We thank Miltenyi Biotec, Germany for providing reagents and CliniMACS Prodigy equipment for evaluation studies. We thank George T. McNamara (Pediatric department, MD Anderson Cancer Center) for proof reading the manuscript. Grant support: Cancer Center Core Grant (CA16672); RO1 (CA124782, CA120956, CA141303; CA141303); R33 (CA116127); P01 (CA148600); Burroughs Wellcome Fund; Cancer Prevention and Research Institute of Texas; CLL Global Research Foundation; Estate of Noelan L. Bibler; Gillson Longenbaugh Foundation; Harry T. Mangurian, Jr., Fund for Leukemia Immunotherapy; Institute of Personalized Cancer Therapy; Leukemia and Lymphoma Society; Lymphoma Research Foundation; MDACC&#8217;s Sister Institution Network Fund; Miller Foundation; Mr. Herb Simons; Mr. and Mrs. Joe H. Scales; Mr. Thomas Scott; National Foundation for Cancer Research; Pediatric Cancer Research Foundation; William Lawrence and Blanche Hughes Children's Foundation.

1. Préparation du matériel dans des conditions stériles (Voir Tableau Matériaux et Équipement) <ol><li> Préparation 3 L de tampon PBS / EDTA additionné de sérum-albumine humaine (HSA) à une concentration finale de 0,5% (p / v). </li><li> Préparer 1 L sac de classe 0,9% de chlorure de sodium (NaCl) clinique et 2 L de qualité GMP milieu de culture cellulaire. </li><li> Préparer 60 nmol de CMV spécifiques à l&#39;antigène peptidique cocktail en reconstituant un flacon de CMV pp65 avec 8 ml d&#39;eau stérile. </li><li> Transfert CMV pp65 peptide cocktail dans un sac de congélation de 50 ml de volume en utilisant un / de Spike interconnexion Luer et serrer avec une pince de verrouillage pour éviter une distribution ultérieure de cocktail dans le jeu de tuyaux. Ouvrir cellule tube enrichissement jeu (TS 500) dans des conditions stériles. </li><li> Utilisant un tube stérile soudeur, connectez peptide cocktail sac de congélation en connexion de tube pour valve 2 du jeu de tuyaux TS 500. Ne pas ouvrir le sac pince peptide cocktail à cette époque. </li><li> Retirer 1 x 10 9 TNC à partir de produit de départ et de suspension cellulaire dans du tampon PBS / EDTA contenant 2,5% de HSA à un volume total de 50 ml. Injecter le produit cellulaire dans un sac de transfert de 150 ml. </li></ol> 2. Préparation et utilisation de cellules automatisé Système d&#39;enrichissement (Voir Tableau Matériaux et Équipement) <ol><li> Allumez le système d&#39;enrichissement de cellules (figure 1B) et sélectionnez le programme &quot;CCS_IFN-γ enrichissement&quot;. Observez une interface utilisateur montrant des écrans avec des instructions et des photos directeurs l&#39;opérateur à travers la procédure. </li><li> Entrez le paramètre &quot;Opérateur&quot; et &quot;Tubing Set P / N Non&quot;. Ensuite, installez le dispositif Tubing Set 500 à l&#39;enrichissement de cellules automatisé selon les instructions affichées sur l&#39;écran du moniteur interactif. </li><li> Suivez les instructions étape par étape affichées sur l&#39;écran pour connecter le moyen et les tampons à l&#39;appareil. Numéro de catalogue et le numéro de lot des réactifs avant connecting à l&#39;instrument. </li><li> Après le contrôle final de l&#39;ensemble de tubes, ouvrir la pince du sac peptide cocktail. Ouvrez le sac moyen et initier amorçage automatique de l&#39;ensemble de tubes. </li><li> Après l&#39;étape d&#39;amorçage est terminée, compléter HSA (2,5%) dans un tampon de NaCl dans le sac de réservoir (200 ml) à l&#39;aide de la soudeuse tube stérile. Transférer le produit cellulaire à partir dans le &quot;sac de l&#39;application&quot; en utilisant le tube soudeur stérile. </li><li> Connectez CCS (IFN) réactifs dans les tubes respectifs via des adaptateurs. Entrez le temps préféré pour recueillir une fraction de matériau cellulaire avant que le processus d&#39;enrichissement. Examiner et de vérifier l&#39;exactitude de toutes les données / paramètres entrés. Lancer le processus. </li><li> Avant le début du processus automatisé d&#39;enrichissement de cellules, enlever le sac Quality Control (BRC, la fraction originale (ori) contient environ 1,3 ml de 100 ml le contenu de la chambre diluée avec un tampon / EDTA PBS). Sceller le BRC, peser et stocker à 4 ° C. </li><li> Démarrez le enrichmeprocessus nt. A la fin du processus, les cellules cibles sont éluées avec un volume approximatif de tampon d&#39;élution à partir de la poche de réservoir. </li><li> Sceller la cellule cible Sac non (NTCB, fraction négative = neg) et Sac cellulaire cible (TCB, positif fraction = pos) et peser chaque sac. Les poids seront utilisés ultérieurement pour le calcul des nombres de cellules. </li><li> Immédiatement après la procédure d&#39;enrichissement recueillir deux aliquotes par fraction de cytométrie de flux, et stocker le reste des échantillons à 4 ° C. Utilisez une aliquote de l&#39;échantillon pour la détermination de la numération cellulaire et l&#39;autre partie aliquote de l&#39;échantillon pour l&#39;analyse des performances de l&#39;enrichissement (tableau 1). </li><li> Retirer le tube fixé à partir de l&#39;instrument d&#39;enrichissement cellulaire. Transférez le fichier journal sur un lecteur USB pour une utilisation future. REMARQUE: Tous les réactifs doivent être préparés dans des conditions stériles. L&#39;utilisation d&#39;une hotte de biosécurité de type II est fortement recommandé. Utilisez aphérèse produit cellulaire à l&#39;état stable (non-mobilisés) isolé à partir d&#39;une bonne santéCMV-séropositif donateurs pour enrichir les cellules T spécifiques de l&#39;antigène CMV. Seulement autorisé par la FDA HSA doit être utilisé. Le tampon pour la préparation de la cellule doit être maintenue à 19 ° C à 25 ° C alors que les températures ambiantes inférieures ou supérieures se traduira par la pureté réduite et un rendement réduit des cellules cibles. </li></ol> 3. Détermination comte cellulaire <ol><li> Prenez les aliquotes de QCB, NTCB et TCB pour la numération des cellules, comme indiqué dans le tableau 1. Ajouter CD45-VoBlue à chaque aliquote (titre 01h11) et incuber dans l&#39;obscurité pendant 10 min à 4 ° C. </li><li> Ajouter 1,5 ml de solution de globules rouges de lyse fraîchement préparé (1x) à la fraction initiale et la fraction négative, à 450 ul fraîchement préparé solution de lyse des globules rouges à la fraction positive, et incuber toutes les fractions de 15 min à température ambiante. </li><li> Juste avant l&#39;analyse, ajouter l&#39;iodure de propidium à une concentration finale de 1 pg / ml (dilution 1: 100 de 100 ug / ml). Utilisez compteur de cellules automatique pour déterminer le nombre de cellules et vicapacité. Utilisez dispositif de compteur de cellules logiciel recommandé pour cytométrie de flux. Déterminer les valeurs absolues de leucocytes pour les fractions originaux, négatifs et positifs. NOTE: Le nombre de cellules de leucocytes viables par ml des échantillons prélevés pour l&#39;analyse de numération cellulaire est déterminée en utilisant le logiciel de l&#39;analyseur recommandé cellulaire. </li><li> Réglez la région comme représenté sur la Figure 2 (région 5, leucocytes viables). Utilisez la stratégie de déclenchement suivant pour déterminer le nombre de cellules. Une fraction de leucocytes viables d&#39;origine est représenté sur la figure 2. </li><li> Les régions indiquées (figure 2, 6.1) sont hiérarchiquement comme suit: 1: Temps porte → 2: cellules individuelles → 3: CD45 + cellules → 4: leucocytes (débris exclu) → 5: leucocytes viables → 6: lymphocytes viables </li><li> Répétez les mêmes étapes pour déterminer le nombre de cellules pour les fractions positives et négatives. Calculer le nombre de cellules de la fraction ensembleen considérant le facteur de dilution de l&#39;échantillon et le volume total de la fraction (tableau 2). </li></ol> 4. Examen de la performance de séparation <ol><li> Laver les aliquotes de QCB, NTCB et les cellules de la fraction TCB avec / EDTA mémoire tampon de pré-réfrigérés PBS / 0,5% de sérum AB. Centrifuger les cellules à 300 g pendant 5 min à 4 ° C et aspirer le surnageant. </li><li> Resuspendre les cellules dans le mélange de coloration 100 pi anticorps fluorochrome contenant: CD3-FITC, CD4-APC, CD8-APC-Vio770, CD14-PerCP, CD20-PerCP, CD45-VioBlue et anti-IFN-PE (titre 01h11) et incuber dans l&#39;obscurité pendant 10 min à 4 ° C. </li><li> Ajouter 1 ml de solution de globules rouges de lyse fraîchement préparé (1x) et incuber pendant 15 min à température ambiante. Centrifugeuse à 300 g pendant 5 min à 4 ° C et aspirer le surnageant. Remettre en suspension les cellules dans un volume suffisant de PBS / EDTA / 0,5% de sérum AB. </li><li> Ajouter l&#39;iodure de propidium à une concentration finale de 1 pg / ml juste avant l&#39;analyse (1: 100 dilutisur 100 ug / ml). Effectuer l&#39;analyse de cytométrie de flux pour évaluer la pureté de l&#39;échantillon. </li><li> Utilisez la stratégie de déclenchement suivante pour calculer les lymphocytes T CD3 + dans la stratégie de vannage de leucocytes viables pour déterminer les lymphocytes T CD3 + est montré dans la fraction positive après processus d&#39;enrichissement de CCS. Les régions indiquées (Figure 3A et 3B, 1-6) sont liés hiérarchiquement comme suit: 1: Temps de porte → 2: cellules individuelles → 3: CD45 + cellules → 4: Cellules (débris exclu) → 5: leucocytes viables → 6: CD3 + cellules viables population </li><li> Déterminer les fréquences de CD4 +, CD8 +, CD4 + IFN-γ + et CD8 + IFN-γ + cellules T après processus d&#39;enrichissement CCS (tableau 2). </li><li> Utiliser la stratégie de déclenchement pour déterminer les fréquences de cellules CD4 +, CD8 +, CD4 + IFN-γ + et CD8 + IFN-γ + cellules T au-dessous fou un (capturé) fraction positive original et enrichi après le processus de CCS. Les régions indiquées sont hiérarchiquement liés et nommés comme suit: 1: Temps de porte → 2: cellules individuelles → 3: CD45 + cellules → 4: Cellules (débris exclu) → 5: leucocytes viables → 6: CD3 + cellules viables → 7: cellules CD4 + → 7a: CD4 + IFN-γ + cellules (encadré) → 8: cellules CD8 + → 8a: CD8 + IFN-y + cellules (encadré) </li></ol> REMARQUE: Les 6 premières régions des liens hiérarchiques indiqués sont les mêmes que sur la figure 3, (6.1) et des 2 dernières régions sont présentés dans la figure 4 (6-8a).

<ol>
	<li>Syed, B. A., Evans, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+the+Analyst's+Couch+Stem+Cell+Therapy+Market.">From the Analyst's Couch Stem Cell Therapy Market.</a> Nat Rev Drug Discov. 12 (3), 185-186 (2013).</li><li>Maus, M. V., 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=Adoptive+Immunotherapy+for+Cancer+or+Viruses.">Adoptive Immunotherapy for Cancer or Viruses.</a> Annu Rev Immunol. 32, 189-225 (2014).</li><li>Kumaresan, P. R., 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=Bioengineering+T+cells+to+target+carbohydrate+to+treat+opportunistic+fungal+infection.">Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection.</a> Proc Natl Acad Sci U S A. 111 (29), 10660-10665 (2014).</li><li>Singh, H., 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=Redirecting+specificity+of+T-cell+populations+for+CD19+using+the+Sleeping+Beauty+system.">Redirecting specificity of T-cell populations for CD19 using the Sleeping Beauty system.</a> Cancer Res. 68 (8), 2961-2971 (2008).</li><li>Kumaresan, P. 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Rise of Cell-Based Immunotherapy : Personalized Medicine Takes Next Step Forward.</a> Genetic Engineering &amp; Biotechnology News. 33 (5), 12-13 (2013).</li><li>Apel, 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=Integrated+Clinical+Scale+Manufacturing+System+for+Cellular+Products+Derived+by+Magnetic+Cell+Separation,+Centrifugation+and+Cell+Culture.">Integrated Clinical Scale Manufacturing System for Cellular Products Derived by Magnetic Cell Separation, Centrifugation and Cell Culture.</a> Chem-Ing-Tech. 85 (1-2), 103-110 (2013).</li><li>Brestrich, G., 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=Adoptive+T-Cell+Therapy+of+a+Lung+Transplanted+Patient+with+Severe+CMV+Disease+and+Resistance+to+Antiviral+Therapy.">Adoptive T-Cell Therapy of a Lung Transplanted Patient with Severe CMV Disease and Resistance to Antiviral Therapy.</a> Am J Transplant. 9 (7), 1679-1684 (2009).</li><li>Feuchtinger, T., 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=Clinical+grade+generation+of+hexon-specific+T+cells+for+adoptive+T-cell+transfer+as+a+treatment+of+adenovirus+infection+after+allogeneic+stem+cell+transplantation.">Clinical grade generation of hexon-specific T cells for adoptive T-cell transfer as a treatment of adenovirus infection after allogeneic stem cell transplantation.</a> J Immunother. 31 (2), 199-206 (2008).</li><li>Peggs, K. 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Les deux MD Anderson Cancer Center et le Dr Cooper ont un intérêt financier dans ZIOPHARM Oncology, Inc., et Intrexon Corporation. Le 7 mai, 2015, le Dr Cooper a été nommé chef de la direction au ZIOPHARM oncologie. Dr Cooper est maintenant un chercheur invité au MD Anderson. Le Dr Cooper a fondé et possède InCellerate, Inc. Il possède des brevets avec Sangamo BioSciences avec nucléases artificielles. Il consulte Targazyme, Inc. (cellules souches anciennement américains, Inc.), GE Healthcare, Ferring Pharmaceuticals, le destin Therapeutics, Janssen Pharmaceuticals et Bristol-Myers Squibb. Il est membre du conseil consultatif scientifique de Cellectis. Il reçoit des honoraires de Miltenyi Biotec.

La thérapie des cellules T adoptive a émergé comme une option viable pour traiter les tumeurs malignes à cellules B 4. Son potentiel thérapeutique dépend de l&#39;infusion le nombre désiré de cellules T spécifiques de l&#39;antigène cible sénescence réplicative qui manquent deux. Ceci peut être obtenu par le tri d&#39;une population pure de cellules T spécifiques de l&#39;antigène de lymphocytes T expansés en conformité avec les pratiques de fabrication actuelles bons. Deux procéd...

Hématopoïétiques transplantation de cellules souches (HSCT) 1 peut être combiné avec la thérapie des cellules T adoptive pour améliorer l&#39;effet du greffon contre la tumeur et de fournir l&#39;immunité aux infections opportunistes 2. Génération de cellules T donateurs dérivés spécifiques de l&#39;antigène pour perfusion a historiquement nécessaire du personnel et l&#39;utilisation des installations spécialisées qui sont conformes aux BPF qualifiés. La livraison de ces cellules T a abouti à la résolution des infections opportunistes 3 ​​ainsi que le traitement de la maladie sous-jacente 4. Récemment, des chercheurs ont démontré que le transfert adoptif de seulement quelques milliers de cellules T spécifiques du virus (~ 1 x 10 4 à 2,5 x 10 5 cellules / kg de poids corporel du receveur) peuvent réussir à traiter les infections à CMV opportunistes après allogéniques HSCT 5-9. Un nombre limité d&#39;installations GMP aux exigences de fabrication qualifiés associés et le coût élevé associé à la production de cellules a, cependant, restrl&#39;accès des patients à des thérapies prometteuses igés cellules T 10. Une approche pour isoler des cellules T spécifiques d&#39;antigène est basée sur le CCS en utilisant un réactif bi-spécifique pour reconnaître CD45 et IFN-γ. Comme on le voit, cette méthodologie peut être utilisée pour générer des cellules T spécifiques de CMV de qualité clinique utilisant un dispositif d&#39;enrichissement de cellules automatisé CCS (figure 1B). Des cellules T spécifiques de CMV sont générés par incubation de peptides chevauchants à partir de l&#39;antigène pp65 de CMV avec des cellules de leucaphérèse totales (TNC) nucléaires provenant de donneurs CMV-séropositif. Ces peptides, affichés dans le contexte de l&#39;antigène de leucocyte humain (HLA), activent les cellules T spécifiques de CMV pp65 au sein du TNC à sécréter IFN-γ. Ces cellules T peuvent alors être &quot;capturés&quot; et séparées magnétiquement. Le fonctionnement du dispositif d&#39;enrichissement de cellules de première génération (figure 1A) requis personnel qualifié dans la culture cellulaire dans des conditions GMP, et la coordination du personnel pour entreprendre les multiples sTeps nécessaire pour générer un produit &quot;capturé&quot;. La procédure généralement requis 10 à 12 h de fonctionnement continu, et donc le personnel probablement besoin de travailler sur deux quarts de travail dans l&#39;établissement GMP. Ces contraintes sont désormais évités par la mise en œuvre d&#39;un dispositif de deuxième génération (le montre la figure 1B). Ce dispositif engage enrichissement magnétique, similaire au premier dispositif de génération, mais automatise les autres aspects de la SCC dans une approche unbreached. Cela réduit considérablement le fardeau de l&#39;équipe GMP que la plupart des étapes peut être accompli sans surveillance par le personnel. En outre, puisque le dispositif fonctionne comme un système fermé, les cellules T spécifiques de l&#39;antigène peuvent être capturées et traitées sur la paillasse à l&#39;exception des étapes impliquées dans l&#39;isolement de leucaphérèse et préparation des matières de départ avant de l&#39;instrument. Détails de l&#39;instrumentation complète et la fonctionnalité de ce dispositif d&#39;enrichissement cellulaire de deuxième génération ont été pubblies 11. Ici, nous décrivons les étapes pour enrichir les cellules T spécifiques de pp65 CMV d&#39;un produit d&#39;aphérèse l&#39;état d&#39;équilibre en utilisant le système de CCS d&#39;enrichissement de cellules automatisé. Une fois isolé, ces cellules T spécifiques de CMV peuvent être immédiatement perfusé dans un patient.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>CliniMACS PBS/EDTA Buffer 3 L bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>700-29</td>
			<td></td>
		</tr>
		<tr>
			<td>CliniMACS Prodigy Tubing Set TS 500</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-097-182</td>
			<td></td>
		</tr>
		<tr>
			<td>5 L waste bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>110-004-067</td>
			<td></td>
		</tr>
		<tr>
			<td>CliniMACS Cytokine Capture System (IFN-gamma)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>279-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin (Human) 25%&#160;</td>
			<td>Grifols</td>
			<td>58516-5216-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer/Spike Interconnector</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-018-701</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9 % NaCl Solution (1 L)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MACS GMP PepTivator HCMV pp65</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>170-076-109</td>
			<td></td>
		</tr>
		<tr>
			<td>Water for injections</td>
			<td>Hospira, inc, Lake Forest, IL</td>
			<td>NDC-0409-4887-10</td>
			<td></td>
		</tr>
		<tr>
			<td>MILLEX GV Filter Unit 0.22 &#956;m&#160;</td>
			<td>Millipore</td>
			<td>SLGV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>TexMACS GMP Medium 2 L bag</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>170-076-306</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Bag, 150 mL (for cellular starting material)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-018-301</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoMACS Freezing Bag 50</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>200-074-400</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mL Syringes, sterile</td>
			<td>BD, Laagstraat, Temse, Belgium</td>
			<td>309653</td>
			<td></td>
		</tr>
		<tr>
			<td>CMV sero positive apheresis product</td>
			<td>Key Biologics, LLC, Memphis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry Materials</td>
			<td>Manufacturer</td>
			<td>Catalog number</td>
			<td></td>
		</tr>
		<tr>
			<td>AB Serum, GemCell</td>
			<td>Gemini Bio-Products, West Sacramento, USA</td>
			<td>100-512</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-FITC</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-080-401</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4-APC</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-033</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8-APC-Vio770</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-065</td>
			<td></td>
		</tr>
		<tr>
			<td>CD14-PerCP</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-072</td>
			<td></td>
		</tr>
		<tr>
			<td>CD20-PerCP</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-077</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45-VioBlue</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-098-136</td>
			<td></td>
		</tr>
		<tr>
			<td>aIFN-&#947;-PE, human</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-097-940</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3-PE</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-091-374</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide Solution (100 &#181;g/mL)</td>
			<td>Miltenyi Biotec GmbH</td>
			<td>130-093-233</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>Manufacturer</td>
			<td>Catalog Number</td>
			<td></td>
		</tr>
		<tr>
			<td>CliniMACS Prodigy Device&#160;</td>
			<td rowspan="2">Miltenyi Biotec GmbH</td>
			<td rowspan="2">200-075-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Software V1.0.0.RC</td>
			<td></td>
		</tr>
		<tr>
			<td>MACSQuant Analyzer 10</td>
			<td rowspan="2">Miltenyi Biotec GmbH</td>
			<td rowspan="2">130-096-343</td>
			<td></td>
		</tr>
		<tr>
			<td>Software 2.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5415R&#160;</td>
			<td>Eppendorf AG</td>
			<td>22331</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellometer K2</td>
			<td>Nexelom Bioscience, Lawrence, MA</td>
			<td>LB-001-0016</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tubing welder SCDIIB</td>
			<td>Terumo Medical Corp., Elkton, MA</td>
			<td>7811</td>
			<td></td>
		</tr>
	</tbody>
</table>

automated cell enrichment of cytomegalovirus-specific t cells for clinical applications using the cytokine-capture system

Le transfert adoptif de lymphocytes T spécifiques de l&#39;agent pathogène peut être utilisé pour prévenir et traiter les infections opportunistes telles que le cytomégalovirus (CMV) survenant après allogéniques hématopoïétiques transplantation de cellules souches. Cellules T virales spécifiques de donneurs allogéniques, y compris les bailleurs de fonds tiers, peuvent être propagés ex vivo en conformité avec les bonnes pratiques de fabrication (cGMP), employant des cycles répétés de stimulation conduit antigène pour propager sélectivement les cellules T souhaités. L&#39;identification et l&#39;isolement des cellules T spécifiques de l&#39;antigène peuvent également être effectuées sur la base du système de capture de cytokine des cellules T qui ont été activées pour sécréter de l&#39;interféron-gamma (IFN-γ). Cependant, l&#39;application humaine généralisée du système cytokine de capture (CCS) pour aider à rétablir l&#39;immunité a été limitée que le processus de production prend du temps et nécessite un opérateur qualifié. Le développement d&#39;un dispositif d&#39;enrichissement cellulaire de deuxième génération tels que CliniMACS Prodigy maintenantpermet aux enquêteurs de générer des cellules T-viraux spécifiques en utilisant un système de main-d&#39;œuvre moins automatisé. Ce dispositif sépare magnétiquement marqué cellules de cellules non marquées à l&#39;aide de la technologie de tri activé magnétique de cellules pour générer des produits de qualité clinique, est conçu comme un système fermé et peut être consulté et utilisé sur la paillasse. Nous démontrons le fonctionnement de ce nouveau dispositif d&#39;enrichissement de cellules automatisé pour la fabrication de cellules T spécifiques de pp65 CMV obtenus à partir d&#39;un produit d&#39;aphérèse l&#39;état d&#39;équilibre obtenu à partir d&#39;un donneur séropositif CMV. Ces cellules T isolées peuvent ensuite être directement perfusé à un patient sous la supervision réglementaire et institutionnel fédéral. Toutes les étapes de traitement, y compris la bio-élimination des cellules rouges du sang, la stimulation des cellules T, la séparation des cellules T spécifiques de l&#39;antigène, la purification et le lavage sont entièrement automatisés. Les dispositifs tels que ce soulèvent la possibilité que les cellules T pour application humaine peuvent être fabriqués à l&#39;extérieur de bonnes pratiques de fabrication dédié (GMP) Et des installations au lieu être obtenus dans des installations bancaires de sang où le personnel peut superviser les protocoles automatisés pour produire des produits multiples.

Dans cette étude, une cellule d&#39;enrichissement du système automatisé CCS a été utilisée pour la production automatisée de cellules T spécifiques de pp65 du CMV. Des cellules T spécifiques de CMV ont été enrichies à partir de trois produits d&#39;aphérèse de cellules. Le produit d&#39;aphérèse l&#39;état d&#39;équilibre a été récolté plus de 2 h à partir d&#39;un donneur CMV-séropositif et a généré 10 10 cellules nucléaires totales (TNC). 10 9 TNC ont ensuite été ac...

Watch this Scientific Journal Video about Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System at JoVE.com

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

Cellules T d&#39;enrichissement de cellules automatisé de cytomégalovirus spécifique pour des applications cliniques utilisant le système de capture de cytokine

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection ...

automated-cell-enrichment-cytomegalovirus-specific-t-cells-for

Research

JoVE Journal

Immunology and Infection

12.3K vues.  U.T. MD Anderson Cancer Center. The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

Vidéo: Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Transduction rétrovirale des récepteurs de cellules T dans les lymphocytes T de souris

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Recherche

Immunologie et infection

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Après Cell-sort<em&gt; E. coli</em&gt; Après infection par le phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

piggyBac Transposon System Modification of Primary Human T Cells

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

piggyBac Transposon System Modification of Primary Human T Cells

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

Modification piggyBac système transposon des cellules T humaines primaires

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

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

Peptide: CMH tétramère basée sur l&#39;enrichissement des cellules T spécifiques d&#39;épitopes

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Quantitative à haut débit Test de cytotoxicité cellulaire unique pour les cellules T

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

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

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

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

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

Application clinique de<em&gt; Belle au Bois Dormant</em&gt; Et artificielle cellules présentatrices d&#39;antigènes pour modifier génétiquement des cellules T du sang de cordon ombilical et périphériques

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

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

La migration des cellules dendritiques de surveillance utilisant l&#39;imagerie par résonance 19F / 1H magnétique

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

L&#39;évaluation de l&#39;Innate détection du VIH-1 CD4 infectés<sup&gt; +</sup&gt; Cellules T par les cellules dendritiques plasmacytoïdes Utilisation d&#39;un<em&gt; Ex vivo</em&gt; Système de co-culture.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&amp;#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Souris Naïf CD4<sup&gt; +</sup&gt; Isolement des cellules T et<em&gt; In vitro</em&gt; Différenciation en sous-ensembles de cellules T

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

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

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

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