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 border="0" cellpadding="0" cellspacing="0" width="907">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Name of Reagent/ Equipment</td>
			<td style="width:223px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:292px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Sodium heparin venous blood collection tube 16 x 100 mm</td>
			<td style="width:223px;">Becton, Dickenson and Company</td>
			<td style="width:119px;">366480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Lymphoprep</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">7801</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:273px;">Rosette Sep Human T Cell Enrichment Kit</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">15061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Dulbecco&#39;s PBS, 1x without Ca or Mg</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Bovine serum albumin</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EDTA</td>
			<td style="width:223px;">Sigma-Aldrich</td>
			<td style="width:119px;">E6635</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Fluorophore-labeled pMHC tetramer</td>
			<td style="width:223px;">NIH Tetramer Facility</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EasySep Biotin Selection Kit</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">18553</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EasySep Selection magnet</td>
			<td style="width:223px;">Stemcell Technologies</td>
			<td style="width:119px;">18000</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:273px;">TruStain FcX Human Fc blocking solution</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">422301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD5 PE-Cy7 (clone UCHT2)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">300621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD14 FITC (clone HCD14)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">325603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Anti-CD19 FITC (clone HIB19)</td>
			<td style="width:223px;">Biolegend</td>
			<td style="width:119px;">302205</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Iscove&#39;s DMEM, without b-ME or L-glutamine</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">15-016-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HEPES</td>
			<td style="width:223px;">Corning</td>
			<td style="width:119px;">25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">b-Mercaptoethanol&#160;</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">21985-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Glutamax</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gentamicin sulfate (50 mg/ml)</td>
			<td style="width:223px;">Omega Scientific</td>
			<td style="width:119px;">GT-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Human AB serum, male donor</td>
			<td style="width:223px;">Omega Scientific</td>
			<td style="width:119px;">HS-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Recombinant human IL-2</td>
			<td style="width:223px;">Peprotech</td>
			<td style="width:119px;">AF 200-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Dynabeads Human T-Activator CD3/CD28</td>
			<td style="width:223px;">Life Technologies</td>
			<td style="width:119px;">11131D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Media</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Cell sorting buffer</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">PBS, pH 7.4</td>
			<td style="width:223px;">1 L</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">BSA</td>
			<td style="width:223px;">10g</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">EDTA (0.5 M)</td>
			<td style="width:223px;">2 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;"></td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Human T Cell Culture Medium</td>
			<td style="width:223px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Iscove&#39;s DMEM</td>
			<td style="width:223px;">351.6 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Heat-inactivated human AB serum</td>
			<td style="width:223px;">40 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HEPES (1 M)</td>
			<td style="width:223px;">4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Glutamax (100 x)</td>
			<td style="width:223px;">4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Gentamicin (50 mg/ml)</td>
			<td style="width:223px;">0.4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">b-mercaptoethanol (14.3 M)</td>
			<td style="width:223px;">1.4 ml</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Recombinant human IL-2 (1 mg/ml)</td>
			<td style="width:223px;">1 ml</td>
			<td style="width:119px;"></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

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Espansione, di purificazione, e la valutazione funzionale delle cellule del sangue umano periferico NK

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Ricerca

Immunologia e infezioni

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 ...

Expansion of Human Peripheral Blood  &gamma;&delta; T Cells using Zoledronate

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and anti-infective activity.1 The majority of &gamma;&delta; T cells in peripheral blood have the V&gamma;9V&delta;2 T cell receptor. These cells recognize antigen in a major histocompatibility complex-independent manner and develop strong cytolytic and Th1-like effector functions.1Therefore, &gamma;&delta; T cells are attractive candidate effector cells for cancer immunotherapy. V&gamma;9V&#x03B4;2 T cells respond to phosphoantigens such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is synthesized in bacteria via isoprenoid biosynthesis;2 and isopentenyl pyrophosphate (IPP), which is produced in eukaryotic cells through the mevalonate pathway.3 In physiological condition, the generation of IPP in nontransformed cell is not sufficient for the activation of &gamma;&delta; T cells. Dysregulation of mevalonate pathway in tumor cells leads to accumulation of IPP and &gamma;&delta; T cells activation.3 Because aminobisphosphonates (such as pamidronate or zoledronate) inhibit farnesyl pyrophosphate synthase (FPPS), the enzyme acting downstream of IPP in the mevalonate pathway, intracellular levels of IPP and sensitibity to &gamma;&delta; T cells recognition can be therapeutically increased by aminobisphosphonates. IPP accumulation is less efficient in nontransfomred cells than tumor cells with a pharmacologically relevant concentration of aminobisphosphonates, that allow us immunotherapy for cancer by activating &gamma;&delta; T cells with aminobisphosphonates. 4 Interestingly, IPP accumulates in monocytes when PBMC are treated with aminobisphosphonates, because of efficient drug uptake by these cells. 5 Monocytes that accumulate IPP become antigen-presenting cells and stimulate V&gamma;9V&delta;2 T cells in the peripheral blood.6 Based on these mechanisms, we developed a technique for large-scale expansion of &gamma;&delta; T cell cultures using zoledronate and interleukin-2 (IL-2).7 Other methods for expansion of &gamma;&delta; T cells utilize the synthetic phosphoantigens bromohydrin pyrophosphate (BrHPP)8 or 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP).9 All of these methods allow ex vivo expansion, resulting in large numbers of &gamma;&delta; T cells for use in adoptive immunotherapy. However, only zoledronate is an FDA-approved commercially available reagent. Zoledronate-expanded &gamma;&delta; T cells display CD27-CD45RA- effector memory phenotype and thier function can be evaluated by IFN-&gamma; production assay. 7

Expansion of Human Peripheral Blood  &amp;#947;&amp;#948; T Cells using Zoledronate

A method to expand &gamma;&delta; T cells from peripheral blood mononuclear cells (PBMC) is described. PBMC-derived &gamma;&delta; T cells are stimulated and expanded using zoledronate and interleukin-2 (IL-2). Large scale expansion of &gamma;&delta; T cells can be applied to autologous cellular immunotherapy of cancer.

Espansione del sangue periferico umano γδ cellule T con Zoledronato

Human &gamma;&delta; T cells can recognize and respond to a wide variety of stress-induced antigens, thereby developing innate broad anti-tumor and ...

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&amp;#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 ...

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

Applicazione clinica della<em&gt; Bella Addormentata</em&gt; Artificiale e cellule presentanti l&#39;antigene per modificare geneticamente le cellule T da sangue del cordone ombelicale e periferico

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

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 ...

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 ...

Flotation-Based T Cell Isolation, Activation, and Expansion from Human Peripheral Blood Mononuclear Cell Samples Using Microbubbles

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical testing, and cell-based therapies. In this study, a simple, novel protocol to isolate, activate, and expand T cells from PBMCs ex vivo is presented. This study utilizes functionalized buoyancy-activated cell sorting (BACS) technology to isolate and activate T cells. Briefly, the protocol involves the positive selection of CD3+ cells from leukopak-derived PBMCs, followed by a 48 h co-stimulation with pre-conjugated anti-CD28-bound streptavidin microbubbles (SAMBs) prior to transduction in 24-well plates. Functionalized microbubbles offer a unique opportunity to buoyantly activate cells, leading to proliferative phenotypes that allow for expansion with minimal exhaustion. This technique offers reduced exhaustion because the co-stimulatory microbubbles remain buoyant and return to the top of the culture medium, thus reducing the amount of time that the expanding cells are in contact with the co-stimulatory factors. The results indicate that the isolated and cultured T cells receive enough stimulation to activate and proliferate but not to an extent that leads to overactivation, which then leads to exhaustion, as demonstrated by the presence of excessive PD-1.

The objective of this study is to demonstrate the feasibility of flotation-based separation to isolate, activate, and expand primary human T cells.

Isolamento, attivazione ed espansione delle cellule T basate sulla flottazione da campioni di cellule mononucleate del sangue periferico umano utilizzando microbolle

The process of isolating T cells from peripheral blood mononuclear&#160;cells (PBMCs) to establish ex vivo cultures is crucial for research, clinical ...

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14&#8722; mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

Differenziazione degli osteoclasti funzionali dai monociti CD14+ del sangue periferico umano

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...