Diese Arbeit wurde vom amerikanischen Gesellschaft für Gastroenterologie (AGA) Forschung Wissenschaftler-Preis und Crohn und Colitis Foundation of America (CCFA) Career Development Award (den SZS), NIH NIDDK F30 DK089692 (ECS) und Universität von North Carolina Center for Gastrointestinal Biology unterstützt und Disease Grants P30 DK34987 (Histologie Core). Der UNC-Durchflusszytometrie Core Facility ist teilweise durch eine NCI-Center Core-Unterstützung Grant (P30CA016086) an den UNC Lineberger Comprehensive Cancer Center unterstützt. Wir danken Lukas B. Borst von North Carolina State University College of Veterinary Medicine für seine Hilfe bei histopathologischen Analyse und Immunhistochemie.

HINWEIS: Stellen Sie sicher, dass alle Tier Protokolle werden von und in Übereinstimmung mit Institutional Animal Care und Verwenden Committee (IACUC) Vorschriften und der National Research Council Handbuch für die Pflege und Verwendung von Labortieren zugelassen. Spendermäusen kann entweder männlich oder weiblich ist, aber Empfängermäuse sollte männlich. Wenn weibliche Empfänger zu verwenden sind, müssen die Spendermäuse female 5 sein. Pflegen Kolonien regelmäßig, unsteril Betten und nicht angesäuerten Wasser mit, da diese die Darmflora auswirken, und damit die Colitis Phänotyp der Mäuse 5,6. 1. Versuchsvorbereitung <ol><li> Verwenden eiskalte Medien und Puffer. Halten Zellen auf Eis während des Experimentes. </li><li> Führen Sie das Experiment in sterile Biohazard Haube mit steriler Technik. </li></ol> 2. Isolierung von Milz-T-Zellen <ol><li> Einschläfern Spendermaus / Mäuse in CO 2 Kammer durch zervikale Dislocatio gefolgtn. Spray Abdomen mit 70% Ethanol. </li><li> Machen Sie einen horizontalen Schnitt im Bauch und abziehen Haut Bauchfell aus. Halten Bauchfell entfernt von der inneren Organe mit der Zange und einen Einschnitt in der linken Bauchfell zu entlarven und die Milz herausschneiden. </li><li> Legen Sie die Milz in 10 ml Komplett Medien in einer Petrischale. Entfernen und entsorgen Sie überschüssiges Gewebe von Milz. </li><li> Verwenden Sie 2 sterilisiert Glasobjektträger zu zerkleinern und necken neben der Milz in Einzelzellsuspension. Filterzellensuspension durch ein 70 um Sieb in einem 50 ml konischen Röhrchen und spülen Sieb mit 5 ml Vollmedium. Platzieren Sie bis zu 5 Milz in einem 50 ml konischen Röhrchen. </li><li> Zentrifuge Zellen bei 450 g für 7 min. Überstand verwerfen durch Abgießen oder durch Vakuumsauger in Abfallbehälter geben. </li><li> Vorsichtig resuspendieren Zellen in 5 ml pro Milz Lysispuffer an Erythrozyten für 10 Minuten bei Raumtemperatur lysiert. Gleiches Volumen Vollmedium (5 ml pro Milz) zu dem Rohr. </li><li&gt; Centrifuge Zellen bei 450 g für 7 min. Überstand verwerfen durch Abgießen oder durch Vakuumsauger in Abfallbehälter geben. </li><li> Vorsichtig in 10 ml Labeling Buffer resuspendieren Zellen. </li><li> Zählen von Zellen durch Trypanblau-Ausschluss. <ol><li> Entfernen Sie 20 ul Zellsuspension und fügen Sie 180 ul Trypanblau auf ein Mikrozentrifugenröhrchen geben und gut mischen. Nach 5 Minuten, fügen Sie 10 ul markierte Zellen zu Hämozytometer und zählen nicht-blauen Zellen unter dem Mikroskop. Ermittlung der Gesamtzahl der lebensfähigen Zellen. Entsorgen Sie Zell / Trypanblau-Mix. </li></ol></li><li> Zentrifuge Zellen in 10 ml Markierungspuffer bei 450 × g für 7 min. Überstand verwerfen durch Abgießen oder durch Vakuumsauger in Abfallbehälter geben. </li></ol> 3. Anreicherung von CD4 + T-Zellen Hinweis: Folgen Sie den Anweisungen des Herstellers für bestimmte Produkte in diesem Abschnitt verwendet. <ol><li> Vorsichtig resuspendieren Zellen zu einer Einzelzell-Suspension von 20 x 10 6 Zellen / ml in kaltem Etikettening Buffer. </li><li> Mit 5 &amp; mgr; l pro 1 × 10 6 Zellen von biotinyliertem CD4 T-Zell-Anreicherung Antikörper. Inkubieren Zellen auf Eis für 15 min. </li><li> Fügen 10x Volumen Labeling Buffer. Zentrifuge Zellen bei 450 · g für 7 min. Sorgfältig absaugen all des Überstandes mit Vakuumsauger in Abfallbehälter geben. </li><li> Gründlich vortexen Streptavidin-konjugierten magnetischen Partikeln. Mit 5 ul der Teilchen pro 1 x 10 6 Zellen. </li><li> Mischen Sie gründlich, aber vorsichtig. Halten Gemisch bei 6-12 ° C für 30 min. </li><li> Hinzufügen Markierungspuffer auf eine Konzentration von 20-80 x 10 6 Zellen / ml. Übertragung von bis zu 1,0 ml pro 12 x 75 mm Rund Reagenzglas markierten Zellen (als &quot;positive-Bruch Rohr&quot; bezeichnet). </li><li> Platzieren Sie jede positive Fraktion Rohr auf Magneten für 6-8 min. </li><li> Mit den positiven Fraktion Rohre noch auf den Magneten, Überstand vorsichtig Transfer vom positiven Bruchrohr mit Glas Pasteur-Pipette in ein neues steriles 50 ml konischen Röhrchen (referred als &quot;angereicherte Fraktion&quot;). Diese angereicherte Fraktion enthält CD4 + T-Zellen. Achten Sie darauf, um die markierten Zellen mit dem Magneten angezogen zu stören. </li><li> Die Zellen in der positiven Fraktion Rohre in dem gleichen Volumen des Markierungspuffer durch Auf- und Abpipettieren kräftig nach links, wie in Schritt 3.6. Zeigen positive Fraktion Rohr wieder auf Magneten für 6-8 min. </li><li> Mit positiven Bruchrohre noch auf den Magneten, sorgfältig überStand (angereicherte Fraktion, CD4 +) von positiv-Bruchrohr mit Glaspasteurpipette auf 50 ml ohne Unterbrechung Zellen Magneten befestigt sterile konische Röhrchen aus Schritt 3.8. </li><li> Die Schritte 3,9-3,10 um die Ausbeute an CD4 + T-Zellen erhalten erhöhen. Weiter Protokoll mit angereicherten Fraktion (CD4 + Zellen). </li></ol> 4. Beschriften und Sortieren von Zellen 7 <ol><li> Zentrifuge angereicherten Zellen bei 450 g für 7 min. Überstand verwerfen durch Abgießen oder Vakuum SUCTIonen in die Abfallbehälter. </li><li> Zellen in 1 ml Markierungspuffer. Entfernen Aliquot von Zellen zu zählen und um die Lebensfähigkeit der Zellen durch Trypanblau-Ausschluss, wie in Schritt 2.9 zu beurteilen. </li><li> Hinzufügen eines Volumens von Markierungspuffer bis 10 x 10 6 Zellen / ml; Wenn Zellen bereits &lt;10 x 10 6, Zentrifuge bei 450 · g für 7 min, verwerfen Stand durch Abgießen oder Vakuumansaugung in den Abfallbehälter und fügen Volumen Markierungspuffer bis 10 x 10 6 Zellen / ml. <ol><li> Getrennte Teilmengen von etwa 5-10 x 10 5 Zellen je Set für ungefärbte, Isotyp-gefärbt und einzelne befleckten Kontrollzellen in Mikrozentrifugenröhrchen. </li></ol></li><li> Mit 5 &amp; mgr; g / ml CD4-FITC und 1 ug / ml CD45RB-PE an Zellen. Hinzufügen Isotypkontrolle Flecken und einzelne Flecken in der gleichen Konzentration zu Aliquots in Mikrozentrifugenröhrchen eignen. Gut mischen, aber sanft und Inkubation auf Eis vor Licht für 30 Minuten geschützt. </li><li> Fügen 10x Volumen Labeling Buffer, um Zellen und Steuers. Zentrifuge 450 g für 7 min. Überstand verwerfen durch Vakuumansaugung in Abfallbehälter geben. </li><li> Resuspendieren in Markierungspuffer zu Volumen in Schritt 4.5. Zentrifuge 450 g für 7 min. Überstand verwerfen durch Vakuumansaugung in Abfallbehälter geben. </li><li> Resuspendieren in Markierungspuffer auf 10 x 10 6 Zellen / ml. Pass Zellen durch 70 &amp; mgr; m Sieb in FACS-Röhrchen. Halten Sie auf dem Eis vor Licht geschützt, bis sie zur FACS. </li><li> Einzurichten und zu bestimmen, eine angemessene Entschädigung für die Zellsortierer mit ungefärbten Zellen und Single-gefärbten Kontrollen. </li><li> Ausschließen nonviable Zellen mit Vorwärts- und Seitenstreuung Gating (1A). Up Gating für CD4 + und CD45RB + -Zellen mit Isotyp-gefärbten Bedienelemente ein. Gate-Zellen auf CD4 + -Population. </li><li> Sortieren CD4 + Zellen in CD45RB hoch und niedrig CD45RB Populationen mit einem einfachen Histogramm für PE-gefärbten Zellen in Röhrchen mit 2 ml Komplett Medien. Die CD45RB hoch </sup&gt; Bevölkerung stellt die höchsten 40% der CD4 + CD45RB + -Zellen (CD45RB hoch), und die CD45RB niedrig Bevölkerung ist die untersten 20% der CD4 + CD45RB + -Zellen (CD45RB niedrig; 1B). </li></ol><img alt="Abbildung 1" src="/files/ftp_upload/52533/52533fig1.jpg" /> Abbildung 1: Repräsentative Durchflusszytometrie Plots von CD4 + CD45RB T-Zell-Populationen während der FACS-Analyse (A - C) FITC CD4- und PE CD45RB-gefärbten Splenozyten aus Spender C57BL / 6-Mäuse wurden durch FACS sortiert in CD4 + CD45RB hoch und CD4 +. CD45RB geringe T-Zell-Populationen. (A) Doublet Ereignisse auf der vorderen Streudiagramm ausgeschlossen. (B) Lymphozyten wurden in Vorwärts- und Seitenstreudiagramm gated. (C) CD4 + </sup&gt; T-Zellen wurden gated, und (D) CD4 + CD45RB + T-Zellen wurden auf einer PE gegen Events Histogramm aufgetragen. CD4 + CD45RB niedrigen Zellen wurden als die niedrigste 20% der CD45RB + Zellen sein. CD4 + CD45RB hoch Zellen wurden als die höchste 40% CD45RB + -Zellen bestimmt. <ol start="11"><li> Führen Sie ein Aliquot jeder Zellpopulation auf dem FACS-Maschine, um die Reinheit der Bevölkerung zu bewerten. </li><li> Zentrifuge sortierten Zellen bei 450 g für 7 min. Resuspendieren in 1 ml PBS. Entfernen Aliquot von Zellen zu zählen und um die Lebensfähigkeit der Zellen durch Trypanblau-Ausschluss, wie in Schritt 2.9 zu beurteilen. </li></ol> 5. Die Injektion von Zellen in Empfänger <ol><li> Resuspendieren sortierten Zellen auf 4 x 10 6 Zellen / ml (CD45RB hoch) oder 2 x 10 6 Zellen / ml (CD45RB niedrig) in PBS. </li><li> Übertragen Sie 100 ul von CD45RB hoch Zellen pro Empfänger zu neuen sterilen Röhrchen. Geben Sie 100 ul PBS proEmpfänger dieser Röhre. So ist die Gesamtinjektionsmenge pro Tier 200 ul und Gesamtbetrag der Zellen pro Empfänger 4 x 10 5 CD4 + CD45RB hoch naiven T-Zellen. </li><li> Wenn Versuchsgruppe, die regulatorischen T-Zellen erwünscht ist, dann 100 ul der CD45RB hoch Zellen pro Empfänger zu neuen sterilen Röhrchen. 100 l CD45RB niedrigen Zellen pro Empfänger auf die gleiche Röhre. Gesamtinjektionsmenge pro Tier ist 200 ul; Verhältnis von CD45RB hoch: CD45RB niedrigen Zellen ist 2: 1. </li><li> Injizieren Sie 100 ul von CD45RB hoch oder CD45RB hoch / niedrig CD45RB CD4 + Zellen intraperitoneal in der rechten und linken Seite des Bauches (insgesamt 200 ul) jedes Empfängers. </li></ol> 6. Überwachung der Krankheitsprogression in Empfängertiere <ol><li> Um den klinischen Zustand der Empfängertiere zu bewerten, weisen Gesamt klinischen Scores für das folgende parameters 8 am Tag der Injektion, danach wöchentlich und bei der Opferung: <ol><li> Bestimmen Sie verschwenden durch Messung Gewichtsverlust: 0 - keine Gewichtsabnahme; 1 - 0,1 bis 10% Verlust der ursprünglichen Körpergewichts; 2 - mehr als 10% Verlust der ursprünglichen Körpergewichts (Abbildung 2A). </li></ol></li></ol><img alt="Abbildung 2" src="/files/ftp_upload/52533/52533fig2highres.jpg" width="650" /> Abbildung 2: Klinische und pathologische Anzeichen einer Entzündung nach der Übertragung von Wildtyp-CD4 + CD45RB hoch -T-Zellen in Rag1 auftreten - / - und NRDKO Empfängermäuse 11 (A) NRDKO Empfänger verloren im Durchschnitt 10% ihres ursprünglichen Körpergewicht um 5. Wochen nach der Übertragung, während Rag1- / - Empfänger nicht klinischen Anzeichen einer Krankheit aufweisen, zu diesem Zeitpunkt. Jeder Punkt stellt den Mittelwert Prozentsatz der anfänglichen Körpergewicht für die Kohorte ± SEM. ** P &lt;0.005. (B) Einige Mäuse entwickelten eine schwere Darmentzündung, wie durch die Anwesenheit von Rektumprolaps demonstriert. Dies ist ein repräsentatives Bild der Darmvorfall in einem NRDKO Empfängermaus. (C) Stark, Doppelpunkte sowohl Rag1 - / - und NRDKO Empfängermäuse sind verdickt und verkürzt im Vergleich zu Doppelpunkte von Rag1 - / - und NRDKO Mäuse ohne T-Zell-adoptiven Transfer. Colons von NRDKO Empfängermäuse zeigen schwere Entzündung und erhöhte Dickgewichte (Daten nicht gezeigt). <a href="https://www.jove.com/files/ftp_upload/52533/52533fig2highres.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieses Bild anzuzeigen.</a> <ol><li><ol start="2"><li> Bestimmen Sie die Qualität des Stuhls, indem Tier in sauberen Behälter, bis sie durchlaufen hatHocker: 0 - keine; 1 - weicher Stuhl; 2 - tränende und / oder Blut im Stuhl. </li><li> Bestimmen Sie die allgemeine Gesundheit des Tieres durch die Anwesenheit der folgenden Anzeichen einer Krankheit: 0 - keine gekrümmte Haltung, Borsten Fell oder Hautläsionen; 1 - einem der folgenden vorhanden: gekrümmte Haltung, Borsten Fell oder Hautveränderungen. </li><li> Bestimmen Vorhandensein Rektumprolaps: 0 - nicht vorhanden; 1 - vorhanden ist (2B). </li></ol></li><li> Opfern Tiere durch CO 2 Inhalation gefolgt durch Genickbruch, wenn sie 20% ihres Ausgangskörpergewicht verloren gehen oder in gewünschten Zeitpunkt, je nachdem was zuerst eintritt. Klinischen Erkrankung ist in der Regel erkennbar ab Woche 5 post Fülle. </li><li> Beurteilen der Schwere der Erkrankung, wie oben beschrieben 5,7. <ol><li> Vergeben klinischen Scores, wie in Schritt 6.1 8. </li><li> Messen Kolon Länge und das Gewicht (2C). </li><li> Führen Sie die histologische Analyse der Entzündung5. </li><li> Bestimmen spontane Zytokinexpression in Darmgewebe Explantatkulturen 9, Mesenteriallymphknoten 10 und / oder Serum 11. <ol><li> Kurz gesagt, für Explantatkulturen 9, entfernen Sie Doppelpunkte nach der Tötung offenen Längsrichtung, und sauber mit PBS. Doppelpunkte Inkubieren auf einem Orbitalschüttler bei 250 Upm in vollständige Medien für 30 Minuten bei Raumtemperatur eingestellt. </li><li> Hacken Sie Gewebe in kleine Stücke und bei 37 ° C in vollständige Medien für 24 Stunden inkubiert. Die überstehende Flüssigkeit und verwenden Sie für die Quantifizierung von Zytokinen auf 100 mg Gewebe mittels ELISA. </li></ol></li><li> Führen ex vivo Charakterisierung der T-Zell-Phänotypen und / oder Funktion 10. </li></ol></li></ol>

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J Immunol. 175 (8), 5412-5422 (2005).</li><li>Wang, 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=IFN-gamma-induced+TNFR2+expression+is+required+for+TNF-dependent+intestinal+epithelial+barrier+dysfunction.">IFN-gamma-induced TNFR2 expression is required for TNF-dependent intestinal epithelial barrier dysfunction.</a> Gastroenterology. 131 (4), 1153-1163 (2006).</li></ol>

The authors declare that they have no competing financial interests.

Hier beschreiben wir eine Schritt-für-Schritt-Protokoll Induzieren Kolonentzündung in Mäusen durch adoptiven Transfer von CD4 + CD45RB + T-Zellen in immundefizienten Mäusen. Wir verwendeten C57BL / 6 Spender Milz und syngenen Rag1 - / - Empfängermäuse, obwohl auch andere Stämme (zB BALB / c, 129S6 / SvEv, nicht-übergewichtigen diabetischen (NOD)) und genetische Modelle der Immunschwäche (zB SCID, Rag2 - / -), können ebenfalls verwe...

The inflammatory bowel diseases (IBD) Crohn&#8217;s disease and ulcerative colitis result from an incompletely defined and complex interaction between host immune responses, genetic susceptibility, environmental factors, and the enteric luminal contents1. Recent genome-wide association studies report associations between immune cell regulatory genes and IBD susceptibility2,3. Both innate and adaptive immune cell intrinsic genes are represented in these studies, indicating a central role for these cell populations in IBD pathogenesis.
There currently exist more than 50 animal models of human IBD. While no one model perfectly phenocopies human IBD, many are useful for studying various aspects of human disease, including disease onset and progression and the wound-healing response. In the method described here, intestinal inflammation is initiated with syngeneic splenic CD4+CD45RBhigh T cell adoptive transfer into T and B cell deficient recipient mice4. The CD4+CD45RBhigh T cell population contains mainly na&#239;ve T cells primed for activation that are capable of inducing chronic small bowel and colonic inflammation. This method allows the researcher to modify key experimental variables, including both innate and adaptive immune cell populations, to answer biologically relevant questions relating to disease pathogenesis. Additionally, this method provides precise initiation of disease onset and a well-characterized experimental time course. This permits the kinetic study of clinical features of disease progression in mice. Intestinal inflammation induced by this method shares many features with human IBD, including chronic large and small bowel transmural inflammation, pathogenesis driven by cytokines such as TNF and IL-12, and systemic symptoms such as wasting5. Thus, it is an ideal model system for studying the pathogenesis of human IBD.
The method here describes in detail the protocol for inducing experimental colitis by adoptive transfer of CD4+CD45RBhigh T cells into Rag1-/- mice. We discuss key technical steps, expected results, optimization, and trouble-shooting. We address the required elements for the successful development of this murine model of intestinal inflammation for research purposes.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Name of Reagent/ Equipment</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments/Description</td>
		</tr>
		<tr >
			<td >10x PBS</td>
			<td >Gibco</td>
			<td >14200075</td>
			<td></td>
		</tr>
		<tr >
			<td >12x75mm round-bottom tube</td>
			<td >Falcon</td>
			<td >352052</td>
			<td></td>
		</tr>
		<tr >
			<td >15 ml conical</td>
			<td >Corning</td>
			<td >430790</td>
			<td></td>
		</tr>
		<tr >
			<td >26g x 3/8 Needle</td>
			<td >BD Biosciences</td>
			<td >305110</td>
			<td></td>
		</tr>
		<tr >
			<td >50 ml conical</td>
			<td >Corning</td>
			<td >430828</td>
			<td></td>
		</tr>
		<tr >
			<td >70 um Cell Strainer</td>
			<td >Fisherbrand</td>
			<td >22363548</td>
			<td></td>
		</tr>
		<tr >
			<td >BD IMagnet</td>
			<td >BD Biosciences</td>
			<td >552311</td>
			<td></td>
		</tr>
		<tr >
			<td >&#946;-mercaptoethanol</td>
			<td >Thermo Scientific</td>
			<td >35602</td>
			<td></td>
		</tr>
		<tr >
			<td >CD4-FITC IgG2b</td>
			<td >eBioscience</td>
			<td >11-0041</td>
			<td></td>
		</tr>
		<tr >
			<td >CD45RB-PE IgG2a</td>
			<td >BD Pharminogen</td>
			<td >553101</td>
			<td></td>
		</tr>
		<tr >
			<td >Complete Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 10% FBS, 0.0004% &#946;-ME</td>
		</tr>
		<tr >
			<td >FACS tube + strainer</td>
			<td >BD Falcon</td>
			<td >352235</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Microscope Slides</td>
			<td >Fisherbrand</td>
			<td >12550A3</td>
			<td></td>
		</tr>
		<tr >
			<td >Heat-inactivated FBS</td>
			<td >Gemini</td>
			<td >100-106</td>
			<td></td>
		</tr>
		<tr >
			<td >Labeling Buffer</td>
			<td ></td>
			<td ></td>
			<td >1x PBS, 0.5% BSA, 2 mM EDTA</td>
		</tr>
		<tr >
			<td >Lysis Buffer</td>
			<td ></td>
			<td ></td>
			<td >0.08% NH4Cl, 0.1% KHCO3, 1 mM EDTA</td>
		</tr>
		<tr >
			<td >MoFlo XDP</td>
			<td >Beckman Coulter</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse CD4 T lymphocyte Enrichment Set - DM</td>
			<td >BD Biosciences</td>
			<td >558131</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2a-PE</td>
			<td >BD Pharminogen</td>
			<td >553457</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2b-FITC</td>
			<td >eBioscience</td>
			<td >11-4732</td>
			<td></td>
		</tr>
		<tr >
			<td >Pasteur pipet</td>
			<td >Fisherbrand</td>
			<td >13-678-20D</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin Solution, 100X</td>
			<td >Corning Cellgro</td>
			<td >30-002-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Petri Dish</td>
			<td >Fisherbrand</td>
			<td >875713</td>
			<td></td>
		</tr>
		<tr >
			<td >Pure Ethanol 200 Proof</td>
			<td >Decon Labs</td>
			<td >2705-HC</td>
			<td></td>
		</tr>
		<tr >
			<td >RPMI-1640</td>
			<td >Gibco</td>
			<td >11-875-093</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringe</td>
			<td >BD Biosciences</td>
			<td >309597</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypan blue</td>
			<td >Corning Cellgro</td>
			<td >25-900-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Wash Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 0.0004% &#946;-ME</td>
		</tr>
	</tbody>
</table>

induction of murine intestinal inflammation by adoptive transfer of effector cd4+cd45rbhigh t cells into immunodeficient mice

Es gibt viele verschiedene Tiermodelle zum Untersuchen der Pathogenese von menschlichen entzündlichen Darmerkrankungen (IBD), die jeweils ihre eigenen Vor- und Nachteile vorhanden. Wir beschreiben hier eine experimentelle Colitis-Modell, das durch adoptiven Transfer von syngenen Milz CD4 + CD45RB hoch -T-Zellen in T-und B-Zell-defizienten Mäusen Empfänger eingeleitet wird. Die CD4 + CD45RB hoch T-Zellpopulation, die größtenteils aus naiven Effektorzellen induzieren kann chronische Darmentzündung, sehr ähnlich wichtige Aspekte der menschlichen IBD. Dieses Verfahren kann manipuliert werden, um Aspekte der Krankheit Beginn und Fortschreiten zu untersuchen. Zusätzlich kann es für die Funktion der angeborenen, adaptive und regulatorische Immunzellpopulationen, und die Rolle von Umweltfaktoren, das heißt, die Mikroflora im Darmentzündung zu untersuchen. In diesem Artikel erläutern wir die Methode zur Induzierung ulcerosa mit einer Schritt-für-Schritt-Protokoll. Diese includes eine Video-Demonstration der wichtigsten technischen Aspekte erforderlich, um diese Mausmodell der experimentellen Kolitis zu Forschungszwecken erfolgreich zu entwickeln.

Ca. 10 x 10 6 CD4 + CD45RB hoch -T-Zellen aus 10 Milz von erwachsenen C57BL / 6 Spendermäusen sind zuverlässig isoliert. Diese Anzahl wird in Abhängigkeit von dem Alter und dem Stamm des Spendermaus und dem Können des Forschers variiert. Beim 4 x 10 5 C57BL / 6 CD4 + CD45RB hoch -T-Zellen in C57BL / 6 Rag1 übertragen - / - Empfängermäuse, klinischen Anzeichen einer Krankheit entstehen rund 5 Wochen nach der Sättigung oder früh...

Watch this Scientific Journal Video about Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice at JoVE.com

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 ...

induction-murine-intestinal-inflammation-adoptive-transfer-effector

Research

JoVE Journal

Immunology and Infection

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

16.2K Aufrufe.  University of North Carolina at Chapel Hill. 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.

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

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimentelle Metastasierung und CTL adoptiven Transfer Immuntherapie Maus-Modell

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Forschung

Immunologie und Infektion

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

Durchflusszytometrie-Analyse von Immunzellen innerhalb Murine Aorten

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.

We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.

Accelerated Typ 1 Diabetes Induktion in Mäusen durch adoptiven Transfer von CD4 + T diabetogene Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer&#039;s Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

Die Beurteilung der Entwicklung der Murine Plasmazytoide dendritischen Zellen in Peyer-Patches mit Adoptiv Übertragung von hämatopoetischen Vorläuferzellen

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Generierung von CAR T-Zellen für die adoptive Therapie im Kontext von Glioblastoma Standardtherapie

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Analyse von<em&gt; Yersinia enterocolitica</em&gt; Effektor Translokation in Wirtszellen unter Verwendung Beta-Lactamase-Effektor-Fusionen

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

Ein optimiertes Verfahren zur Isolierung und Ausbau unveränderlichen natürlichen Killer T-Zellen aus der Milz der Maus

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

Ein Nicht-invasive und in technischer Hinsicht nicht intensive Verfahren zur Induktion und Phänotypisierung für experimentelle bakterielle Pneumonie bei Mäusen

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

Induktion von Lähmung und visuelles System Verletzungen bei Mäusen durch T-Zellen spezifisch für Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Stereotaktische Adoptiv Übertragung von zytotoxischen Immunzellen in murinen Modellen der Orthotopen Human Glioblastoma Multiforme Xenotransplantate

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...