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. Herstellung des Materials unter sterilen Bedingungen (siehe Materialien und Geräte Tabelle) <ol><li> Vorbereitung 3 l PBS / EDTA-Puffer mit humanem Serumalbumin (HSA) ergänzt, um eine Endkonzentration von 0,5% (w / v). </li><li> Bereiten Sie 1 L Tasche von klinischen Grade 0,9% Natriumchlorid (NaCl) -Lösung und 2 l GMP Grade-Zellkulturmedium. </li><li> Bereiten Sie 60 nmol von CMV-spezifischen Peptid-Antigen-Cocktail durch Rekonstitution eines Fläschchens CMV pp65 mit 8 ml sterilem Wasser. </li><li> Bringen CMV pp65-Peptid-Cocktail in ein 50 ml Volumen Gefrierbeutel mit einem Luer / Spike Verbindungsleitung und klemmen mit Sicherungszange an nachfolgende Verteilung der Cocktail in das Schlauchsystem zu vermeiden. Offene Zellanreicherung Schlauchsatz (TS 500) unter sterilen Bedingungen. </li><li> Mit einer sterilen Schlauchschweißgerät, schließen Peptidcocktail Gefrierbeutel in Rohranschluss für Ventil 2 der Schlauchsatz TS 500, die das Peptid-Cocktail Beutelklemme zu diesem Zeitpunkt nicht öffnen. </li><li> 1 Entfernen x 10 9 TNC aus Ausgangszellprodukt und setzt in PBS / EDTA-Puffer, enthaltend 2,5% HSA mit einem Gesamtvolumen von 50 ml. Spritzen Sie das Zellprodukt in einen 150 ml Transferbeutel. </li></ol> 2. Herstellung und Einsatz von automatisierten Zellanreicherung System (siehe Materialien und Geräte Tabelle) <ol><li> Schalten Sie den Zellanreicherungssystem (1B), und wählen Sie das Programm &quot;CCS_IFN-γ Enrichment&quot;. Beobachten eine Benutzerschnittstelle, die Siebe mit Anweisungen und Abbildungen Führung der Bedienungsperson durch das Verfahren. </li><li> Geben Sie den Parameter &quot;Operator&quot; und &quot;Tubing Set P / N Nein&quot;. Als nächstes installieren Sie das Tubing Set 500 um automatisierten Zellanreicherung Gerät gemäß den Anweisungen auf dem interaktiven Bildschirm angezeigt. </li><li> Befolgen Sie die Anweisungen Schritt für Schritt auf dem Bildschirm angezeigt, um das Medium und Puffer an das Gerät anschließen. Nehmen Sie Katalognummer und Chargennummer der Reagenzien vor connecting an das Instrument. </li><li> Nach der letzten Überprüfung der Schlauchsatz, öffnen Sie die Klemme des Peptids Cocktail Tasche. Öffnen Sie das Medium Tasche und initiieren automatischer Entlüftung des Schlauchsets. </li><li> Nach dem Entlüftungsschritt abgeschlossen ist, ergänzen HSA (2,5%) in NaCl-Puffer in der Reservoirbeutel (200 ml) mit Hilfe der sterilen Schlauchschweißgerät. Übertragen Sie die Ausgangszellprodukt in die Verwendung der sterilen Schlauchschweißgerät &quot;Application-Tasche&quot;. </li><li> Schließen CCS (IFNy) Reagenzien in entsprechende Rohr über Adapter. Geben Sie bevorzugte Zeit, um eine Fraktion von Zellmaterial vor dem Anreicherungsprozess zu sammeln. Bewertung und Überprüfung der Richtigkeit aller Daten / eingegebenen Parameter. Starten Sie den Prozess. </li><li> Vor dem Start der automatisierten Zellanreicherungsprozess, entfernen Sie die Qualitätskontrolle Bag (QCB, original Fraktion (ori) enthält etwa 1,3 ml von 100 ml Inhalt Kammer mit PBS / EDTA-Puffer verdünnt). Dichten Sie das QCB, wiegen, und bei 4 ° C. </li><li> Starten Sie den enrichment-Prozess. Am Ende des Prozesses werden die Zielzellen mit einem ungefähren Volumen Elutionspuffer aus dem Reservoirbeutel eluiert werden. </li><li> Dichten Sie das Nichtzielzelle Bag (NTCB, negative Fraktion = neg) und Zielzelle Bag (TCB, positive Fraktion = pos) und wiegen jede Tasche. Die Gewichte werden später für die Berechnung der Zellzahl verwendet werden. </li><li> Unmittelbar nach dem Anreicherungsverfahren sammeln zwei Aliquots pro Fraktion für die Durchflußzytometrie-Analyse, und der Rest der Proben bei 4 ° C. Verwenden Sie eine Probenaliquot zur Zellzahlbestimmung und die andere Probenaliquot zur Anreicherung Performance-Analyse (Tabelle 1). </li><li> Entfernen Sie den Schlauch von der Zellanreicherung Instrument festgelegt. Übertragen Sie die Protokolldatei auf einem USB-Laufwerk für die zukünftige Verwendung. HINWEIS: Alle Reagenzien werden unter sterilen Bedingungen hergestellt werden. Die Verwendung eines Biosafety Typ-II-Haube ist sehr zu empfehlen. Verwenden stationären Apherese Zellprodukt (nicht mobilisiert) von einem gesunden isoliertCMV-seropositiven Spender zum CMV-Antigen-spezifischen T-Zellen anzureichern. Nur FDA lizenziert HSA verwendet werden. Der Puffer für Zellpräparation sollte +19 ° C bis +25 ° C, niedrigere oder höhere Umgebungstemperaturen führt zu verminderter Reinheit und einer reduzierten Ausbeute der Zielzellen führen, gehalten werden. </li></ol> 3. Zellzahlbestimmung <ol><li> Nehmen die Portionen von QCB, NTCB und TCB für Zellzahlen, wie in Tabelle 1 gezeigt. In CD45-VoBlue zu jedem Aliquot (Titer 1,11) und Inkubieren im Dunkeln für 10 min bei 4 ° C. </li><li> 1,5 ml frisch hergestelltem Lyse der roten Blutkörperchen-Lösung (1x) auf die ursprüngliche Fraktion und negativen Fraktion, 450 &amp; mgr; l frisch hergestellte Lyse roter Blutkörperchen Lösung der positiven Fraktion und Inkubieren aller Fraktionen für 15 min bei RT. </li><li> Gerade vor der Analyse hinzuzufügen Propidiumiodid in einer Endkonzentration von 1 ug / ml (1: 100 Verdünnung von 100 ug / ml). Verwenden Sie die automatische Zellzähler auf Zellzahl und vi zu bestimmenFähigkeit. Verwenden Sie Zellzähler Gerät empfohlene Software für die Durchflusszytometrie-Analyse. Bestimmen Sie die absolute Zählungen von Leukozyten für original, negative und positive Fraktionen. HINWEIS: Die Zellzahl der lebensfähigen Leukozyten pro ml der Proben zur Analyse entnommen Zellzahl wird unter Verwendung des Zellanalyse empfohlene Software bestimmt. </li><li> Stellen Sie den Bereich, wie in Abbildung 2 (Bereich 5, lebensfähigen Leukozyten) gezeigt. Verwenden Sie die folgenden Gating-Strategie zur Zellzahl zu bestimmen. Ein lebensfähiger Leukozyten in Original Fraktion wird in Abbildung 2 dargestellt. </li><li> Die genannten Bereiche (2, 1-6) sind hierarchisch, wie folgt: 1: Zeit Gate → 2: Einzelzellen → 3: CD45 + Zellen → 4: Leukozyten (Ablagerungen ausgeschlossen) → 5: Lebensfähige Leukozyten → 6: Lebensfähige Lymphozyten </li><li> Wiederholen Sie die gleichen Schritte, um Zellzahlen für negative und positive Fraktionen zu bestimmen. Berechnung der Zellzahl von der gesamten Fraktionunter Berücksichtigung des Verdünnungsfaktors der Probe, und das Gesamtvolumen der Fraktion (Tabelle 2). </li></ol> 4. Prüfung der Trennleistung <ol><li> Mit vorgekühlten PBS / EDTA-Puffer / 0,5% AB-Serum Waschen Sie die Teilmengen von QCB, NTCB und TCB Bruchzellen. Zentrifugieren der Zellen bei 300 g für 5 min bei 4 ° C und saugt den Überstand. </li><li> Die Zellen in 100 &amp; mgr; l Antikörper-Fluorochrom-Färbung Mischung enthaltend: CD3-FITC, CD4-APC CD8-APC-Vio770, CD14-PerCP, CD20-PerCP, CD45-VioBlue und anti-IFN-PE (Titer 1,11) und Inkubieren im Dunkeln für 10 min bei 4 ° C. </li><li> 1 ml frisch hergestelltes Lyse der roten Blutkörperchen-Lösung (1x) und Inkubation für 15 min bei RT. Zentrifuge bei 300 × g für 5 min bei 4 ° C und saugt den Überstand. Resuspendieren der Zellen in einem geeigneten Volumen von PBS / EDTA-Puffer / 0,5% AB-Serum. </li><li> Hinzufügen Propidiumiodid in einer Endkonzentration von 1 ug / ml unmittelbar vor der Analyse (1: 100 dilutiauf 100 &amp; mgr; g / ml). Zuführen Durchflusszytometrie, um die Reinheit der Probe zu bewerten. </li><li> Verwenden Sie die folgenden Gating-Strategie, um die CD3 + T-Zellen in lebensfähiger Leukozyten-Gating-Strategie zur Bestimmung der CD3 + T-Zellen in positiven Fraktion nach CCS Anreicherungsprozess gezeigt, zu berechnen. Die angegebenen Bereiche (3A und 3B, 1-6) sind hierarchisch wie folgt verbunden: 1: Zeit Gate → 2: Einzelzellen → 3: CD45 + Zellen → 4: Zellen (Ablagerungen ausgeschlossen) → 5: Lebensfähige Leukozyten → 6: Lebensfähige CD3 + Zellen Bevölkerung </li><li> Bestimmen die Frequenz von CD4 +, CD8 +, CD4 + IFN-γ + und CD8 + IFN-γ + T-Zellen nach CCS Anreicherungsverfahren (Tabelle 2). </li><li> Verwenden die Gating-Strategie, die Frequenzen der CD4 +, CD8 +, CD4 + IFN-γ + und CD8 + IFN-γ + T-Zellen unter f dargestellten bestimmenoder ein Original und angereicherten (gefangenen) positive Fraktion nach CCS-Verfahren. Die angegebenen Bereiche sind hierarchisch verknüpft und wie folgt benannt: 1: Zeit Gate → 2: Einzelzellen → 3: CD45 + Zellen → 4: Zellen (Ablagerungen ausgeschlossen) → 5: Lebensfähige Leukozyten → 6: Lebensfähige CD3 + Zellen → 7: CD4 + -Zellen → 7a: CD4 + IFN-γ + Zellen (box) → 8: CD8 + -Zellen → 8a: CD8 + IFN-γ + Zellen (Kasten) </li></ol> HINWEIS: Die ersten 6 angegebenen Regionen der Hierarchieverbindungen sind die gleichen wie in Figur 3, (1-6) und zuletzt 2-Regionen sind in Abbildung 4 (6-8a) gezeigt.

<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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Sowohl MD Anderson Cancer Center und Dr. Cooper haben ein finanzielles Interesse an ZIOPHARM Oncology, Inc., und Intrexon Corporation. Am 7. Mai 2015 wurde Dr. Cooper als Chief Executive Officer bei ZIOPHARM Onkologie ernannt. Dr. Cooper heute ist er Gastwissenschaftler am MD Anderson. Dr. Cooper gegründet und besitzt InCellerate, Inc. Er hat Patente mit Sangamo BioSciences mit künstlichen Nukleasen. Er berät sich mit Targazyme, Inc. (ehemals amerikanischen Stammzellen, Inc.), GE Healthcare, Ferring Pharmaceuticals, Fate Therapeutics, Janssen Pharmaceuticals und Bristol-Myers Squibb. Er ist auf dem wissenschaftlichen Beirat von Cellectis. Er erhält Honorare von Miltenyi Biotec.

Adoptive T-Zell-Therapie hat sich als praktikable Option, um B-Zell-Malignitäten 4 Behandlung entstanden. Sein therapeutisches Potential ist abhängig von der Infusion der gewünschten Anzahl von Ziel-Antigen spezifischen T-Zellen, die replikative Seneszenz 2 fehlt. Dies kann durch Aussortieren eine reine Population von Antigen-spezifischen T-Zellen aus expandierten T-Zellen in Übereinstimmung mit den geltenden guten Herstellungspraxis erreicht werden kann. Zwei Sortierverfahren sind weit verbrei...

Hämatopoetischen Stammzelltransplantation (HSCT) 1 kann mit Adoptiv-T-Zell-Therapie kombiniert werden, um Graft-versus-Tumor-Wirkung zu verbessern und Immunität gegen opportunistische Infektionen 2 bereitzustellen. Erzeugung von Antigen-spezifischen Spender abgeleiteten T-Zellen für die Infusion hat historisch erforderlichen Fachkräfte und die Verwendung von spezialisierten Einrichtungen, die GMP-konform sind. Die Lieferung solcher T-Zellen wurde in der Entschließung des opportunistischen Infektionen 3 sowie Behandlung der zugrunde liegenden malignen 4 geführt. Vor kurzem haben Forscher gezeigt, dass der adoptive Transfer von nur wenigen tausend Virus-spezifischen T-Zellen (~ 1 x 10 4 bis 2,5 x 10 5 Zellen / kg Körpergewicht des Empfängers) opportunistische CMV Infektionen nach alloHSZT 5-9 erfolgreich zu behandeln. Eine begrenzte Anzahl von GMP-Anlagen mit zugehörigen Fachfertigungsanforderungen und der hohen Kosten, die mit der Zellproduktion assoziiert hat jedoch restricted Patienten den Zugang zu vielversprechenden T-Zelltherapien 10. Ein Ansatz zum Isolieren von antigenspezifischen T-Zellen basiert auf den CCS Verwendung eines bispezifischen Reagenzes an CD45 und IFN-γ erkennt beruht. Wie gezeigt ist, kann diese Methode verwendet werden, um klinische Studien zu CMV-spezifische T-Zellen unter Verwendung eines automatisierten Zellanreicherung CCS Einrichtung (1B) zu erzeugen. CMV-spezifische T-Zellen werden durch Inkubation von überlappenden Peptiden von CMV pp65 Antigens mit Leukapherese Gesamtkernzellen (TNC) von CMV-seropositiven Spendern erzeugt. Diese Peptide im Kontext von Human-Leukozyten-Antigen (HLA) angezeigt wird, aktivieren die CMV pp65-spezifischen T-Zellen innerhalb der TNC zu IFN-γ sezernieren. Diese T-Zellen können dann &quot;eingefangen&quot; werden und magnetisch getrennt. Der Betrieb der ersten Generation Zellanreicherungseinrichtung (1A) unter GMP-Bedingungen erforderliche Personal Mann Zellkultur, und die Koordination der Mitarbeiter, um die Mehrfach s verpflichtenTEPs notwendig, eine &quot;eingefangen&quot; Produkt zu erzeugen. Das Verfahren in der Regel erforderlich, 10 bis 12 h Dauerbetrieb, und damit Personal wahrscheinlich benötigen, um mehr als zwei Schichten in der GMP-Anlage zu arbeiten. Diese Beschränkungen werden nun durch die Durchführung einer zweiten Erzeugungsvorrichtung (in 1B gezeigt) vermieden. Diese Vorrichtung übernimmt magnetische Anreicherung, ähnlich zu der ersten Erzeugungseinrichtung, aber automatisiert andere Aspekte der CCS in einem unbreached Ansatzes. Dies reduziert die Belastung für die GMP-Team als die meisten Schritte können unbeaufsichtigt von Mitarbeitern durchgeführt werden. Da ferner die Vorrichtung arbeitet als geschlossenes System, können die Antigen-spezifischen T-Zellen, aufgenommen und gegen die Arbeitsfläche mit Ausnahme der Schritte in Leukapherese Isolierung und Herstellung von Materialien vor dem Starten des Instruments eingebundenen verarbeitet werden. Details des kompletten Instrumentierung und Funktionalität dieser zweiten Generation Zellanreicherungsvorrichtung wurden published 11. Hier beschreiben wir die Schritte zum CMV pp65-spezifischen T-Zellen aus einer stationären Aphereseprodukt Verwendung des automatischen Zellanreicherung CCS-System anzureichern. Nachdem sie isoliert wurde, kann diese CMV-spezifische T-Zellen sofort in einen Patienten infundiert werden.

<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

Der adoptive Transfer von Pathogen-spezifische T-Zellen können verwendet werden zur Vorbeugung und Behandlung opportunistischer Infektionen, wie Cytomegalievirus (CMV) nach allogener hämatopoetischer Stammzelltransplantation auftritt. Viral-spezifischen T-Zellen von allogenen Spendern, einschließlich Dritter Spender kann ex vivo vermehrt werden in Übereinstimmung mit den geltenden Guten Herstellungspraxis (cGMP) und beschäftigt wiederholte Runden von Antigen-angetriebenen Stimulation, um die gewünschten T-Zellen selektiv zu verbreiten. Die Identifizierung und Isolierung von Antigen-spezifischen T-Zellen kann auch auf der Grundlage der Zytokin-Capture-System der T-Zellen, die aktiviert wurden, um gamma-Interferon (IFN-γ) sekretieren vorgenommen werden. Doch weit verbreiteten Menschen Anwendung der Zytokin-Capture-System (CCS) zur Wiederherstellung der Immunität wurde begrenzt, wie der Produktionsprozess ist zeitaufwendig und erfordert einen erfahrenen Bediener. Die Entwicklung einer zweiten Generation Zellanreicherung Vorrichtung wie CliniMACS Prodigy jetztermöglicht Ermittler viral-spezifischen T-Zellen unter Verwendung eines automatisierten, weniger arbeitsintensiv System zu erzeugen. Diese Vorrichtung trennt magnetisch von unmarkierten Zellen durch magnetische Zellsortierung Technologie klinischem Produkte erzeugen markierten Zellen wird als ein geschlossenes System konstruiert und zugegriffen werden kann und gegen die Arbeitsfläche betrieben werden. Wir demonstrieren die Funktionsweise dieser neuen automatisierten Zellanreicherungsvorrichtung zur CMV pp65-spezifischen T-Zellen, die aus einer stationären Apherese Produkt von einem CMV-seropositiven Spender erhalten erhalten herzustellen. Diese isolierten T-Zellen können dann direkt in einen Patienten unter institutionellen und bundesstaatlichen Regulierungsaufsicht infundiert werden. Alle Bio-Verarbeitungsschritten einschließlich der Entfernung der roten Blutkörperchen, die Stimulation von T-Zellen, die Trennung von Antigen-spezifischen T-Zellen, Reinigung und Waschen sind voll automatisiert. Geräte wie dies die Möglichkeit, dass T-Zellen für die Anwendung beim Menschen kann außerhalb gewidmet guten Herstellungspraxis (GMP hergestellt werden) Einrichtungen und stattdessen in Blutbanken Einrichtungen, in denen Mitarbeiter können automatisierte Protokolle überwachen, um mehrere zu produzieren werden.

In dieser Studie wurde eine automatisierte Zellanreicherung CCS System zur automatisierten Erzeugung von CMV pp65-spezifischen T-Zellen verwendet. CMV-spezifische T-Zellen wurden aus drei Apherese Zellprodukten angereichert. Die Steady-State-Apherese-Produkt wurde über 2 Stunden von einem CMV-seropositiven Spender 10 10 Gesamtkernzellen (TNC) geerntet und erzeugt. 10 9 TNC wurden dann mit CMV pp65 abgeleitete Peptide (60 nmol) für 4 Stunden und die IFN-γ sezernierenden T-Zellen wurden mit Hilfe ...

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.

Automatisierte Zellanreicherung des Cytomegalovirus-spezifischen T-Zellen für die klinische Anwendung mit der Zytokin-Capture-System

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 Aufrufe.  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.

Serie: 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

Retrovirale Transduktion von T-Zell-Rezeptoren in Maus-T-Zellen

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 ...

Forschung

Immunologie und Infektion

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.

Nach Cell-Schicksal<em&gt; E. coli</em&gt; Nach Infektion mit Phagen

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.

piggyBac Transposon-System Modifikation von primären menschlichen T-Zellen

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.

Peptid: MHC-Tetramer-basierte Anreicherung von Epitop-spezifischer T-Zellen

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 High-throughput Single-cell Zytotoxizitätstest für T-Zellen

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.

Klinische Anwendung der<em&gt; Dornröschen</em&gt; Und künstliche Antigen präsentierenden Zellen genetisch zu verändern T Zellen aus dem peripheren und Nabelschnurblut

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.

Überwachung dendritische Zellen mit Migration<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Magnetic Resonance Imaging

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.

Beurteilung der Innate Sensing von HIV-1-infizierten CD4<sup&gt; +</sup&gt; T-Zellen durch Plasmazytoide Dendritische Zellen unter Verwendung eines<em&gt; Ex-vivo-</em&gt; Co-Kultursystem.

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.

Maus Naive CD4<sup&gt; +</sup&gt; T-Zell-Isolierung und<em&gt; In-vitro-</em&gt; Differenzierung in T-Zell-Populationen

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.