Siamo in debito con Alice Gaughan e-Jiao Jing Wang cui eccellente supporto tecnico, apporto intellettuale, e sostegno morale hanno contribuito a spostare in avanti questi studi. Questi studi sono stati sostenuti dalla concessione NIH AI036532 a GAH.

1. Irradiazione letale <ol><li> Inserire fino a 10 topi riceventi in una gabbia microisolator compatibile con l&#39;irradiatore da utilizzare. </li><li> Irradiare in 2 dosi uguali sommatori dose totale (dose totale = 9 cGy per i destinatari BALB.B). Irraggiamento secondo dovrebbe essere di 3 ore dopo la prima. L&#39;iniezione deve avvenire tra 4-6 ore dopo l&#39;irradiazione finale. Topi irradiano in entrambi 137 sorgente Cs o RS2, 000, irradiatore. </li><li> Dopo la seconda dose di radiazioni, i topi in gabbia negozio microisolator con acqua acidificata fino al momento del trapianto. </li></ol> 2. Preparazione Splenocyte <ol><li> Eutanasia un topo donatore secondo le linee guida istituzionali. Ogni donatore produce generalmente 75x10 6 - 125x10 6 splenociti. Utilizzando CD8 kit di purificazione, ciascun topo produce generalmente 6x10 6 - 12x10 6 cellule T CD8. </li><li> Rimuovere la milza facendo prima una verticale 2 centimetri di incisione 2 centimetri a destra della linea mediana direttamentesotto la gabbia toracica. Tagliare la pelliccia, la pelle, e la membrana viscerale. </li><li> Rimuovere milza e creare singola sospensione cellulare mettendo milza in 40 microlitri mesh in una piastra di Petri con RPMI 1640 con 5% di FBS. Utilizzare un stantuffo della siringa per spezzare la milza fino a quando la milza intero è stato passato attraverso mesh. </li><li> Ripetere questa procedura per ogni WT e L2G85.B6 donatore. Raccogliere tutte le sospensioni WT monocellulari in 1-50 ml tubetto conico, e raccogliere tutte le L2G85.B6 sospensioni di cellule singole in un apposito tubo da 50 ml. </li><li> Centrifugare le cellule a 1.200 rpm per 10 minuti a 4 ° C. Risospendere pellet in RPMI 1640 con 5% di FBS e le cellule di conteggio. </li></ol> 3. Preparazione di midollo osseo <ol><li> Togliere la pelle da una dell&#39;arto posteriore di un topo donatore. </li><li> Tagliare con cautela via come tessuto muscolare più possibile da femore e tibia / perone. </li><li> Rimuovere dell&#39;arto posteriore, tagliando via al femore dell&#39;anca. Tagliare zampa posteriore appena sotto tibia / perone intersezione. Tutti i tagli ossei devono essere effettuate utilizzando le forbici robuste. </li><li> Rimuovere con cautela la restante tessuto muscolare. Tagliare perone-relativamente poco midollo osseo si trova in fibula e non vale la pena. </li><li> Mettere arto posteriore a freddo RPMI 1640 5% FBS processo e ripetere con la seconda parte posteriore. Ogni mouse dovrebbe produrre tra 20x10 6 - 40x10 6 cellule del midollo osseo. </li></ol> 4. Midollo osseo rimozione <ol><li> Rimuovere un arto posteriore da un supporto e mettere in grande piastra di Petri con una piccola quantità di supporti a freddo (~ 1 ml). </li><li> Tagliare l&#39;articolazione del ginocchio. Utilizzando una siringa (volume&gt; 5 ml), inserire l&#39;ago sottocutanea a tibia e deprimere siringa finché tutto il materiale rosso viene rimosso dall&#39;interno della tibia. Ripetere il processo con il femore. Eliminare le ossa rimaste prive di midollo osseo. Ripetere il processo con il secondo degli arti posteriori. </li><li> Crea singola sospensione cellulare da parte dei media di pipettaggio contenenti midollo osseo in grande piastra di Petri e stantuffo della siringa uso e 40 microlitrimaglia dello schermo. Pipettare sospensione singola cellula in tubo da 50 ml conica e tenere in ghiaccio. </li></ol> 5. CD3 esaurimento Ci sono una varietà di modi per riducono cellule CD3 + dal midollo osseo. Il nostro laboratorio utilizza un kit composto da Miltenyi Biotec (CD3-biotina - 130-093-021). Riducono cellule CD3 + nel midollo osseo seguendo il protocollo del produttore. Il buffer per i kit Miltenyi sarà ora in poi denominato MACS Buffer (2 mM EDTA, 0,5% BSA in PBS, pH 7,2). <ol><li> Lavare le cellule per centrifugazione splenociti e le cellule del midollo osseo a 1200 rpm per 10 min a 4 ° C e cellule conteggio. </li><li> Rimuovi tutto il surnatante. Risospendere le cellule del midollo osseo in 100 pl per le cellule ossee 10000000 midollo in Buffer MACS. </li><li> Continuare con CD3 esaurimento produce utilizzando il protocollo &#39;. </li><li> Lavare CD3 impoverito cellule del midollo osseo 3 volte in PBS sterile. Conte CD3 impoverito cellule del midollo osseo e risospendere in un volume adeguato di iniettare 10 7 cellule. </li></ol> Ci sono una varietà di modi per purificare le cellule T CD8 da L2G85.B6 topi. Inoltre, ci sono diversi modi per riducono le cellule T CD8 da WT donatori splenocyte. Il nostro laboratorio utilizza i kit da Miltenyi Biotec (CD8 esaurimento - 130-049-401, CD8 purificazione - 130-095-236). <ol><li> Risospendere il pellet in 90 WT Buffer MACS microlitri per 10 7 cellule. Continuare con deplezione delle cellule T CD8 secondo produce &#39;protocollo (CD8 esaurimento - 130-049-401). </li><li> Risospendere il pellet L2G85.B6 in 40 microlitri di buffer MACS per 10 7 cellule. Continuare con la purificazione delle cellule T CD8 secondo produce &#39;protocollo (CD8 depurazione - 130-095-236). </li><li> Contare ogni popolazione di cellule e lavare ogni popolazione 3 volte con PBS sterile. Risospendere ogni popolazione in volume sufficiente per iniettare 18x10 6 WT (CD8 impoverito) splenociti e 2x10 6 L2G85.B6 purificato le cellule T CD8. </li></ol>7. Iniezione di preparazione <ol><li> Combinare 10 7 CD3 impoverito cellule di midollo osseo, 18x10 6 WT (CD8 impoverito) splenociti e 2x10 6 L2G85.B6 purificate cellule T CD8 in una provetta da microcentrifuga. Lavare con PBS sterile e risospendere in 300 microlitri. </li><li> Iniettare il preparato cellulare nella vena della coda di topi riceventi. Le iniezioni devono essere fatto utilizzando aghi di calibro 28. </li><li> Conservare i topi in gabbia microisolator con acqua acidulata. Topi punteggio giornaliero con sistema di punteggio GVHD scoring sviluppato da Cooke et al 1. </li></ol> 8. Bioluminescent Imaging <ol><li> Sei giorni dopo il trapianto, iniettare topi riceventi con 4 mg di D-luciferina. 5 minuti per consentire di reagire con luciferina luciferasi. </li><li> Anestetizzare il mouse nella camera di isoflurano imager bioluminescenza e dei beneficiari immagine per 5 min con binning piccolo. Questo creerà una immagine ad alta risoluzione, mentre la raccolta di quanti più eventi possibili. </li><li> Analizzare i dati con LivingImmagine software. La scala di pseudo-immagine a colori può essere modificato per ottenere i migliori risultati. Tuttavia, è essenziale che la stessa scala da utilizzare in tutti gli esperimenti. </li><li> Regioni di interesse possono essere creati utilizzando il software Image vita e di emittanza luce può essere quantificata calcolando il flusso (fotoni / sec) essendo emessa da ciascuna regione di interesse. </li></ol> 9. Risultati rappresentativi Circa 7-10 giorni dopo il trapianto, i topi cominciano a mostrare segni clinici di GVHD. I topi sembrano trasandato a causa della mancanza di governare. Destinatari inizierà anche a perdere peso dopo il trapianto tra 7-10 giorni. Attività e la postura dei topi riceventi rimarrà relativamente normale fino a circa 12-14 giorni dopo il trapianto. Punteggi cumulativi GVHD saranno costantemente migliorate attraverso il primo 2-3 dopo il trapianto settimane (Figura 1A). Il decorso della malattia è molto variabile tra i topi, tuttavia, i beneficiari dovrebbero uniformemente soccombere alla GVHD del 30-40 days dopo il trapianto (Figura 1B). La Figura 2 mostra topi riceventi che sono stati impressi sei giorni dopo il trapianto. Pseudo-color scala mostra diverse emittanza luce in tutto il corpo con la massima intensità di luce emessa nella milza e l&#39;intestino. Cellule T CD8 accumulo nell&#39;intestino è coerente con i risultati precedenti 6. Topi riceventi può essere rimesso in gabbia microisolator da acquisire in un secondo momento o punto di eutanasia per l&#39;imaging ex vivo. <table border="1"><tbody><tr><td> Criteri </td><td> Grado 0 </td><td> Grade 1 </td><td> Grado 2 </td></tr><tr><td> Perdita di peso (wkly.) </td><td> &lt;10% </td><td> &gt; 10% - &lt;25% </td><td> &gt; 25% </td></tr><tr><td> Posizione </td><td> Normale </td><td> Incurvando solo a riposo </td><td> Diversi incurvando; altera movement </td></tr><tr><td> Attività </td><td> Normale </td><td> Da lieve a moderata diminuzione dell&#39;attività </td><td> Stazionario a meno stimolato </td></tr><tr><td> Pelliccia tessitura </td><td> Normale </td><td> Da lieve a moderata scompigliava </td><td> Arruffarsi Grave / poveri grooming </td></tr><tr><td> Struttura della pelle </td><td> Normale </td><td> Scala di zampe / coda </td><td> Evidenti aree di pelle denudata </td></tr></tbody></table> Tabella 1. Cooke et al ha sviluppato questo sistema di punteggio nel 1996 1. I topi, deve essere catalogato ogni giorno su ciascuno dei criteri a sinistra. Ogni mouse viene dato un punteggio di 0-2 per ciascun criterio e il punteggio totale è la somma di tutti i punteggi individuali. <img alt="Figura 1" src="/files/ftp_upload/3697/3697fig1.jpg" /> Figura 1. Letale BALB.B irradiati sono stati trapiantati con 10 7 ossocellule del midollo da soli o con 18x10 6 cellule T CD8 splenociti impoverito WT e 2x10 6 purificate L2G85.B6 cellule T CD8. A) Dati clinici punteggio dei destinatari di midollo osseo da solo o con cellule T CD8 impoverito splenociti WT e purificato L2G85.B6 cellule T CD8. B) I dati di sopravvivenza dei destinatari di midollo osseo da solo o con cellule T CD8 impoverito splenociti WT e purificato L2G85.B6 cellule T CD8. <a href="http://www.jove.com/files/ftp_upload/3697/3697fig1large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img alt="Figura 2" src="/files/ftp_upload/3697/3697fig2.jpg" /> Figura 2. BALB.B topi letalmente irradiati sono stati trapiantati con 10 cellule di midollo osseo 7 da soli o con 18x10 6 cellule T CD8 splenociti impoverito WT e 2x10 6 purificate L2G85.B6 cellule T CD8. Destinatari sono stati iniettati con 4 mg D-luciferina tramite iniezione intraperitoneale e sono stati ripresi utilizzando IVIS Xenogen per 5 min a binning piccola. Pseudo di colore le immagini vengono visualizzate in cui viola rappresenta bassa intensità e rosso rappresenta regioni ad alta intensità di interesse sono stati elaborati intorno tutto il mouse e il flusso totale (fotoni / sec) sono stati quantificati. <img alt="Figura 3" src="/files/ftp_upload/3697/3697fig3.jpg" /> Figura 3. BALB.B topi letalmente irradiati sono stati trapiantati con 10 cellule di midollo osseo 7 da soli o con 18x10 6 cellule T CD8 splenociti impoverito WT e 2x10 6 purificate L2G85.B6 cellule T CD8. Destinatari sono stati iniettati con 4 mg D-luciferina tramite iniezione intraperitoneale e sono stati ripresi utilizzando IVIS Xenogen per 5 min a binning piccola. Pseudo-color immagini vengono visualizzate in cui viola rappresenta bassa intensità e rosso rappresenta ad alta intensità. I destinatari sono stati ripreso su A) giorno 4, B) giorno 6, e C) giorno 8 vaglia postale.

<ol>
	<li>Cooke, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+of+idiopathic+pneumonia+syndrome+after+bone+marrow+transplantation:+The+roles+of+minor+H+antigens+and+endotoxin.">An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation: The roles of minor H antigens and endotoxin.</a> Blood. 88, 3230-3239 (1996).</li><li>Berger, M. <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+subsets+involved+in+lethal+graft-versus-host+disease+directed+to+immunodominant+minor+histocompatibility+antigens.">T cell subsets involved in lethal graft-versus-host disease directed to immunodominant minor histocompatibility antigens.</a> Transplantation. 57, 1095-1102 (1994).</li><li>Nimer, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+depletion+of+CD8++cells+for+prevention+of+graft-versus-host+disease+after+bone+marrowtransplantation.+A+randomized+controlled+trial.">Selective depletion of CD8+ cells for prevention of graft-versus-host disease after bone marrowtransplantation. A randomized controlled trial.</a> Transplantation. 57, 82-87 (1994).</li><li>Korngold, R., 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=Surface+markers+of+T+cells+causing+lethal+graft-vs-host+disease+to+class+I+vs+class+II+H-2+differences.">Surface markers of T cells causing lethal graft-vs-host disease to class I vs class II H-2 differences.</a> Journal of Immunology. 135, 3004-3010 (1985).</li><li>Cao, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+using+labeled+donor+tissues+reveals+patterns+of+engraftment,+rejection,+and+survival+in+transplantation.">Molecular imaging using labeled donor tissues reveals patterns of engraftment, rejection, and survival in transplantation.</a> Transplantation. 80, 134-139 (2005).</li><li>Asady, R. E. l. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-{beta}-dependent+CD103+expression+by+CD8(+)+T+cells+promotes+selective+destruction+of+the+host+intestinal+epithelium+during+graft-versus-host+disease.">TGF-{beta}-dependent CD103 expression by CD8(+) T cells promotes selective destruction of the host intestinal epithelium during graft-versus-host disease.</a> J. Exp. Med. 201, 1647-1657 (2005).</li><li>Larson, R. S., Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+leukocyte+integrins.">Structure and function of leukocyte integrins.</a> Immunol Rev. 114, 181-217 (1990).</li><li>Karecla, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+E-cadherin+on+epithelial+cells+by+the+mucosal+T+cell+integrin+alpha+M290+beta+7+(alpha+E+beta+7).">Recognition of E-cadherin on epithelial cells by the mucosal T cell integrin alpha M290 beta 7 (alpha E beta 7).</a> Eur. J. Immunol. 25, 852-856 (1995).</li><li>Cepek, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+between+epithelial+cells+and+T+lymphocytes+mediated+by+E-cadherin+and+the+alpha+E+beta+7+integrin.">Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the alpha E beta 7 integrin.</a> Nature. 372, 190-193 (1994).</li><li>Malarkannan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+and+functional+characterization+of+a+dominant+minor+H+antigen,+H60.">The molecular and functional characterization of a dominant minor H antigen, H60.</a> J. Immunol. 161, 3501-3509 (1998).</li></ol>

Nessun conflitto di interessi dichiarati.

Il protocollo per indurre GVHD nei topi qui presentato rappresenta un modello clinicamente rilevante di GVHD murino. Fondato da Berger et al. Nel 1994, il C57Bl / 6 in combinazione ceppo BALB.B è MHC-abbinato, con una mortalità GVHD mediata da CD4, CD8 dipendenti effettori T 2, molto simili allo scenario clinico più comune 3. E &#39;noto che trapiantando cellule T CD8 sola non causa GVHD in questo modello, tuttavia, la progressione della malattia è significativamente peggiore quando si...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 12633-012 </td><td></td></tr><tr><td> Fetal Calf Serum </td><td> Invitrogen </td><td> 10439016 </td><td></td></tr><tr><td> 40 Filtro Cella pM </td><td> BD Biosciences </td><td> 352340 </td><td></td></tr><tr><td> CD3e-Biotina </td><td> Miltenyi Biotech </td><td> 130-093-021 </td><td></td></tr><tr><td> Anti-biotina Microsfere </td><td> Miltenyi Biotech </td><td> 130-091-147 </td><td></td></tr><tr><td> CD8a Microsfere </td><td> Miltenyi Biotech </td><td> 130-049-401 </td><td> Utilizzato per esaurire le cellule T CD8 da milza. </td></tr><tr><td> CD8a Purificazione cocktail di anticorpi </td><td> Miltenyi Biotech </td><td> 130-095-236 </td><td> Usato per purificareCellule T CD8 di milza. </td></tr><tr><td> D-Luciferin </td><td> Caliper Life Sciences </td><td> 122796 </td><td></td></tr></tbody></table>

induction of graft-versus-host disease and in vivo t cell monitoring using an mhc-matched murine model

Graft-versus-host disease (GVHD) è la barriera che limita l&#39;ampio utilizzo di trapianto di midollo osseo come terapia curativa per una serie di carenze ematologiche. GVHD è causata da cellule T mature alloreattive presenti nel trapianto midollo osseo che sono infuse nel ricevente e causare danni agli organi di accoglienza. Tuttavia, nei topi, cellule T deve essere aggiunto al inoculo midollo osseo a causare GVHD. Sebbene un ampio lavoro è stato fatto per caratterizzare le risposte delle cellule T dopo trapianto, bioluminescente tecnologia di imaging è un metodo non invasivo per monitorare il traffico delle cellule T modelli in vivo. A seguito di irradiazione letale, topi riceventi sono trapiantati con cellule del midollo osseo e splenociti di topi donatori. Sottopopolazioni di cellule T da L2G85.B6 (topi transgenici che esprimono costitutivamente luciferasi) sono incluse nel trapianto. Da trapiantare solo alcune sottopopolazioni di cellule T, è in grado di monitorare specifici sottogruppi di cellule T in vivo,e in base alla loro posizione, sviluppare ipotesi sul ruolo di specifici sottogruppi di cellule T nel promuovere la GVHD in vari momenti. A intervalli predeterminati dopo il trapianto, topi riceventi vengono esposte con una Xenogen IVIS camera CCD. L&#39;intensità della luce può essere quantificato utilizzando il software Image Vivere per generare una pseudo-immagine a colori in base all&#39;intensità fotone (rosso = alta intensità, viola intensità = bassa). Tra 4-7 dopo il trapianto giorni, topi riceventi cominciano a mostrare segni clinici di GVHD. Cooke et al. 1 messo a punto un sistema di valutazione per quantificare la progressione della malattia in base al destinatario pelliccia trama topi, l&#39;integrità della pelle, attività, perdita di peso, e la postura. I topi sono segnati tutti i giorni, e l&#39;eutanasia quando diventano moribondi. Topi riceventi diventano in genere moribondo 20-30 giorni dopo il trapianto. Modelli murini sono strumenti preziosi per lo studio della immunologia di GVHD. Selettivamente il trapianto delle cellule T sottoinsiemi particolare alminimi per l&#39;identificazione accurata dei ruoli ogni sottoinsieme gioca. Non invasivo di monitoraggio risposte delle cellule T in vivo aggiunge un ulteriore livello di valore per i modelli murini GVHD.

Watch this Scientific Journal Video about Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model at JoVE.com

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine trapianto di midollo osseo è una tecnica ampiamente utilizzata per studiare i meccanismi immunologici che regolano graft-versus-host disease negli esseri umani. La capacità di monitorare il traffico delle cellule T modelli<em&gt; In vivo</em&gt; Consente per l&#39;analisi dettagliata dello sviluppo e la perpetuazione delle risposte delle cellule T durante la graft-versus-host disease.

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

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

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JoVE Journal

Immunology and Infection

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

15.6K Views.  The Ohio State University Medical Center. Murine trapianto di midollo osseo è una tecnica ampiamente utilizzata per studiare i meccanismi immunologici che regolano graft-versus-host disease negli esseri umani. La capacità di monitorare il traffico delle cellule T modelli In vivo Consente per l'analisi dettagliata dello sviluppo e la perpetuazione delle risposte delle cellule T durante la graft-versus-host disease.

Video: Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Trasduzione retrovirale di recettori delle cellule T nel topo le cellule T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

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Immunologia e infezioni

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Un<em&gt; Ex vivo</em&gt; Modello di un attacco T-Cell Oligodendrocyte-diretto nel acute cervello fette

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

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

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

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

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

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

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

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

Femore finestra del modello Camera per<em&gt; In Vivo</em&gt; Monitoraggio delle cellule nel midollo osseo murino

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Un<em&gt; In Vitro</em&gt; Modello della barriera emato-encefalica Utilizzando spettroscopia di impedenza: un focus sui T Cell-endoteliale cellulare Interazione

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

In Vivo Test per la determinazione della funzione citolitica di cellule T antigene-specifiche utilizzando un modello di vaccinazione

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Un protocollo di aspirazione Blister per studiare T-cellula umana richiamo Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Un modello di cultura Bursal-cellula primaria di pollo Ex Vivo per studiare la patogenesi Virus bursite infettiva

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

Quantificazione di basata su citometria a flusso multicolor dei mitocondri e lisosomi in cellule di T

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

Determination of Regulatory T Cell Subsets in Murine Thymus, Pancreatic Draining Lymph Node and Spleen Using Flow Cytometry

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, thymic derived Treg (tTreg) cells and peripherally induced Treg (pTreg) cells. They are present in different organs of our body and can be distinguished by specific markers, such as Helios and Neuropilin 1. It has been reported that tTreg cells are functionally more suppressive than pTreg cells. Therefore, it is important to determine the proportion of both tTreg and pTreg cells when investigating heterogeneous cell populations. Herein, we collected thymic glands, pancreatic draining lymph nodes and spleens from normoglycemic non-obese diabetic mice to distinguish tTreg cells from pTreg cells using flow cytometry. We manually prepared single cell suspensions from these organs. Fluorochrome conjugated surface CD4, CD8, CD25, and Neuropilin 1 antibodies were used to stain the cells. They were kept in the fridge overnight. On the next day, the cells were stained with fluorochrome conjugated intracellular Foxp3 and Helios antibodies. These markers were used to characterize the two subsets of Treg cells. This protocol demonstrates a simple but practical way to prepare single cells from murine thymus, pancreatic draining lymph node and spleen and use them for subsequent flow cytometric analysis.

Herein, we present a protocol to prepare single cells from murine thymus, pancreatic draining lymph node and spleen to further study these cells using flow cytometry. In addition, this protocol was used for determining the subsets of regulatory T cells using flow cytometry.

Determinazione dei sottoinsiemi di regolamentazione delle cellule T nel timo murino, pancreatico drenante linfonodi e milza mediante citometria a flusso

Our immune system consists of a number and variety of immune cells including regulatory T cells (Treg) cells. Treg cells can be divided into two subsets, ...