Diese Arbeit wurde durch Zuschüsse von NIH (5U01CA137070) und der American Cancer Society (RSG-09-070 bis 01-LIB), einem Cancer Research Institute Investigator Award (MK), eine American Heart Association vor-Stipendium (KM) und unterstützt einem spanischen Ministerium für Bildung und Wissenschaft Fellowship (APG). Wir würden das danken Dr. Nicholas Restifo und Dr. Zhiya Yu (NIH / NCI) für hilfreiche Anregungen, um das Protokoll wie.

1. Bereiten Retrovirale Construct <ol><li> Sub-Klon des T-Zell-Rezeptor (TCR) Gen von Interesse in einen retroviralen Vektor (Abbildung 1, Beispiel Vektoren pMSG 7, pMIGII 5,8, pMXs aus Cellbiolabs). Die TCR α-und β-Kette-Gen sollte auf dem gleichen Vektor unter der Kontrolle des gleichen Promotors werden, um gleich Expression gewährleisten. Wenn die TCR von Interesse ist menschlich, benötigen Sie den humanen konstanten Domänen von Maus konstanten Domänen 9 ersetzt werden. Unsere Beobachtung ist, dass in voller Länge menschlichen TCRs nicht stabil auf Maus-T-Zellen zu exprimieren. </li><li> Bereiten Sie qualitativ hochwertige Plasmid-DNA mittels Qiagen Maxi Prep Kit oder ein ähnliches Produkt. Die Konzentration der Plasmid sollte bei etwa 1 ug / ul werden. </li></ol> 2. Transfektion und T-Zell-Isolation Tag -1 (Platte Verpackung Zellen) <ol><li> Verdrängen Platinum-E (Plat-E, Cellbiolabs) retroviralen Verpackung Zellen mit Trypsin-EDTA und neutralisieren mit DMEM-Medium. </li><li> Zentrifugieren Sie die Zellen bei 1000 xg für 5 min. Saugen Sie den Überstand und Zellpellet in DMEM-Medium. </li><li> Bestimmen Sie die Anzahl der Zellen und löst die Zellen in 0.6x10 6 / mL. Plate 10 ml Zellsuspension in einem 10 mm Poly-Lysin beschichtet Gewebekulturplatte und wachsen über Nacht in einer Zelle Brutschrank bei 37 ° C, 5% CO 2. </li></ol> Tag 0 (Transfektion) <ol start="4"><li> Am nächsten Morgen, untersuchen die Zellen unter einem Lichtmikroskop. Die Zellen sollten ca. 80% konfluent. </li><li> Entfernen Sie vorsichtig das Medium von der Platte. Waschen Sie die Zellen einmal mit 1x PBS und 10 ml vorgewärmte, frische DMEM-Medium ohne Penicillin-Streptomycin (Pen / Strep). (Hinweis: Pen / Strep kann mit der Transfektion stören) </li><li> Bereiten Transfektion komplex. Bereiten Mix A und B wie folgt: <table border="0"><tbody><tr><td> Mix A: Plasmid-DNA </td><td> 9 ug </td></tr><tr><td> PCL-Eco Helferplasmids </td><td> 6,3 pg </td></tr><tr><td> OptiMEM </td><td> 1,5 ml </td></tr><tr><td colspan="2"></td></tr><tr><td> Mix B: Lipopfectime 2000 </td><td> 60 &amp;mgr; l </td></tr><tr><td> OptiMEM </td><td> 1,5 ml </td></tr></tbody></table> Inkubieren A und B getrennt für 5 Minuten bei Raumtemperatur Mix A und B zusammen vorsichtig und inkubieren für 20 Minuten bei Raumtemperatur auf die Transfektion Komplex zu bilden. </li><li> Langsam tropft das Gemisch (insgesamt 3 ml) in den Plat-E-Zellen. Schütteln Sie die Platte hin und her, um die Transfektion Mischung gleichmäßig zu verteilen. </li><li> Inkubieren Zellen bei 37 ° C / 5% CO 2 in einer Zelle Inkubator. </li><li> Nach 6-8 Stunden, ersetzen Medium mit 10 ml frischem DMEM-Medium (mit Pen / Strep) für die virale Produktion. </li></ol> Coat Platte mit Antikörpern <ol start="10"><li> Maus T-Zellen müssen vor dem viralen Transduktion aktiviert werden. Es gibt eine Vielzahl Möglichkeiten, um T-Zellen zu aktivieren. Hier verwenden wir Platte gebundenen Anti-CD3e und anti-CD28 Antikörper. Bereiten Sie ein Antikörper Mischung von 1 pg / ml anti-CD3e und 2 pg / ml anti-CD28 in sterile PBS. </li><li> Dispense 250 ul des Antikörpers Mischung in jede Vertiefung einer 24-well Zellkulturschale. Die Platte kann über Nacht bei 4 ° C oder 2 Stunden beschichtet werden bei 37 ° C. </li></ol> Erstellung von Einzel-Zellsuspension aus Maus-Milz <ol start="12"><li> Zwergmaus Milz und Transfer zum RPMI-Medium. </li><li> Legen Sie die Milz in einer Zelle Sieb. Mash die Milz mit einer Spritze Kolben in eine 5 cm Gewebekulturplatten. Rinse-Zellen aus der Zelle Sieb mit sterilem PBS. </li><li> Centrifuge 1000 xg, 5 min. </li><li> Überstand. Resuspendieren Zellpellet in ACK Lysepuffer (2 mL pro Milz). Inkubieren von 2 bis 3 min bei Raumtemperatur. Add RPMI-Medium bis zu 20 ml und Zentrifuge 1000 xg für 5 min. </li><li> Überstand. Resuspendieren Zellpellet in sterile PBS. Bestimmen der Zellzahl und Zentrifuge 1000 xg für 5 min bei 4 ° C. Gehen Sie auf die Isolierung von T-Zellen. </li></ol> Isolation und Aktivierung von Maus-T-Zellen <ol start="17"><li> Magnetisch-Label die Zellen nach dem Produkt-Handbuch (CD8 + T-Zell-Isolations-Kit von Miltenyi Biotec). </li><li> Legen Sie eine LS-Säule (Miltenyi Biotec) in der magnetic Feld. Übernehmen markierte Zellsuspension auf die Säule. Sammeln Flow-Through-(angereichert Maus CD8 + T-Zellen). Die Säule wird dreimal mit 3 ml Puffer nach dem Handbuch und verbinden sich mit früheren Durchströmung. </li><li> Stain die Zellen mit anti-CD3e und anti-CD8a Antikörper und zu analysieren, mittels Durchflusszytometrie (Abbildung 2a). Für eine typische Trennung kann ~ 8-10% der gesamten Zellen isoliert werden. ~ 90% der isolierten Zellen sind CD8 + T-Zellen. </li><li> Zentrifugieren Sie die Zellen bei 1000 xg, 5 min. Überstand verwerfen. Zellpellet in RPMI-Medium mit rekombinantem humanem IL-2 (rhIL2, 20 ng / ml) bei 1x10 6 / ml. </li><li> Kurz vor Zugabe der Zellen, zu entfernen Antikörper-Lösung von der Platte (vorher in 2.11) hergestellt. Spülen Sie jede Vertiefung mit sterilem PBS und entfernen PBS. </li><li> 1 ml der Zellsuspension in jede Vertiefung. Legen Sie die Platte bei 37 ° C / 5% CO 2 in einer Zelle Inkubator. </li></ol> 3. (Tag 2 und Tag 3) Die Infektion von aktivierten T-Zellen <ol><li> Nach 2 Tagen Virus-Produktion sollte das Medium in den Plat-E Zellplatte gelb werden. Übertragung viraler Überstand in ein 15 mL konischen Rohr. Fügen Sie 10 ml frisches DMEM-Medium auf die Platte für weitere virale Produktion (Optional). </li><li> Centrifuge Virusüberstand bei 1000 xg für 5 min auf Zellen Ablagerungen zu entfernen. Sorgfältig Transfer Virusüberstand in ein neues Röhrchen. Lassen Sie etwas Flüssigkeit am Boden des Röhrchens und stören nicht die Zellen Schutt. </li><li> Aktivierten T-Zellen von der Platte in eine 50 ml konischen Rohr Collect. Bestimmen Sie die Anzahl der Zellen. Sparen Sie einige Zellen in ein separates Röhrchen als negativ (un-transduzierten) eingesetzt werden. Zentrifugation bei 1000 xg für 5 min und Überstand verwerfen. </li><li> Zellpellet in Virusüberstand bei 10 6 Zellen pro 1 ml mit 20 ng / mL rhIL2 und 10 pg / ml Protaminsulfat. 1 ml der Zellsuspension in jede Vertiefung einer 24-Well-Platte. Resuspendieren der Zellen in das Steuerrohr in RPMI Medium bei gleicher Zelldichte mit der gleichen Menge an rhIL2 und Protaminsulfat. Fügen Sie die Zellen auf die gleiche Platte. </li><li> Wickeln Sie die Platte mit Kunststoff-Folie. Zentrifuge 90 Minuten bei 2000 xg, bei 32 ° C ohne Bremse. </li><li> Nach der Zentrifugation, entfernen Sie die Kunststoff-Folie. 1 ml frisches RPMI-Medium mit 20 ng / mL rhIL2 in jede Vertiefung. Legen Sie die Platte wieder in den Inkubator. </li><li> (Optional) Höhere Expression von TCR kann durch das Sammeln der Virus-Überstand und Wiederholung der Infektion Verfahren am Tag 3 erreicht werden. </li></ol> 4. Expansion von Zellen und Evaluierung von Transduktionseffizienz <ol><li> Untersuchen Sie die transduzierten T-Zellen täglich. Split der Zellen bei 1:3-Verhältnis, wenn nötig in RPMI-Medium rhIL2. Lassen Sie sich nicht die Zellen überwuchern (medium gelb). </li><li> Am Tag 6 oder Tag 7, färben die Zellen mit dem Antikörper und MHC-Tetramer spezifisch für den TCR auf den Ausdruck des TCR auf T-Zell-Oberfläche zu beurteilen. Verwenden Sie die un-transduzierten T-Zellen als Kontrolle (Abbildung 2c). </li><li> Die transduzierten T-Zellen sind bereit für weitere Downstream-Anwendungen. </li></ol> 5. Repräsentative Ergebnisse Oft ist es vorteilhaft, T-Zell-Untergruppen vor Transduktion zu reinigen, obwohl Splenozyten können ohne Reinigung transduziert werden. Hohe Reinheit T-Zell-Untergruppen erhalten Sie über kommerzielle magnetische Beads (Abb. 2a). Zur Beurteilung der Expression von TCR auf T-Zellen können Antikörper, die spezifisch für TCR Alpha-oder Beta-Ketten verwendet werden. In der Regel 30% bis 80% der Zellen ausdrücken können transduzierten TCR (Abb. 2b) in Abhängigkeit von der TCR-Gene und Virustiter. MHC-Tetramer spezifisch für den TCR kann auch verwendet werden, um weiter zu verifizieren funktionelle Expression von TCR (Abbildung 2c) werden. <img src="/files/ftp_upload/2307/2307fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Beispiel T-Zell-Rezeptor-Gen-Konstrukt sub-kloniert in einen retroviralen Vektor. Volle Länge alpha und beta-Kette-Gene sind durch eine Selbst-spaltbare 2A Peptidlinker 8 verbunden. <img src="/files/ftp_upload/2307/2307fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Beispiel für erfolgreiche Isolierung und Transduktion von Maus-T-Zellen. (A) CD8 T-Zellen wurden aus Milzzellen mit CD8a + T-Zell-Isolation Kit (Miltenyi Biotec) und gefärbt mit anti-CD3e und anti-CD8a Antikörper isoliert. 209 -217 4-Peptid und gefärbt mit anti-human Vbeta 8-Antikörper (b) und HLA-A2kb gp100-Tetramer (c): Isolierte CD8 T-Zellen wurden mit R6C12 TCR spezifisch für humanes gp100 transduziert.

<ol>
	<li>Krogsgaard, M., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+T+cells+'see'+antigen.">How T cells 'see' antigen.</a> Nat. Immunol. 6, 239-245 (2005).</li><li>Berg, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen/MHC-specific+T+cells+are+preferentially+exported+from+the+thymus+in+the+presence+of+their+MHC+ligand.">Antigen/MHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand.</a> Cell. 58, 1035-1046 (1989).</li><li>Hogquist, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+antagonist+peptides+induce+positive+selection.">T cell receptor antagonist peptides induce positive selection.</a> Cell. 76, 17-27 (1994).</li><li>Yu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poor+immunogenicity+of+a+self/tumor+antigen+derives+from+peptide-MHC-I+instability+and+is+independent+of+tolerance.">Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance.</a> J. Clin. Invest. 114, 551-559 (2004).</li><li>Holst, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+T-cell+receptor+retrogenic+mice.">Generation of T-cell receptor retrogenic mice.</a> Nat Protoc. 1, 406-417 (2006).</li><li>Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+regression+in+patients+after+transfer+of+genetically+engineered+lymphocytes.">Cancer regression in patients after transfer of genetically engineered lymphocytes.</a> Science. 314, 126-129 (2006).</li><li>Hawley, R. G., Lieu, F. H., Fong, A. Z., Hawley, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+retroviral+vectors+for+potential+use+in+gene+therapy.">Versatile retroviral vectors for potential use in gene therapy.</a> Gene Ther. 1, 136-138 (1994).</li><li>Szymczak, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+multi-gene+deficiency+in+vivo+using+a+single+'self-cleaving'+2A+peptide-based+retroviral+vector.">Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector.</a> Nat. Biotechnol. 22, 589-594 (2004).</li><li>Cohen, C. J., Zhao, Y., Zheng, Z., Rosenberg, S. A., Morgan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+antitumor+activity+of+murine-human+hybrid+T-cell+receptor+(TCR)+in+human+lymphocytes+is+associated+with+improved+pairing+and+TCR/CD3+stability.">Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability.</a> Cancer Res. 66, 8878-8886 (2006).</li><li>Letourneur, F., Malissen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+a+T+cell+hybridoma+variant+deprived+of+functional+T+cell+receptor+alpha+and+beta+chain+transcripts+reveals+a+nonfunctional+alpha-mRNA+of+BW5147+origin.">Derivation of a T cell hybridoma variant deprived of functional T cell receptor alpha and beta chain transcripts reveals a nonfunctional alpha-mRNA of BW5147 origin.</a> Eur. J. Immunol. 19, 2269-2274 (1989).</li><li>Bendle, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lethal+graft-versus-host+disease+in+mouse+models+of+T+cell+receptor+gene+therapy.">Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy.</a> Nat. Med. 16, 565-570 (2010).</li></ol>

Mehrere Schritte sind entscheidend, um optimale Ergebnisse zu erzielen. Plat-E Verpackungs-Zelllinie sollte in gesundem Zustand gehalten werden. Falls erforderlich, können die Zellen ein paar Mal in DMEM-Medium mit 1 pg / mL Puromycin, 10 pg / ml Blasticidin zu einem Verlust der retroviralen Struktur Proteinexpression verhindern passagiert werden. Am Tag der Transfektion sollten die Zellen ca. 80% konfluent sein. Zu wenige oder zu viele Zellen reduzieren die Virustiter. Das Virus Qualität kann durch Tests an Zelllinien, die leicht zu transduzierten überprüft werden. Da TCRs CD3-Komplexe auf der Zelloberfläche exprimiert werden müssen, verwenden wir routinemäßig 58α-/β- Hybridomzellen 10 (a-Zelllinie, die nicht exprimiert endogenen TCR α-oder β-Kette, aber nicht ausdrücken CD3-Komplex). Wir erhalten fast 100% Transduktionseffizienz von Hybridomzellen, wenn transduzierenden Zellen mit 1 ml Virus-Überstand. Schließlich müssen die T-Zellen aktiviert werden und wuchernden, weil Retrovirus nur infizieren können sich aktiv teilenden Zellen. Die hier vorgestellte Methode zur Herstellung von Antigen-spezifischen T-Zellen hat einige Einschränkungen. Erstens ist es schwierig, 100% Transduktionseffizienz für primäre Maus T-Zellen zu erreichen. Ein Teil der Zellen exprimieren nicht das TCR von Interesse. Zweitens, die transduzierten TCR α-und β-Ketten können mit endogenen TCR Fehlpaarung 9, die die Expression Niveau des TCR von Interesse zu reduzieren. Darüber hinaus können die fehlgepaarten TCRs Ursache Autoimmunität in vivo 11. Schließlich gibt es nur einen begrenzten Zeitrahmen der transduzierten T-Zellen verwendet werden kann. Nach ca. 1 Woche durchlaufen die Zellen Apoptose sei denn, er re-stimuliert. Allerdings können die Zellen erschöpft und verlieren Funktionen mit sich wiederholenden re-Stimulationen.

Media: DMEM-Medium (DMEM +10% hitzeinaktiviertem FBS + Pen / Strep + L-Glu + Natriumpyruvat + Non-essentielle Aminosäure). DMEM-Medium ohne Pen / Strep (Wie DMEM-Medium, jedoch ohne Zusatz von Pen / Strep). RPMI-Medium (RPMI +10% hitzeinaktiviertem FBS + L-Glu + Non-essentielle Aminosäure + β-Mercaphoethanol + Pen / Strep + Natriumpyruvat).

retroviral transduction of t-cell receptors in mouse t-cells

T-Zell-Rezeptoren (TCR) spielen eine zentrale Rolle im Immunsystem. TCRs auf T-Zell-Oberflächen können spezifisch erkennen Peptid-Antigenen durch Antigen-präsentierende Zellen (APC) 1 vorgestellt. Diese Anerkennung führt zur Aktivierung von T-Zellen und eine Reihe von funktionellen Ergebnisse (zB Produktion von Zytokinen, die Tötung der Zielzellen). Das Verständnis der funktionellen Rolle des TCR ist entscheidend für die Leistung des Immunsystems zu nutzen, um eine Vielzahl von immunologischen Erkrankungen (zB Krebs oder Autoimmunität) zu behandeln. Es ist zweckmäßig, TCRs in Maus-Modellen, die auf verschiedene Weise erreicht werden kann studieren. Machen TCR transgenen Mausmodellen ist kostspielig und zeitaufwändig und derzeit gibt es nur eine begrenzte Anzahl von ihnen zur Verfügung 2-4. Alternativ Mäusen mit Antigen-spezifischen T-Zellen können durch Knochenmark-Chimäre generiert werden. Auch diese Methode dauert mehrere Wochen und erfordert Know-how 5. Retrovirale Transduktion von TCRs in in vitro aktivierten Maus-T-Zellen ist eine schnelle und relativ einfache Methode, um T-Zellen des gewünschten Peptid-MHC-Spezifität zu erhalten. Antigen-spezifische T-Zellen können in einer Woche erzeugt werden und in jedem Downstream-Anwendungen. Studieren transduzierten T-Zellen hat auch unmittelbare Anwendung für die menschliche Immuntherapie, wie adoptiven Transfer von menschlichen T-Zellen transduziert mit Antigen-spezifischen TCR ist eine neue Strategie für Krebs Behandlung 6. Hier präsentieren wir ein Protokoll zur retroviral transduzieren TCRs in in vitro aktivierten T-Zellen der Maus. Sowohl Mensch und Maus TCR-Gene verwendet werden können. Retroviren tragen spezifische TCR-Gene erzeugt und verwendet, um Maus-T-Zellen mit anti-CD3 und anti-CD28 Antikörper aktiviert infizieren. Nach In-vitro-Expansion transduzierten T-Zellen mittels Durchflusszytometrie analysiert.

Watch this Scientific Journal Video about Retroviral Transduction of T-cell Receptors in Mouse T-cells at JoVE.com

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

Wir präsentieren ein Protokoll zur Antigen-spezifischen Maus-T-Zellen produzieren unter Verwendung von retroviralen Transduktion

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 ...

retroviral-transduction-of-t-cell-receptors-in-mouse-t-cells

Research

JoVE Journal

Immunology and Infection

34.8K Aufrufe.  New York University School of Medicine. Wir präsentieren ein Protokoll zur Antigen-spezifischen Maus-T-Zellen produzieren unter Verwendung von retroviralen Transduktion

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

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 ...

Forschung

Immunologie und Infektion

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 ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

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

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

Adenovirale Transduktion von Naive CD4 T-Zellen zu Treg Differenzierung Studieren

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

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Mouse embryonic stem cells can be differentiated to T cells in vitro using the OP9-DL1 co-culture system. Success in this procedure requires careful attention to reagent/cell maintenance, and key technique sensitive steps. Here we discuss these critical parameters and provide a detailed protocol to encourage adoption of this technology.

Ableitung von T-Zellen<em&gt; In-vitro-</em&gt; Aus embryonalen Stammzellen der Maus

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

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&#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 ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Retrovirale Transduktion von Knochenmark-Progenitorzellen zu generieren T-Zell-Rezeptor Retrogenic Mäuse

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

Generieren<em&gt; De Novo</em&gt; Antigen-spezifische Zellrezeptoren humanen T von Retrovirale Transduktion von Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.

Many experimental systems have been utilized to understand the mechanisms regulating T cell development and function in an immune response. Here a genetic approach using retroviral transduction is described, which is economic, time efficient, and most importantly, highly informative in identifying regulatory pathways.

Retrovirale Transduktion von T-Helferzellen als genetischer Ansatz zur Untersuchung Mechanismen der Kontrolle ihrer Differenzierung und Funktion

Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

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

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

In Vivo Augmentation der Gut-Homing Regulatory T Cell Induction

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

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the key steps to investigate TRMs. There are slight variations in lymphocyte isolation protocols for different organs. Kidney is an essential non-lymphoid organ with numerous immune cell infiltration especially after pathogen exposure or autoimmune activation. In recent years, multiple labs including our own have started characterizing kidney resident CD8+ T cells in various physiological and pathological settings in both mouse and human. Due to the abundance of T lymphocytes, kidney represents an attractive model organ to study TRMs in non-mucosal or non-barrier tissues. Here, we will describe a protocol commonly used in TRM-focused labs to isolate CD8+ T cells from mouse kidneys following systemic viral infection. Briefly, using an acute lymphocytic choriomeningitis virus (LCMV) infection model in C57BL/6 mice, we demonstrate intravascular CD8+ T cell labeling, enzymatic digestion, and density gradient centrifugation to isolate and enrich lymphocytes from mouse kidneys to make samples ready for the subsequent flow cytometry analysis.

Isolation of Mouse Kidney-Resident CD8+ T cells for Flow Cytometry Analysis

Following viral infection, kidney harbors a relatively large number of CD8+ T cells and offers an opportunity to study non-mucosal TRM cells. Here, we describe a protocol to isolate mouse kidney lymphocytes for flow cytometry analysis.

Isolierung von Maus-Kidney-Resident-CD8+ T-Zellen für die Durchflusszytometrie-Analyse

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...