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 at takke Jennifer Strange og Greg Bauman for deres assistance med flowcytometri analyse og sortering. Dette arbejde blev støttet af NIH Grant Number 2P20 RR020171 fra NCRR og ved University of Kentucky start midler til FM, GIE anerkender støtte af præsidentens Graduate Fellowship og Kentucky Opportunity Fellowship.</p

1. Isolering af humane perifere mononukleære blodceller (PBMC&#39;er) fra fibrinlag <ol><li> Opnå en enhed af buffy coat fra hospitalet eller i nærheden af ​​blod centrum. Vores blod center giver os med omkring 40-60 ml buffy coat pr opnået fra normale bloddonorer. </li><li> Hæld blodet i en autoklaveret 500 ml glasflaske indeholdende sterilt PBS til fortynding buffy coat. Det endelige volumen af ​​PBS + buffy coat være 250 ml. </li><li> Fylde ti 50 ml koniske rør med hver 20 ml Lymphoprep opløsning. <...

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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 overførsel besidder enorm terapeutisk løfte i bekæmpelsen af ​​autoimmunitet transplantatafvisning og andre immune eller inflammatoriske-medieret sygdomme, metoder til deres effektive generation og stabil vedligeholdelse er endnu ikke blevet udviklet. Da kun 1-5% af cirkulerende humane T-celler er tregs, kontrolleret ekspansion og differentiering overvinder denne mangel som en stor afskrækkende for gennemførelsen af ​​tregs ind på klinikken. På den anden side, som vi lærte fra TGN1412 forsøget...

<table border="1"><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognr </td><td> Kommentarer </td></tr><tr><td> Lymphoprep </td><td> Axis-Shield </td><td> 1114547 </td><td> Opbevares ved stuetemperatur </td></tr><tr><td> Afkølet bordcentrifuge </td><td> Eppendorf </td><td> 5810R </td><td></td></tr><tr><td> Bright-Line hæmocytometer </td><td> Hausser Videnskabelig </td><td> 3110 </td><td></td></tr><tr><td> EasySep humane CD4 + T celle Enrichment Kit </td><td> Stem Cell Technologies </td><td> 19052 </td><td></td></tr><tr><td> EasySep menneskelige hukommelse CD4 + T-celle Enrichment Kit </td><td> Stem Cell Technologies </td><td> 19157 </td><td></td></tr><tr><td> EasySep Humane naive CD4 + T-celle Enrichment Kit </td><td> Stem Cell Technologies </td><td> 19155 </td><td></td></tr><tr><td> The Big Easy EasySep Magnetto </td><td> Stem Cell Technologies </td><td> 18001 </td><td> Sølv </td></tr><tr><td> 5 ml rundbundet polystyrenrør </td><td> BD Falcon </td><td> 352008 </td><td></td></tr><tr><td> 15 ml polypropylencentrifugerør </td><td> VWR </td><td> 89004-368 </td><td></td></tr><tr><td> 14 ml polypropylen rundbundet rør </td><td> BD Falcon </td><td> 352059 </td><td></td></tr><tr><td> 50 ml polypropylencentrifugerør </td><td> VWR </td><td> 89004-364 </td><td></td></tr><tr><td> Humant anti-CD3-antistof </td><td> Bio X Cell </td><td> BE0001-2 </td><td> Klon: OKT3 </td></tr><tr><td> 24-brønds Cell Polystyren dyrkningspladen </td><td> BD Falcon </td><td> 353047 </td><td></td></tr><tr><td> 96-brønds rundbundet vævsdyrkningsplade </td><td> Grenier Bio-One </td><td> 650180 </td><td></td></tr><tr><td> RPMI 1640 med glutamax-I og HEPES-puffer </td><td> Gibco </td><td> 72400 </td><td></td></tr> &lt;tr&gt; <td> Føtalt bovint 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 human TGF-β1 </td><td> eBioscience </td><td> 14-8348-62 </td><td></td></tr><tr><td> Rekombinant human IL-2 </td><td> eBioscience </td><td> 14-8029-63 </td><td></td></tr><tr><td> Bovint serumalbumin (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> Muse-anti-humant CD25-PE </td><td> Miltenyi Biotec </td><td> 130-091-024 </td><td> Klon: 4E3 </td></tr><tr><td> Muse anti-human CD45RA-PE-Cy5 </td><td> eBioscience </td><td> 15-0458-42 </td><td> Klon: HI100 </td></tr><tr><td> Muse anti-human CD127-APC </td><td> Miltenyi Biotec </td><td> 130-094-890 </td><td> Klon: MB15-18C9 </td></tr><tr><td> DNase II </td><td> MP Biomedicals Inc. </td><td> 190370 </td><td></td></tr><tr><td> MoFlo Flow Cytometer </td><td> Beckman Coulter </td><td></td><td></td></tr><tr><td> FlowJo Software </td><td> Tree 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> Serum fra nyfødt kalv </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 Cell Proliferation 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 uM Nylon cellestammeis </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

Udvikling og vedligeholdelse af immunosuppressive CD4 + regulatoriske T-celler (tregs) bidrager til den perifere tolerance er nødvendig for at forblive i immunologisk homeostase med den store mængde af selvstændige og kommensale antigener i og på den menneskelige krop. Forstyrrelser i balancen mellem tregs og inflammatoriske konventionelle T-celler kan resultere i immunopatologi eller cancer. Selv terapeutisk injektion af tregs har vist sig at være effektiv i murine modeller af colitis 1, type I diabetes 2, rheumatoid arthritis og graft versus host-sygdom, 4 flere fundamentale forskelle i human versus muse Treg biologi 5 hidtil udelukket klinisk anvendelse. Manglen på tilstrækkeligt mange renhed, stabilitet og homing specificitet terapeutisk tregs nødvendiggjorde en dynamisk platform af human Treg udvikling som at optimere betingelserne for deres ex vivo-ekspansion 6. Her d kanescribe en fremgangsmåde til differentiering af induceret tregs (iTregs) fra et enkelt humant perifert blod donor, som kan opdeles i fire faser: isolering af perifere mononucleære blodceller, magnetisk udvælgelse af CD4 + T-celler, in vitro celledyrkning og fluorescens-aktiveret cellesortering (FACS) af T-celledelmængder. Idet Treg signaturen transkriptionsfaktoren gaffelhoved boks P3 (foxp3) er en aktiverings-induceret transkriptionsfaktor i mennesker 7 og ingen anden unik markør eksisterer et kombinatorisk panel af markører skal anvendes til at identificere T-celler med suppressor-aktivitet. Efter seks dage i kultur, kan celler i vores system kan afgrænses i naive T-celler, hukommelses-T-celler eller iTregs baseret på deres relative ekspression af CD25 og CD45RA. Som hukommelses-og naive T-celler har forskellige rapporterede polarisationsniveauer krav og plasticities 8, forsortering af den oprindelige T-cellepopulation i CD45RA + og CD45RO + delmængder kan Be bruges til at undersøge disse forskelle. I overensstemmelse med andre vores CD25 Hi CD45RA - iTregs eksprimerer høje niveauer af foxp3 9, GITR og CTLA-4 11 og lave niveauer af CD127 12. Efter FACS for hver population, kan resulterende celler anvendes i et suppressor assay, som evaluerer den relative evne til at forsinke udbredelsen af ​​carboxyfluorescein succinimidylester (CFSE)-mærkede autologe 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

Beskriver vi en fremgangsmåde til generering af regulering, hukommelses-og naive T-celler fra en enkelt human bloddonor. Polariseret tregs kan derefter sammenlignes med andre delmængder i forskellige genetiske og funktionelle anvendelser med genetisk ensartethed, herunder suppression assay også beskrevet her.

Generering af inducerede regulatoriske T-celler fra primære humane naive og memory 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 Ændring af 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 ...

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Nye værktøjer til at udvide regulatoriske T celler fra HIV-1-inficerede personer

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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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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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 differentiering af humane CD4 + Foxp3 + Induced Regulatory T-celler (iTregs) fra naive CD4 + T-celler under anvendelse et TGF-β-holdige protokol

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