<p class="jove_content"&gt; Gli autori desiderano ringraziare Jennifer Strange and Greg Bauman per la loro assistenza con l&#39;analisi di Citometria a Flusso e smistamento. Questo lavoro è stato sostenuto dal numero di sovvenzione del NIH 2P20 RR020171 dal NCRR e dalla University of Kentucky di avvio dei fondi di FM; GIE riconosce il sostegno della Compagnia Presidenziale e il Graduate Fellowship Opportunity Kentucky.</p

1. Isolamento di cellule mononucleari del sangue periferico (PBMC) provenienti da Buffy Coat <ol><li> Acquisire una unità di buffy coat dall&#39;ospedale e dintorni posizione centrale sangue. Il nostro centro di sangue ci fornisce circa il 40-60 mL di buffy coat per unità ottenute da donatori di sangue normali. </li><li> Versare il sangue in una bottiglia di vetro sterilizzato 500 mL contenente PBS sterile per diluire buffy coat. Il volume finale di PBS + buffy coat deve essere di 250 mL. </li><li> Riempire dieci provette da 50 ml coniche ciascuno con 20 mL di soluzione Lymphoprep. </li><li> Sovrapporre delicatamente la soluzione Lymphoprep con 25 mL di buffy coat diluito, facendo attenzione a non disturbare la soluzione Lymphoprep. </li><li> Spin provette per 30 minuti a temperatura ambiente a 500 x g. Assicurati di spegnere freno della centrifuga, in modo da non disturbare la frazione dei linfociti. </li><li> Raccogliere le PBMC all&#39;interfaccia tra il Lymphoprep e plasma medio strati con una pipetta. Aspirare il plasma medio-off fino a circa 1 mLricopre ancora il livello di buffy coat contenente le PBMC. Utilizzare una pipetta 10 mL di trasferire PBMC a un nuovo tubo da 50 ml. </li><li> Lavare le cellule due volte con PBS e poi risospendere le cellule in 50 ml di RPMI freddo per contare su un emocitometro. Tipico è il recupero 8 x 10 8 - 1 x 10 9 PBMC. </li></ol> Questa procedura può essere ridotta per i minori volumi di sangue. Diluizione dei campioni di sangue intero è di 1:1 in PBS. 2. Magnetic selezione negativa di cellule CD4 + T totali, CD4 + CD45RA + cellule T naive o CD45RO + CD4 + di memoria delle cellule T da PBMC utilizzando kit di arricchimento EasySep (Cellule Staminali Technologies) Passi da seguire per 1 x 10 8 a 4,25 x 10 8 PBMC. <ol><li> Risospendere le PBMC ad una concentrazione finale di 5 x 10 7 per ml in PBS contenente 2% FBS e 1 mM EDTA. Spostare le celle in una nuova 14 tubo di polipropilene fondo mL. <ol class = &quot;lalpha&quot;&gt; <li> Isolamento del totale delle cellule CD4 + T umani per selezione negativa: aggiungere umani CD4 + Cocktail cellule T Arricchimento di anticorpi (50 microlitri per ml di PBMC), mescolare e incubare per 10 minuti a temperatura ambiente. </li><li> L&#39;isolamento di cellule T naive per selezione negativa: si aggiungono 50 pl di anticorpo anti-CD45RO per mL di sospensione di cellule PBMC, mescolare e incubare per 15 minuti a temperatura ambiente. Aggiungi cocktail arricchimento fornito con il kit (50 pl di mL per delle PBMC), mescolare e incubare a temperatura ambiente per 10 minuti. </li><li> L&#39;isolamento di cellule umane T di memoria per selezione negativa: aggiungere umani CD4 + di memoria Cocktail cellule T Arricchimento di anticorpi (50 microlitri per ml di PBMC), mescolare e incubare per 10 minuti a temperatura ambiente. </li></ol></li><li> Mescolare particelle magnetiche bene anche li distribuiscono in tutta la soluzione. Sei nanoparticelle non vortice dal kit ingenuo. </li><li> Aggiungere le particelle magnetiche (100 pl per mL di PBMC per CD4 + totali e naif selezione di cellule T, 50 pl per mL di PBMC per la selezione della cella di memoria T). Mix pipettando gentilmente 2-3 volte e incubare a temperatura ambiente per 10 minuti per la selezione delle cellule T naive o 5 minuti per CD4 + di memoria o totale di selezione delle cellule T. </li><li> Aggiungi PBS contenente 2% di FBS e 1 mM EDTA per portare il volume fino a 10 ml per provetta. Mescolare con delicatezza pipettando 2-3 volte prima di tubo non livellata in argento EasySep calamita per i minuti 5, 10 o 2.5 per complessivi CD4 + cellule T naive, sottoinsiemi e la memoria, rispettivamente. </li><li> Con tubo ancora in magnete EasySep, versare il liquido in un nuovo tubo da 50 ml a isolare le cellule di interesse. </li><li> Ripetere i punti 2.5 e 2.6 per un miglior recupero. </li></ol> 3. Condizioni della coltura cellulare per indurre cellule T regolatorie <ol><li> Il giorno prima passaggi 1 e 2, cappotto piastre di coltura tissutale da prima diluendo anticorpo anti-CD3 (OKT3 clone) ad una concentrazione di 1 ug / ml in PBS sterile. Per un piatto ben 6 aggiungere 2 mL di PBS + anti-CD3 per bene, per un piatto ben 12 aggiungere 1 ml o per una piastra a 24 pozzetti aggiungere 500 ul per pozzetto. Mantenere la piastra rivestita a 4 ° C fino all&#39;utilizzo. </li><li> Preparare un mezzo di polarizzazione aggiungendo 10% inattivato al calore siero fetale di bovino (FBS), 100 U / ml penicillina, 100 pg / mL di streptomicina e 50 pM β-mercaptoetanolo a RPMI-1640 pre-integrato con 2,06 mM Glutamax-I e 25 mM HEPES tampone. Successivamente, aggiungere 2 ng / mL TGF-β e 5 ng / ml IL-2. Qui, si può aggiungere ligandi o inibitori di interesse per i media. Risospendere le cellule dal passaggio 2 alla concentrazione di 2 x 10 6 cellule / mL di mezzo di polarizzazione. </li><li> Pre-riscaldare le piastre a 37 ° C e aspirare il PBS di anti-CD3-pozzetti rivestiti prima di aggiungere le cellule. Aggiungere 4 mL per pozzetto di sospensione cellulare per una piastra da 6 pozzetti, 2 mL di cellule per una piastra da 12 pozzetti o 1 mL di cellule per una piastra di ben 24. Riempire i pozzi vuoti con PBS per ridurre l&#39;evaporazione delmedia. Incubare per 3 giorni a 37 ° C / 5% CO 2. </li><li> Il giorno 3 placcatura, spin down piastra in una centrifuga per 5 minuti a 500 x g. Senza disturbare cellule T sul fondo del piatto, togliere la metà di media e sostituirli con mezzi freschi. In alternativa, se il pozzo diventa sovraffollati con cellule (concentrazione superiore 3 x 10 6 / ml), suddividere il volume equamente in un altro anti-CD3 piastra rivestita e aggiungere mezzi freschi indietro fino al volume iniziale. Incubare per due o tre giorni più a 37 ° C / 5% CO 2. </li></ol> 4. Fluorescence-Activated cell sorting (FACS) di tre popolazioni di cellule T <ol><li> Preparare tampone FACS lavaggio aggiungendo 0,5% (w / v) albumina di siero bovino (BSA) e 2 mM EDTA a PBS sterile. Luogo tampone a 4 ° C a fare freddo. </li><li> Rimuovere le cellule di coltura cellulare ben e risciacquare ciascun pozzetto con 2 ml di PBS per assicurare un completo recupero delle cellule. Mettere tutte le celle in tubi di polipropilene da 50 ml e centrifugare a 500 xg per 10minuti a 4 ° C. </li><li> Contare le cellule su un emocitometro per determinare la densità cellulare. </li><li> Luogo 3-5 x 10 5 cellule per tubo in quattro distinte 5 provette a fondo tondo polistirene mL per i controlli di compensazione. Porre le cellule rimanenti in provette da 50 ml di polipropilene, non più di 35 x 10 6 cellule per provetta. </li><li> Centrifugare le cellule per 5 minuti a 4 ° C e supporti aspirato. Risospendere le cellule da ordinare in 90 microlitri di tampone di lavaggio a freddo per 1 x 10 6 cellule. Risospendere in 100 pl di tampone di lavaggio a freddo dei tubi di compensazione di controllo. </li><li> Per le celle da ordinati, combinare 2 microlitri di anti-CD25-PE, 3 pl di anti-CD45RA-PE-Cy5 e 1 ul di anti-CD127-APC per 1 x 10 6 cellule. Aggiungere l&#39;assenza di anticorpi al primo tubo controllo compensazione, 2 microlitri di anti-CD25-PE al secondo tubo, 3 pl di anti-CD45RA - PE-Cy5 al terzo tubo e1 pl di anti-CD127-APC al tubo quarto. Incubare tutte le provette in ghiaccio per 45 minuti al buio. </li><li&gt; Aggiungere 10 mL di tampone di lavaggio a freddo alle cellule per essere ordinati e 1 ml di provette controllare la compensazione. Centrifugare tutte le celle a 500 xg per 5 minuti a 4 ° C. </li><li> Aspirare celle buffer e risospendere ad una concentrazione di 1 x 10 7 cellule per ml di tampone di lavaggio. Aggiungere 1,5 microlitri di DNasi II per ml di cellule prima di filtrare attraverso un colino 40 cella nylon pM. Spostare le celle a più 5 tubi in polipropilene mL a fondo tondo con non più di 3,5 mL per provetta. Risospendere le cellule di controllo di compensazione in 300 microlitri di tampone di lavaggio. </li><li> Impostare la compensazione al citofluorimetro MoFlo per ridurre al minimo il rilevamento incrociato da parte, PE APC e PE-Cy5 filtri. Impostare porte per ordinare iTregs (CD25 CD127 Hi -/LOW CD45RA - cellule), naïve (CD25-CD45RA +) e la memoria (CD25 - CD45RA -) le cellule T in 5 provette a fondo tondo di polistirolo contenenti 1 ml di siero di vitello neonato mL. </li></ol> 5. Soppressione Assay <ol><li> Fai supprmezzi di analisi ESSIONE aggiungendo 100 U / mL di penicillina, 100 pg / ml di streptomicina, 5 ng / ml di IL-2 e 2 ng / ml di TGF-β per AIM-V. </li><li> Il giorno del saggio, purificare le cellule CD4 + T eterologhi da buffy coat come indicato nei punti 1 e 2. Questi saranno le cellule bersaglio per il saggio soppressione e non contiene CD25 + Treg, come visibile in figura 2, giorno 0. </li><li> Cellule etichette con CellTrace kit come da istruzioni del produttore, ad eccezione usando solo 1 ml di soluzione madre 5 mM per mL di cellule invece di 2 pl. Tenere fuori dalla luce diretta, aggiungere 18 microlitri della DMSO fornito dal kit CellTrace ad un flaconcino di CFSE per ottenere una soluzione stock 5 mM. Risospendere il numero di cellule bersaglio (ad un massimo di 1 x 10 7) preriscaldato in PBS + 0,1% (w / v) BSA ad una concentrazione finale di 1 x 10 6 cellule / ml. Aggiungere 1 ml di 5 mM CFSE per mL di cellule e incubare in un bagno d&#39;acqua a 37 ° C per 5 minutes. Aggiungere 5 volumi di completo, ghiacciata RPMI con 10% FBS per placare la colorazione ed incubare in ghiaccio per 5 minuti. Lavare le cellule due volte con freddo RPMI completo e risospendere 1 x 10 5 cellule per 100 pl di mezzi di analisi di soppressione. </li><li> Treg perle ispettore di soppressione sono ad una concentrazione stock di 2 x 10 7 perle / mL. Pellet un certo numero di perline pari al numero totale di cellule per centrifugazione esperimento pratica in una provetta Eppendorf. Lavare perline una volta con RPMI e ri-pellet. Dopo l&#39;aspirazione di RPMI, Risospendere le sfere in modo che la quantità appropriata di perline per pozzetto sono in 8 microlitri di mezzi di soppressione di saggio. </li><li> Per un pozzo rotondo 96 piastra di coltura tissutale inferiore, aggiungere CFSE cellule colorate (1 x 10 5 cellule / ml), perline ispezione e cellule polarizzate e ordinati (1 x 10 5 cellule / ml) in mezzi freschi analisi soppressione di un bersaglio desiderato (tinto CFSE): effector (ordinato) il rapporto in un volume finale di 200 pl. Tutte le condizioni sono impostati in tripliCates. </li><li> Preparare la prima delle due condizioni di controllo aggiungendo 100 pl di cellule colorate CFSE, 8 microlitri di perline di ispezione e 1 x 10 5 di cellule fresche, non marcate in 92 pl dosaggio medio soppressore per pozzetto. Preparare il secondo controllo con gli stessi componenti cellulari come sopra ma senza Treg perline di ispezione. </li><li> Piastra di copertura in foglio di alluminio e incubare a 37 ° C / 5% di CO 2 per cinque giorni. </li><li> Nel buio, raccogliere le cellule di ogni bene da pipettaggio e posto in una provetta da 5 ml fondo rotondo polistirolo. Centrifugare le cellule a 500 xg per 5 minuti a 4 ° C, media aspirato, e risospendere in 300 FACS ul tampone di lavaggio a freddo dal punto 4. Analizzare i primi 3 x 10 4 CFSE + eventi dal cancello dal vivo dei linfociti che rappresentano le cellule bersaglio in un istogramma con il software Cell Quest. </li></ol> 6. Risultati rappresentativi Esempio di pseudocolori flussocitometrici punteggiano trame nell&#39;arco di cinque giorni di tempo couRSE differenziazione iTreg monitoraggio basato sul relativo co-espressione di CD25 con FoxP3, CTLA-4 e CD45RA può essere visto in Figura 2. L&#39;istogramma in figura 3 mostra un saggio di successo soppressione in cui ordinati iTregs (CD25 Hi CD45RA - cellule CD127 -/LOW). Sono il sottoinsieme solo da cinque giorni coltura che ha acquisito la capacità di regolamentazione / soppressore Figura 4 mostra l&#39;induzione di Tregs da un pool di cellule T naïve (pannelli superiori) e un pool di memoria delle cellule T (pannelli inferiori), dopo cinque giorni di cultura medio iTreg standard. CFSE colorazione delle cellule iniziali dimostrano che sia le cellule T naive (pannello superiore a destra) o la memoria (in basso pannello di destra) differenziarsi iTregs (più alto FoxP3-cellule che esprimono) solo dopo diversi cicli di divisione cellulare. <img alt="Figura 1" src="/files/ftp_upload/3738/3738fig1.jpg" /> Figura 1. Schema di procedura sperimentale. PBMC are separati dal sangue periferico umano tramite centrifugazione in gradiente prima magnetico selezione negativa di CD4 + CD25 - cellule T. Dopo cinque o sei giorni in coltura, le cellule subiscono FACS e sono co-incubate con eterologhe cellule bersaglio CFSE etichettati per misurare l&#39;attività di soppressione. <img alt="Figura 2" src="/files/ftp_upload/3738/3738fig2.jpg" /> Figura 2 purificati umani primari CD4 + CD25 -. Cellule T vengono coltivate in iTreg media. Un&#39;aliquota di cellule viene raccolto subito dopo isolamento (giorno 0) e nei giorni 1, 3 e 5 di coltura cellulare per monitorare il progresso di CD45RA, FoxP3, CTLA-4 e marker CD25. Il profilo corrisponde a iTreg CD45RA -, FoxP3 Hi, CTLA-4 e CD25 Hi Hi (evidenziato nel grafico in-finestra). <img alt="Figura 3" src="/files/ftp_upload/3738/3738fig3.jpg" /> Figura 3. Purificato CD4 + CFSE lacellule Beled (1 x 10 5 / pozzetto) sono coltivate con Treg perle ispettore di soppressione in presenza di cellule ordinati ingenuo, di memoria o iTreg (3 x 10 4 / pozzetto). Dopo cinque giorni, le cellule vengono raccolte e il profilo CFSE delle cellule colorate sono analizzate mediante citometria di flusso. La presenza di cellule iTreg abolisce completamente la proliferazione delle cellule T CD4 +. I numeri sono indicativi della percentuale di cellule CFSE marcati che sono stati sottoposti divisione. <img alt="Figura 4" src="/files/ftp_upload/3738/3738fig4.jpg" /> Figura 4 purificata umana primaria naïve. (CD4 + CD25 - CD45RA +) e la memoria (CD4 + CD25 - CD45RO +) le cellule T sono colorate con CFSE e coltivate in iTreg media. Dopo cinque giorni, le cellule sono fenotipizzati e le aliquote divisione cellulare stimato. Come indicato nella figura 2, il sottoinsieme iTreg differenziato da entrambi i sottoinsiemi corrispondeCD45RA - CD25 Hi FoxP3 Hi e CTLA-4 Hi (ultime due non mostrato). Profili comparativi di colorazione CFSE identificare iTregs (qui come le cellule che esprimono alti FOXP3 T), come le cellule più proliferativi durante i cinque giorni di cultura.

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Mentre il trasferimento Treg detiene un enorme promessa terapeutica nella lotta contro il rigetto del trapianto autoimmunità e altri disturbi del sistema immunitario o infiammatorio-mediata, i metodi per la loro generazione efficiente e stabile di mantenimento non sono ancora state sviluppate. Poiché solo il 1-5% delle cellule T circolanti sono Tregs, la loro espansione e la differenziazione controllata supera questa carenza come un deterrente importante di attuazione del Tregs nella clinica. D&#39;altra parte, come a...

<table border="1"><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Lymphoprep </td><td> Axis-Shield </td><td> 1114547 </td><td> Conservare a temperatura ambiente </td></tr><tr><td> Refrigerato da banco Centrifuga </td><td> Eppendorf </td><td> 5810R </td><td></td></tr><tr><td> Brillante-Line emocitometro </td><td> Hausser Scientific </td><td> 3110 </td><td></td></tr><tr><td> EasySep CD4 + Kit cellule umane T Enrichment </td><td> Stem Cell Technologies </td><td> 19052 </td><td></td></tr><tr><td> CD4 + di memoria EasySep umani Kit cellule T Enrichment </td><td> Stem Cell Technologies </td><td> 19157 </td><td></td></tr><tr><td> EasySep umani CD4 + naive Kit cellule T Enrichment </td><td> Stem Cell Technologies </td><td> 19155 </td><td></td></tr><tr><td> The Big Easy EasySep Magrete </td><td> Stem Cell Technologies </td><td> 18001 </td><td> Argento </td></tr><tr><td> 5 mL di fondo in polistirolo Tubes </td><td> BD Falcon </td><td> 352008 </td><td></td></tr><tr><td> 15 ml in polipropilene provette da centrifuga; </td><td> VWR </td><td> 89004-368 </td><td></td></tr><tr><td> 14 ml in polipropilene a fondo rotondo Tubes </td><td> BD Falcon </td><td> 352059 </td><td></td></tr><tr><td> 50 ml in polipropilene provette da centrifuga; </td><td> VWR </td><td> 89004-364 </td><td></td></tr><tr><td> Umana anti-CD3 Antibody </td><td> Bio X cella </td><td> BE0001-2 </td><td> Clone: ​​OKT3 </td></tr><tr><td> 24 Beh piastra di coltura cellulare in polistirolo </td><td> BD Falcon </td><td> 353047 </td><td></td></tr><tr><td> 96 Beh fondo sferico Tissue piastra di coltura </td><td> Grenier Bio-One </td><td> 650180 </td><td></td></tr><tr><td> RPMI 1640 con Glutamax-I e tampone HEPES </td><td> Gibco </td><td> 72400 </td><td></td></tr> &lt;tr&gt; <td> Siero bovino fetale (FBS) </td><td> Gibco </td><td> 16000 </td><td></td></tr><tr><td> β-mercaptoetanolo </td><td> Sigma </td><td> M7522 </td><td></td></tr><tr><td> Ricombinante umano TGF-β1 </td><td> eBioscience </td><td> 14-8348-62 </td><td></td></tr><tr><td> Umano ricombinante IL-2 </td><td> eBioscience </td><td> 14-8029-63 </td><td></td></tr><tr><td> Albumina sierica bovina (BSA) </td><td> MP Biomedicals Inc. </td><td> 810531 </td><td></td></tr><tr><td> 0,5 M EDTA </td><td> Amresco </td><td> E177 </td><td></td></tr><tr><td> Mouse Anti-Human CD25-PE </td><td> Miltenyi Biotec </td><td> 130-091-024 </td><td> Clone: ​​4E3 </td></tr><tr><td> Mouse Anti-Human CD45RA-PE-Cy5 </td><td> eBioscience </td><td> 15-0458-42 </td><td> Clone: ​​HI100 </td></tr><tr><td> Mouse Anti-Human CD127-APC </td><td> Miltenyi Biotec </td><td> 130-094-890 </td><td> Clone: ​​MB15-18C9 </td></tr><tr><td> DNasi II </td><td> MP Biomedicals Inc. </td><td> 190370 </td><td></td></tr><tr><td> MoFlo Citofluorimetro </td><td> Beckman Coulter </td><td></td><td></td></tr><tr><td> FlowJo Software </td><td> Albero Star, Inc. </td><td></td><td></td></tr><tr><td> Cella Quest Software Pro </td><td> BD Biosciences </td><td></td><td></td></tr><tr><td> Newborn di siero di vitello </td><td> Gibco </td><td> 16010 </td><td></td></tr><tr><td> L-glutammina </td><td> Gibco </td><td> 25030 </td><td></td></tr><tr><td> AIM-V </td><td> Gibco </td><td> 0870112 </td><td></td></tr><tr><td> CellTrace CFSE Kit proliferazione cellulare </td><td> Invitrogen </td><td> C34554 </td><td></td></tr><tr><td> Treg Beads Inspector soppressione </td><td> Miltenyi Biotec </td><td> 130-092-909 </td><td></td></tr><tr><td> Penicillina-Streptomicina </td><td> Gibco </td><td> 15140 </td><td></td></tr><tr><td> 40 micron Nylon ceppo cellulareer </td><td> BD Falcon </td><td> 352340 </td><td></td></tr></table>

generation of induced regulatory t cells from primary human na&iuml;ve and memory t cells

Lo sviluppo e la manutenzione di immunosoppressori CD4 + cellule T regolatorie (Tregs) contribuiscono alla tolleranza periferica necessario per rimanere in omeostasi immunologica con la grande quantità di antigeni self e commensali nel e sul corpo umano. Perturbazioni in bilico tra Tregs e infiammatorie cellule T convenzionali può portare a immunopatologia o il cancro. Sebbene iniezione terapeutico di Tregs ha dimostrato di essere efficace in modelli murini di colite 1, diabete di tipo I 2, artrite reumatoide e malattia dell&#39;innesto verso l&#39;ospite, 4 varie differenze fondamentali umano contro topo Treg biologia 5 ha finora precluso l&#39;uso clinico. La mancanza di numero sufficiente, la purezza, la stabilità e la specificità di homing terapeutica Tregs necessaria una piattaforma dinamica di sviluppo umano su cui Treg per ottimizzare le condizioni per la loro espansione ex vivo 6. Qui D TuttoEscribe un metodo per la differenziazione di indotto Tregs (iTregs) da un singolo donatore di sangue periferico umano che può essere suddiviso in quattro fasi: isolamento di cellule mononucleate del sangue periferico, selezione magnetico di cellule T CD4 +, in coltura cellulare in vitro e fluorescenza attivato di separazione delle cellule (FACS) di sottoinsiemi di cellule T. Poiché la firma fattore di trascrizione Treg P3 forkhead scatola (FoxP3) è una attivazione indotta da fattore di trascrizione nell&#39;uomo 7 e nessun altro marcatore unico esiste, un pannello combinatoria di marcatori devono essere utilizzati per identificare cellule T con attività soppressiva. Dopo sei giorni di coltura, le cellule del nostro sistema può essere delimitato in ingenui cellule T, cellule T di memoria o iTregs in base alla loro relativa espressione di CD25 e CD45RA. Mentre le cellule T di memoria e ingenuo hanno diversi requisiti di polarizzazione segnalati e plasticities 8, pre-selezione della popolazione iniziale delle cellule T in CD45RA + e + CD45RO sottoinsiemi possono be utilizzato per esaminare queste discrepanze. Coerentemente con gli altri, il nostro CD25 Hi CD45RA - iTregs esprimono alti livelli di FoxP3 9, GITR e CTLA-4 11 e bassi livelli di CD127 12. Dopo FACS di ciascuna popolazione, le cellule risultanti possono essere utilizzati in un saggio soppressore che valuta la relativa capacità di ritardare la proliferazione di carbossifluoresceina succinimmidil estere (CFSE)-marcato cellule T autologhe.

Watch this Scientific Journal Video about Generation of Induced Regulatory T Cells from Primary Human NaÃ¯ve and Memory T Cells at JoVE.com

Generation of Induced Regulatory T Cells from Primary Human Na&amp;#239;ve and Memory T Cells

Noi descriviamo un metodo per generare le cellule T normativo, di memoria e naif da un singolo donatore di sangue umano. Polarizzato Tregs può essere quindi confrontato con altri sottoinsiemi in una varietà di applicazioni genetiche e funzionali con omogeneità genetica, comprendente un saggio di soppressione anche dettagliati qui.

Generazione di cellule T regolatrici indotte da cellule primarie umane T naive e memoria

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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Research

JoVE Journal

Immunology and Infection

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

23.9K Views.  University of Kentucky. Noi descriviamo un metodo per generare le cellule T normativo, di memoria e naif da un singolo donatore di sangue umano. Polarizzato Tregs può essere quindi confrontato con altri sottoinsiemi in una varietà di applicazioni genetiche e funzionali con omogeneità genetica, comprendente un saggio di soppressione anche dettagliati qui.

Video: Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac Transposon modifica del sistema di cellule primarie umane T

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Ricerca

Immunologia e infezioni

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

Nuovi strumenti per espandere cellule T regolatorie di individui HIV-1-infetti

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Derivazione di cellule T<em&gt; In Vitro</em&gt; Da cellule staminali embrionali di topo

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Generazione di Human alloantigene-specifiche cellule T da sangue periferico

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

Cellule T Capture batteri da Transinfection da cellule dendritiche

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Fenotipica e analisi funzionale di cellule T regolatorie attivati ​​Isolato dal linfocitica cronica coriomeningite topi infettati da virus

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Lo sviluppo di cellule staminali derivate antigene-specifiche cellule T regolatorie contro autoimmunità

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

&lt;em&gt;In Vitro&lt;/em&gt; Differentiation of Human CD4&lt;sup&gt;+&lt;/sup&gt;FOXP3&lt;sup&gt;+&lt;/sup&gt; Induced Regulatory T Cells (iTregs) from Na&amp;#239;ve CD4&lt;sup&gt;+&lt;/sup&gt; T Cells Using a TGF-&amp;#946;-containing Protocol

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

La differenziazione in vitro di cellule CD4 + umana FOXP3 + indotta da T regolatorie (iTregs) da cellule T CD4 Naïve utilizzando un protocollo TGF-β contenenti

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

Valutazione dell'interfaccia sinaptica primaria umana delle cellule di T dal sangue periferico e tessuto linfoide

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.