Die Autoren möchten sich Dr. Boris Reizis von der Abteilung für Mikrobiologie und Immunologie an der Columbia University danken für hilfreiche Diskussionen. Wir möchten auch Dr. Margaret Bynoe, Dr. Rodman Getchell und Deequa Mahamed vom Department of Microbiology and Immunology danken an der College of Veterinary Medicine, Cornell University für die technische Unterstützung bei der Durchflusszytometrie und Lu Huang für die Unterstützung bei Videobearbeitung. Die Autoren wollen auch die Mitglieder des Duhamel und Weiss Labors für die kritische Durchsicht des Manuskripts und Video-Produktion, und Stephanie Yazinski danken aus dem Weiss-Labor für die Zucht und Genotypisierung p53 - / - Mäusen. Tierversuche wurden in Übereinstimmung mit den Richtlinien und Vorschriften her von der Cornell University Institutional Animal Care und Verwenden Ausschusses durchgeführt. Diese Arbeit wurde teilweise durch Mittel von der US Department of Agriculture, Cooperative State Research, Education, and Extension Service, Animal Health and Disease Research Program (um GED und RSW) und NIH gewährt R03 HD058220 und R01CA108773 (zum RSW) zur Verfügung gestellt unterstützt. Das Video wurde unter Verwendung von in-house Einrichtungen durch das Personal aus dem Duhamel &amp; Weiss Labors.

1. Dissection der Tumor <ol><li> Für Tumor Dissektion, ist ein sauberes Papiertuch oder Einweg-OP-Tuch auf dem Sezieren Pad gelegt und versprüht mit 70% Ethanol. </li><li> Mäuse für dieses Protokoll werden routinemäßig mit CO 2 getötet. Legen Sie die eingeschläfert Maus auf die Dissektion Pad und Spray beiden Seiten des Tieres mit Ethanol. Die Maus ist in Ort auf die Dissektion Pad mit vier oder mehr Push-Pins durch die Beine gesteckt und gründlich mit 70% Ethanol besprüht statt. </li><li> Die Haut über der Mittellinie ist vom Schambein an der Basis des Kopfes mit einer sterilen Schere, unter Vermeidung der darunter liegenden Muskeln und Eindringen in die Körperhöhlen geschnitten. </li><li> Öffnen der Bauchhöhle mit einem neuen Paar sterile Schere und Pinzette zu vorsichtig, um nicht Schaden der inneren Organe. </li><li> Öffnen Sie die Brusthöhle durch Sezieren und Ablenkung des Brustkorbs, um den Tumor zu offenbaren, ohne Beschädigung der Blutgefäße. </li><li> Separate den Tumor von anderen Organen, und übertragen ein Stück ca. 40-50% des gesamten Tumors (ca. bis zu 1,5 x 0,8 x 0,8 cm) in kaltem PBS auf Eis gehalten. </li><li> Der verbleibende Teil des Tumors kann auch für andere Tests gespeichert werden und eine vollständige Autopsie kann zu diesem Zeitpunkt durchgeführt werden. </li></ol> 2. Anzucht-Lymphom-Zellen <ol><li> Spray das Rohr mit dem sezierten Tumor mit 70% Ethanol und Transfer zu einem Biosicherheitswerkbank. </li><li> Fügen Sie 10 ml Kulturmedium in ein steriles 150 mm 2 Petrischale. </li><li> Übertragen Sie die Stück Tumor in der Petrischale durch vorsichtiges Absaugen mit einer Pipette. </li><li> Beugen zwei sterilen 18-Gauge-Nadel mit Blick auf den abgeschrägten Seite nach außen. </li><li> Distanzieren der Tumor mit der gebogenen Nadeln. </li><li> Swirl der Schale, klopfen Sie leicht und in einem Winkel zu der Zellsuspension auf eine Seite zu sammeln, bei gleichzeitiger Vermeidung große Stücke des Tumors. </li><li> Saugen Sie die Zellsuspension und Überführung in eine 50 ml konischen Rohr unter Vermeidung große Stücke des Tumors. </li><li> Waschen Sie die Platte mit 10 ml Medium und Überführung in das gleiche Rohr unter Vermeidung große Stücke des Tumors. </li><li> Mischen Sie die Zellsuspension von Lymphozyten und Stromazellen sanft zusammen und Transfer 5 mL in einen T-25 Zellkulturflasche und 10 ml in einen T-75 Zellkulturflasche. Vorsichtig schwenken die Flaschen. </li><li> Verwenden Sie die restlichen Zellsuspension auf 2, 4, 10.100 und 1000 Verdünnungen in 2,5 ml Endvolumen (Kultivieren von Zellen in verschiedenen Dichten mit verschiedenen Arten von Kulturgefäße maximiert die Wahrscheinlichkeit der Schaffung einer T-Zell-Linie) zu machen. </li><li> Platte 2,0 ml von jeder Verdünnung in einzelne Vertiefungen einer 6-Well-Platte. Vorsichtig schwenken der Platte. </li><li> Inkubieren Sie die Zellen für 48 h in feuchter CO 2-Atmosphäre in der Luft. </li></ol> 3. Feeding-Lymphom-Zellen <ol><li> Nach 48 h Inkubation mit 5 ml Medium, um die T-25 Kolben und 10 ml Medium, um die T-75 Kolben an der Wand entlang mit minimaler Störung der Zellen. </li><li> Add 2 ml Medium in jede Vertiefung der 6-Well-Platte an der Wand entlang mit minimaler Störung der Zellen. </li><li> Inkubieren Sie die Zellen für weitere 72 h. </li></ol> 4. Die Passage-Lymphom-Zellen <ol><li> Erste Passage <ol><li> Am Ende des fünften Tages der Inkubation werden die Zellen im Verhältnis 1:1 passagiert, unter Beibehaltung der ursprünglichen Flaschen und Teller (Beibehaltung der Zelldichte bei einer Konzentration von etwa 3x106 / mL in den ersten Passagen fördert erfolgreiche Etablierung eines kontinuierliche Zelllinie). <ol class="lalpha"><li> Für die T-25 Kolben, Transfer 5 ml Zellsuspension in eine neue Flasche mit 5 ml Medium. 5 ml Medium in den ursprünglichen Kolben. </li><li> Für die T-75 Kolben, Transfer 10 ml Zellsuspension in eine neue Flasche mit 10 ml Medium. Fügen Sie 10 ml Medium in den ursprünglichen Kolben. </li><li> Für die 6-Well-Platte, Transfer 2 mL Zellsuspension aus jeder Vertiefung in entsprechende Vertiefungen einer neuen Platte mit 2 ml Medium in jede Vertiefung. Add 2 ml Medium in jede Vertiefung der ursprünglichen Brunnen. </li></ol></li><li> Inkubieren Sie alle Flaschen und Platten für weitere 72 h. In unserer Erfahrung die meisten Zelllinien werden aus diesen Gang sind Kulturen (das Vorhandensein von lose anhaftenden Federung Zellaggregate über die angeschlossenen Stromazellen ist ein frühes Anzeichen einer etablierten Zelllinie) gegründet. </li><li> Für die Kulturen mit weniger oder gar keine lose anhaftenden Federung Zellaggregate, füttern die Flaschen oder Platten durch vorsichtiges ersetzt ca. 50% des Mediums alle 72 h. Um zu vermeiden, Entfernung von Zellen aus dem ursprünglichen Kultur, Kultur Schiffe stehen gelassen und ungestört für mindestens 5 min zu den Zellen Sediment am Boden lassen durch die Schwerkraft, bevor sanfte Aspiration des Überstandes Medium. </li></ol></li><li> Die Aufrechterhaltung etablierten Zelllinien <ol><li> Etablierte Zelllinien werden in neuen Flaschen und Platten passagiert. Für T-25 und T-75 Flaschen, 5 ml des Originals oderDurchgang eine Suspension in 5 ml Medium. Für 6-Well-Platten, 3 ml des Originals oder Passage einer Zelle Suspension in 7 ml Medium in einem T-25 Kolben. Feed und bleibt das Original oder eine Passage einer Kultur durch den Austausch des Volumens mit frischem Kulturmedium entfernt. </li><li> Passage etablierten Zelllinien in neue T-25 Flaschen. Die Zell-Linien haben eine Verdoppelung der Zeit von 18-24 h. Die Kulturen werden routinemäßig bei einer Dichte von 1-3x106 / mL gepflegt und passagiert alle 3 Tage. Fügen Sie die benötigte Menge an früherer Stelle Kultur in das neue Medium zu machen bis zu 10 mL. Futter-und behält die ursprüngliche Kultur. </li><li> Nach ein paar Passagen, die nicht haftenden Zellen selbst ohne die anhaftenden Stromazellen erhalten und eine kleinere Anzahl von adhärenten Zellen werden über mit jedem Durchgang durchgeführt. Über diesen Punkt können die Kulturen in nicht-Gewebekultur gehalten werden behandelt T-25 Flaschen. </li></ol></li></ol> 5. Freezing und Wiederherstellen von Lymphom-Zelllinien <ol><li> Die Zelllinien sind in frisch zubereiteten RPMI 1640 Medium mit 20% FBS und 10% DMSO eingefroren. </li><li> Zellen aus einer 48 h alten Kultur in T-25-Kolben (etwa 1.5x10 6 Zellen / ml) werden bei 1500 rpm für 5 min bei Raumtemperatur zentrifugiert. Nach Verwerfen des Überstandes werden die Zellen in 2 ml kaltem Einfriermedium resuspendiert. Zwei 1,0 ml Volumen der Zellsuspension aus jedem Kolben sind in Einfrierröhrchen platziert, sofort bei -80 ° C, und in flüssigem Stickstoff am folgenden Tag. </li><li> Die Zellen werden aus flüssigem Stickstoff Lagerung von schnellen Auftauen, und Resuspendieren der Inhalt in 10 ml frisches Kulturmedium gewonnen. Alternativ sind frisch aufgetauten Zellen einmal in warmem Kulturmedium gewaschen, um die DMSO vor Resuspendieren in 10 ml frisches Kulturmedium zu entfernen. Die Zellen werden für mindestens 72 h vor der anschließenden Passage kultiviert. </li></ol> 6. Repräsentative Ergebnisse Ein ATM - / - T-Zell-Linie entwickelt, die dieses Protokoll verwenden, und bezeichnet DWJ-ATM-1 wurde durch Färbung mit anti-CD3-FITC, anti-CD4-APC und anti-CD8-PE und Durchflusszytometrie (Abbildung 1) aus. Mehr als 90% der T-Zellen wurden CD3, CD4-und CD8-Triple-positiv. <img src="/files/ftp_upload/2598/2598fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Charakterisierung eines T-Zell-Linie entwickelt, die dieses Protokoll verwenden. Die Zelloberflächenmarker eines etablierten Atm - / --Zelllinie bezeichnet DWJ-ATM-1 durch Färbung mit geprägt waren anti-CD3-FITC, anti-CD4-APC, anti-CD8-PE und Durchflusszytometrie. Kurz gesagt, wurden 1x10 6 frische Zellen einmal mit kaltem PBS (1500 rpm / 5 min) gewaschen und bei 4 ° C für 10 min mit 200 ul Antikörper-Cocktail (jeder Antikörper bei 1:200-Verdünnung in PBS mit 1% BSA. Die Zellen können sofort untersucht werden, während frisch oder fixiert für eine spätere Analyse. fixierten Zellen durch Zugabe von neutral gepuffertem Formalin bei 4% (vol / vol) Endkonzentration bereit sind und über Nacht bei 4 ° C für 30 min, durch Zentrifugation geerntet (1500 rpm / 5 min) und erneut in 200 ul PBS mit 1% BSA. Insgesamt 1 X 10 4 Zellen wurden unter Verwendung eines BD FACS Calibur Analysator und CellQuest Software (BD Biosciences, San Jose, CA) analysiert.

<ol>
	<li>Barlow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atm-deficient+mice:+a+paradigm+of+ataxia+telangiectasia.">Atm-deficient mice: a paradigm of ataxia telangiectasia.</a> Cell. 86, 159-171 (1996).</li><li>Elson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+defects+in+ataxia-telangiectasia+protein-deficient+mice.">Pleiotropic defects in ataxia-telangiectasia protein-deficient mice.</a> Proc Natl Acad Sci U S A. 93, 13084-13089 (1996).</li><li>Kuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Atm-/-+thymic+lymphoma+cell+proliferation+in+vitro+and+in+vivo+by+dexamethasone.">Control of Atm-/- thymic lymphoma cell proliferation in vitro and in vivo by dexamethasone.</a> Cancer Chemother Pharmacol. 55, 203-212 (2005).</li><li>Browne, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+a+catalytic+antioxidant+corrects+the+neurobehavioral+defect+in+ataxia-telangiectasia+mice.">Treatment with a catalytic antioxidant corrects the neurobehavioral defect in ataxia-telangiectasia mice.</a> Free Radic Biol Med. 36, 938-942 (2004).</li><li>Jacks, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+spectrum+analysis+in+p53-mutant+mice.">Tumor spectrum analysis in p53-mutant mice.</a> Curr Biol. 4, 1-7 (1994).</li><li>Chervinsky, D. S., Lam, D. H., Zhao, X. F., Melman, M. P., Aplan, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+T+cell+leukemia+cell+lines+established+from+SCL/LMO1+double+transgenic+mice.">Development and characterization of T cell leukemia cell lines established from SCL/LMO1 double transgenic mice.</a> Leukemia. 15, 141-147 (2001).</li><li>Sharma, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1+contributes+to+mouse+T-cell+leukemia+by+directly+inducing+the+expression+of+c-myc.">Notch1 contributes to mouse T-cell leukemia by directly inducing the expression of c-myc.</a> Mol Cell Biol. 26, 8022-8031 (2006).</li><li>Maser, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomally+unstable+mouse+tumours+have+genomic+alterations+similar+to+diverse+human+cancers.">Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers.</a> Nature. 447, 966-971 (2007).</li></ol>

In diesem Protokoll, bieten wir detaillierte Verfahren, um murine T-Zell-Linien aus zwei verschiedenen Maus-Genotypen zu schaffen; Atm - / - [FVB/N- Atm tm1Led / J] und p53 - / - [129/S6- Trp53 tm1Tyj / J] . Durch dieses Protokoll verwenden, insgesamt 6 (von 7 Versuche) Atm - / - und drei (von 5 Versuche) p53 - / - Maus T-Zell-Linien haben in unserem Labor etabliert. Repräsentative Daten zeigen, dass ...

<ul><li> Kühler </li><li> 70% Ethanol </li><li> Dissection Pad aus Schaumstoff-Block mit Aluminiumfolie abgedeckt und Stifte </li><li> Zwei Sätze von kleinen sterilen Schere und Pinzette </li><li> Sterile 150 mm 2 Petrischale (Thermo Fisher Scientific, Waltham, MA;. Cat # 08-757-13) </li><li> Sterile 18-Gauge-Nadel </li><li> 50 ml konische Röhrchen (Corning Inc., Corning NY, cat # 430829) </li><li> 10% gepuffertem Formalin </li><li> Zellkulturflaschen <ul><li> T-25 (TPP, Trasadingen, Schweiz; cat # 90025) </li><li> T-75 (TPP, Trasadingen, Schweiz; cat # 90075) </li></ul></li><li> 6-well-Platten (Corning Inc., Corning NY, cat # 3506) </li><li> Sterile Phosphat-gepufferter Kochsalzlösung pH 7,2 (PBS) </li><li> Nährboden <ul><li> RPMI 1640 mit 25 mM HEPES, 200 mM L-Glutamin (Lonza, Walkersville, MD; cat # 12-115F) mit den folgenden ergänzt: <ul><li> 10% hitzeinaktiviertem (56 ° C, 30 min) fötalem Rinderserum (FBS; Hyclone, Logan, UT, cat # SH30088.03) </li><li> 1% Penicillin und Streptomycin (Mediatech, Manassas, VA; cat # 30-002-CI) </li><li> 1% nicht-essentiellen Aminosäuren (Mediatech; cat # 25-025-CI) </li><li> 55 mM 2β-Mercaptoethanol (Sigma, St Louis, MO, cat # M752) </li></ul></li></ul></li><li> Dimethylsulfoxid (DMSO; Calbiochem, La Jolla, CA, cat # 317275) </li><li> Antikörper (eBioscience, San Diego, CA) <ul><li> anti-CD3-FITC (Kat. # 11-0031-85) </li><li> anti-CD4-APC (Kat. # 17-0041-83) </li><li> anti-CD8-PE (Kat. # 12-081-85) </li></ul></li><li> Rinderserumalbumin (BSA; Life Technologies, Grand Island, NY;. Cat # 11018-017) </li><li> Concanavalin A (Sigma cat # C5275) </li><li> 1,8 ml Einfrierröhrchen (Nunc, Roskilde, Dänemark, cat # 368632) </li></ul>

derivation of thymic lymphoma t-cell lines from atm-/- and p53-/- mice

Etablierte Zelllinien sind ein wichtiger Forschungs-Tool, das den Einsatz von Versuchstieren in der Forschung verringern kann. Bestimmte Stämme von genetisch veränderten Mäusen, wie ATM - / - und p53 - / - konsequent zu entwickeln Thymuslymphom früh im Leben 1,2, und damit kann als zuverlässige Quelle für die Ableitung von murinen T-Zell-Linien dienen. Hier präsentieren wir Ihnen ein detailliertes Protokoll für die Entwicklung der etablierten murinen Thymuslymphom T-Zell-Linien, ohne die Notwendigkeit, Interleukine wie in früheren Protokollen 1,3 beschrieben hinzufügen. Die Tumoren wurden von Mäusen im Alter von drei Minuten vor sechs Monaten geerntet, frühestens Hinweis auf sichtbare Tumoren auf der Beobachtung der gekrümmte Haltung basiert, erschwerte Atmung, schlechte Pflege und Verschwendung in einer anfälligen Stamm 1,4. / - - [FVB/N- Atm tm1Led / J] 2 und p53 - / - [129/S6- Trp53 tm1Tyj / J] 5 Mäusen Wir haben bereits mehrere T-Zell-Linien mit diesem Protokoll und Inzuchtstämme von ATM gegründet. Wir zeigen ferner, dass mehr als 90% der etablierten T-Zell-Population CD3, CD4 und CD8 ausdrückt. In Übereinstimmung mit stabil etablierten Zelllinien, die T-Zellen, die durch Verwendung der vorliegenden Protokoll generiert seit über einem Jahr wurden passagiert.

Watch this Scientific Journal Video about Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice at JoVE.com

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In diesem Video zeigen wir ein Protokoll zur Maus Thymuslymphom Zelllinien zu etablieren. Und p53-/ - - Mäusen mit Thymuslymphom Durch Einhalten dieses Protokolls haben wir erfolgreich mehrere T-Zell-Linien von Atm-/ etabliert.

Ableitung von Thymuslymphom T-Zell-Linien aus Atm - / -</sup Und P53 - / -</sup Mäuse

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

derivation-of-thymic-lymphoma-t-cell-lines-from-atm-and-p53-mice

Research

JoVE Journal

Immunology and Infection

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

12.2K Aufrufe.  Cornell University. In diesem Video zeigen wir ein Protokoll zur Maus Thymuslymphom Zelllinien zu etablieren. Und p53-/ - - Mäusen mit Thymuslymphom Durch Einhalten dieses Protokolls haben wir erfolgreich mehrere T-Zell-Linien von Atm-/ etabliert.

Serie: Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Transnuclear Mäuse mit vordefinierten T-Zell-Rezeptor Spezifitäten gegen Toxoplasma gondii Via SCNT Erhalten

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Forschung

Immunologie und Infektion

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 ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

Prüfung Thymic positive und negative Selektion mittels Durchflusszytometrie

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Echtzeit-Live-Imaging von T-Zell-Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

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 ...

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 ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

Herstellung und Bedeutung von Organotypischen Thymic Scheibe Kulturen

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed feeder cells, purified NK or T cells with as much as 10 mL of blood, or a high-dose of IL-2. This study presents a new method with a powerful and simple strategy to establish NK and T cell lines by culturing the peripheral blood mononuclear cells (PBMC) with the addition of recombinant human IL-2 (rhIL-2), and uses as little as 2 mL of whole blood. The cells can proliferate quickly in two weeks and be maintained for more than 3 months. With this method, 7 NK or T cell lines have been established with a high success rate. This method is simple, reliable, and applicable to establishing cell lines from more cases of CAEBV or NK/T cell lymphoma.

A simple method for obtaining NK and T cell clones from CAEBV patients was developed with high efficiency, a small amount of peripheral blood, and a low-dose of IL-2.

Eine effiziente und einfache Methode, NK und T-Zell-Linien von Patienten mit chronischen aktiven Epstein - Barr-Virus-Infektion zu etablieren

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...

Ableitung von Thymuslymphom T-Zell-Linien aus Atm - / - Und P53 - / - Mäuse

Ableitung von Thymuslymphom T-Zell-Linien aus Atm^{- / - Und P53^{- / - Mäuse}}