Questo lavoro è stato sostenuto da Gastroenterologiche American Association (AGA) Research Scholars Award e morbo di Crohn e Colite Foundation of America (CCFA) Career Development Award (a SOI), NIH NIDDK F30 DK089692 (ECS), e University of North Carolina Centro di Biologia gastrointestinale e la malattia di Grant P30 DK34987 (Istologia Core). Il Fondo UNC Citometria a flusso Core è sostenuto in parte da un nucleo di supporto NSC Centro Grant (P30CA016086) alla UNC Lineberger Comprehensive Cancer Center. Ringraziamo Luca B. Borst da North Carolina State University College di Medicina Veterinaria per il suo aiuto con analisi istopatologica e immunoistochimica.

NOTA: Assicurarsi che tutti i protocolli animali sono approvati dal e nel rispetto Istituzionale Animal Care and Use Committee (IACUC) regolamenti e guida del Consiglio Nazionale delle Ricerche per la cura e l&#39;uso di animali da laboratorio. Topo donatore può essere maschio o femmina, ma topi riceventi devono essere di sesso maschile. Se i destinatari femmina devono essere utilizzati, i topi donatori devono essere femmina 5. Mantenere le colonie con regolare, biancheria da letto non sterili e acqua non acidificato, in quanto potrebbero avere un impatto del microbiota intestinale, e, quindi, il fenotipo colite dei topi 5,6. Preparazione 1. Sperimentale <ol><li> Utilizzare supporti e tamponi ghiacciate. Mantenere cellule in ghiaccio durante l&#39;esperimento. </li><li> Eseguire l&#39;esperimento in cappa di rischio biologico sterile con tecnica sterile. </li></ol> 2. Isolamento delle cellule della milza T <ol><li> Euthanize topo donatore / mice in CO 2 da camera seguiti da dislocatio cervicalen. Spray addome con etanolo al 70%. </li><li> Fare un incisione orizzontale nell&#39;addome e staccarsi la pelle per esporre il peritoneo. Tenere peritoneo lontano dagli organi interni con le pinze e fare un&#39;incisione nel peritoneo addominale sinistro per esporre e asportare la milza. </li><li> Posizionare la milza in 10 ml di completo media in una capsula di Petri. Rimuovere e gettare il tessuto in eccesso dalla milza. </li><li> Utilizzare 2 vetrini sterilizzati per schiacciare e prendere in giro a parte milza in cella singola sospensione. Sospensione cellulare Filtrare attraverso un filtro 70 micron in una provetta conica da 50 ml e risciacquare il filtro con 5 ml di completo media. Luogo fino a 5 milze in un tubo da 50 ml conica. </li><li> Cellule centrifugare a 450 xg per 7 minuti. Gettare il surnatante versando off o per aspirazione di vuoto nel contenitore dei rifiuti. </li><li> Risospendere delicatamente le cellule in 5 ml per milza di tampone di lisi per lisare i globuli rossi per 10 min a temperatura ambiente. Aggiungere un volume uguale di Complete mezzi (5 ml per milza) al tubo. </li><li&gt; cellule centrifugare a 450 xg per 7 minuti. Gettare il surnatante versando off o per aspirazione di vuoto nel contenitore dei rifiuti. </li><li> Risospendere delicatamente le cellule in 10 ml di Labeling Buffer. </li><li> Contare le celle di trypan esclusione blu. <ol><li> Rimuovere 20 ml di sospensione cellulare e aggiungere 180 microlitri blu trypan ad una provetta per microcentrifuga e mescolare accuratamente. Dopo 5 minuti, aggiungere 10 ml di cellule emocitometro e contare le cellule non-blu al microscopio etichettati. Determinare il numero totale di cellule vitali. Eliminare cell / trypan blue mix. </li></ol></li><li> Centrifugare le cellule in 10 ml di Labeling Buffer a 450 xg per 7 minuti. Gettare il surnatante versando off o per aspirazione di vuoto nel contenitore dei rifiuti. </li></ol> 3. Arricchimento di cellule CD4 + T NOTA: Seguire le istruzioni del produttore per i prodotti specifici utilizzati in questa sezione. <ol><li> Risospendere delicatamente le cellule a una cella singola sospensione di 20 x 10 6 cellule / ml in freddo LabelBuffer ing. </li><li> Aggiungere 5 ml per 1 x 10 6 cellule di anticorpi arricchimento delle cellule T CD4 biotinilati. Incubare le cellule in ghiaccio per 15 min. </li><li> Aggiungere 10x volume di Labeling Buffer. Cellule centrifugare a 450 g per 7 min. Aspirare accuratamente tutto il surnatante con aspirazione di vuoto nel contenitore dei rifiuti. </li><li> Vortice accuratamente particelle magnetiche streptavidina coniugato. Aggiungere 5 ml di particelle per 1 x 10 6 cellule. </li><li> Mescolare accuratamente, ma delicatamente. Tenere mix a 6-12 ° C per 30 min. </li><li> Aggiungere Labeling Buffer ad una concentrazione di 20-80 x 10 6 cellule / ml. Trasferimento fino a 1,0 ml cellule marcate per 12 x 75 mm provetta a fondo tondo (denominato &quot;tubo positivo frazione&quot;). </li><li> Collocare ogni provetta positiva frazione sulla calamita per 6-8 min. </li><li> Con i tubi positivo frazione ancora sul magnete, trasferire accuratamente surnatante dal tubo positiva frazione con Pasteur vetro pipetta ad un nuovo tubo conico sterili 50 ml (referred come la &quot;frazione arricchita&quot;). Questa frazione arricchita contiene cellule CD4 + T. Fare attenzione a non distruggere le cellule marcate attratti al magnete. </li><li> Risospendere le cellule lasciati nei tubi positivo frazione nello stesso volume di Labeling Buffer come al punto 3.6 pipettando su e giù vigorosamente. Posizionare il tubo positivo frazione indietro calamita per 6-8 min. </li><li> Con i tubi positiva frazione ancora sul magnete, trasferire attentamente surnatante (arricchito frazione, CD4 +) dal tubo di positivo-frazione con vetro Pasteur pipetta per sterile tubo da 50 ml da punto 3.8 senza interrompere le cellule attaccate a magnete. </li><li> Ripetere i passaggi 3,9-3,10 per aumentare la resa delle cellule CD4 + T ottenuti. Continuare protocollo utilizzando frazione arricchita (cellule CD4 +). </li></ol> 4. L&#39;etichettatura e Ordinamento Cells 7 <ol><li> Centrifugare arricchito cellule a 450 xg per 7 minuti. Gettare il surnatante versando off o SUCT vuotoione nel contenitore dei rifiuti. </li><li> Risospendere le cellule in 1 ml di Labeling Buffer. Rimuovere aliquota di cellule per contare e valutare per la vitalità cellulare del trypan blu come al punto 2.9. </li><li> Aggiungere un volume di Labeling Buffer a 10 x 10 6 cellule / ml; se le cellule sono già &lt;10 x 10 6, centrifugare a 450 xg per 7 minuti, scartare il surnatante versando off o di aspirazione di vuoto nel contenitore dei rifiuti, e aggiungere il volume di Labeling Buffer a 10 x 10 6 cellule / ml. <ol><li> Impostare aliquote separate di circa 5-10 × 10 5 celle ciascuno per le cellule di controllo non colorate, isotipo macchiati, e single-macchiato in tubi microcentrifuga. </li></ol></li><li> Aggiungere 5 ug / ml CD4-FITC e 1 mg / ml CD45RB-PE alle cellule. Aggiungere macchie di controllo isotipico e macchie singoli alla stessa concentrazione di appropriarsi aliquote in tubi microcentrifuga. Mescolare bene ma delicatamente e incubare su ghiaccio al riparo dalla luce per 30 min. </li><li> Aggiungere 10x volume di Labeling Buffer alle cellule e di controllos. Centrifuga 450 xg per 7 minuti. Gettare il surnatante mediante aspirazione di vuoto nel contenitore dei rifiuti. </li><li> Risospendere in Labeling Buffer di volume in fase 4.5. Centrifuga 450 xg per 7 minuti. Gettare il surnatante mediante aspirazione di vuoto nel contenitore dei rifiuti. </li><li> Risospendere in Labeling Buffer a 10 x 10 6 cellule / ml. Passare cellule attraverso 70 micron filtro nel tubo FACS. Tenere su ghiaccio al riparo dalla luce fino al momento FACS. </li><li> Impostare e stabilire un&#39;adeguata compensazione sul cell sorter con le cellule non colorate e controlli singoli macchiati. </li><li> Escludi cellule non vitali con forward e side-scatter gating (Figure 1A). Impostare gating per CD4 + e + cellule CD45RB con controlli isotipici macchiato. Cellule CD4 + cancello sulla popolazione. </li><li> Ordina CD4 + cellule in CD45RB alta e CD45RB basse popolazioni utilizzando una semplice istogramma per le cellule PE macchiate in tubi con 2 ml di completo media. Il CD45RB alta </sup&gt; popolazione rappresenta la più alta del 40% di CD4 + CD45RB + cellule (CD45RB alta), e la popolazione bassa CD45RB è il più basso del 20% + CD45RB cellule CD4 + (CD45RB basso; Figura 1B). </li></ol><img alt="Figura 1" src="/files/ftp_upload/52533/52533fig1.jpg" /> Figura 1: flusso rappresentante citometria appezzamenti di popolazioni di cellule CD4 + T CD45RB durante l&#39;analisi FACS (A - C) FITC CD4- e splenociti PE CD45RB-macchiato da donatori C57BL / 6 topi sono stati ordinati per FACS in CD4 + CD45RB alta e CD4 +. CD45RB popolazioni di cellule T basso. Eventi (A) Doppietti stati esclusi nella rappresentazione della dispersione in avanti. (B) I linfociti sono stati gated nella trama in avanti e scatter laterale. (C) CD4 + </sup&gt; cellule T sono stati gated, e cellule (D) CD4 + CD45RB + T sono state tracciate su un PE contro Eventi istogramma. Cellule CD4 + bassi CD45RB sono stati considerati i più bassi del 20% delle cellule + CD45RB. Cellule di alta CD4 + CD45RB sono stati definiti come la più alta del 40% delle cellule + CD45RB. <ol start="11"><li> Eseguire un&#39;aliquota di ciascuna popolazione cellulare sulla macchina FACS per valutare la purezza delle popolazioni. </li><li> Centrifugare allineati cellule a 450 xg per 7 minuti. Risospendere in 1 ml di PBS. Rimuovere aliquota di cellule per contare e valutare per la vitalità cellulare del trypan blu come al punto 2.9. </li></ol> 5. iniezione di cellule in Destinatari <ol><li> Risospendere allineati alle cellule di 4 x 10 6 cellule / ml (CD45RB alta) o 2 x 10 6 cellule / ml (CD45RB basso) in PBS. </li><li> Trasferire 100 ml di cellule di alta CD45RB per destinatario di nuova provetta sterile. Aggiungere 100 ml di PBS perdestinatario di questo tubo. Così, il volume di iniezione totale per animali è di 200 ml, e l&#39;importo totale delle cellule per destinatario è di 4 alti cellule T naive x 10 5 CD4 + CD45RB. </li><li> Se si desidera gruppo sperimentale che riceve le cellule T regolatorie, trasferire 100 ml di cellule di alta CD45RB per destinatario di nuova provetta sterile. Aggiungere 100 ml di CD45RB bassi cellule per destinatario lo stesso tubo. Volume di iniezione totale per animali è di 200 ml; Rapporto di alto CD45RB: CD45RB cellule bassi è di 2: 1. </li><li> Iniettare 100 ml di CD45RB alta o CD45RB alto / basso CD45RB CD4 + cellule per via intraperitoneale nel lato destro e sinistro dell&#39;addome (totale di 200 ml) di ciascun destinatario. </li></ol> 6. Monitoraggio della progressione della malattia in animali destinatari <ol><li> Per valutare lo stato clinico degli animali beneficiari, assegnare punteggi clinici aggregati per le seguenti parameters 8 il giorno dell&#39;iniezione, poi settimanalmente e al momento del sacrificio: <ol><li> Determinare sprecare misurando la perdita di peso: 0 - nessuna perdita di peso; Perdita 0,1-10% del peso corporeo iniziale - 1; 2 - perdita più del 10% del peso corporeo iniziale (Figura 2A). </li></ol></li></ol><img alt="Figura 2" src="/files/ftp_upload/52533/52533fig2highres.jpg" width="650" /> Figura 2: segni patologici clinici e lordi di infiammazione si verificano dopo il trasferimento di tipo selvaggio cellule T CD4 + alta CD45RB in Rag1 - / - e NRDKO topi riceventi 11 (A) i destinatari NRDKO perso in media il 10% del loro peso corporeo iniziale del 5. settimane dopo il trasferimento, mentre Rag1- / - I beneficiari non hanno mostrato segni clinici di malattia in questo momento. Ogni punto rappresenta la percentuale media del peso corporeo iniziale per la coorte ± SEM. **, P &lt;0.005. (B) Alcuni topi hanno sviluppato una grave infiammazione intestinale, come dimostrato dalla presenza di prolasso rettale. Questo è un quadro rappresentativo di prolasso rettale in un topo ricevente NRDKO. (C) fortemente, i due punti sia da Rag1 - / - e NRDKO topi riceventi sono ispessite e accorciati rispetto a due punti dal Rag1 - / - mice e NRDKO senza T cell trasferimento adottivo. I due punti di NRDKO topi riceventi mostrano grave infiammazione e aumento del peso del colon (dati non riportati). <a href="https://www.jove.com/files/ftp_upload/52533/52533fig2highres.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <ol><li><ol start="2"><li> Determinare la qualità delle feci mettendo animali in recipiente pulito fino a quando non è passatosgabello: 0 - nessuno; 1 - feci molli; 2 - feci acquose e / o sanguinosa. </li><li> Determinare la salute generale di animale presenza dei seguenti segni di malattia: 0 - no postura ingobbita, setole pelliccia, pelle o lesioni; 1 - uno qualsiasi dei seguenti presente: curvo postura, setole di pelliccia, o lesioni cutanee. </li><li> Determinare la presenza di prolasso rettale: 0 - assente; 1 - presente (Figura 2B). </li></ol></li><li> Sacrifica animali da CO 2 inalazione seguita da dislocazione cervicale quando hanno perso il 20% del loro peso corporeo iniziale o nel punto desiderato momento, quello che viene prima. Malattia clinica è di solito evidente a partire da settimana 5 post-sazietà. </li><li> Valutare per la gravità della malattia, come descritto in precedenza 5,7. <ol><li> Assegnare punteggi clinici come al punto 6.1 8. </li><li> Misurare la lunghezza del colon e del peso (Figura 2C). </li><li> Eseguire l&#39;analisi istologica di infiammazione5. </li><li> Determinare citochine spontanea in intestinali culture espianto dei tessuti 9, la linfa mesenterica nodi 10, e / o siero 11. <ol><li> In breve, per le culture espianto 9, rimuovere i due punti dopo il sacrificio, aperta longitudinalmente, e pulire con PBS. Incubare due punti in un agitatore orbitale a 250 rpm impostato in completa media per 30 minuti a temperatura ambiente. </li><li> Tritare il tessuto in piccoli pezzi e incubate a 37 ° C in media completa per 24 ore. Raccogliere il surnatante e utilizzare per la quantificazione di citochine per 100 mg di tessuto di ELISA. </li></ol></li><li> Eseguire caratterizzazione ex vivo di fenotipi di cellule T e / o la funzione 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.

Qui si descrive un protocollo step-by-step indurre infiammazione del colon nei topi mediante trasferimento adottivo di cellule CD4 + T + CD45RB in topi immunodeficienti. Abbiamo usato milza C57BL / 6 donatori e Rag1 singenico - / - topi riceventi, anche se altri ceppi (ad esempio, BALB / c, 129S6 / SvEv, non-obesi diabetici (NOD)) Modelli e genetici di immunodeficienza (ad esempio, SCID, Rag2 - / -) può essere utilizzato anche 4,14-16....

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.

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	<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

Ci sono molti modelli animali differenti disponibili per lo studio della patogenesi delle malattie infiammatorie croniche intestinali umane (IBD), ognuno con i suoi vantaggi e svantaggi. Descriviamo qui un modello di colite sperimentale che è iniziata da trasferimento adottivo di cellule della milza alta T CD4 + CD45RB singenici in T e cellule B topi riceventi deficienti. La popolazione di cellule T CD4 + alta CD45RB che consiste in gran parte di cellule effettrici naïve è in grado di indurre infiammazione intestinale cronica, molto simili aspetti chiave di IBD umana. Questo metodo può essere manipolato per studiare gli aspetti di insorgenza della malattia e la progressione. Inoltre può essere utilizzata per studiare la funzione di innata, adattabile e popolazioni regolamentazione cellule immunitarie, e il ruolo delle esposizioni ambientali, cioè, il microbiota, infiammazione intestinale. In questo articolo si illustra il metodo per indurre la colite con un protocollo passo-passo. Questo incLudes un video dimostrativo di aspetti tecnici chiave necessari per sviluppare con successo questo modello murino di colite sperimentale per scopi di ricerca.

Circa 10 x 10 6 CD4 + CD45RB cellule di alta T da 10 milza di adulti C57BL / 6 topi donatori sono affidabile isolati. Questo numero varia a seconda dell&#39;età e la tensione del mouse donatore e la competenza del ricercatore. Quando 4 x 10 5 C57BL / 6 celle ad alta T CD4 + CD45RB vengono trasferiti in C57BL / 6 Rag1 - / - topi riceventi, i segni clinici della malattia emergono intorno settimana 5 post-sazietà o prima, se i topi sono...

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.

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

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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 Views.  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.

Video: 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.

Metastasi sperimentale e CTL adottivi Immunoterapia modello di trasferimento del mouse

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

Ricerca

Immunologia e infezioni

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.

Analisi Citometria a flusso di cellule immunitarie all&#39;interno aorte Murine

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 diabete di tipo 1 nei topi induzione trasferimento adottivo di cellule CD4 + T Cells diabetogenici

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.

Valutare lo sviluppo delle cellule murine plasmocitoidi dendritiche in placche del Peyer Utilizzando trasferimento adottivo di ematopoietiche Progenitori

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.

Generazione di cellule CAR T per adottiva terapia nel contesto di Glioblastoma standard di cura

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.

Analisi di<em&gt; Yersinia enterocolitica</em&gt; Effector traslocazione in cellule ospiti Utilizzando beta-lattamasi Effector Fusions

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.

Un metodo ottimizzato per l&#39;isolamento ed espansione invariante Natural Killer cellule T da Spleen mouse

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.

Un metodo non invasivo e tecnicamente non intensiva per l&#39;induzione e Fenotipizzazione Sperimentale polmonite batterica nei topi

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.

Induzione di paralisi e di lesioni del sistema visivo in topi da specifiche cellule T per neuromielite ottica Autoantigen acquaporina-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.

Trasferimento adottivo stereotactic di citotossici cellule immunitarie nei modelli murini di xenotrapianti Multiforme di Glioblastoma umano ortotopico

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