Name: development of stem cell-derived antigen-specific regulatory t cells against autoimmunity
Uploaded: 2016-11-08T15:00:00.000+00:00
Duration: 10 min 10 s
Description: Watch this Scientific Journal Video about Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity at JoVE.com

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

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+T+cell+development+and+establishment+of+T+cell+competence+from+embryonic+stem+cells+differentiated+in+vitro.">Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro.</a> Nat Immunol. 5, 410-417 (2004).</li><li>La Motte-Mohs, R. N., Herer, E., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+T-cell+development+from+human+cord+blood+hematopoietic+stem+cells+by+Delta-like+1+in+vitro.">Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro.</a> Blood. 105, 1431-1439 (2005).</li><li>Lei, F., Haque, R., Weiler, L., Vrana, K. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry:+paraffin+sections+using+the+Vectastain+ABC+kit+from+vector+labs.">Immunohistochemistry: paraffin sections using the Vectastain ABC kit from vector labs.</a> J Vis Exp. , (2007).</li><li>Lu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+of+all-trans+retinoic+acid+in+stabilizing+human+natural+regulatory+T+cells+under+inflammatory+conditions.">Critical role of all-trans retinoic acid in stabilizing human natural regulatory T cells under inflammatory conditions.</a> Proc Natl Acad Sci U S A. 111, E3432-E3440 (2014).</li><li>Wu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galectin-9-CD44+interaction+enhances+stability+and+function+of+adaptive+regulatory+T+cells.">Galectin-9-CD44 interaction enhances stability and function of adaptive regulatory T cells.</a> Immunity. 41, 270-282 (2014).</li><li>Di Stasi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+apoptosis+as+a+safety+switch+for+adoptive+cell+therapy.">Inducible apoptosis as a safety switch for adoptive cell therapy.</a> N Engl J Med. 365, 1673-1683 (2011).</li><li>Ramos, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inducible+caspase+9+suicide+gene+to+improve+the+safety+of+mesenchymal+stromal+cell+therapies.">An inducible caspase 9 suicide gene to improve the safety of mesenchymal stromal cell therapies.</a> Stem Cells. 28, 1107-1115 (2010).</li><li>Haque, R., Lei, F., Xiong, X., Wu, Y., Song, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxP3+and+Bcl-xL+cooperatively+promote+regulatory+T+cell+persistence+and+prevention+of+arthritis+development.">FoxP3 and Bcl-xL cooperatively promote regulatory T cell persistence and prevention of arthritis development.</a> Arthritis Res Ther. 12, R66 (2010).</li><li>van Loenen, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixed+T+cell+receptor+dimers+harbor+potentially+harmful+neoreactivity.">Mixed T cell receptor dimers harbor potentially harmful neoreactivity.</a> Proc Natl Acad Sci U S A. 107, 10972-10977 (2010).</li><li>Kim, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+antigen-specific+human+regulatory+T+cells:+immunosuppression+of+FVIII-specific+T-+and+B-cell+responses.">Engineered antigen-specific human regulatory T cells: immunosuppression of FVIII-specific T- and B-cell responses.</a> Blood. 125, 1107-1115 (2015).</li><li>Himburg, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotrophin+regulates+the+expansion+and+regeneration+of+hematopoietic+stem+cells.">Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells.</a> Nat Med. 16, 475-482 (2010).</li></ol>

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

Universidad San Sebastian

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

Regulatory T cells: Therapeutic Potential for Treating Transplant Rejection and Type I Diabetes

My name's Jeff Bluestone. I'm the director of the UCSF Diabetes Center. We're at a laboratory here and oversee the immune tolerance network.My work is involved in basic understanding of how the immune system distinguishes self from non-self and how we can use therapies to try to induce what we call immune tolerance, which is the ability of the immune system being modulated and protect itself from recognizing itself as foreign and attacking things like its own pancreas or brain or joints. That happens in many autoimmune diseases. We're particularly interested in therapies, both drug and cellular therapies that might be used to educate or reeducate the immune system so that we don't block or destroy our own ability To protect our own tissue.So whenever A a T cell comes through the thymus or B cell comes out of the bone marrow, it has to learn to distinguish self from non-self, and this is what we call immune tolerance, which is that ability to tolerate one's own tissue and not destroy it or attack it. What often happens in many disease settings is that the immune system because of environmental and genetic reasons, starts attacking one's own tissue and tolerance is broken and it's that system that we're trying to reestablish with some of our therapies. And the ultimate goal of immune tolerance therapies as opposed to generalized immunosuppression is to try to develop therapies or drugs that will in fact reestablish tolerance without requirement for ongoing therapy.So that today where somebody with a kidney transplant or a liver transplant might require 20 or 30 pills a day to keep the immune system from attacking the, the kidney or pancreas or, or or liver in the future. And we might be able to just provide some kind of therapy that would change the immune system so that it becomes tolerant to the kidney or liver And doesn't reject it. So one of the more Interesting elements of immune tolerance has been to discovery that there are specialized cells in the body whose job it is to protect the organs of the body and and to regulate the immune system so that it doesn't attack those tissues.These so-called regulatory T cells have become a very important and central part of understanding immune tolerance over the last decade or so. In fact, regulatory T cells are often known as suppressor. T cells have been around for 30 years since early discovery and and descriptions by people like Dick Han.Unfortunately, in the eighties, these cells came in some disrepute because of the absence of unique biological or biochemical markers that could distinguish them from other cells and data that was difficult to interpret given our current understanding of how T cells recognize proteins and how the immune system works. But then starting in the mid nineties through seminal work by investigators like Shimon Succi and others, we started to understand both the cellular as well as biochemical basis for regulatory T cells, such that when a transcription factor called Fox P three was discovered in early two thousands, we're able to finally put a specialized tag on these cells and really understand them. And ever since we've really been able to exploit those cells by expanding em, isolating em, and reintroducing em into animals to demonstrate that they are capable of inducing immune tolerance, which is what we are All about.One of the Most important questions that is challenge the field is trying to understand what the mechanism of tregs are, both in immune responses as well as during tolerance induction. And there have been a number of questions related to do the cells act on other T cells that are pathogenic? Do they act on antigen presenting cells or do they make cytokines that ultimately regulate the immune response?Our own studies have suggested based on in vi, in vivo imaging that in fact these cells act directly on the major antigen presenting cell in in the pancreas, and that is a dendritic cell. And we demonstrated that in fact we could label regulatory T cells one color green while the dendritic cells were labeled another color, and show that in fact these regulatory T cells interacted directly with the dendritic cells and by doing so, fundamentally altered the ability of those dendritic cells to activate the pathogenic T cell response. So we think that either directly or through some intermediate cytokine like IL 10 or TGF beta that the tregs educate or change the dendritic cells to make them less able to activate a pathogenic T cell response.Yeah, So one of the more interesting aspects of regulatory T cells has been the notion that in addition to this very specialized subset that exists in the thymus and develops during thymic development, the so-called natural T-Rex that during the course of an immune response, other T cells may in fact turn into regulatory T cells either through direct upregulation of Fox P three due to cytokines like TGF beta, which are known to turn on FOX P three or even other cells which are not Fox P three positive, but take on regulatory T-cell characteristics. Those so-called adaptive tregs are generally very important in in regulating an inflammatory response because they're interacting with the antigens that are present locally in that inflamed tissue. And those adaptive cells are probably critical in maintaining tolerance locally in a targeted tissue, whereas the natural tregs are probably more useful or important in maintaining a, a, a immune homeostasis or generalized suppression of auto reactivity in the whole animal Or patient.So now that we know That these regulatory cells are essential in in maintaining immune homeostasis, we can imagine what would happen if they're disrupted. And in fact, if you destroy Fox P three and either mouse models using knockout technology, or in fact there's a human disease called I pex, the individual animals die in a short period of time of a massive autoimmune disease where each targeted tissue, liver, kidney, heart are in infiltrated with T cells and the tissues are destroyed. So that's the fundamental immuno immune homeostasis that's required that these cells Really protect the individual.One of the important Discoveries with regards to regulatory T cells is the possibility that we might be able to both isolate them and expand them in a specific way to reintroduce into individuals with diseases to bring the immune system back into a homeostatic equilibrium. And in fact, work in our laboratory and others have been able to take these cells out and expand them up to a thousand fold and show that they retain their suppressor activity and can be reintroduced into individual animals to induce an a regulatory response. Where that's been most successful at, at this time has been in treating autoimmune disease and animal models of diabetes, for instance, we can prevent or even reverse diabetes by putting in small numbers of these antigen specific eyelid antigen specific regulatory T cells that we've expanded ex vivo and we hope that we can get into people soon with similar populations that have been expanded in in a similar manner.Now the future hopefully will go beyond autoimmune diseases and into more broad based immunological settings and perhaps the most daunting of which is transplantation. Today we transplant over a half a dozen organs into people, but still are required to give them lifelong immunosuppression to prevent those transplants from being rejected. The hope is that we can take regulatory cells that see the donor tissue from these kidney, liver, pancreas, eyelet transplants and be able to target those regulatory cells to those tissues and prevent the immune system from recognizing those tissues as far and now take this alloimmune response and convert it into a safe self response and prevent rejection without ongoing immunosuppressive Drug therapy.So What we hope is, is that this particular therapy is going to be very appropriate, appropriate for treating people with type one diabetes. Once all of the insulin producing beta cells have been destroyed, the only way we're ultimately gonna be able to treat the disease is by both reconstituting the individual with an eyelet or some insulin producing cell, and at the same time prevent the autoimmune and alloimmune responses from destroying it. So our approach is to take people with long-term diabetes and transplant eyelets at present from cric donors, but in the future, perhaps from stem cells or other sources to actually replace the insulin producing capability, we hope to introduce regulatory T cells into that setting to prevent those eyelets that are coming from foreign sources, from being rejected.As I mentioned, this notion that we can induce tolerance in these individuals will require not just the source of an insulin producing cell, but some combination of drugs and cellular therapies. We hope like regulatory cells to put the individual into a state of tolerance in homeostasis so that these foreign tissues are not recognized and destroyed once introduced into the diabetic patient. Specifically, our approach to actually reconstituting insulin producing beta cells in patients starts out with the kind of experiments we're doing in animal models, which is to take eyelets that have been prepared from a pancreas, remembering that only about 0.5%of all of the cells in the pancreas actually are the eyelets to make insulin to isolate those cells and purify them in a way that we can reintroduce them into a recipient.We reintroduce them either by putting them under the kidney capsule, which is routinely the way we do it in animal models are directly into the liver, which is the way we do it in humans. And once those cells go into that site, either kidney or liver, they then seed that site and start getting the blood and other nutrients that they need in order to survive and make the insulin that then gets circulated to other tissues and organs as needed. And the goal is to obviously, once those cells are in that tissue, to then treat with our various immunotherapeutics to prevent the immune system from attacking the cells Within those tissues.So of course there are a Number of challenges for all novel immunotherapies and tregs are no exception. We need to worry about a number of things as we try to this for a therapeutic. We need to worry about cell numbers at present, that we can only identify small numbers of cells that have the antigen specificity that we need to block both auto and allo immunity.So we'll need to figure out ways to both isolate those cells and expand them to sufficient numbers to treat an individual. In addition, we need to be careful that these cells are truly pure regulatory T cells. We don't wanna make the mistake of putting in cells that have the ability to be pathogenic and attack the foreign tissue as opposed to the regulatory cells which protect.And so understanding the stability and the robustness of these cells and how how they survive in various inflammatory settings will be critical. The third thing we need to worry about is non-specific immunosuppression that could be caused. These regulatory cells are designed to shut down broadly in immune response.And so we don't want to, at the same time we're blocking graft rejection, block the ability of an individual to recognize a bacteria or a virus or cancer cells that might come up, but instead stay targeted and focused on the tissue that we've transplanted into the individual. And then finally, I think the, the other challenge with any cellular therapy is being able to do it robustly and routinely many, many, many times over because each patient is gonna require their own set of cells. This is not a drug you can just take off the shelf.And so we're gonna have to work technologies out that are, use good manufacturing practices that are approved by the food and drug administration that allow us to grow these up the same way every time and be sure that we're working with a, a truly homogeneous and and well-defined population of Cells. When one Thinks about funding for projects like this, it's always a mix between the federal funding that we get through the NIH to support the basic science that's important in moving this forward. And then for us, fortunately, other organizations such as the Juvenile Diabetes Foundation who have been very supportive having raised an enormous amount of money from the community to help in these highly risky projects.And so we've been fortunate in being able to partner with both the NIH and JDRF as well as with pharmaceutical companies on occasion to be able to move these kinds of projects along. So at present, I think the funding is actually adequate and we really hope that we will be able to drive this project forward Effectively. We've been lucky in the diabetes community having such a strong, both federal and non-federal interest in the disease.Of course, that's not inappropriate given the fact that disease is really growing and reaching epidemic proportions, but we hope that the funding that exists will be sufficient for us to move This project. So Like all scientists, we always feel as if we are underfunded and underappreciated when you look at how much money is being spent to, to deal with problems around the world, to deal with war in Iraq, to deal with our, our military, and know that just a couple of days of that funding could support the NIH for a year. It's hard to imagine how we've placed our priorities because we think as scientists that there are a lot of things that we can do to change human health, human, the human state.So we only continue to hope that the advances in the science look in in 50 years. We're not gonna be talking about health insurance, we're not gonna be talking about HMOs and management. We're gonna talk about the amazing discoveries that have been made that are gonna change health in in our country that's gonna change health in the world.And just like the industrial revolution of turn of the last century, that in this century the, the major impact is gonna be on how we've changed diseases, both acute and chronic diseases. And so we hope that the public appreciates that and appreciates our efforts to try to make that kind of Difference.

Cellule T regolatorie: potenziale terapeutico per terapia antirigetto nei trapianti e diabete di tipo I

Ricerca

Biologia

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

Un sistema di coltura timica tridimensionale per generare emigrati timici derivati da murine

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Ricerca sul cancro

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 ...

Immunologia e infezioni

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Differenziazione Regia di cellule staminali pluripotenti indotte verso Linfociti T

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

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

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

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

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

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

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

Trasduzione Adenoviral di CD4 T naive Cells allo Studio Differenziazione Treg

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

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 ...

Generation of Human Regulatory T Cell Clones

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

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

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

<meta charset="utf8"></head><body>Ingegnerizzazione ed espansione delle Treg CAR umane per terapie di regolazione immunitaria

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4+ T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs&#39; immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.

<meta charset="utf8"></head><body>Generazione di cellule T regolatorie del recettore dell'antigene chimerico umano

This protocol provides a streamlined workflow to generate and test human chimeric antigen receptor regulatory T cells (CAR Tregs).

Generazione di cellule T regolatorie del recettore dell'antigene chimerico umano

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable ...