Sterile</td>
		</tr>
	</tbody>
</table>

isolation and transplantation of different aged murine thymic grafts.

Die Mechanismen, die die Wirksamkeit von Thymus Auswahl, zu regeln schlecht definiert. Die hier vorgestellte Methode ermöglicht in vivo-Analysen von der Entwicklung und Auswahl bestimmter für Selbst- und Fremdantigene T-Zellen. Der Ansatz beinhaltet die Implantation von Thymus-Transplantate aus verschiedenen alten Mäusen in immundefiziente SCID-Empfänger ab. Über einen relativ kurzen Zeitraum die Empfänger sind mit T-Zellen aus dem Thymus implantierten Transplantats abgeleitet rekonstituiert. Nur Thymozyten Impfen der Thymus zum Zeitpunkt der Trennung zu unterziehen Auswahl und entwickeln sich zu reifen T-Zellen. Als solches können Änderungen in der Art und Spezifität der transplantierten T-Zellen als Funktion der altersabhängigen Thymus Ereignisse bewertet werden. Obwohl technische Know-how für erfolgreiche Thymus-Transplantation erforderlich ist, stellt dieses Verfahren eine einzigartige Strategie, um in vivo zu untersuchen eine breite Palette von Krankheiten, die durch oder aufgrund der abweichenden Thymus-Funktion und / oder sind homeostasist.

Der Erfolg dieses Verfahrens hängt von minimalen chirurgischen Traumas sowie die genaue Positionierung der Prothese unterhalb der Nierenkapsel. Der Thymus Transplantat sollte geschnitten angemessen sicherzustellen bemessen Thymus Abschnitte für eine nachfolgende Transplantation, wie in Abbildung 1 dargestellt. Nach der schematischen Darstellung in Figur 1 kann Thymi aus neugeborenen oder erwachsenen Mäusen für eine erfolgreiche Transplantation subkapsuläre mit eindeutigen und repro...

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

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

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

Isolation und Transplantation von Different Aged Murine Thymic Grafts.

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

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

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Immunology and Infection

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

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

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Mausmodell der CD40-Aktivierung von B-Zellen

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Forschung

Immunologie und Infektion

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

Prüfung Thymic positive und negative Selektion mittels Durchflusszytometrie

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

Isolation, Verarbeitung und Analyse von Murine Gingivazellen

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

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

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

Isolierung, Identifizierung und Reinigung von Murine Thymusepithelzellen

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

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Isolierung von murinen Lymphknoten Stromazellen

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of various fields in biomedical science. The method described below is a simple and effective way to isolate murine monocytes from heterogeneous bone marrow.
Bone marrow from the femur and tibia of Balb/c mice is harvested by flushing with phosphate buffered saline (PBS). Cell suspension is supplemented with macrophage-colony stimulating factor (M-CSF) and cultured on ultra-low attachment surfaces to avoid adhesion-triggered differentiation of monocytes. The properties and differentiation of monocytes are characterized at various intervals. Fluorescence activated cell sorting (FACS), with markers like CD11b, CD115, and F4/80, is used for phenotyping. At the end of cultivation, the suspension consists of 45%&#177; 12% monocytes. By removing adhesive macrophages, the purity can be raised up to 86%&#177; 6%. After the isolation, monocytes can be utilized in various ways, and one of the most effective and common methods for in vivo delivery is intravenous tail vein injection. 
This technique of isolation and application is important for mouse model studies, especially in the fields of inflammation or immunology. Monocytes can also be used therapeutically in mouse disease models.

Here we present a protocol that generates large amounts of murine monocytes from heterogeneous bone marrow for translational applications. In comparison to others, this new method helps reduce the number of sacrificed animals and lowers costs by avoiding expensive methods such as high gradient magnetic cell separation (MACS).

Isolierung und intravenöse Injektion von Maus-Knochenmark stammenden Monozyten

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

Isolierung und Durchflusszytometrie Charakterisierung von murinen Dünndarms Lymphozyten

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

Herstellung und Bedeutung von Organotypischen Thymic Scheibe Kulturen

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Isolation und Aktivierung von murinen Lymphozyten

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

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 ...