Name: adenoviral transduction of naive cd4 t cells to study treg differentiation
Uploaded: 2013-08-13T13:00:00
Description: Watch this Scientific Journal Video about Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation at JoVE.com

Forfatterne vil gerne takke Lirui Du til konstruktion af pCAGAdDU vektor og Oliver Gorka for levering af fikseringen protokollen.

1.. Kloning af et gen af ​​interesse i en indtastning Vector <ol><li> Klone genet af interesse ind i en post vektor. PCR-amplifikation af genet efterfulgt af stump-ende-ligering ind i en topoisomerase-koblet vektor (f.eks pENTR / D-TOPO) eller restriktionsenzym-medieret kloning kan anvendes til denne procedure. </li></ol> 2.. Overførsel af genet af interesse i pCAGAdDu Destination Vector <ol><li> Overfør genet af interesse fra posten vektor i destinationen vektor af LR rekombin...

<ol>
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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roquin+binds+inducible+costimulator+mRNA+and+effectors+of+mRNA+decay+to+induce+microRNA-independent+post-transcriptional+repression.">Roquin binds inducible costimulator mRNA and effectors of mRNA decay to induce microRNA-independent post-transcriptional repression.</a> Nature Immunology. 11 (8), 725-733 (2010).</li><li>Russell, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+adenovirus+and+its+vectors.">Update on adenovirus and its vectors.</a> The Journal of General Virology. 81 (Pt. 11), 2573-2604 (2000).</li><li>Graham, F. L., Smiley, J., Russell, W. 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Virus Generation og titrering For at opnå optimale transfektion resultater vises kvaliteten og mængden af ​​lineariseret vektor vigtigste. Vi har ikke observere negative virkninger på den primære lysat produktion fra en indledende overvækst af kulturen da infektionen vil hurtigt videre, når effektiv virus produktionen finder sted. Dog kan virusproduktion ved HEK293A celler upåvirket af lange skær, der sænker effektiviteten. Nogle åbne læserammer blev faktisk fundet at interferere...

Tregs er afgørende at fastholde immun tolerance og til at dæmpe overskridelse immunrespons. Tregs undertrykke tilskuer T-celleaktivering. Derfor ablation af tregs fører til fatal autoimmunitet og selvdestruktion drevet af aktiverede T celler 1.. Tregs udvikle sig i thymus under negativ selektion af CD4 enkelt-positive prækursorer, men de kan også differentiere i periferien fra naive CD4 T-celler upon lavdosis antigenstimulering med suboptimal co-stimulation 1,2. Thymisk tregs synes at undertrykke væv autoimmunitet mod selv-antigener, mens perifer tregs har været impliceret i at yde tolerance i tarmen eller lungerne. Disse inducerede tregs potente forhindre T-celle aktivering efter indregning af udenlandske antigener i slimhinden, herunder miljø-antigener fra fødevarer og luft, kommensale bakterier og allergener 3,4. Desuden er tregs afgørende at etablere maternel tolerance over føtale peptider 5 og til preaftræk graft-versus-host sygdom 6.. På samme tid, også tregs medierer uønskede virkninger ved at dæmpe immunovervågning af tumorceller 7,8. Kendetegnet træk tregs er udtryk for delmængde-angivelse transskription faktor Foxp3, en gaffel-head domæne-holdige transskription faktor, der er nødvendig og tilstrækkelig til at give Treg funktion 9,10. Nogle signalveje, der kan fremkalde Foxp3 udtryk er kendt. Imidlertid er de molekylære processer, der styrer, regulerer eller modulerer Treg differentiering i respons på T-celle receptoren udløser mindre godt forstået. Tregs kan meget effektivt induceres in vitro ved stimulering af naive CD4 T-celler med anti-CD3 og anti-CD28-antistoffer i nærvær af TGFp og IL-2 11. Som det spirende tregs er funktionelle in vivo, manipulation af molekyler, der fremmer Treg differentiering bærer et enormt potentiale for fremtidig therapies, f.eks. behandling af astma, Crohns sygdom, og transplantation 11,12 Omvendt kan terapeutisk modulation af molekyler til at blokere Treg differentiering giver fordele i fremtidige tilgange kombineret behandling af tumor patienter. In vitro differentiering assays har været medvirkende til beskrivelse af molekylære ændringer, der er forbundet med T-celle delmængde differentiering. I øjeblikket. Eksperimentelle forsøg på søgning eller screene for genprodukter, der styrer T-celle differentiering er hæmmet af, at de mest almindelige metoder til ektopisk genekspression mislykkes i naive T-celler For eksempel er elektroporation og retroviral transduktion kun effektiv i aktiverede T-celler. I modsætning til de oprindelige forventninger, lentiviral transduktion, som typisk er effektiv i hvilende celler, kræver forudgående aktivering af naive T-celler ved cytokiner 13.. Endvidere overførsel af cDNA eller mRNA under elektroporationinvolverer depolarisering af plasmamembranen, som selv giver funktioner af T-celleaktivering, og kan endda mobilisere Ca 2 +-signalering og aktivere NFAT proteiner (upubliceret observation og ref. 14). Tilsvarende for retroviral transduktion, har den naive T-celler, der skal aktiveres i 18-40 timer. I løbet af denne tid sker en opdeling af kernemembranen under celledeling og muliggør efterfølgende genomisk integration af den retrovirale vektor 15.. Disse metoder er derfor ikke i stand til at løse den tidlige molekylære regulering af indledende T-celle møde med antigen, der er den afgørende fase af hjælper-T-celle differentiering. Adenoviral transduktion er kendt for at give forbigående ektopisk genekspression i en række humane celletyper, der udtrykker den humane Coxsackie adenovirus receptor (CAR). Den fortsætter uden krav om celleaktivering eller cellecyklusfremadskriden. Overfladen ekspression af CAR er afgørende for effektiv virus fastgørelse og internalisering, og transgene ekspression af den trunkerede version, CARΔ1 under en T-celle-specifik promotor blev fundet at gøre musethymocytter og T-celler er modtagelige for adenovirusinfektion 16. Vigtigt er transgenet ikke ændre thymocyt udvikling eller in vitro differentiering af naive CD4 T-celler i forskellige undergrupper (data ikke vist, ref 17..). Adenovirus-medieret transduktion af T-celler er tidligere blevet anvendt til overekspression 17,18 og knock-down-tilgange 19,20. De transgene T-celler kan oprenses fra kommercielt tilgængeligt DO11.10 TG, CARΔ1 tg (Taconic, Inc. og ref 17..). Vigtigere er, adenoviral transduktion muliggør høj ekspression af et gen af ​​interesse i naive T-celler uden at inducere tydelige tegn på aktivering. T-cellerne forbliver naive (CD44 lav, CD62L høj) og hvile (CD25 -, CD69 -) efter infectipå og kan aktiveres og differentierede til Treg minder om ikke-inficerede celler. Produktion af rekombinante adenovira kan opnås efter transfektion af HEK293A celler med adenovirale plasmider (figur 1). Disse plasmider indeholder typisk human type 5 adenovirus genomet med E1-og E3 deleteret at gøre rekombinante adenovira replikation-inkompetente 21. HEK293A celler supplerer replikation mangel, som de er blevet udødeliggjort gennem stabil integration af klippet adenovirus 22.. Da adenovirusvektorer er store (~ 40 kb) og følgelig ikke velegnet til traditionel restriktionsenzym-medieret kloning, anvendte vi Gateway systemet. Genet af interesse er først klonet ind i en mindre post vektor, hvorfra det let kan overføres til den adenovirale destination vektor via lambda rekombinationsreaktion (LR) 23. Vi konstrueret pCAGAdDu vektorenved at kombinere CAG promotor (kylling actinpromotoren og CMV-enhanceren) med en ekspressionskassette indeholdende LR flankerer den prokaryote CCDB selektionsmarkør 24. Denne ekspressionskassette fusioneret til et internt ribosomindgangssted (IRES) element, der tillader coekspression af eukaryote infektion markør forstærket grønt fluorescerende protein (eGFP), der er fusioneret til en sekvens indeholdende bovint væksthormon poly (A)-signal. Vi valgte CAG cis-regulatoriske sekvenser, da prototypisk CMV-promotoren blev fundet at være meget aktiveringen afhængige og derfor ugunstig for genekspression i naive T-celler. Her giver vi en protokol til effektiv in vitro Treg differentiering og en fremgangsmåde til at transducere naive CD4 T-celler uden aktivering (fig. 2). Metoden giver mulighed for ektopisk genekspression eller vælte foregående CD4 T-celle differentiering på naive tilstand. Det gør det muligt at teste virkningen af ​​en overexpressed gen af ​​interesse i den tidlige signaleringsbegivenheder upon indledende TCR stimulering indtil T-celle delmængde engagement. Vores validering eksperimenter også give et grundlag for at etablere tilsvarende adenovirus anvendelse i differentieringen af ​​andre T-celle delmængder såsom Th1, Th2, Th9, Th17, Th22, eller TFH celler.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>pENTR/D-TOPO Cloning Kit</td>
			<td>Invitrogen</td>
			<td>K240020</td>
		</tr>
		<tr>
			<td>Gateway LR Clonase II Enzyme mix</td>
			<td>Invitrogen</td>
			<td>11791020</td>
		</tr>
		<tr>
			<td>PacI</td>
			<td>New England Biolabs</td>
			<td>R057S</td>
		</tr>
		<tr>
			<td>jetPEI</td>
			<td>Polyplus-transfection</td>
			<td>101-10N</td>
		</tr>
		<tr>
			<td>HEK293A Cell Line</td>
			<td>Invitrogen</td>
			<td>R705-07</td>
		</tr>
		<tr>
			<td>A549</td>
			<td>ATCC</td>
			<td>CCL-185</td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>BD Falcon</td>
			<td>353046</td>
		</tr>
		<tr>
			<td>14 cm tissue culture dish</td>
			<td>Nunc</td>
			<td>168381</td>
		</tr>
		<tr>
			<td>BALB/cJ-Tg(DO11.10)10Dlo Tg(CAR&Delta;1)1Jdgr</td>
			<td>Taconic Farms</td>
			<td>Model Nr. 4285</td>
		</tr>
		<tr>
			<td>Naive CD4+ T Cell Isolation Kit II</td>
			<td>Miltenyi Biotech</td>
			<td>130-094-131</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>41966-052</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>PAN Biotech</td>
			<td>1502-P110704</td>
		</tr>
		<tr>
			<td>PenStrep</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Lonza</td>
			<td>BE12-167F</td>
		</tr>
		<tr>
			<td>NaPyruvate</td>
			<td>Lonza</td>
			<td>BE13-115E</td>
		</tr>
		<tr>
			<td>NEAA 100x</td>
			<td>Lonza</td>
			<td>BE13-114E</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030</td>
		</tr>
		<tr>
			<td>HEPES Buffer Solution (1 M)</td>
			<td>Invitrogen</td>
			<td>15630-056</td>
		</tr>
		<tr>
			<td>&beta;-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M-7522</td>
		</tr>
		<tr>
			<td>MEM Essential vitamin mixture (100x)</td>
			<td>Lonza</td>
			<td>13-607C</td>
		</tr>
		<tr>
			<td>Dynabeads Mouse T-Activator CD3/CD28 for Cell Expansion and Activation</td>
			<td>Invitrogen</td>
			<td>114-56D</td>
		</tr>
		<tr>
			<td>Recombinant Human TGF-beta 1</td>
			<td>R&amp;D Systems</td>
			<td>240B</td>
		</tr>
		<tr>
			<td>Proleukin S (18 x 106IE)</td>
			<td>Novartis</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit</td>
			<td>Invitrogen</td>
			<td>L-23105</td>
		</tr>
		<tr>
			<td>Mouse BD Fc BlockT</td>
			<td>BD Pharmingen</td>
			<td>553141</td>
		</tr>
		<tr>
			<td>Anti-Mouse/Rat Foxp3 PE</td>
			<td>eBioscience</td>
			<td>12-5773-82</td>
		</tr>
		<tr>
			<td>Mmu-miR-155 TaqMan MicroRNA Assay</td>
			<td>Roche Applied Biosystems</td>
			<td>4427975</td>
		</tr>
		<tr>
			<td>LightCycler 480 Probes Master</td>
			<td>Roche Applied Biosystems</td>
			<td>04902343001</td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Reverse Transcription Kit</td>
			<td>Roche Applied Biosystems</td>
			<td>4366596</td>
		</tr>
	</tbody>
</table>

adenoviral transduction of naive cd4 t cells to study treg differentiation

Regulatoriske T-celler (tregs) er vigtigt at give immuntolerance over for selv samt til visse fremmede antigener. Tregs kan genereres fra naive CD4 T-celler in vitro med TCR-og co-stimulering i nærvær af TGFp og IL-2. Det bærer et enormt potentiale for fremtidige behandlinger dog molekylerne og signalveje, der kontrollerer differentiering er stort set ukendte. Primære T-celler kan manipuleres gennem ektopisk genekspression, men fælles metoder undlader at målrette den vigtigste naive tilstand af T-celle forud for primær antigengenkendelse. Her giver vi en protokol til at udtrykke ektopiske gener hos naive CD4 T-celler in vitro, før induktion Treg differentiering. Den anvender transduktion med replikationsdefekt adenovirus og forklarer dets produktion og produktion. Adenovirus kan tage op store indsatser (op til 7 kb), og kan udstyres med initiativtagere at opnå en høj og forbigående overexpression i T-celler. Det effektivt transducerer naive mus T-celler, hvis de udtrykker en transgen Coxsackie adenovirus receptor (CAR). Vigtigere er det, efter infektion af T-cellerne forbliver naiv (CD44 lav, CD62L høj) og hvile (CD25 -, CD69 -) og kan aktiveres og differentierede til tregs minder om ikke-inficerede celler. Således er denne metode giver mulighed manipulation af CD4 T-celle differentiering lige fra begyndelsen. Det sikrer, at ektopisk genekspression er allerede på plads, når tidlige signalering begivenheder i det oprindelige TCR stimulering inducerer cellulære ændringer, der i sidste ende føre til Treg differentiering.

Virusproduktion For generation af high virustitere er timingen af ​​HEK293A cellehøst i den primære virus produktion eller virus forstærkning afgørende. Repræsentative fluorescerende og fasekontrast billeder med visuelle tegn på virus produktion er vist i figur 3.. CPE blev observeret 10 dage efter transfektion af HEK293A celler med en kontrolgruppe pCAGAdDu vektor uden indsats. CPE er kendetegnet ved fremkomsten af ​​områder med forstørrede og round-up celler, ...

Watch this Scientific Journal Video about Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation at JoVE.com

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Adenoviral genoverføring i naive CD4 T-celler med transgen ekspression af Coxsackie adenovirus receptoren muliggør molekylær analyse af regulatoriske T-celle differentiering<em&gt; In vitro</em&gt;.

Adenoviral transduktion af Naive CD4 T-celler til at studere Treg Differentiering

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 ...

Research

JoVE Journal

Immunology and Infection

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 ...

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

Accelerated Type 1 Diabetes Induktion i mus ved adoptiv overførsel af diabetogene CD4 + T-celler

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Isolation and Th17 Differentiation of NaÃƒÆ’Ã‚Â¯ve CD4 T Lymphocytes

Isolering og Th17 Differentiering af Naïve CD4 T-lymfocytter

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

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

Transplantation af Tail Hud til Study Allogen CD4 T-celle respons i mus

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

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induktion af murine tarmbetændelse ved adoptiv overførsel af effektor CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Høj</sup&gt; T celler i immunsvækkede mus

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

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

Mouse NaÃƒÆ’Ã‚Â¯ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Mus Naïve CD4<sup&gt; +</sup&gt; T Cell Isolation og<em&gt; In vitro</em&gt; Differentiering til T-celledelmængder

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

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Isolation and Ex Vivo Culture of VÃƒÅ½Ã‚Â´1+CD4+ÃƒÅ½Ã‚Â³ÃƒÅ½Ã‚Â´ T Cells, an Extrathymic ÃƒÅ½Ã‚Â±ÃƒÅ½Ã‚Â²T-cell Progenitor

Isolering og<em&gt; Ex vivo</em&gt; Kultur Vδ1<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt; γδ T-celler, en Extrathymic αβT-celleprogenitor

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

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

Retroviral transduktion af knoglemarven til at generere T-celle receptor Retrogenic Mus

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 of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

Fremstilling af CD4<sup&gt; +</sup&gt; T-celler til analyse af GD3 og GD2 gangliosidet Membrane Expression ved mikroskopi

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...