Questo progetto è stato finanziato, in parte, sotto sovvenzioni dal National Institutes of Health (R01AI121180, R21AI109239 e K18CA151798), l&#39;American Diabetes Association (1-16-IBS-281), e il Dipartimento della Salute della Pennsylvania (tabacco Settlement Funds) a JS

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The authors have nothing to disclose.

In questo protocollo, un passaggio fondamentale è la differenziazione in vitro di TCR / FOXP3 iPSCs gene-trasdotte. In vitro Notch induce lo sviluppo verso la linea cellulare T. Per differenziare iPSCs in CD4 + FoxP3 + Tregs, abbiamo utilizzato le cellule B OP9-DL1 / DL4 / Ia, altamente espressa molecole MHC II (IA b). La maggior parte dei iPSCs differenziano in cellule CD4 +. Tuttavia, dopo l&#39;espressione del TCR di superficie, molte cellule pre-T differenziati perdono la capacità di differenziare e alla fine muoiono. Come risultato, il numero di cellule dei Tregs funzionali COPSI-derivati riduce drasticamente dopo quattro settimane di differenziazione in vitro. Per evitare questo, l&#39;aggiunta di IL-2 può migliorare la sopravvivenza cellulare in quei punti temporali. Un metodo alternativo per guidare la maturazione e la sopravvivenza di Ag-specifici pre-Tregs è di trasferire i pre-Tregs che sono differenziate in vitro per una settimana in topi. Questi pre-Tregs può continue differenziazione e maturazione in vivo per altre tre settimane. Utilizzando questo metodo, una popolazione Treg Ag-specifica funzionale può essere generata da iPSCs, che sono nTregs-come e hanno la capacità di sopprimere l&#39;artrite autoimmune nel modello murino. Per migliorare l&#39;efficacia della vivo sviluppo di Ag-specifici iPSC-Tregs, composti diversi (ad esempio, l&#39;acido retinoico, gelectin) 13,14 o soppressiva citochine (ad esempio, TGF-β, IL-10) possono essere utilizzati dopo il trasferimento adottivo del pre-Tregs. Oltre ad aumentare la sopravvivenza cellulare, questo approccio combinato può mantenere espressione FoxP3 14,15 e migliorare la qualità del Ag-specifica COPSI-Tregs. Un potenziale problema che potrebbe sorgere per lo sviluppo in vivo Treg è immunosoppressione globale, con conseguente complicazioni durante infezioni o conseguente perdita di peso. Un gran numero di Ag-specifica Tregs in via di sviluppo nel VIvo può peggiorare le infezioni. In questo caso, un gene suicida, la caspasi 9 inducibile (ICasp9) 15,16, può essere incorporato nel vettore TCR / FoxP3. Questo approccio permette la rimozione dei Tregs cellule staminali derivate dalla iniezione di una piccola molecola bioinerti dimerizing agente (AP1903) per &quot;spegnere&quot; la generazione di cellule staminali derivate da Tregs, che permette di superare questo potenziale problema. Un self-Ag è una proteina tipica riconosciuta dal sistema immunitario di ospiti che soffrono di una malattia autoimmune individuale. Questa auto-Ag è l&#39;obiettivo del sistema immunitario, e le cellule T correlate non vengono eliminati. Per generare auto-Ag Tregs specifici, l&#39;uso di TCR trasduzione è una buona scelta. Un auto-Ag (ad esempio, la proteina heat shock) specifica TCR può essere trasduzione in maturi CD4 + CD25 + Treg dalle cellule mononucleate del sangue periferico (PBMC), e questo approccio è stato utilizzato in studi clinici. In alternativa, come described in questo protocollo, utilizzando la trasduzione genica di auto-Ag TCR specifico con FoxP3 e la stimolazione con segnalazione Notch, staminali Tregs derivati ​​dalle cellule può essere auto-Ag specifica Tregs. Terapie a base di cellule per le malattie autoimmuni che utilizzano Tregs ingegnerizzati possono essere utili 17. Anche se TCR trasduzione nelle cellule T ha dimostrato di essere al sicuro, fattibile, e applicabile negli studi clinici, ci sono ancora notevoli problemi di sicurezza a causa della autoimmunità causata da cross-reattività con i tessuti sani 18. Inoltre, usando i metodi attuali, geneticamente modificato Tregs solito hanno una persistenza breve termine in vivo 19. In alternativa, una fonte promettente per lo sviluppo di un gran numero di monoclonali Ag-specifici Tregs è cellule staminali. Le cellule staminali embrionali (ESC) hanno la migliore pluripotenza e di auto-rinnovamento, ma non è possibile ottenere loro di pazienti. Anche se facilmente isolati dal sangue periferico, le cellule staminali ematopoietiche (CSE) sono multi-Le cellule staminali potenti e possono essere espanse in coltura cellulare in modo simile a CES 20. La tecnologia attuale COPSI è avanzata ad una generazione più efficace di PSC da cellule somatiche dei pazienti da trasduzione di diversi fattori di trascrizione. Un certo numero di miglioramenti metodologici sono stati sviluppati negli ultimi anni per generare iPSCs riducendo al massimo i rischi potenziali, come immunogenicità e tumorigenicità. Rispetto al CES, iPSCs hanno pluripotenza identici e auto-rinnovamento. Come risultato, la tecnologia COPSI può fornire un vantaggio nello sviluppo PSC-paziente e / o malattie specifiche. L&#39;uso di iPSCs per sviluppare Ag-specifica Tregs può avanzare il campo delle terapie a base di cellule per le malattie autoimmuni 10,11.

Autoimmune arthritis is a systemic disease characterized by hyperplasia of synovial tissue and progressive destruction of articular cartilage, bone, and ligaments1. The defective generation or function of Tregs in autoimmune arthritis contributes to chronic inflammation and tissue injury because Tregs play a crucial role in preventing the development of auto-reactive immune cells. 
Manipulation of Tregs is an ideal strategy for the development of therapies to suppress inflammation in an Ag-dependent manner. For Treg-based immunotherapy, the specificity of the transferred Tregs is important for the treatment of ongoing autoimmunity2. To exhibit the suppressive activity, Tregs need to migrate and be retained at the afflicted region, which can be directed by the specificity of the TCR for the Ag at that location3. Although polyclonal Tregs may contain a small population containing this Ag specificity from their TCRs, the numbers of these Ag-specific Tregs are usually low. Consequently, cell-based therapies using polyclonal Tregs against autoimmune disorders require adoptive transfers of a large number of Tregs4,5. Because pluripotent stem cells (PSCs) have the ability to develop into any type of cell, Ag-specific PSC-Tregs may prove to be good candidates for Treg-based immunotherapy. Previous studies have shown the successful development of PSC-derived T cells, including Tregs6-8. 
Here, we describe a protocol to develop Ag-specific iPSC-Tregs. We further describe a cell-based therapy of autoimmune arthritis in a murine model using such Tregs. This method is based upon genetically modifying murine iPSCs with Ag-specific TCRs and the transcriptional factor FoxP3. The engineered iPSCs then differentiate into Ag-specific Tregs on the OP9 stromal cells expressing Notch ligands DL1, DL4, and MHC-II (I-Ab) molecules in the presence of cytokines mFlt3L and mIL-7. These Ag-specific iPSC-Tregs can produce suppressive cytokines, such as TGF-&#946; and IL-10, when stimulated with the Ag, and adoptive transfer of such Tregs has the ability to suppress AIA development in a murine model. The described protocol can be used to develop stem cell-derived Ag-specific Tregs for potential therapeutic interventions.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">C57BL/6j mice</td>
			<td style="width:71px;">Jackson Laboratory</td>
			<td style="width:119px;">664</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">B6.129S7 Rag1tm1Mom/J</td>
			<td style="width:71px;">Jackson Laboratory</td>
			<td style="width:119px;">2216</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Anti-CD3 (2C11) antibody</td>
			<td style="width:71px;">BD Pharmingen</td>
			<td style="width:119px;">553058</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Anti-CD28 (37.51) antibody</td>
			<td style="width:71px;">BD Pharmingen</td>
			<td style="width:119px;">553295</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-CD4 (GK1.5) antibody</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">100417</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-CD8 (53&#8211;6.7) antibody</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">100714</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-CD25 (3C7) antibody</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">101912</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-TCR-&#946; (H57597) antibody</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">109220</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-IL10</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">505010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-TGF&#946;</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">141402</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">DMEM</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">ABCD1234</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">&#945;-MEM</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">A10490-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">FBS</td>
			<td style="width:71px;">Hyclone</td>
			<td style="width:119px;">SH3007.01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Brefeldin A</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">B7651</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Polybrene</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">107689</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Genejammer</td>
			<td style="width:71px;">Integrated science</td>
			<td style="width:119px;">204130</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">ACK Lysis buffer</td>
			<td style="width:71px;">Lonza</td>
			<td style="width:119px;">10-548E</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">mFlt-3L</td>
			<td style="width:71px;">peprotech</td>
			<td style="width:119px;">250-31L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">mIL-7</td>
			<td style="width:71px;">peprotech</td>
			<td style="width:119px;">217-17</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Gelatin</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">G9391</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Paraformaldehyde</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P6148-500G</td>
			<td style="width:167px;">Caution: Allergenic, Carcenogenic, Toxic</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Permeabilization buffer</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">421002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">mBSA</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A7906</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Ova albumin</td>
			<td style="width:71px;">Avantor</td>
			<td style="width:119px;">0440-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">CFA</td>
			<td style="width:71px;">Difco</td>
			<td style="width:119px;">2017014</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Tailveiner restrainer</td>
			<td style="width:71px;">Braintree scientific</td>
			<td style="width:119px;">RTV 150-STD</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

development of stem cell-derived antigen-specific regulatory t cells against autoimmunity

Malattie autoimmuni insorgono a causa della perdita di immunologica auto-tolleranza. cellule T regolatorie (Tregs) sono importanti mediatori di immunologica auto-tolleranza. Tregs rappresentano circa il 5 - 10% del maturo sottopopolazione di cellule T CD4 + in topi e nell&#39;uomo, con circa 1 - 2% di tali Tregs circolanti nel sangue periferico. Le cellule staminali pluripotenti indotte (iPSCs) possono essere differenziati in Tregs funzionale, che hanno un potenziale da utilizzare per le terapie a base di cellule di malattie autoimmuni. Qui, vi presentiamo un metodo per sviluppare antigene (Ag) Treg specifico d&#39;da iPSCs (vale a dire, IPSC-Tregs). Il metodo si basa sulla incorpora il fattore di trascrizione FoxP3 e un recettore delle cellule T Ag-specifica (TCR) in iPSCs e differenziazione di cellule stromali OP9 esprimenti Notch ligandi delta-simili (DL) 1 e DL4. A seguito di differenziazione in vitro, l&#39;IPSC-Treg esprimono CD4, CD8, CD3, CD25, FoxP3, e Ag-specifici TCR e sono in grado di rispondere alla stimolazione Ag.Questo metodo è stato applicato con successo a terapia cellulare di artrite autoimmune in un modello murino. trasferimento adottivo di questi Ag-specifici iPSC-Tregs in artrite Ag-indotta (AIA) topi -bearing ha la capacità di ridurre l&#39;infiammazione articolare e gonfiore e per prevenire la perdita ossea.

Come mostrato qui, il giorno 28, Ag-specifica Treg espresso sostanzialmente CD3 e Ag-specifica TCR, due marcatori di cellule T. La popolazione CD3 + TCRVβ5 + CD4 espresso. La maggior parte delle cellule CD3 + TCRVβ5 + CD4 + CD25 espresse anche, CD127, e CTLA-4, che sono tipicamente espressi a livelli elevati nel naturale REGS T (nTregs) e nelle cellule T che esprimono FoxP3 ectopica. Espressione FoxP3 nelle cellule iPSC-derivate persisteva anche dopo lungo periodo di stimolazione in vitro con il ligando Notch, come rilevato dalla colorazione intracellulare analizzata mediante citometria di flusso (Figura 1). Inoltre, l&#39;Ag-specifica COPSI-Tregs prodotta citochine soppressive, quali TGF-β e IL-10, quando stimolate in vitro con splenociti Ag-pulsata (Figura 2), che indica l&#39;IPSC-Tregs aveva potenziali attività soppressiva. Microscopia a fluorescenza rivelato<html> che più Foxp3 + cellule erano presenti nel OVA trattati con rispetto le ginocchia PBS trattati; non ci sono stati + cellule FOXP3 esistenti nelle ginocchia nei topi trattati con i iPSCs vettore-trasdotte DsRed +. Molti di più CD4 + + + TCRVβ5 cellule FOXP3 presentati nelle ginocchia di topi trattati con iPSCs trasdotte con il MIDR-TCR-FoxP3 rispetto al MIDR-FoxP3 (Figura 3). Queste osservazioni suggeriscono che Ag-specifici IPSC-Tregs migrano al ginocchio AIA dopo il trasferimento adottivo in topi riceventi. Le cellule derivate COPSI trasferiti sostanzialmente diminuiti ginocchio infiammatori gonfiore quando OVA era presente, ma non ha avuto effetto sul ginocchio controllo che è stato iniettato solo con MBSA nel modello murino (figura 4); perdita ossea ridotta in ginocchio trasferimento di cellule visualizzata dal sistema micro-CT ad alta risoluzione (Figura 5). tp_upload / 54720 / 54720fig1.jpg &quot;/&gt; Figura 1:. Differenziazione di Ag-specifici iPSC-Tregs murino iPSCs sono state trasdotte con un costrutto: MIDR-TCRα-2A-TCRβ-2A-FoxP3 contenenti geni di OVA-specifici TCR e FoxP3. Le cellule gene-trasdotte (DsRed +) sono stati co-coltivati su OP9-DL1-DL4-IA cellule B in presenza di mFlt3L e MIL-7. (A) Morfologia di T REGS differenziazione delle cellule al giorno 0, 7, 14 e 22. citometria (B) di flusso per l&#39;espressione della proteina delle cellule iPSC-derivati al giorno 28. CD3 + TCRVβ5 + cellule sono state gated come indicato (R1) e analizzati per l&#39;espressione di CD4 e CD8, con CD25, CD127, CTLA- 4, e FoxP3 dimostrato per le cellule gated come CD4 + CD8 - cellule (R2). (linee scure; aree ombreggiate indicano controlli isotipo) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig1large.jpg" target="_blank">clicca qui per visualizzare un larg</a>versione er di questa figura. <img alt="figura 2" src="/files/ftp_upload/54720/54720fig2.jpg" /> Figura 2: Analisi funzionale di I n Vitro differenziata Ag-specifica Tregs murini IPSC-Treg sono state stimolate da splenociti.;: E pulsata con OVA 323-339 peptide (APC REGS T / APC = 1 4). La produzione di citochine intracellulari (TGF-β e IL-10) è stato analizzato in citometria a flusso dopo gating sulle cellule dal vivo CD4 + CD25 +. (Linee scure; aree ombreggiate indicano controlli isotipo) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/54720/54720fig3.jpg" /> Figura 3: AiPSC-Tregs infiltrarsi articolazioni del ginocchio specifici g. Ag-specifici IPSC-Treg sono stati adoptively trasferiti in topi C57BL / 6. Poco dopo l&#39;artrite induzione (giorni 7 - 14), le ginocchia sono stati rimossi e colorate per Immunoistologia (barre di scala: 20 micron). I topi trattati con OVA-specifici iPSC-Tregs hanno un gran numero di cellule + TCRVβ5 alle ginocchia. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig3large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 4" src="/files/ftp_upload/54720/54720fig4.jpg" /> Figura 4: trasferimento adottivo di Ag-specifici iPSC-Tregs migliora AIA nei topi iPSCs murini sono state trasdotte con il costrutto retrovirale MIDR, MIDR-FoxP3, o MIDR-TCR-FoxP3 e sono stati co-coltura sul OP9-DL1 / DL4 /. cellule B IA. Il giorno 7, le cellule gene-trasdotte (3 x 10 6 / topo) è undoptively trasferiti in femmine C57BL / 6 topi che sono stati indotti con AIA due settimane dopo il trasferimento delle cellule. Il giorno dopo l&#39;induzione artrite, artrite gravità è stata monitorata mediante misurazione del diametro ginocchio. (AC) aumento percentuale del diametro del ginocchio. Aumento del diametro del ginocchio è stato calcolato sulla base di pre-iniezione del diametro ginocchio per ogni mouse prima dell&#39;iniezione al giorno 0. punteggio artrite è stata valutata esaminando entrambe le ginocchia in modo cieco; ogni ginocchio è stato assegnato un punteggio (0: nessun gonfiore visibile o decolorazione; 1: visibile gonfiore con o senza decolorazione; 2: moderata gonfiore con decolorazione; 3: grave gonfiore con scolorimento). In ciascun gruppo, cinque topi sono stati utilizzati, ei dati sono rappresentativi di tre esperimenti indipendenti. I dati sono rappresentati come media ± SD. (D) La media aver eseguito il giorno 7 per entrambe le ginocchia di cinque topi. I dati sono rappresentati come media ± SD di tre esperimenti indipendenti (** p &lt;0,01, *** p &lt;0,001, ANOVA a due vie). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig4large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 5" src="/files/ftp_upload/54720/54720fig5.jpg" /> Figura 5:. Ag-specifici iPSC-Tregs migrare al OVA iniettato ginocchio e ridurre la perdita ossea IPSC-Treg sono stati adoptively trasferito in topi C57BL / 6. Entro 21 giorni post-induzione artrite, i topi sono stati anestetizzati e l&#39;architettura osso intorno alle ginocchia mouse è stato ripreso dal sistema di micro-CT. I topi trattati con OT-II TCR / FoxP3 gene-trasdotte murino IPSC-Tregs presentare una significativa riduzione della perdita ossea, rispetto ai topi di controllo che ricevono iPSCs vettori trasdotte. (A) Le scansioni sono state eseguite con una lunghezza 2,2 millimetri, e le immagini sono state catturate dopo un&#39;ulteriore elaborazione delle immagini (rendering volumetrico e trasformazione;barre di scala:. 1 mm) (B) volume osseo intorno ginocchio è stata valutata utilizzando la ricostruzione tridimensionale di immagini micro-CT, e il volume delle ossa all&#39;interno del volume di interesse è stato fissato (barre di scala:. 1 mm) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig5large.jpg" target="_blank">Si prega di cliccare qui per visualizzare una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity at JoVE.com

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

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

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

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

development-stem-cell-derived-antigen-specific-regulatory-t-cells

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.6K Views.  The Pennsylvania State University College of Medicine. We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model. 

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

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 ...

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Immunologia e infezioni

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

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

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

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

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

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

Killer artificiale cellule presentanti l&#39;antigene (KaAPC) per una efficiente<em&gt; In Vitro</em&gt; L&#39;esaurimento delle Umana antigene-specifiche cellule T

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

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, ...

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

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

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

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

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

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Quantificazione delle cellule CD4 specifiche dell'antigene umano che proliferano utilizzando Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

Generazione ad alta efficienza di cellule T Topo primario specifico dell'antigene per test funzionali in un modello di diabete autoimmune

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8+ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

Le cellule staminali derivate dall'ag virale specifico t linfociti sopprimeno la replica HBV nei topi

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

In Vivo Augmentazione dell'induzione delle cellule T regolatorie Gut-Homing

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

Meningitidis di Neisseria Infezione di cellule endoteliali cerebrali derivate da cellule staminali pluripotenti indotte

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...