Diese Arbeit wird von einer Produktion Assistance for Cellular Therapies (Vertrag NIH-NHLBI (HB-10 bis 03) HHSN26820100000C) (CMR), eine spezialisierte Zentren für Zell-basierten Therapie unterstützt Zuschuss NIH-NHLBI 1 U54 HL081007 (CMR), ein ASBMT Young Investigator Award (UG und JV), ein Leukemia and Lymphoma Society Special Fellow in Clinical Research Award (UG), und eine Amy Strelzer Manasevit Scholar Award (AML).

1. DC Nukleofektion <ol><li> Ernte Monozyten-abgeleiteten DCs, die angereichert sind mit Kunststoff-Haftung, für 5 Tage mit Zell Genix Medien mit IL4 (1000U/ml) ergänzt, GMCSF (800IU/ml) und weitere 24 Stunden lang mit dem DC-Reifung Zytokine IL4 (1000U gereift kultivierten / ml), GMCSF (800IU/ml), IL6100ng/ml, TNF-α 10ng/ml, IL1-β 10ng/ml und PGE2 (1μg/ml) 1, durch vorsichtiges Resuspendieren mit 3ml Transferpipette. </li><li> Graf lebensfähig DCs mit Trypanblau, Transfer in 3x 15ml Röhrchen mit nicht weniger als 0.5x10 6 und nicht mehr als 2x10 6 Zellen / Röhrchen. </li><li> Centrifuge DCs für 10 Minuten @ 200g. 2ml/well in drei Wells einer 12-well Zellkultur behandelte Platte in einem 37 ° C / 5% CO 2-Inkubator - Während dieser Zeit vorwärmen Zelle Genix Medien mit der DC-Reifung Zytokine (DC Reifung media) ergänzt. </li><li> Sobald Zellen fertig sind Spinnen, saugen Sie den Überstand und fügen Sie die entsprechende DNA-Plasmide zu jedem der Rohre in einer finalen Konzentration von 5μg DNA / Rohr. In diesem Fall fügen Sie die Plasmid-Kodierung IE1-pp65 zu Rohr Nr. 1, Hexon-Penton zu Rohr Nr. 2 und EBNA1-LMP2-BZLF1 zu Rohr Nr. 3. </li><li> Resuspendieren DCs und DNA mit 100 &amp;mgr; l der Amaxa nucloefection verdünnen, gut mischen und Transfer zum Nukleofektion Küvetten. </li><li> Legen Sie Küvetten in der 4D Nucleofector, wählen Sie Programm CB150 (Amaxa / Lonza), und drücken Sie Start. </li><li> Unmittelbar nach Nukleofektion 500μl der 2ml vorgewärmten Zelle Genix DC Reifung Medien in die Küvette hinzu, mischen Sie vorsichtig durch Auf-und Abpipettieren 2-3 mal, und übertragen nucleofected DCs auf die vorbereitete 12-Well-Platte mit den restlichen 1,5 ml vorgewärmtem DC Reifung Medien. Transfer zum 37 ° C / 5% CO 2-Inkubator für eine weitere 12-18h. </li></ol> 2. T-Zell-Stimulation <ol><li> Ernte und zählen nucleofected DCs, und bestrahlen bei 30 Gy. Wash einmal mit 10 ml CTL-Medium (45% RPMI, 45% Klicks EHAA, 10% FBS, 2 mM Glutamax) und resuspendieren @ 3 x 10 5 DCs pro ml CTL Medien. </li><li> Pool ein Minimum von 7.5x10 5 (2,5 ml) und einem Maximum von 15x10 5 (5 ml) der DCs mit jedem der Plasmide und Transfer der gepoolten DCs auf die G-Rex Gerät, das dann in den Inkubator platziert werden. </li><li> Für die Herstellung der Responder-Zellen entweder vorher eingefroren PBMCs oder nicht haftende mononukleären Zellen, die nach dem DC-Auswahl (Einhaltung oder CD14 Auswahl) bleiben. Thaw die Zellen, um vorgewärmte Kulturmedium übertragen, einmal abwaschen mit CTL-Medien. Resuspendieren der Zellen in CTL Media, zählen die Zellen und bringt sie zu einer Konzentration von 2x10 6 Zellen pro ml. Nehmen 15x10 6 Zellen oder 7,5 ml und ergänzen mit 30000U IL4 (1000U/ml -. Endkonzentration) und 300ng IL7 (10ng/ml - Endkonzentration.). </li><li> Transfer 7.5ml von PBMC (15x10 6 Zellen), die G-Rex und Aufstockung des Bioreaktors mit CTL Medien bis zu einem Gesamtvolumen von 30ml. </li><li> Kultur der G-Rex für 6-7 Tage in einem 37 ° C / 5% CO 2 befeuchteten Inkubator. </li></ol> 3. T-Zell-Expansion <ol><li> Am Tag 6-7, absaugen 10ml von Medien, dann mischen die Zellen in den verbleibenden 20 ml Medium mit 10ml Pipette und zählen lebensfähigen Zellen unter Verwendung von Trypanblau. Wenn es &lt;50x10 6 mit frischen Medien + Zytokine aufzufüllen. Wenn es&gt; 50x10 6 Zellen zu entfernen 10ml Zellsuspension, um eine neue G-Rex zu übertragen, und dann füttern beide G-Rexs mit frischen CTL media + Zytokine. </li><li> Kultur für weitere 4-6 Tage. Sobald genügend Zellen wurden erweitert, führen phänotypische und funktionelle Charakterisierung der CTL und Kryokonservierung überschüssiger für die zukünftige Verwendung. </li></ol> 4. Repräsentative Ergebnisse: Eine schematische Darstellung unserer FDA-Zulassung multivirus-spezifischen CTL-Generation ist in Abbildung 1 dargestellt. Im Gegensatz zu Konventionen multivirus CTL-Protokolle, die Adenovektoren und EBV-LCL verwenden, um Virus-reaktiven T-Zellen zu stimulieren 2 haben wir infektiösen Virus-Material mit DNA-Plasmiden ersetzt dass kodieren mehrere Antigene aus jedem der Viren 3 abgeleitet. Zur Stimulierung trivirus CTL wir entwarf drei multicistronischen Plasmiden Hexon und Penton von Adenovirus, IE1 und pp65 von CMV und EBNA1, LMP2 und BZLF1 des EBV. Diese Antigene wurden auf der Grundlage ermutigenden klinischen Ergebnisse unserer eigenen und anderer Gruppen zeigen, dass T-Zellen gegen Adv-Hexon und Penton 2,4-6 gerichtet, und CMV-1E1 und CMV-pp65 sind Schutzmaßnahmen in vivo 7 gewählt. Für EBV ist EBNA1 ein immundominantes CD4 + T-Zell-Target-Antigen in allen EBV-assoziierten Tumoren und in normalen EBV-infizierten B-Zellen exprimiert 8,9, LMP2 immunogen ist über mehrere HLA-Typen und ausgedrückt in den meisten EBV Malignome, 10,11, während BZLF1 kodiert für ein immundominante, immediate early lytischen Zyklus Antigen, das sowohl CD4 + und CD8 + T-Zellen stimuliert von den meisten Menschen und ist wahrscheinlich wichtig für die Zusammenarbeitntrol von Zellen replizieren Virus 12. Zur weiteren Optimierung unserer Fertigung Methoden, die wir in Zusammenarbeit mit Nature-Technologie, die minimalized, Antibiotika-freien (FDA-konform) Plasmide für CTL Stimulation 13,14 erzeugt. Mit dieser Strategie setzen wir konsequent erreichen Nukleofektion Wirkungsgrade von&gt; 35% unter Beibehaltung der hohen Zellvitalität (Daten nicht gezeigt) 3. Abbildung 2 zeigt, dass die Häufigkeit von Virus-spezifischen T-Zellen als Reaktion auf optimierten DNA-Plasmide wie IFNy ELIspot gemessen, größer war als Reaktion auf herkömmliche pShuttle-basierten Expressionsplasmiden Ausdruck der gleichen Antigene (n = 8 Adenovirus, n = 4 CMV, und n = 2 EBV). Das optimale Verhältnis von DC: PBMC wurde für potente T-Zell-Stimulation, wie in Abbildung 3, wo ein Verhältnis von 1:50 produziert sub-optimale Aktivierung im Vergleich zu einer 1:20 S gezeigt wichtig: R-Verhältnis (n = 2 Geber). Produktion von ausreichenden CTL Zahlen mit breiten Antigen-Spezifität ist eine Voraussetzung für die klinische Wirksamkeit gegen alle drei Viren. Dies wird durch CTL Kultur in der G-Rex, der überlegene T-Zell-Expansion unterstützt im Vergleich zu herkömmlichen 24-Well-Platten (Abbildung 4A) 15 erreicht, während die Zugabe von IL4 und IL7 die Kulturen steigt das Repertoire und Spezifität, wie in Abbildung 4B dargestellt wobei die Frequenz von T-Zellen reaktiv gegen das CMV-pp65-derived HLA-A2 resticted NLV Peptid wurde in Kulturen in der Gegenwart oder Abwesenheit von IL-4 und / oder IL7 16,17 generiert beurteilt. Zur Beurteilung der Phänotyp und funktionellen Kapazität der expandierten Zellen führen wir durchflusszytometrische Analyse intrazellulärer Zytokin-Färbung / IFNy ELIspot und Cr 51 Release Assays, die auf das Endprodukt für die Kryokonservierung / Infusion. Typischerweise werden die erzeugten Zellen sind polyklonale mit einer gemischten Bevölkerung von CD4 + und CD8 + T-Zellen mit Antigen-Spezifität nachweisbar in beiden T-Zell-Kompartimenten. Die CTL sind in der Lage, virale Antigen-exprimierenden Zielzellen töten, aber nicht negativ Virus teilweise HLA abgestimmt Zielen unter Angabe, dass sie nicht veranlassen Graft-versus-Host Erkrankung (GvHD) in vivo (Abbildung 5). <img src="/files/ftp_upload/2736/2736fig1.jpg" alt="Abbildung 1"/> Abbildung 1. RCTL Generation-Protokoll. Erstens sind DCs mit dem viralen Antigen-kodierenden Plasmiden nucleofected und anschließend mit autologen PBMCs bei einem R: S 10 oder 20:1. Die Zellen werden in der G-Rex für 10-14 Tage in Gegenwart von IL4 und IL7, dann geerntet, gezählt, funktionsgeprüft, Identität und Sterilität, und dann für den klinischen Gebrauch kryokonserviert erweitert. <img src="/files/ftp_upload/2736/2736fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Optimized DNA-Plasmide bewirken überlegene T-Zell-Aktivierung in vitro. DCs wurden mit einer optimierten, FDA-konforme Plasmiden Hexon und Penton (Adv), IE1 und pp65 (CMV), und EBNA1, LMP2 und BZLF1 (EBV) oder konventionelle pShuttle Plasmiden die gleichen Antigene nucleofected. Diese wurden verwendet, um T-Zellen zu stimulieren und Spezifität wurde durch IFNy ELIspot 10 Tage post-Stimulation untersucht. <img src="/files/ftp_upload/2736/2736fig3.jpg" alt="Abbildung 3"/> Abbildung 3 Optimale DC:. T-Zell-Verhältnisse für CTL-Aktivierung. DCs von 2 Spendern wurden mit allen drei optimierten Plasmiden nucleofected und dann zur autologen PBMCs um 1:20 oder 1:50 DC stimulieren: PBMC-Verhältnis. Die Häufigkeit der reaktivierten T-Zellen wurde am Tag 10 nach IFNy ELIspot beurteilt. <img src="/files/ftp_upload/2736/2736fig4.jpg" alt="Abbildung 4"/> Abbildung 4. T-Zell-Expansion in den G-Rex mit Verbesserung Zytokine. Panel A zeigt die G-Rex-Gerät sowie CTL Auftritt auf dem Gas-Membran, durch Mikroskopie untersucht. Ein Vergleich zwischen Messsignaländerung in Konvention Gewebekultur behandelten Platten vs G-Rex erreicht wird ebenfalls angezeigt. Panel B zeigt die Häufigkeit von CMV-Pentamer positive CTL in Kulturen in der Gegenwart keine Zytokin, IL4 allein IL7 allein und IL4 + IL7 erweitert erreicht. <img src="/files/ftp_upload/2736/2736fig5.jpg" alt="Abbildung 5"/> Abbildung 5. Phänotyp und Funktion erweitert CTL. Panel A zeigt ein repräsentatives Beispiel für den Phänotyp des erweiterten multivirus CTL, die polyklonale mit einer Mischung aus CD4 + (45% - Helfer) und CD8 + (42% - zytotoxische) T-Zellen, von denen die Mehrheit (95%) äußerte die Merker CD45RO + / CD62L +. Panel B zeigt, dass diese Zellen, die für alle die stimulierende Antigene sind und polyfunktionellen als durch intrazelluläre Zytokin-Färbung beurteilt, die Produktion von IFN &amp;ggr; und TNF nach Antigen-Stimulation zu erkennen. Panel C zeigt, dass die erweiterte CTL funktional wie gemessen werden von Cr 51-Assay. Autologe LCL, entweder allein oder transduziert mit einem Null-Vektor oder einem adenoviralen Vektor, CMV-pp65 wurden als tar verwendetbekommt. Alloreaktivität wurde mittels allogener PHA Blasten als Ziel.

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+cellular+immunity+against+cytomegalovirus+in+recipients+of+allogeneic+bone+marrow+by+transfer+of+T-cell+clones+from+the+donor.">Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor.</a> N. Engl. J. Med. 333, 1038-1044 (1995).</li></ol>

Juan F. Vera ist als Berater für Wilson Wolf Manufacturing. Die anderen Autoren haben keine entsprechenden Angaben.

Virale Infektionen entfallen erhebliche Morbidität und Mortalität bei Patienten, die durch ihre Krankheit oder deren Behandlung immunsupprimierten. Nach HSCT, sind zum Beispiel Infektionen, die durch anhaltende Herpesviren wie EBV und CMV sowie durch respiratorische Viren wie Respiratory Syncytial Virus (RSV) hervorgerufen, bekannt, während die Bedeutung von Infektionen durch Adv, BK-Virus verursacht, und die menschliche Herpesvirus (HHV) -6 wurden in jüngerer Zeit gewürdigt worden. Während pharmakologische Wirkstoffe Standard-Therapie für einige Infektionen sind, haben sie erhebliche Nebenwirkungen erzeugen resistente Varianten, und sind häufig wirkungslos. Im Gegensatz dazu haben virus-spezifischen T-Zellen aus Stammzellen Spender stammen nachweislich sicher und wirksam für die Prävention und Behandlung von viralen Infektion oder Erkrankung in den blutbildenden Stammzellentransplantation (HSCT) Einstellung 2,5,6,18-21. Allerdings ist die breitere Umsetzung von T-Zell-Immuntherapie letztlich durch die Kosten, Komplexität und Zeit für CTL Produktion erforderliche Maß beschränkt. Unsere neuartige und schnelle Annäherung an multivirus CTL, in der aktuellen Manuskript beschrieben, erzeugen sollte deutlich erhöhen die Machbarkeit von zytotoxischen T-Zell-Therapeutikum für Viruserkrankungen, so dass die Strategie zu einem Standard in der Versorgung der immungeschwächten Host geworden. Die Verwendung von Plasmid nucleofected DCs als APCs ermöglicht Antigen-Präsentation auf beiden MHC-Klasse I und II ohne Konkurrenz durch virale Vektoren oder auch von mehreren viralen Antigenen, die innerhalb einer einzelnen Zelle zum Ausdruck, da verschiedene DC-Populationen für jedes Plasmid 3 sind eingesetzt. Die Verwendung von IL-7.4 erhöht T-Zell-Überleben und die Proliferation, die entsprechend trägt zur Steigerung der Frequenz und des Repertoires zu reagieren Antigen-spezifischen T-Zellen 16,17. Schließlich, Kultur in der G-Rex drastisch reduziert T-Zell-Apoptose während der Kultur. Der Gasaustausch (O 2 und CO 2 in out) tritt über eine gasdurchlässige Silikonmembran am Boden der Flasche und verhindert Hypoxie und ermöglicht eine größere Tiefe des Mediums über den Zellen, die mehr Nährstoffe und Verdünnen Abfallprodukte. Diese Plattform kann auch zusätzliche Viren als wenn schützende Antigene identifiziert erweitert werden.

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th></tr><tr><td> CellGenix </td><td> CellGenix </td><td> 2005 </td></tr><tr><td> IL4 </td><td> R + D Systems </td><td> 204-IL/CF </td></tr><tr><td> IL7 </td><td> PeproTech </td><td> 200-15 </td></tr><tr><td> Hyclone RPMI 1640 </td><td> Thermo Scientific </td><td> SH30096.01 </td></tr><tr><td> Humanem AB-Serum </td><td> Tal Biomedical Inc. </td><td> HP1022 </td></tr><tr><td> Nucleofector </td><td> Amaxa / Lonza </td><td> AAF-1001B &amp; AAF-1001X </td></tr><tr><td> Nucleofection Kit </td><td> Amaxa / Lonza </td><td> V4XP-3012 </td></tr><tr><td> Plasmide </td><td> NTC </td><td> n / a </td></tr><tr><td> GM-CSF </td><td> R &amp; D </td><td> 215-GM/CF </td></tr><tr><td> IL1 </td><td> R &amp; D </td><td> 201-LB-025 </td></tr><tr><td> IL6 </td><td> R &amp; D </td><td> 206-IL-CF </td></tr><tr><td> TNF </td><td> R &amp; D </td><td> 210-TA-010 </td></tr><tr><td> PGE2 </td><td> Sigma </td><td> P6532-1MG </td></tr><tr><td> G-Rex </td><td> Wilson Wolf Fertigung </td><td> AY11-00027 </td></tr></tbody></table>

generation of multivirus-specific t cells to prevent/treat viral infections after allogeneic hematopoietic stem cell transplant

Virale Infektionen verursachen Morbidität und Mortalität in allogene hämatopoetische Stammzelltransplantation (HSCT) Empfänger. Wir und andere haben erfolgreich erzeugt und infundiert T-Zellen spezifisch für Epstein Barr Virus (EBV), Cytomegalovirus (CMV) und Adenovirus (Adv) mit Monozyten und EBV-transformierten lymphoblastische Zellen (EBV-LCL) mit einem Adenovirus-Vektor-Gen-modifiziert Antigen-präsentierende Zellen (APC). So wenig wie 2x10 5 / kg trivirus-spezifischen zytotoxischen T-Lymphozyten (CTL) durch mehrere Protokolle nach der Infusion vermehrt und erschien zur Vorbeugung und Behandlung sogar schwere Viruserkrankung beständig zu anderen verfügbaren Therapien. Vorbereitung - Der breiter gefasste Umsetzung dieser ermutigenden Ansatz wird durch die hohen Produktionskosten, die Komplexität der Herstellung und der längere Zeit (insgesamt 10-14 Wochen 4-6 Wochen für die EBV-LCL-Generation, und 4-8 Wochen für CTL Herstellung) begrenzt. Um diese Einschränkungen zu überwinden haben wir eine neue, GMP-konforme Produktion CTL-Protokoll entwickelt. Erstens, an Stelle des Adenovektoren auf T-Zellen verwenden wir dendritische Zellen (DCs) mit DNA Plasmiden LMP2, EBNA1 und BZLF1 (EBV), Hexon und Penton (Adv) und pp65 und IE1 (CMV) als Antigen-präsentierende nucleofected stimulieren Zellen. Diese APCs aktivieren T-Zellen spezifisch für alle stimulierenden Antigenen. Zweitens, die Kultur von aktivierten T-Zellen in Anwesenheit von IL-4 (1.000 U / ml) und IL-7 (10ng/ml) erhöht und erhält das Repertoire und die Häufigkeit von spezifischen T-Zellen in unseren Linien. Drittens haben wir eine neue, gasdurchlässige Kultur-Gerät (G-Rex), dass die Expansion und das Überleben der großen Zellzahlen fördert nach einer einzigen Stimulation, wodurch der Bedarf an EBV-LCL und die Verringerung der Techniker Intervention verwendet. Durch die Umsetzung dieser Veränderungen können wir jetzt produzieren multispezifische CTL Targeting EBV, CMV, und Adv zu einem Preis pro 10 6 Zellen, die von&gt; 90% reduziert wird, und in nur 10 Tagen statt 10 Wochen mit einem Ansatz, um zusätzliche verlängert werden kann Schutz virale Antigene. Unsere von der FDA zugelassene Ansatz sollte der Wert für die prophylaktische Behandlung und Anwendungen für ein hohes Risiko einer allogenen HSCT Empfängern werden.

Watch this Scientific Journal Video about Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant at JoVE.com

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Eine schnelle, einfache und kostengünstige Protokoll für die Erzeugung von Spender-derived multivirus-spezifische CTLs (RCTL) zur Infusion einer allogenen hämatopoetischen Stammzelltransplantation (HSCT) Empfänger die Gefahr der Entwicklung CMV, Adv. oder EBV-Infektionen. Dieses Herstellungsverfahren ist GMP-konform und sollte die breitere Umsetzung von T-Zell-Immuntherapie über spezialisierte Zentren zu gewährleisten.

Generierung von Multivirus-spezifischen T-Zellen zu verhindern / zu behandeln virale Infektionen nach allogenen hämatopoetischen Stammzelltransplantation

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

generation-multivirus-specific-t-cells-to-preventtreat-viral

Research

JoVE Journal

Immunology and Infection

17.1K Aufrufe.  Baylor College of Medicine. Eine schnelle, einfache und kostengünstige Protokoll für die Erzeugung von Spender-derived multivirus-spezifische CTLs (RCTL) zur Infusion einer allogenen hämatopoetischen Stammzelltransplantation (HSCT) Empfänger die Gefahr der Entwicklung CMV, Adv. oder EBV-Infektionen. Dieses Herstellungsverfahren ist GMP-konform und sollte die breitere Umsetzung von T-Zell-Immuntherapie über spezialisierte Zentren zu gewährleisten.

Serie: Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

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 ...

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

Generation der induzierte regulatorische T-Zellen aus primären humanen naiven und Gedächtnis-T-Zellen

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

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

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

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

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

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 ...

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

Identifizierung von DNA-Mutationen in hämatopoetischen Stammzellen Gereinigtes / Vorläuferzellen

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

Transplantation von Haut-Schwanz zu allogene CD4-T-Zellantworten in Mäusen untersucht

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

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

The adoptive transfer of pathogen-specific T cells can be used to prevent and treat opportunistic infections such as cytomegalovirus (CMV) infection occurring after allogeneic hematopoietic stem-cell transplantation. Viral-specific T cells from allogeneic donors, including third party donors, can be propagated ex vivo in compliance with current good manufacturing practice (cGMP), employing repeated rounds of antigen-driven stimulation to selectively propagate desired T cells. The identification and isolation of antigen-specific T cells can also be undertaken based upon the cytokine capture system of T cells that have been activated to secrete gamma-interferon (IFN-&#947;). However, widespread human application of the cytokine capture system (CCS) to help restore immunity has been limited as the production process is time-consuming and requires a skilled operator. The development of a second-generation cell enrichment device such as CliniMACS Prodigy now enables investigators to generate viral-specific T cells using an automated, less labor-intensive system. This device separates magnetically labeled cells from unlabeled cells using magnetic activated cell sorting technology to generate clinical-grade products, is engineered as a closed system and can be accessed and operated on the benchtop. We demonstrate the operation of this new automated cell enrichment device to manufacture CMV pp65-specific T cells obtained from a steady-state apheresis product obtained from a CMV seropositive donor. These isolated T cells can then be directly infused into a patient under institutional and federal regulatory supervision. All the bio-processing steps including removal of red blood cells, stimulation of T cells, separation of antigen-specific T cells, purification, and washing are fully automated. Devices such as this raise the possibility that T cells for human application can be manufactured outside of dedicated good manufacturing practice (GMP) facilities and instead be produced in blood banking facilities where staff can supervise automated protocols to produce multiple products.

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 ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generation und Multi-phänotypischen High-Content-Screening<em&gt; Coxiella burnetii</em&gt; Transposon-Mutanten

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

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.

Entwicklung von Stammzellen abgeleiteten Antigen-spezifische regulatorische T-Zellen gegen Autoimmunität

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

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

Die Messung der T-Zell-Alloreaktivität Mit Imaging Durchflusszytometrie

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...