Name: generation of induced regulatory t cells from primary human na&iuml;ve and memory t cells
Uploaded: 2012-04-16T12:00:00
Description: Watch this Scientific Journal Video about Generation of Induced Regulatory T Cells from Primary Human NaÃƒÂ¯ve and Memory T Cells at JoVE.com

<p class="jove_content"&gt; Forfatterne ønsker å takke Jennifer Strange og Greg Bauman for deres assistanse med flowcytometrisystemer analyse og sortering. Dette arbeidet ble støttet av NIH Grant Number 2P20 RR020171 fra NCRR og ved University of Kentucky oppstart midler til FM, GIE erkjenner støtte av Presidential Graduate Fellowship og Kentucky Opportunity Fellowship.</p

1. Isolering av humane perifere mononukleære blodceller (PBMCs) fra Buffy Coat <ol><li> Erverve en enhet av Buffy Coat fra sykehuset eller i nærheten blod sentrum. Vårt blod senter gir oss om 40-60 ml av Buffy Coat per enhet hentet fra normale blodgivere. </li><li> Hell blod inn i en autoklaveres 500 ml glass flaske inneholder sterilt PBS å fortynne Buffy Coat. Den endelige volumet av PBS + Buffy Coat bør være 250 ml. </li><li> Fyll ti 50 ml koniske rør hver med 20 ml Lymphoprep løsning. </li><li> Forsiktig ov...

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	<li>Powrie, F., Correa-Oliveira, R., Mauze, S., Coffman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+interactions+between+CD45RBhigh+and+CD45RBlow+CD4++T+cells+are+important+for+the+balance+between+protective+and+pathogenic+cell-mediated+immunity.">Regulatory interactions between CD45RBhigh and CD45RBlow CD4+ T cells are important for the balance between protective and pathogenic cell-mediated immunity.</a> The Journal of Experimental Medicine. 179, 589-600 (1994).</li><li>Tarbell, K. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+infusion+of+ex+vivo+activated+and+expanded+CD4(+)CD25(+)+immune+regulatory+cells+inhibits+graft-versus-host+disease+lethality.">The infusion of ex vivo activated and expanded CD4(+)CD25(+) immune regulatory cells inhibits graft-versus-host disease lethality.</a> Blood. 99, 3493-3493 (2002).</li><li>Ziegler, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FOXP3:+of+mice+and+men.">FOXP3: of mice and men.</a> Annual Review of Immunology. 24, 209-226 (2006).</li><li>Riley, J. L., June, C. H., Blazar, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+T+regulatory+cell+therapy:+take+a+billion+or+so+and+call+me+in+the+morning.">Human T regulatory cell therapy: take a billion or so and call me in the morning.</a> Immunity. 30, 656-6565 (2009).</li><li>Morgan, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+FOXP3+mRNA+is+not+confined+to+CD4+CD25++T+regulatory+cells+in+humans.">Expression of FOXP3 mRNA is not confined to CD4+CD25+ T regulatory cells in humans.</a> Human Immunology. 66, 13-20 (2005).</li><li>Wang, J., Huizinga, T. W. J., Toew, R. E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+Novo+Generation+and+Enhanced+Suppression+of+Human+CD4+CD25++Regulatory+T+Cells+by+Retinoic+Acid.">De Novo Generation and Enhanced Suppression of Human CD4+CD25+ Regulatory T Cells by Retinoic Acid.</a> Journal of Immunology. 183, 4119-4126 (2009).</li><li>Hori, S., Nomura, T., Sakaguchi, S. <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+regulatory+T+cell+development+by+the+transcription+factor+Foxp3.">Control of regulatory T cell development by the transcription factor Foxp3.</a> Science (New York, N.Y.). 299, 1057-1061 (2003).</li><li>McHugh, 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=CD4(+)CD25(+)+immunoregulatory+T+cells:+gene+expression+analysis+reveals+a+functional+role+for+the+glucocorticoid-induced+TNF+receptor.">CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor.</a> Immunity. 16, 311-323 (2002).</li><li>Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+Self-Tolerance+Maintained+by+Cd25+Cd4+Regulatory+T+Cells+Constitutively+Expressing+Cytotoxic+T+Lymphocyte-Associated+Antigen+4.">Immunologic Self-Tolerance Maintained by Cd25+Cd4+Regulatory T Cells Constitutively Expressing Cytotoxic T Lymphocyte-Associated Antigen 4.</a> The Journal of Experimental Medicine. 192, 303-310 (2000).</li><li>Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD127+expression+inversely+correlates+with+FoxP3+and+suppressive+function+of+human+CD4++T+reg+cells.">CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells.</a> The Journal of Experimental Medicine. 203, 1701-1711 (2006).</li><li>Schraven, B., Kalinke, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD28+superagonists:+what+makes+the+difference+in+humans.">CD28 superagonists: what makes the difference in humans.</a> Immunity. 28, 591-595 (2008).</li><li>Reneer, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripherally+induced+human+Regulatory+T+cells+uncouple+Kv1.3+activation+from+TCR-associated+signaling.">Peripherally induced human Regulatory T cells uncouple Kv1.3 activation from TCR-associated signaling.</a> European Journal of Immunology. , (2011).</li></ol>

Mens Treg overføring holder enorm terapeutisk løftet i kampen mot autoimmunitet pode avvisning og andre immun eller provoserende-mediert lidelser, metoder for deres effektiv produksjon og stabil vedlikehold ennå ikke er utviklet. Som bare 1-5% av sirkulerende menneskelige T-celler er Tregs, overvinner deres kontrollert ekspansjon og differensiering dette sparsomt som en stor avskrekkende for gjennomføringen av Tregs inn i klinikken. På den annen side, så vi lært fra TGN1412 rettssaken 13, er det vitens...

<table border="1"><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Lymphoprep </td><td> Axis-Shield </td><td> 1114547 </td><td> Hold ved romtemperatur </td></tr><tr><td> Nedkjølt Benk-toppen Centrifuge </td><td> Eppendorf </td><td> 5810R </td><td></td></tr><tr><td> Bright-Line hemocytometer </td><td> Hausser Scientific </td><td> 3110 </td><td></td></tr><tr><td> EasySep Menneskelige CD4 + T celle Enrichment Kit </td><td> Stilk Cell Technologies </td><td> 19052 </td><td></td></tr><tr><td> EasySep menneskets hukommelse CD4 + T celle Enrichment Kit </td><td> Stilk Cell Technologies </td><td> 19157 </td><td></td></tr><tr><td> EasySep Menneskelige Naiv CD4 + T celle Enrichment Kit </td><td> Stilk Cell Technologies </td><td> 19155 </td><td></td></tr><tr><td> The Big Easy EasySep Magnet </td><td> Stilk Cell Technologies </td><td> 18001 </td><td> Sølv </td></tr><tr><td> 5 ml rund bunn Polystyrene Tubes </td><td> BD Falcon </td><td> 352008 </td><td></td></tr><tr><td> 15 mL polypropylen Sentrifugerør </td><td> VWR </td><td> 89004-368 </td><td></td></tr><tr><td> 14 mL polypropylen rundbunnet Tubes </td><td> BD Falcon </td><td> 352059 </td><td></td></tr><tr><td> 50 ml Polypropylene Sentrifugerør </td><td> VWR </td><td> 89004-364 </td><td></td></tr><tr><td> Menneskelig anti-CD3 antistoff </td><td> Bio X Cell </td><td> BE0001-2 </td><td> Clone: ​​OKT3 </td></tr><tr><td> 24 Vel Cell Isopor Kultur Plate </td><td> BD Falcon </td><td> 353047 </td><td></td></tr><tr><td> 96 Vel rund bunn Tissue Culture Plate </td><td> Grenier Bio-One </td><td> 650180 </td><td></td></tr><tr><td> RPMI 1640 med Glutamax-I og HEPES buffer </td><td> Gibco </td><td> 72400 </td><td></td></tr> &lt;tr&gt; <td> Fetal bovin serum (FBS) </td><td> Gibco </td><td> 16000 </td><td></td></tr><tr><td> β-Mercaptoethanol </td><td> Sigma </td><td> M7522 </td><td></td></tr><tr><td> Rekombinant humant TGF-β1 </td><td> eBioscience </td><td> 14-8348-62 </td><td></td></tr><tr><td> Rekombinant humant IL-2 </td><td> eBioscience </td><td> 14-8029-63 </td><td></td></tr><tr><td> Bovine serum albumin (BSA) </td><td> MP Biomedicals Inc. </td><td> 810531 </td><td></td></tr><tr><td> 0,5 M EDTA </td><td> Amresco </td><td> E177 </td><td></td></tr><tr><td> Mouse Anti-Human CD25-PE </td><td> Miltenyi Biotec </td><td> 130-091-024 </td><td> Clone: ​​4E3 </td></tr><tr><td> Mouse Anti-Human CD45RA-PE-Cy5 </td><td> eBioscience </td><td> 15-0458-42 </td><td> Clone: ​​HI100 </td></tr><tr><td> Mouse Anti-Human CD127-APC </td><td> Miltenyi Biotec </td><td> 130-094-890 </td><td> Clone: ​​MB15-18C9 </td></tr><tr><td> DNase II </td><td> MP Biomedicals Inc. </td><td> 190370 </td><td></td></tr><tr><td> MoFlo Flowcytometer </td><td> Beckman Coulter </td><td></td><td></td></tr><tr><td> FlowJo Software </td><td> Tre Star, Inc. </td><td></td><td></td></tr><tr><td> Cell quest Pro Software </td><td> BD Biosciences </td><td></td><td></td></tr><tr><td> Nyfødt kalv Serum </td><td> Gibco </td><td> 16010 </td><td></td></tr><tr><td> L-glutamin </td><td> Gibco </td><td> 25030 </td><td></td></tr><tr><td> AIM-V </td><td> Gibco </td><td> 0870112 </td><td></td></tr><tr><td> CellTrace CFSE celleproliferasjon Kit </td><td> Invitrogen </td><td> C34554 </td><td></td></tr><tr><td> Treg Suppression Inspector perler </td><td> Miltenyi Biotec </td><td> 130-092-909 </td><td></td></tr><tr><td> Penicillin-Streptomycin </td><td> Gibco </td><td> 15140 </td><td></td></tr><tr><td> 40 mM Nylon Cell StrainER </td><td> BD Falcon </td><td> 352340 </td><td></td></tr></table>

generation of induced regulatory t cells from primary human na&iuml;ve and memory t cells

Utvikling og vedlikehold av immunsuppressive CD4 + regulatoriske T-celler (Tregs) bidra til perifer toleranse for å forbli i immunologisk homeostase med den enorme mengden av selvtillit og Commensal antigener i og på menneskekroppen. Forstyrrelsene i balansen mellom Tregs og inflammatoriske konvensjonelle T-celler kan resultere i immunopathology eller kreft. Selv om terapeutisk injeksjon av Tregs har vist seg å være effektiv i murine modeller av en kolitt, type I 2 diabetes, revmatoid artritt og graft versus host sykdom, 4 flere grunnleggende forskjeller i menneskelige versus mus Treg biologi 5 har hittil utelukket klinisk bruk. Mangelen på tilstrekkelig antall, renhet, stabilitet og homing spesifisitet av terapeutisk Tregs nødvendiggjorde en dynamisk plattform for menneskelig Treg utvikling som å optimalisere forholdene for sin ex vivo ekspansjon 6. Her har vi describe en metode for differensiering av indusert Tregs (iTregs) fra et enkelt menneske perifert blod donor som kan brytes ned i fire stadier: isolasjon av perifere mononukleære blodceller, magnetisk utvalg av CD4 + T celler, in vitro cellekultur og fluorescens aktivert celle sortering (FACS) av T-celle undergrupper. Siden Treg signaturen transkripsjonsfaktor forkhead boksen P3 (FoxP3) er en aktivering-indusert transkripsjonsfaktor i mennesker 7 og ingen andre unike markør eksisterer en kombinatorisk panel av markører må brukes til å identifisere T-celler med suppressor aktivitet. Etter seks dager i kultur, kan celler i systemet vårt skal avgrenses til naive T-celler, minne T-celler eller iTregs basert på deres relative uttrykk for CD25 og CD45RA. Som minne og naive T-celler har forskjellige rapporterte polarisering krav og plasticities 8, pre-sortering av den første T-celle befolkningen inn CD45RA + og CD45RO + undergrupper kan be brukes til å undersøke disse avvikene. Konsistent med andre, vår CD25 Hi CD45RA - iTregs uttrykte høye nivåer av FoxP3 9, GITR og CTLA-4 11 og lave nivåer av CD127 12. Etter FACS av hver populasjon, kan resulterende celler brukes i en Lyddemper analysen som evaluerer den relative evnen til å forsinke spredning av carboxyfluorescein succinimidyl ester (CFSE)-merket autolog T-celler.

Watch this Scientific Journal Video about Generation of Induced Regulatory T Cells from Primary Human NaÃƒÂ¯ve and Memory T Cells at JoVE.com

Generation of Induced Regulatory T Cells from Primary Human NaÃƒÆ’Ã‚Â¯ve and Memory T Cells

Vi beskriver en metode for generering av regulatoriske, minne og naive T-celler fra et enkelt menneske blodgiver. Polarisert Tregs kan deretter sammenlignes med andre undergrupper i en rekke genetiske og funksjonelle applikasjoner med genetisk homogenitet, inkludert en undertrykkelse analysen også beskrevet her.

Generering av Utførte regulatoriske T-celler fra primære humane naiv og Minne T-celler

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

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Immunology and Infection

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

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  ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac transposon System Endring av primære humane T celler

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

Nye verktøy for å utvide Regulatoriske T-celler fra HIV-1-infiserte individer

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

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Avledning av T-celler<em&gt; In Vitro</em&gt; Fra 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 ...

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T celler Capture Bakterier av Transinfection fra dendrittiske celler

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

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Fenotypisk og funksjonell analyse av Aktivert regulatoriske T-celler isolert fra kronisk lymfatisk choriomeningittvirus-infiserte mus

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

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

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells iTregs from NaÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¯ve CD4+ T Cells Using a TGF-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-containing Protocol

In vitro Differensiering av Menneskelig CD4 + foxp3 + Induced regulatoriske T-celler (iTregs) fra naive CD4 + T-celler under anvendelse av et TGF-β holdige Protocol

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...