Christian Welker is funded by a grant provided by the J&#252;rgen-Manchot-Stiftung.

Dichiarazione etica: Tutte le procedure sono state eseguite secondo la Dichiarazione di Helsinki e sono stati approvati dal Comitato Etico Clinica presso l&#39;Università di Tübingen (progetti 38 / 2009B02 e 470 / 2013B02). 1. Isolamento di cellule mononucleate del sangue periferico (PBMC) <ol><li> Prendere 50-100 ml da un volontario sano tramite venipuntura utilizzando una siringa da 50 ml contenente 1.000 UI eparina solfato e diluire il sangue 1: 2 con PBS (pH = 7,2). </li><li> Strato cautela 35 ml di sangue: soluzione PBS soluzione separazione 15 ml di sangue come Biocoll (d = 1.077 g / ml) in un tubo da 50 ml. Centrifugare per 15 minuti a 800 xg a RT. NOTA: Il freno non deve essere utilizzato. </li><li> Raccogliere le cellule dello strato linfocitica con una pipetta e lavare le cellule due volte in almeno 50 ml di PBS (centrifuga per 12 min; a 400 xg). Rimuovere il surnatante dopo centrifugazione con una pipetta. </li><li> Lyse restante eritrociti sospendendo e incubdall&#39;uso della frazione linfocitica con 1-5 ml di una soluzione tampone ipotonico per 2-4 min. NOTA: Durata dell&#39;esposizione e il volume di tampone di lisi dipendono dal tampone di lisi utilizzato e la quantità di globuli rossi lasciati nella frazione linfociti. </li><li> Diluire le cellule con PBS (1,10) e centrifugare a 250 xg per 12 min e poi risospendere e lavare il pellet cellulare una volta in 10 ml di PBS a 300 xg per 12 min. </li><li> Risospendere i linfociti in PBS e contare le cellule in una camera di Neubauer conteggio utilizzando Trypan blue soluzione. NOTA: Di solito,&gt; 1 x 10 8 linfociti vengono ricevuti da 100 ml appena disegnato sangue periferico. </li></ol> 2. Isolamento di Vδ1 cellule T <ol><li> Prendere &lt;1 x 10 6 linfociti isolati per un periodo iniziale di FACS che determinano la frequenza di cellule T CD4 Vδ1. NOTA: le dimensioni della popolazione di questa entità delle cellule T può variare notevolmente tra gli individui e secondo il loro stato immunologico. Typicalleato, circa l&#39;1% delle cellule T sono Vδ1 + e circa il 1-5% del + cellule Vδ1 sono CD4 +. <ol><li> Diluire le cellule con 1 ml FACS-buffer, che consiste di PBS contenente 2% di FCS e 5 mM EDTA. Centrifugare a 660 xg per 2 minuti e decantare il surnatante. Il volume reflusso di circa 100 microlitri tampone FACS è sufficiente per la successiva colorazione. Brevemente vortex e incubare le cellule con 5 ml Reagente Fc-Blocking per 5 minuti a temperatura ambiente per una maggiore specificità della colorazione FACS. </li><li> Aggiungere anticorpi umani monoclonali direttamente alle cellule senza rimuovere il reagente-Fc Blocking. Assicurati di macchiare le cellule con anticorpi contro Vδ1 (01:50), CD3 (01:50), CD4 (1: 200), CD8 (1: 200) e TCRαβ (1,25). Preparare il controllo isotipico con i corrispondenti isotipo di immunoglobulina. Incubare le cellule con anticorpi per 20 min a 4 ° C al buio. </li><li> Lavare le cellule in 1 ml FACS tampone (centrifugare per 2 minuti; a 660 xg), decantare il supernatant e procedere all&#39;acquisizione FACS nel volume buffer residuo. </li></ol></li><li> Pelletizzare cellule (che non vengono utilizzati nell&#39;analisi FACS) mediante centrifugazione per 12 min; a 300 g; 8 ° C. Rimuovere il surnatante e risospendere il cellulare pelletin 60 microlitri MACS-tampone per 1 x 10 7 cellule. Aggiungere 20 ml Reattivo-Fc Blocco per 1 x 10 7 cellule e incubare per 5 minuti a 4 ° C. </li><li> Aggiungere 10 microlitri FITC-coniugato anticorpo anti-umano Vδ1 per 1 x 10 7 cellule direttamente alle cellule. Incubare per 12 minuti a 4 ° C al buio. </li><li> Lavare le cellule con 14 ml MACS-buffer (centrifuga per 12 minuti; a 300 g; 8 ° C). Gettare il surnatante completamente e risospendere le cellule in 80 microlitri di buffer MACS per 1x10 7 cellule. </li><li> Prendere 1-2 x 10 5 cellule e diluirli in 100 microlitri di buffer FACS per la verifica dell&#39;etichettatura mediante analisi FACS. Nessun ulteriore additivi sono necessari per questa operazione. Assicurarsi che ci sia una suffidifferenza ciente di luminosità per Vδ1 - e le cellule Vδ1 +. Se questo non è il caso, ripetere i passaggi 2.3) e 2.4). </li><li> Aggiungere 20 microlitri microsfere anti-FITC per 1 x 10 7 cellule alle cellule rimanenti, mescolare bene e incubare per 15 minuti al buio a 4 ° C. Successivamente, lavare le cellule utilizzando almeno 10 ml MACS tampone (centrifuga per 12 minuti, a 300 xg; 8 ° C). </li><li> Durante la centrifugazione, equilibrare la colonna magnetica pre-raffreddata collocato in un magnete con tampone MACS 500 microlitri. </li><li> Risospendere le cellule in 500 microlitri MACS-buffer (per il numero di cellule superiore 1 x 10 8, scalare questo volume di conseguenza) e applicare le cellule con attenzione sulla colonna. Raccogliere il flow-through contiene il Vδ1 - cellule. </li><li> Lavare la colonna tre volte con 500 microlitri MACS-buffer e raccogliere nuovamente il flusso continuo nello stesso tubo. Si noti che il serbatoio deve essere vuoto prima di applicare tampone sulla colonna. </li><li> Rimuovere la colonna dal dispositivo magnetico e posizionarlo in un tubo da 15 ml. Svuotare rapidamente la colonna con tampone 1 ml MACS con fermezza spingendo lo stantuffo nella colonna. Raccogliere l&#39;eluato in un tubo. NOTA: Per ottenere una purezza&gt; 98%, di solito è necessario ripetere i passaggi 2,7-2,9) con una seconda colonna. </li><li> Verificare la purezza delle cellule mediante analisi FACS 18 con lo stesso pannello di anticorpi come prima (vedi 2.1.2) e contare le cellule. </li></ol> 3. Isolamento di Vδ1CD4 + cellule T <ol><li> Risospendere 25 ml di CD4 perle con un vortice per&gt; 30 sec. Lavare le perline (la quantità minima come descritto dal produttore) in 1 ml di tampone di MACS in una provetta da 1,5 ml di reazione ponendo la provetta in un magnete per 1 min. Eliminare il surnatante con cura con una pipetta su piccola scala (200 ml) e risospendere le sfere nel volume originale di MACS-buffer. </li><li> Pelletizzare Vδ1 cellule + (centrifugare per 12 min; a 300 xg) e aspirare il surnatante e risospendere le cellule + Vδ1 in 500 microlitri MACS-buffer e aggiungerli alla provetta contenente le perline. Mescolare vigorosamente e incubare per 20 minuti a 4 ° C con costante inclinazione (esempio, in un rotatore). </li><li> Posizionare il tubo contenente le cellule in un magnete per 2 min. Assicurarsi che non ci siano residui nel coperchio. NOTA: cellule CD4 + sarà vincolato sfere magnetiche e allegare al lato del tubo rivolto verso il magnete. </li><li> Aprire con cautela il coperchio, mantenendo il tubo all&#39;interno del dispositivo magnetico e raccogliere il surnatante che contiene il CD4 - + cellule Vδ1 utilizzando una pipetta su piccola scala. Posizionare CD4 - + cellule Vδ1 in un tubo separato e collocare questo tubo nel magnete di nuovo per evitare possibilmente rimanendo cellule CD4 o perline da parte della popolazione. <ol><li> Lavare il CD4 - le cellule in 5 ml MACS-buffer (centrifuga per 12 minuti; a 300 xg) unand risospendere in una concentrazione di 1 x 10 5 cellule per 100 microlitri mezzi. CD4 - le cellule possono essere facilmente coltivate secondo le condizioni riportate di seguito (4.2). </li></ol></li><li> Posizionare il tubo con gli obiettivi di cellule CD4 + al di fuori del campo magnetico, sospendere nuovamente le cellule in 500 microlitri MACS-buffer e rimetterli nel dispositivo magnetico. Ripetere i passaggi 3,3-3,5 due volte per ottenere una maggiore purezza. Rimuovere il surnatante pipettando </li><li> Risospendere le cellule CD4 + in 100 ml di coltura (RPMI 1640, il 10% FCS, 1% di L-glutammina, 1% penicillina / streptomicina) e aggiungere 10 ml di una soluzione stallonatore. Incubare a temperatura ambiente per 45 min con inclinazione costante (ad esempio, in un rotatore). </li><li> Collocare le cellule in un magnete per 1 min. Raccogliere accuratamente il surnatante contenente Vδ1 cellule CD4 + + senza tallone con una pipetta su piccola scala. </li><li> Al di fuori del magnete, risospendere le sfere con i terreni di coltura 100 microlitri e ripeterepassi 3.6 e 3.7 due volte per ottenere il numero di cellule più alti di cellule CD4 +. </li><li> Pelletizzare le cellule (centrifugare a 300 xg per 12 minuti) ed eliminare completamente il surnatante pipettando. Risospendere le cellule in mezzi freschi, preriscaldata e contarli. Esaminare la purezza delle Vδ1 + CD4 + cellule mediante analisi FACS raffigurati nei passaggi 2.1.1-2.1.3. </li></ol> Clonazione 4. Single-cella limitata diluizione <ol><li> Isolare le cellule mononucleate del sangue periferico (PBMC) da un donatore allogenico come illustrato in 1,1-1,6. </li><li> Irradiare 2,5 x 10 7 PBMC allogenici con 80 Gy in 25 ml di coltura utilizzando γ-radiazioni. </li><li> Aggiungere IL-2 (200 U / ml), IL-7 (20 ng / ml) e PHA (2 mg / ml) per le cellule feeder irradiate e distribuirli in piastre da 96 pozzetti ad U, 5 x 10 4 alimentatore cellule in 50 pl per pozzetto. </li><li> Diluire le cellule Vδ1 + CD4 + a una concentrazione di 0,3 cellule per 50 ml. Dispensarete 50 microlitri della soluzione di cellule in ciascun pozzetto ospitare le cellule irradiate e citochine. NOTA: Così, le citochine sono diluiti alla concentrazione finale di 100 U / ml di IL-2, 10 ng / ml di IL-7 e 1 mg / ml PHA. Incubare le piastre a 96 pozzetti in un incubatore a 37 ° C, 5% CO 2 atmosfera umidificata. <ol><li> Facoltativamente, coltivare rimanendo purificato + cellule CD4 + Vδ1 come la cultura di massa, alle stesse condizioni di coltura, come i cloni. </li></ol></li><li> Fornire le cellule ogni 3-4 giorni con citochine e mezzi freschi. Per questo, la metà scambio del mezzo nei pozzetti con 50 microlitri fresca media pipettando. Aggiungere la concentrazione di 2 volte di citochine a ogni integrazione. Aggiungere 5x10 4 cellule di alimentazione irradiati per bene ogni altro giro di alimentazione. </li><li> Utilizzando un microscopio, osservare primi cloni diventano visibili dopo 3-4 settimane, in quanto metabolizzano e quindi modificare il colore delle colonie di coltura e di forma. NOTA: per pelliccether coltivazione, risospendere i cloni bene e trasferire loro di separare piastre a 96 pozzetti. Analizzare le cellule tramite FACS, come indicato in 2.1.1-2.1.3. Cloni conservati in queste condizioni possono finalmente cambiare il loro TCR da Vδ1 a αβTCR. Il punto di tempo per questo transdifferenziazione varia da clone per clonare. </li></ol>

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The authors have nothing to disclose.

Per studiare il fenotipo, la biologia e la funzione di una scarsa (T) entità delle cellule, ovvero cellule Vδ1 + T CD4 +, abbiamo usato due marcatori: Vδ1 e CD4 per il suo isolamento positivo cella magnetica. Vδ1 è un recettore orfano, considerando CD4 è espresso sulle cellule T helper, ad un livello inferiore su monociti e cellule dendritiche, e ad un livello molto basso sulle cellule progenitrici ematopoietiche. Tecniche per l&#39;arricchimento e la selezione di celle a el...

Nei vertebrati, immunità adattativa che si struttura nel cellulare e una parte umorale di immunità svolge un ruolo importante nella difesa contro gli agenti patogeni. Il riconoscimento di una vasta gamma di antigeni è mediata da T- hyperpolymorphic e recettori cellulari B (TCR / BCR), che per quanto riguarda le cellule T sono assunti essere prodotta principalmente nel timo 1. A tale scopo, le cellule staminali ematopoietiche (CSE), derivate dal midollo osseo, il timo seme e differenziare lungo fasi ben definite finalmente danno origine a tutte le linee cellulari T. Thymus progenitori semina sono CD4 - e CD8 - e costituiscono quindi la frazione timocita immaturo, doppio negativo (DN). Segnali Thymus derivate poi inducono il loro impegno lignaggio e la differenziazione in cellule T αβ sia o γδ. L&#39;espressione di funzionalmente riarrangiati TCR-gamma e TCR-δ catena geni in DN2 / 3 timociti porta a γ δTCR complessi, che guidano la proliferazione cellulare und promuovere la differenziazione in cellule γ DT 2,3. Al contrario, il riarrangiamento di un funzionale catena TCR-β, che può accoppiarsi con preTα costruire una preTCR pT, induce il silenziamento trascrizionale della catena TCR-γ in timociti DN3 e il loro inserimento CD4 + CD8 + doppio positive timociti 4 . In questa fase, la ricombinazione della catena TCR-α si verifica, eliminando il locus TCR-δ che annida nel locus TCR-α, abrogando quindi la produzione di un γδTCR in queste cellule irrevocabilmente 5-9. ΑβTCRs riarrangiati sono successivamente selezionati per la loro capacità di legarsi auto-MHC debolmente (selezione positiva), che non può superare una certa soglia per evitare autoimmunità (selezione negativa). Secondo la loro capacità di I o II legame MHC di classe, le celle selezionate αβT si sviluppano in cellule singole positive CD4 + o CD8 +, che uscita il timo cellule T come naïve. Tuttavia, involuzione del timo inizia presto nella vita che porta a esponenzialmente ridotto la produzione di cellule T naive che è quasi estinta dopo l&#39;adolescenza 10. Tuttavia, la dimensione del pool di cellule T rimane costante per tutta la vita, che può essere spiegato solo in parte dal post-timica proliferazione omeostatica di cellule T e la proliferazione di lunga durata memoria immunologica 11. Di conseguenza, deve avvenire lo sviluppo extrathymic cellule T. La ricerca recente ha acquisito notevole attrazione che caratterizza progenitori delle cellule αβT che-a-extrathymic siti hanno dato origine alla αβ funzionale cellule T 12-17. Eppure, conoscenza dettagliata extrathymic precursori delle cellule αβT che indipendente da un timo differenziarsi in cellule αβT è frammentaria come i precedenti che abbiamo sulla rotta prendono così. Recentemente abbiamo identificato la piccola impresa a cellule T di Vδ1 + </sup&gt; CD4 + γδT come un extrathymic prognitor cella αβT 18, che una volta isolato dal sangue periferico di donatori umani sani possono transdifferenziarsi in cellule αβT in un ambiente infiammatorio lieve. È interessante notare che e contrario alla proliferazione omeostatica delle cellule T post-timo, transdifferenziazione di cellule CD4 + Vδ1 genera nuovi recettori delle cellule T, ampliando così la diversità repertorio, in modo che potenzialmente nuovi antigeni possono essere riconosciuti e può tutela dell&#39;ospite contro gli agenti patogeni di nuova acquisizione. Questo aggiunge alla plasticità delle cellule T e aggiunge un nuovo percorso finora unappreciated per lo sviluppo delle cellule T extrathymic. L&#39;isolamento quantitativo da fonti linfocitari, la generazione di cloni unicellulari e la loro espansione efficiente sono essenziali per l&#39;obiettivo di individuare i marcatori e molecole che innescano questa cella αβT precursor`s sviluppo extrathymic.

<table border="0" cellpadding="0" cellspacing="0" width="789">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biocoll Solution</td>
			<td style="width:93px;">Biochrom</td>
			<td style="width:119px;">L 6113</td>
			<td style="width:361px;">lymphocyte separating solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lysing Buffer</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>555899</td>
			<td>lysis of erythrocytes&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phosphate-buffered Saline</td>
			<td style="width:93px;">Sigma Aldrich</td>
			<td style="width:119px;">D8537&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MACS buffer</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-091-222</td>
			<td>supplement with BSA and pre-cool before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BSA</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-091-376</td>
			<td>not mandatorily from this supplier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human Vd1 FITC (clone:&#160; TS8.2)</td>
			<td style="width:93px;">Thermo Scientific</td>
			<td>TCR2730</td>
			<td>not mandatorily from this supplier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD3 PerCP (clone: SK7)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>345766</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human TCRab PE (clone: T10B9.1A-31)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>555548</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD4 VioBlue (clone: M-T466)&#160;</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-097-333</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-human CD8 APC-H7 (clone: SK1)</td>
			<td style="width:93px;">BD BioSciences</td>
			<td>641400</td>
			<td>not mandatorily from this supplier or this flurochrome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Anti-FITC MultiSort Kit</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-058-701</td>
			<td>yields better results than anti-FITC MicroBeads</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MS columns</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-042-201</td>
			<td>pre-cool before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">MiniMACS Separator</td>
			<td style="width:93px;">Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CD4 Positive Isolation Kit</td>
			<td style="width:93px;">life technologies</td>
			<td>11331D</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and ex vivo culture of v&#948;1+cd4+&#947;&#948; t cells, an extrathymic &#945;&#946;t-cell progenitor

Il timo, l&#39;organo principale per la generazione di cellule T αβ e spina dorsale del sistema immunitario adattativo in vertebrati, è stata a lungo considerata come unica fonte di cellule αβT. Eppure, involuzione timica inizia presto nella vita che porta ad un&#39;uscita drasticamente ridotto di cellule αβT naïve in periferia. Tuttavia, anche centenari possono costruire immunità contro gli agenti patogeni di nuova acquisizione. Recenti ricerche suggeriscono lo sviluppo delle cellule αβT extrathymic, tuttavia la nostra comprensione dei percorsi che possono compensare la perdita della funzione timica sono ancora rudimentali. γδ cellule T sono linfociti innate che costituiscono il principale sottoinsieme cellule T nei tessuti. Recentemente abbiamo attribuito un finora incompreso importante funzione di un sottoinsieme delle cellule T γδ mostrando che la scarsa entità dei CD4 + Vδ1 + γδ cellule T possono transdifferenziarsi in cellule αβT in condizioni infiammatorie. Qui, si provide il protocollo per l&#39;isolamento di questo progenitrici da sangue periferico e la sua successiva coltivazione. Cellule Vδ1 sono positivamente arricchiti da PBMC di donatori umani sani utilizzando sfere magnetiche, seguita da una seconda fase in cui puntiamo la scarsa frazione di cellule CD4 + con un ulteriore tecnica di etichettatura magnetica. La forza magnetica del secondo nell&#39;etichettatura supera quello della prima etichetta magnetica, e quindi permette efficiente, quantitative e specifiche isolamento positivo della popolazione di interesse. Abbiamo poi introdurre la condizione tecnica e la cultura necessaria per la clonazione e in modo efficiente l&#39;espansione delle cellule e per l&#39;identificazione dei cloni generati mediante analisi FACS. Quindi, mettiamo a disposizione un protocollo dettagliato per la purificazione, la cultura e l&#39;espansione ex vivo di cellule CD4 + Vδ1 + γδ cellule T. Questa conoscenza è condizione indispensabile per gli studi che riguardano questo αβT progenitor`s biologico cellula e per coloro che mirano a identify i trigger molecolari che sono coinvolti nel suo transdifferenziamento.

La figura 1 illustra le diverse fasi e il risultato l&#39;isolamento delle cellule T Vδ1 da sangue periferico. La figura 1A mostra una tipica distribuzione di + cellule CD3 + Vδ1 in linfociti, così come l&#39;espressione co-recettore della popolazione Vδ1 +. In questo donatore, la frequenza di cellule Vδ1 + (rosso) è 2,3% del totale delle conta dei linfociti CD4 e l&#39;espressione (verde) di Vδ1 + linfociti è del 2,6%. Comp...

Watch this Scientific Journal Video about Isolation and Ex Vivo Culture of VÎ´1+CD4+Î³Î´ T Cells, an Extrathymic Î±Î²T-cell Progenitor at JoVE.com

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral V&#948;1+CD4+ T cells that is, as we showed recently, an extrathymic &#945;&#946; T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

Isolamento e<em&gt; Ex Vivo</em&gt; Cultura di Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; Celle γδ T, un Extrathymic αβT-cellule progenitrici

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

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Research

JoVE Journal

Immunology and Infection

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

9.2K Views.  Children's University Hospital Tübingen. Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral Vδ1+CD4+ T cells that is, as we showed recently, an extrathymic αβ T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.

Video: Isolation and Ex Vivo Culture of Vδ1+CD4+γδ T Cells, an Extrathymic αβT-cell Progenitor

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

Isolamento linfociti T umani, la cultura e l&#39;analisi delle migrazioni In Vitro</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

Ricerca

Immunologia e infezioni

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Espansione ex vivo di tumore cellule T reattive per mezzo di 1/Ionomycin Bryostatin e il Comune catena gamma Citochine Formulazione

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Imaging ex vivo di cellule T nel Fette murini Linfonodo con grandangolari e microscopi confocale

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in various immune responses. DN T cells are a unique immune cell type that has been implicated in regulating immune and autoimmune responses and tolerance to allotransplants1-6. DN T cells are, however, rare in peripheral blood and secondary lymphoid organs (spleen and lymph nodes), but are major residents of the normal kidney. Very little is known about their pathophysiologic function7 due to their paucity in the periphery. We recently described a comprehensive phenotypic and functional analysis of this population in the kidney8 in steady state and during ischemia reperfusion injury. Analysis of DN T cell function will be greatly enhanced by developing a protocol for their isolation from the kidney.
Here, we describe a novel protocol that allows isolation of highly pure ab CD4+ CD8+ T cells and DN T cells from the murine kidney. Briefly, we digest kidney tissue using collagenase and isolate kidney mononuclear cells (KMNC) by density gradient. This is followed by two steps to enrich hematopoietic T cells from 3% to 70% from KMNC. The first step consists of a positive selection of hematopoietic cells using a CD45+ isolation kit. In the second step, DN T cells are negatively isolated by removal of non-desired cells using CD4, CD8, and MHC class II monoclonal antibodies and CD1d &#945;-galcer tetramer. This strategy leads to a population of more than 90% pure DN T cells. Surface staining with the above mentioned antibodies followed by FACs analysis is used to confirm purity.

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine kidney.

Isolamento di Double Negative Cellule T αβ dal rene

There is currently no standard protocol for the isolation of DN T cells from the non-lymphoid tissues despite their increasingly reported involvement in ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Valutare l&#39;innato rilevamento dell&#39;infezione da HIV-1 CD4 infetti<sup&gt; +</sup&gt; Cellule T dalle cellule dendritiche plasmacitoidi Utilizzando un<em&gt; Ex vivo</em&gt; Sistema di co-coltura.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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 ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Linfociti Isolamento da Pelle umana per l&#39;analisi fenotipica e<em&gt; Ex Vivo</em&gt; Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Il<em&gt; Ex Vivo</em&gt; Cultura e Pattern Recognition recettore stimolazione del mouse intestinale organoidi

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Trasduzione retrovirale di cellule progenitrici del midollo osseo per generare cellule T del recettore Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

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, ...

Isolamento e

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Isolamento linfociti T umani, la cultura e l'analisi delle migrazioni In Vitro

Espansione ex vivo di tumore cellule T reattive per mezzo di 1/Ionomycin Bryostatin e il Comune catena gamma Citochine Formulazione

Imaging ex vivo di cellule T nel Fette murini Linfonodo con grandangolari e microscopi confocale

Isolamento di Double Negative Cellule T αβ dal rene

Valutare l'innato rilevamento dell'infezione da HIV-1 CD4 infetti

Un

Linfociti Isolamento da Pelle umana per l'analisi fenotipica e

Il

Trasduzione retrovirale di cellule progenitrici del midollo osseo per generare cellule T del recettore Retrogenic Mice

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