Name: generation of human alloantigen-specific t cells from peripheral blood
Uploaded: 2014-11-21T15:00:00.000+00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Generation of Human Alloantigen-specific T Cells from Peripheral Blood at JoVE.com

The author would like to thank the NIH Tetramer Core Facility for tetramer production. The author would also like to thank E.D. O&#8217;Connor and K.E. Marquez at the UCSD Human Embryonic Stem Cell Core Facility flow cytometry laboratory for assistance in cell sorting. This work was funded by National Institutes of Health grant K08AI085039 (G.P.M.).

NOTA: Questo protocollo richiede l&#39;utilizzo di campioni di sangue periferico di volontari umani. Tutta la ricerca con soggetti umani deve essere esaminato e approvato da un studi sull&#39;uomo Institutional Review Board per garantire la conformità con la Dichiarazione di Helsinki (2013) e l&#39;Health Insurance Portability e Accountability Act del 1996. 1. Isolamento dei linfociti T da sangue intero <ol><li> Prima di iniziare, riscaldare il mezzo gradiente di densità a temperatura ambiente. Aliquota 4 ml di terreno gradiente di densità in 2-4 sterili da 15 ml provette coniche (1 tubo verrà utilizzato per ogni 10 ml di volume totale di sangue diluito). </li><li> Ottenere 10-20 ml di sangue in 1-2 sodio eparina a spruzzo con rivestimento (verde-top) provette di raccolta del sangue venoso. Raccogliere campioni umani sotto la supervisione di persone addestrate e secondo un Institutional Review Board ha approvato il protocollo. </li><li> Pulire la parte esterna dei tubi con il 70% di etanolo. Rimuovere con attenzione le cime della firiempito provette per il prelievo e decantare il sangue in una sterile 50 ml provetta da centrifuga. </li><li> Aggiungere 10 ml sterile tampone fosfato salino (PBS) per ogni tubo di raccolta del sangue. Raccogliere il PBS, aggiungere al sangue intero decantato, e mescolare delicatamente. </li><li> Aggiungere 25 microlitri T umano di arricchimento cellulare Cocktail / 2 ml di volume totale. Incubare a temperatura ambiente per 20 min. </li><li> Usando una pipetta 10 ml, sovrapporre fino a 10 ml di 1: 1 sangue diluito delicatamente sulla sommità del mezzo gradiente di densità. Fare attenzione a non disturbare la superficie del terreno gradiente di densità. </li><li> Centrifugare il sangue strati e media densità gradiente a 1.200 xg per 20 min a 20 ° C. </li><li> Rimuovere le provette dalla centrifuga, avendo cura di non interrompere l&#39;interfaccia tra il mezzo gradiente di densità e lo strato di leucociti all&#39;interfaccia tra il mezzo gradiente di densità e il plasma diluito. Usando una pipetta da 5 ml, raccogliere con cura lo strato di leucociti e trasferimento in una nuova sterile 50 ml vasca conicae. </li><li> Aggiungere PBS per portare il volume della raccolta PBL a 50 ml e mescolare delicatamente. </li><li> Centrifugare a 600 xg per 5 min a 20 ° C. Decantare. </li><li> Risospendere le cellule in pellet in 10 ml sterile flusso citometria ordinamento tampone (sterile filtrata PBS contenente 1% BSA). Esempio 10 ml di sospensione cellulare, diluire 1:10 (aggiungere a 90 ml) trypan blu per contare le cellule utilizzando un emocitometro (rendimento atteso 1 - 4 x 10 6 cellule / tubo di sangue intero). <ol><li> Rimuovere 1 ml un&#39;aliquota, trasferire 5 ml di tubo, e tenere in ghiaccio per l&#39;analisi delle cellule T non etichettati da tetramero. Tenere sospensione cellulare su ghiaccio. </li></ol></li></ol> 2. magnetico arricchimento di cellule T alloantigene-specifici <ol><li> Diluire alloantigene pMHC tetramero a 1: 100 in tampone tipo sterile. </li><li> Sospensione cellulare Centrifuga (9 ml dal punto 1.11) a 600 xg per 5 min a 20 ° C. Decantare. </li><li> Aggiungere 50 ml di diluito alloantigene pMHC tetramero a pelletcellule (fino a 10 7 cellule). Mescolare delicatamente pipettaggio. Trasferimento in una provetta sterile da 5 ml. Incubare per 30 minuti a temperatura ambiente. </li><li> Aggiungere 2 ml di tampone di sorta. Centrifugare a 600 xg per 5 min a 20 ° C. Decantare. </li><li> Risospendere le cellule in 100 microlitri di buffer sorta. Aggiungere 10 ml biotina selezione di cocktail e incubare per 15 minuti a temperatura ambiente. </li><li> Aggiungere 5 ml Nanoparticelle magnetiche e incubare per 10 minuti a temperatura ambiente. </li><li> Aggiungere 2,5 ml sorta tampone e mescolare delicatamente con una pipetta. Togliere 100 ml un&#39;aliquota, trasferimento in 5 ml di tubo, e tenere in ghiaccio per l&#39;analisi di cellule pre-arricchimento. </li><li> Posizionare il tubo 5 ml, contenente la sospensione cellulare nel magnete separazione cellulare. Il tappo deve essere vagamente in cima al tubo. Incubare per 5 minuti a temperatura ambiente. </li><li> Rimuovere delicatamente il tappo dal tubo. Mantenendo il tubetto e magnete insieme, decantare il contenuto del tubo in una nuova provetta 5 ml. Non urtare o scuotere il contenuto della provetta perrimuovere le ultime gocce di liquido. </li><li> Togliere la provetta dal magnete. Aggiungere 2 ml di tampone di tipo freddo e mescolare delicatamente con una pipetta. Esempio 10 ml di sospensione cellulare, diluire 1: 2 (aggiungere a 10 ml) trypan blu per contare le cellule utilizzando un emocitometro. </li></ol> 3. Preparazione delle cellule T per la singola cella di Citometria a Flusso Ordinamento cellulare <ol><li> Aggiungi buffer di tipo fredda a tutte le provette di cellule tetramero (senza etichetta, tetramero marcato non-arricchito, e tetramero marcati arricchito) per portare il volume a 3 ml. </li><li> Centrifugare a 600 xg per 5 minuti a 4 ° C e tampone decantare per recuperare le cellule. </li><li> Risospendere le cellule pellet in 25 microlitri di buffer tipo freddo. Aggiungere 5 ml di blocco Fc umana. Incubare in ghiaccio per 20 min. </li><li> Preparare anticorpi per citometria a flusso ordinamento. Mescolare 15 ml di buffer sorta, 15 microlitri anti-CD5, 15 microlitri anti-CD14, e 15 microlitri anti-CD19. </li><li> Aggiungere 20 ml di miscela di anticorpi a ciascuna provetta di cellule. Incubare in ghiaccio per 20 min. </li><li> Aggiungere 2 ml di tampone di tipo freddo ad ogni provetta. </li><li> Centrifugare a 600 xg per 5 minuti a 4 ° C e tampone decantare per recuperare le cellule. </li><li> Risospendere le cellule ad una concentrazione di 1 -2 x 10 6 cellule / ml in tampone di ordinamento. </li><li> Un&#39;aliquota di 100 ml di terreno di coltura delle cellule T umane (DMEM di Iscove integrati con 2 Glutamax mM, HEPES 10 mM, 50 mg / ml di gentamicina, 50 mM 2-mercaptoetanolo, siero umano AB 10% inattivato al calore, e 2,5 ng / ml umana ricombinante IL-2) a ciascun pozzetto di una piastra di coltura cellulare a fondo rotondo da 96 pozzetti. Tenere su ghiaccio. </li></ol> 4. L&#39;isolamento di cellule T tetramero marcato da Single-cell Citometria a flusso Ordinamento <ol><li> Stabilire citometria a flusso parametri di gating per identificare etichettati cellule T alloantigene-pMHC tetramero (Figura 2.a). Utilizzare la frazione tetramero di pre-arricchimento etichettati per stabilire gating strategie per eliminare doppietti, porta sui linfociti, cellule che esprimono CD19 escludere B e CD14esprimendo monociti, e identificare le cellule T dall&#39;espressione CD5. Utilizzare il campione non etichettati con tetramero come una fluorescenza meno un controllo per stabilire il gating per l&#39;identificazione di cellule tetramero legame T (con lo 0% di CD5 + CD14 - CD19 - le cellule del campione non etichettati da tetramero dovrebbero rientrare nell&#39;ambito di questa porta) . NOTA: Soppressione strategie che non sono sufficientemente stringenti comporterà isolamento di cellule non-T o cellule T non antigene specifico, mentre le strategie di gating eccessivamente restrittive riducono il numero di cellule T isolate. </li><li> Programmare i parametri di targa del singolo tipo cellulare. Dirigere il sorter di inserire 1 CD5 + CD14 - CD19 - tetramero + cellule in ciascun pozzetto. Dirigere il piatto creato per contenere 1 controllo negativo (assenza di cellule ordinate nel pozzo) e 1 controllo positivo (100 CD5 + CD14 - CD19 - tetramero - cellule) in ogni riga (Figura 2.B </strong&gt;). </li><li> Utilizzando il flusso stabilito citometria strategia di gating e messa a punto piatto, isolare singole cellule tetramero vincolante T direttamente nella piastra a 96 pozzetti con un cell sorter citometria a flusso. </li></ol> 5. Cultura ed espansione di alloantigene-specifici cloni di cellule T <ol><li> Dopo il completamento della separazione delle cellule, piastra centrifugare a 600 xg per 5 min a 20 ° C. Luogo piastra di coltura a 37 ° C in un incubatore CO 2 al 6%. </li><li> Vortex stimolatori microsfere anti-CD3 / anti-CD28-per 30 secondi per risospendere le sfere. </li><li> Calcolare il volume di perline attivatore necessari per la stimolazione delle cellule T raccolti (0,5 ml di attivatore microsfere / pozzetto). Trasferire volume calcolato di perline stimolatore di una provetta sterile da 5 ml. Aggiungere 1 ml di cellule T umane terreno di coltura e vortice. </li><li> Posizionare il tubo con sospensioni microbead magnete. Il tappo deve essere vagamente in cima al tubo. Incubare per 2 minuti a temperatura ambiente. </li><li> Rimuovere delicatamenteil tappo dal tubo. Tenendo il tubo e magnete insieme, contenuto della provetta decantare in una nuova provetta da 5 ml. Non urtare o scuotere il contenuto della provetta. </li><li> Togliere la provetta dal magnete. Aggiungere mezzo di coltura delle cellule T umane (100 microlitri medio / 0,5 ml di microsfere inizialmente aggiunto). </li><li> Aliquotare 100 microlitri di sospensione attivatore tallone a ciascun pozzetto della piastra a 96 pozzetti. Incubare piastra di coltura a 37 ° C in un incubatore CO 2 al 6%. </li><li> Monitorare la crescita cellulare tutti i giorni con l&#39;esame microscopico. I piccoli gruppi di cellule proliferanti possono essere osservati al microscopio dopo 5 - 7 giorni di coltura (Figura 3.a). NOTA: visualizzazione di colture cellulari T a questo punto può essere difficile, e l&#39;assenza di gruppi di cellule osservabili in questa fase può non essere indicativo di una mancanza di cellule T in crescita. </li><li> A 14 giorni dopo l&#39;isolamento delle cellule, le culture di alimentazione rimuovendo con cura 100 ml di mezzi fuori della parte superiore della cultura e sostituirli con 100 μ; L fresca umano T terreno di coltura cellulare. Continuare l&#39;incubazione piastra di coltura a 37 ° C in un incubatore CO 2 al 6%. </li><li> Monitorare la crescita cellulare giornalmente mediante controllo e valutazione del colore del mezzo di coltura microscopica. I grandi gruppi di cellule dovrebbero essere visibili microscopicamente 2 - 3 giorni dopo il cambio medio. Macroscopicamente, pellet cellulari dovrebbero diventare visibili durante i 14 giorni successivi al cambio medio. </li><li> A 28 giorni dopo l&#39;isolamento delle cellule, individuare i cloni che crescono con l&#39;esame microscopico. Trasferire il volume di 200 microlitri della piastra a 96 pozzetti culture crescita positivi a singoli pozzetti di una piastra di coltura tissutale a 48 pozzetti contenente 200 microlitri terreno di coltura delle cellule T umane. </li><li> Aggiungere 100 ml contengono 12,5 ml di microsfere di stimolo anti-CD3 / anti-CD28 (preparato come descritto nelle sezioni 5,2-5,6). Incubare piastra di coltura a 37 ° C in un incubatore CO 2 al 6%. </li><li> Monitorare la crescita della cultura ogni giorno da microscopici examminazione e valutazione del colore del mezzo di coltura. Quando la cultura delle cellule T raggiunge&gt; 2x10 6 / ml (in genere 3-5 giorni dopo la ri-stimolazione, può essere stimato dal giorno in cui i media inizia a trasformare giallo paglierino), la cultura trasferimento ad un pozzetto di una piastra di coltura da 24 pozzetti tessuto e aggiungere 500 media delle cellule T umane ml. </li><li> A 10-14 giorni seguito ri-stimolazione, raccogliere 200 ml di coltura di cellule T per valutare la specificità alloantigene citometria a flusso di pMHC tetramero vincolante (utilizzando la strategia di etichettatura e gating descritto nelle sezioni 3 e 4.1). </li></ol> 6. a lungo termine Re-stimolazione e la cultura di cloni di cellule T <ol><li> Continuamente ri-stimolare ed espandere colture di cellule T clonali con la specificità desiderata può essere ogni 14 giorni. Raccogliere colture di cellule T in una provetta sterile da 5 ml al giorno 14 dopo l&#39;ultima stimolazione microbead anti-CD3 / CD28. Combinare pozzi multipli dello stesso clone di cellule T in un unico tubo, fino ad un volume di 2,5 ml. </li&gt; <li> Aggiungere mezzo per portare il volume finale di 2,5 ml e mescolare delicatamente con una pipetta. </li><li> Posizionare il tubo 5 ml contenente la coltura di cellule T nel magnete separazione cellulare per rimuovere le microsfere dalla cultura. Il tappo deve essere vagamente in cima al tubo. Incubare per 5 minuti a temperatura ambiente. </li><li> Rimuovere delicatamente il tappo dal tubo. Mantenendo il tubetto e magnete insieme, decantare la sospensione cellulare in una nuova provetta 5 ml. Non urtare o scuotere il contenuto della provetta per rimuovere le ultime gocce di liquido. </li><li> Aggiungere mezzo per portare il volume finale a 4 ml. Campione di 10 microlitri per contare le cellule utilizzando un emocitometro. </li><li> Recupero cellule T mediante centrifugazione a 600 xg per 5 minuti a temperatura ambiente. </li><li> Decantare il surnatante. Risospendere le cellule T a 10 6 cellule / ml T umano mezzo di coltura cellulare. Trasferire 1 ml aliquote a ciascun pozzetto di una piastra di coltura tissutale a 24 pozzetti. </li><li> Re-stimolare le cellule T con l&#39;aggiunta di 100 microlitri supporti contenenti 12,5 ml di stimolazione anti-CD3 / amicrosfere NTI-CD28 (come descritto nelle sezioni 5,2-5,6). Incubare a 37 ° C in un incubatore CO 2 al 6%. </li></ol>

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The authors declare no competing financial interests.

T cell alloreactivity is a long-studied and clinically-relevant phenomenon. The robust proliferative and effector responses of T cells to allogeneic stimulation has enabled extensive analyses of human T cell responses in vitro through relatively straightforward mixed lymphocyte reactions of peripheral blood T cells against inactivated allogeneic cells. However, these primary alloreactive T cell responses are oligoclonal, comprised of a large number of individual T cells responding to specific alloantigens. This ...

I linfociti T sono componenti critici del sistema immunitario adattativo. Le cellule T sono responsabili non solo mediare direttamente risposte immunitarie protettive a patogeni attraverso una varietà di meccanismi effettori, ma anche mantenere attivamente immunologica auto-tolleranza e dirigere le risposte di altre cellule del sistema immunitario. Queste funzioni sono diretti attraverso una serie di segnali integrati, compreso recettore delle cellule T (TCR) legatura, citochine e chemochine, e metaboliti 1. Di questi segnali, il TCR è di particolare importanza, in quanto fornisce la specificità caratteristica che definisce il ruolo delle cellule T nell&#39;immunità adattativa. Un TCR interagisce con antigeni peptidici lineari presentati da MHC (HLA ortologo umano) molecole (complessi pMHC) in modo altamente specifico e sensibile per fornire i segnali che attivano la funzione delle cellule T effettrici. I parametri biochimici delle interazioni TCR con leganti pMHC non solo forniscono la specificità per Tattivazione delle cellule, ma anche avere un impatto qualitativo sulla funzione delle cellule T 2 successiva. Così, studiando la funzione delle cellule T richiede spesso di esaminare le risposte delle cellule T clonali con specificità antigenica definito. Il vano umano delle cellule T, che contiene circa 10 12 cellule T αβ, contiene, secondo le stime 10 luglio-10 agosto αβTCRs distinte 3-4. Questo vasto repertorio offre opportunità per il riconoscimento della vasta gamma di peptidi da potenziali agenti patogeni tali da richiedere una risposta delle cellule T per l&#39;immunità protettiva. Si stima che la frequenza delle cellule T rispondere ad un dato antigene estraneo presentata dalla auto-MHC è dell&#39;ordine di 10 -4 - 10 -7 in assenza di prima risposta immunitaria a questo antigene 5. L&#39;ingenuo repertorio delle cellule T è modellato dalla selezione timica per garantire la capacità di riconoscere auto-MHC presentano antigeni peptidici e limite reagisconoivity contro antigeni self-peptide, massimizzando l&#39;utilità potenziale di mediare l&#39;immunità protettiva 2. Tuttavia, in violazione del presente progettato reattività relativamente grande frequenza, 10 -3 - 10 -4, di cellule T da individui immunologicamente naive rispondere alla stimolazione con cellule allogeniche, riconoscendo entrambe le molecole MHC estere nonché peptidi endogeni che presentano 6. Il riconoscimento di allogenici ligandi pMHC è strutturalmente simile al riconoscimento di antigeni estranei presentate da auto-MHC; TCR rende interazioni biochimiche critici sia con la molecola MHC allogeniche e il peptide presentato 7. La natura robusta della risposta delle cellule T allogeniche ai risultati della stimolazione della diversità dei pMHC complessi presenti sulla superficie delle cellule allogeniche 8. Si stima che ogni MHC presenta circa 2 x 10 4 differenti antigeni peptidici endogeni 9. Questo breadth di risposta alla stimolazione allogenico è un aspetto significativo della patologia clinicamente rilevanti, come il rigetto o di malattia da trapianto contro ospite (GVHD), derivanti da alloreattività cellule T. Studio delle cellule T risposte alloreattivi umani ha tradizionalmente invocato l&#39;esame delle risposte policlonali di cellule T naive in seguito alla stimolazione con cellule allogeniche. Stimolazione ripetuta con la stessa linea di cellule allogeniche combinato con diluizione limite analisi è in grado di generare cellule T clonali con riconoscimento definito di allogenico HLA 10. Tuttavia, questo approccio è problematico per l&#39;esame delle risposte ai singoli ligandi allogenici pMHC, come il grande e variegato repertorio di endogena pMHC complessi presenti per un determinato allogenico HLA stimola un ampio repertorio di cellule T. Questa stimolazione popolazione di massa e di limitare la diluizione approccio richiederebbe lo screening di un gran numero di cloni per isolare le cellule T con il riatti desideratovità contro un singolo ligando pMHC. Inoltre, la frequenza delle cellule T che rispondono ad un individuo allogenico pMHC ligando è relativamente bassa tra popolazioni di cellule T naive, che presenta una barriera efficace generazione di cloni di cellule T umane sensibili ad un dato antigene. Identificazione e isolamento di cellule T antigene-specifiche da popolazioni policlonali sono stati abilitati dallo sviluppo di fluoroforo marcata pMHC multimeri 11. Questo approccio utilizza specifici antigeni peptidici caricati in molecole MHC ricombinanti solubili biotinilate, etichettati legandosi ad un fluoroforo streptavidina marcata. Multimerizzazione di pMHC aumenta l&#39;avidità, compensando la intrinsecamente basso (mM) affinità del TCR per ligandi solubili pMHC. Cellule marcate possono essere identificati e isolati mediante citometria a flusso. Tuttavia, questo approccio è ancora limitata dalla bassa frequenza di cellule T antigene-specifiche tra naive popolazioni di cellule T, che sono tipicamenteordini di grandezza inferiore al limite di identificazione accurata e quantificazione sulla maggior citofluorimetri. Per affrontare questa limitazione, un metodo di etichettatura tetramero pMHC e successiva tallone arricchimento magnetico per cellule tetramero-marcato è stato sviluppato 12. Questo metodo ha dimostrato rilevamento affidabile, l&#39;enumerazione, e l&#39;isolamento di cellule T antigene-specifiche bassa frequenza. Questo protocollo descrive un protocollo efficace per la generazione di cloni di cellule T umane che rispondono specificamente ai singoli ligandi allogenici pMHC. Il protocollo si applica pMHC (HLA) etichettatura Multimer e di arricchimento per l&#39;isolamento di cellule T umane specifiche per alloantigene con citometria a flusso delle cellule di ordinamento e un metodo robusto per la coltura in vitro di cellule T umane per consentire la produzione di cloni di cellule T da cellule ordinati singole ( panoramica nella Figura 1).

<table><tbody><tr><td>Sodium heparin venous blood collection tube 16 x 100 mm</td><td>Becton, Dickenson and Company</td><td>366480</td><td></td></tr><tr><td>Lymphoprep</td><td>Stemcell Technologies</td><td>7801</td><td></td></tr><tr><td>Rosette Sep Human T Cell Enrichment Kit</td><td>Stemcell Technologies</td><td>15061</td><td></td></tr><tr><td>Dulbecco&#039;s PBS, 1x without Ca or Mg</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E6635</td><td></td></tr><tr><td>Fluorophore-labeled pMHC tetramer</td><td>NIH Tetramer Facility</td><td>NA</td><td></td></tr><tr><td>EasySep Biotin Selection Kit</td><td>Stemcell Technologies</td><td>18553</td><td></td></tr><tr><td>EasySep Selection magnet</td><td>Stemcell Technologies</td><td>18000</td><td></td></tr><tr><td>TruStain FcX Human Fc blocking solution</td><td>Biolegend</td><td>422301</td><td></td></tr><tr><td>Anti-CD5 PE-Cy7 (clone UCHT2)</td><td>Biolegend</td><td>300621</td><td></td></tr><tr><td>Anti-CD14 FITC (clone HCD14)</td><td>Biolegend</td><td>325603</td><td></td></tr><tr><td>Anti-CD19 FITC (clone HIB19)</td><td>Biolegend</td><td>302205</td><td></td></tr><tr><td>Iscove&#039;s DMEM, without b-ME or L-glutamine</td><td>Corning</td><td>15-016-CV</td><td></td></tr><tr><td>HEPES</td><td>Corning</td><td>25-060-CI</td><td></td></tr><tr><td>b-Mercaptoethanol&amp;nbsp;</td><td>Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>Glutamax</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>Gentamicin sulfate (50 mg/ml)</td><td>Omega Scientific</td><td>GT-50</td><td></td></tr><tr><td>Human AB serum, male donor</td><td>Omega Scientific</td><td>HS-30</td><td></td></tr><tr><td>Recombinant human IL-2</td><td>Peprotech</td><td>AF 200-02</td><td></td></tr><tr><td>Dynabeads Human T-Activator CD3/CD28</td><td>Life Technologies</td><td>11131D</td><td></td></tr><tr><td>Media</td><td></td><td></td><td></td></tr><tr><td>Cell sorting buffer</td><td></td><td></td><td></td></tr><tr><td>PBS, pH 7.4</td><td>1 L</td><td></td><td></td></tr><tr><td>BSA</td><td>10 g</td><td></td><td></td></tr><tr><td>EDTA (0.5 M)</td><td>2 ml</td><td></td><td></td></tr><tr><td>Human T Cell Culture Medium</td><td></td><td></td><td></td></tr><tr><td>Iscove&#039;s DMEM</td><td>351.6 ml</td><td></td><td></td></tr><tr><td>Heat-inactivated human AB serum</td><td>40 ml</td><td></td><td></td></tr><tr><td>HEPES (1 M)</td><td>4 ml</td><td></td><td></td></tr><tr><td>Glutamax (100x)</td><td>4 ml</td><td></td><td></td></tr><tr><td>Gentamicin (50 mg/ml)</td><td>0.4 ml</td><td></td><td></td></tr><tr><td>b-mercaptoethanol (14.3 M)</td><td>1.4 ml</td><td></td><td></td></tr><tr><td>Recombinant human IL-2 (1 mg/ml)</td><td>1 ml</td><td></td><td></td></tr></tbody></table>

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.

Questo protocollo descrive la generazione di colture di cellule T clonale umani con definito alloantigene specificità attraverso un arricchimento tallone magnetico e flusso a cella singola citometria di ordinamento strategia. La Figura 1 fornisce una panoramica del processo. <img alt="Figura 1" src="/files/ftp_upload/52257/52257fig1highres.jpg" /> Figura 1: Panoramica del protocollo Il protocollo qui descritto forn...

Watch this Scientific Journal Video about Generation of Human Alloantigen-specific T Cells from Peripheral Blood at JoVE.com

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

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 ...

generation-of-human-alloantigen-specific-t-cells-from-peripheral-blood

Research

JoVE Journal

Immunology and Infection

12.9K Views.  University of California, San Diego. 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.

Video: Generation of Human Alloantigen-specific T Cells from Peripheral Blood

Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.

This paper describes a simple technique to induce alloantigen-specific anergy in human peripheral blood mononuclear cells. The technique can be applied clinically to generate non-alloreactive donor cells. Infusion of these cells could improve immune reconstitution and reduce toxicity after allogeneic hematopoietic stem cell transplantation.

L&#39;induzione di anergia Alloantigen specifici nelle cellule mononucleari del sangue periferico di stimolazione Alloantigen con co-stimolatorie blocco del segnale

Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic ...

Ricerca

Immunologia e infezioni

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

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

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

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

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 ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Generazione di integrazione-liberi cellule staminali pluripotenti indotte dalle cellule mononucleari di sangue periferico umano Utilizzando episomiale Vettori

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Biologia dello sviluppo

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

Utilizzo di cellule staminali pluripotenti indotte umane per la generazione di cellule T specifiche dell'antigene tumorale

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Ricerca sul cancro

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Generazione di Human CD40 delle cellule B attivate

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Biologia

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

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 considered as the only source of &#945;&#946;T cells. Yet, thymic involution begins early in life leading to a drastically reduced output of na&#239;ve &#945;&#946;T cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic &#945;&#946;T cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. &#947;&#948; T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a &#947;&#948; T cell subset by showing that the scarce entity of CD4+ V&#948;1+&#947;&#948; T cells can transdifferentiate into &#945;&#946;T cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. V&#948;1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ V&#948;1+&#947;&#948; T cells. This knowledge is prerequisite for studies that relate to this &#945;&#946;T cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

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 ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

Generazione<em&gt; De Novo</em&gt; Antigene-specifici T umana recettori cellulari di trasduzione retrovirale di Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Generazione di derivate da monociti cellule dendritiche umane da sangue intero

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

Generazione di anticorpi monoclonali umani discriminante da rari B antigene-specifiche cellule circolanti nel sangue

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Generation of Human Regulatory T Cell Clones

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based on the identification of Treg through several activation-dependent cellular surface markers with varying expression levels in different physiologic and pathologic conditions. Populations isolated as &#8220;Treg&#8221; therefore often contain considerable numbers of non-Treg effector cells (i.e., Teff) which hamper the precise phenotypic and functional characterization of these cells, their genomic and proteomic characterization, their reliable enumeration in different states of health and disease, as well as their isolation and expansion for therapeutic purposes. The latter, in particular, remains a major hurdle, as the inadvertent expansion of effector cells homing in Treg-relevant cellular compartments (e.g., CD4+CD25+ T cells) may render Treg-based immunotherapy ineffective, or even harmful. This work presents a method that circumvents the problems associated with population-based isolation and expansion of Treg and shows that the generation of Treg candidate clones with the subsequent selection, culture, and expansion of only carefully vetted, monoclonal cells, enables the generation of an ultrapure Treg cell product that can be kept in culture for many months, enabling downstream investigation of these cells, including for possible therapeutic applications.

This protocol describes the cloning and expansion of human regulatory T cells for the generation of ultra-high purity viable human Treg with stable demethylation at the Treg-specific demethylated region (TSDR) and Treg-specific phenotypic features.

Human regulatory T cells (Treg) are notoriously difficult to isolate in high purity given the current methods of Treg enrichment. These methods are based ...

Generazione di Human alloantigene-specifiche cellule T da sangue periferico

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L'induzione di anergia Alloantigen specifici nelle cellule mononucleari del sangue periferico di stimolazione Alloantigen con co-stimolatorie blocco del segnale