Name: 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
Uploaded: 2016-12-30T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells iTregs from NaÃƒÂ¯ve CD4+ T Cells Using a TGF-ÃŽÂ²-containing Protocol at JoVE.com

Nina Nagel is gratefully acknowledged for technical assistance during the video shoot and experimental preparation. We thank Eva-Maria Weiss for help with the intracellular FOXP3 staining protocol and Elisabeth Suri-Payer and Nina Oberle for establishing the nTreg isolation protocol. Matilda Eriksson and Peri Noori are acknowledged for laboratory management. 
Funding: A.S. was supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme, the Dr. &#197;ke Olsson Foundation and KI research foundations; A.S. and J.T. were supported by a CERIC (Center of Excellence for Research on Inflammation and Cardiovascular disease) grant, J.T. was supported by Vetenskapsr&#229;det Medicine and Health (Dnr 2011-3264), Torsten S&#246;derberg Foundation, FP7 STATegra, AFA Insurance and Stockholm County Council.

Humane perifere mononukleære blodceller (PBMC) ble nylig isolert fra anonymiserte sunn giver buffy coats kjøpt fra Karolinska universitetssykehus, Sverige. Etisk tillatelse for eksperimentene ble innhentet fra Regional Etisk Review Board i Stockholm (Regionala etikprövningsnämnden i Stockholm), Sverige (godkjenningsnummer: 2013 / 1458-1431 / 1). 1. T Cell Isolasjon fra perifert blod <ol><li> PBMC Isolation <ol><li> Pre-lå 15 ml tetthetssentrifugering medium (for eksempel Ficoll) oppløsni...

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Den beskrevne protokollen gjør den robuste induksjon av menneskelig CD4 + foxp3 + iTregs fra menneskelige naive CD4 + T-celler. Det inkluderer en ny protokoll som vi nylig beskrevet, ved hjelp av en kombinasjon av TGF-β, ATRA og rapamycin, for induksjon av iTregs med overlegen in vitro undertrykkende funksjon 22. Sammenlignet med andre publiserte protokoller, er en annen fordel induksjon av forskjellige iTreg populasjoner i parallell med forskjellige protokoller, som muliggjør direkte s...

CD4 + CD25 + foxp3 + regulatoriske T-celler (tregs) undertrykke andre immunceller og er kritiske formidlere av perifer toleranse, hindrer autoimmunitet og overdreven betennelse en. Betydningen av Tregs er eksemplifisert ved den humane sykdommen immunodysregulation polyendocrinopathy enteropati X-bundet syndrom (IPEX), i hvilken tap av Tregs på grunn av mutasjoner i `master&#39; Treg transkripsjonsfaktor gaffelhodeboks P3 (foxp3) fører til alvorlig systemisk autoimmun sykdom, dødelig i tidlig alder. Men Tregs fungere som et tveegget sverd i immunsystemet som de kan også hemme anti-tumor immunitet i visse innstillinger 2. Terapeutisk manipulering av treg antall og funksjon er derfor underlagt en rekke kliniske undersøkelser. I kreft, kan uttømming av tregs være ønskelig og noen suksess av kliniske tilnærminger oppmuntrer til videre forskning tre. I autoimmune og inflammatoriske sykdommer, i tillegg til terapeutiske effektene av Tregs i seveRAL mus sykdomsmodeller, nye første i manns forsøk med adoptiv Treg overføring for å hindre graft- versus -host sykdom (GvHD) 4-7 og vurdere sikkerheten i behandling av type 1 diabetes 8 viste svært lovende resultater. Naturlig forekommende Tregs (nTregs) omfatter thymus-avledet tTregs og perifert indusert pTregs, med ikke-redundante viktige funksjoner i å opprettholde helse 9-11. Men nTreg tallene er begrenset, oppmuntre til komplementær tilnærming som induserer tregs (iTregs) in vitro fra naive T-celle forløpere 12. Fortsatt stabilitet av iTregs, antagelig på grunn av manglende demetylering i den såkalte treg-spesifikke demetylerte region (TSDR) i foxp3 gen locus 13, forblir et problem og flere studier tyder på at in vivo-indusert Tregs er mer 14 stabil. Til dags dato, foxp3 fortsatt den beste protein mArker for Tregs men det er ikke helt bestemt, fordi menneskekonvensjonelle CD4 + CD25- T-celler forbigående uttrykker middels nivå av foxp3 ved aktivering 15,16. Selv om betydelige fremskritt har blitt gjort i å belyse regulering av foxp3 uttrykk, er fortsatt mye å bli oppdaget med induksjon, stabilitet og funksjon av foxp3 særlig i humane celler. Til tross for forskjeller i nTregs, in vitro indusert foxp3 + CD4 + T-celler kan brukes som et modellsystem for å studere molekylære mekanismer for foxp3 induksjon og som et utgangspunkt for å utvikle protokoller i fremtiden som tillater generering av iTregs som er mer lik den i vivo genererte Tregs, noe som kan være aktuelt for adoptiv overføring strategier i fremtiden. Det er ingen `gull standard&#39;-protokollen for å indusere humane iTregs, og nåværende protokoller er utviklet basert på å etterligne treg-induserende betingelser in vivo: interleukin 2 (IL-2) Og transformerende vekstfaktor β (TGF-β) signalering er avgjørende for foxp3 induksjon in vivo 17, og all-trans retinsyre (ATRA) - som er produsert in vivo ved tarmassosiert dendrittiske celler - blir ofte brukt for å forbedre foxp3 induksjon in vitro 18 - 21. Vi har utviklet flere menneskelige Treg-induserende protokoller ved hjelp av smørsyre 22, en gut microbiota-avledet kortkjedet fettsyre som nylig ble vist seg å forsterke murine Treg induksjon 23,24. Vi har også nylig etablert en ny protokoll for generering av iTregs med overlegen undertrykkende funksjon in vitro ved hjelp av en kombinasjon av TGF-β, ATRA og rapamycin 22, sistnevnte er en klinisk godkjent pattedyr målet for rapamycin (mTOR) inhibitor som er kjent for å fremme foxp3 vedlikehold under menneskelig treg utvidelse 25,26. Denne metoden beskriver reproduserbar ivitro generering av humane CD4 + foxp3 + iTregs ved hjelp av et sett av forskjellige forhold, og deres etterfølgende fenotyping ved flow-cytometri og kvantitativ revers transkripsjon polymerasekjedereaksjon (QRT-PCR) for å avsløre protokoll-spesifikke mønstre av ekspresjon av foxp3 og andre treg signatur molekyler slike som CD25, CTLA-4, EOS, såvel som undertrykkelse av IFN-γ og SATB1 uttrykk 22. De genererte cellepopulasjoner kan anvendes for funksjonelle analyser vedrørende undertrykkende aktivitet eller for molekylære studier, enten om generell foxp3 regulatorer eller for å studere effekter som er spesifikke for visse forbindelser slik som butyrat eller rapamycin. Videre forståelse av molekylære mekanismer som driver Treg differensiering er svært relevant for fremtidige terapeutiske tilnærminger i autoimmunitet eller kreft spesifikt mot molekyler involvert i Treg generasjon og funksjon.

<table border="0" cellpadding="0" cellspacing="0" width="1689">
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			<td height="20" style="height:21px;width:501px;">All-trans retinoic acid</td>
			<td style="width:176px;">Sigma Aldrich</td>
			<td style="width:248px;">R2625-50MG&#160;&#160;</td>
			<td style="width:764px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anit-human Foxp3-APC clone 236A/E7</td>
			<td>eBioscience</td>
			<td>17-4777-42</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anti-human CD25 microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-983</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anti-human CD25-PE</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anti-human CD28 antibody, LEAF Purified&#160;</td>
			<td>Biolegend</td>
			<td>302914</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anti-human CD3 Antibody, LEAF Purified&#160;</td>
			<td>Biolegend</td>
			<td>317315</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">anti-human CD45RA , FITC</td>
			<td>Miltenyi Biotec</td>
			<td style="width:248px;">130-092-247</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anti-human CD45RO PE clone UCHL1</td>
			<td>BD Biosciences</td>
			<td>555493</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anti-human CD4-PerCP clone SK3; mIgG1</td>
			<td>BD Biosciences</td>
			<td>345770</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anti-human CD8-eFluor 450 (clone OKT8), mIgG2a</td>
			<td>eBioscience</td>
			<td>48-0086-42&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">anti-human CTLA-4 (CD152), clone BNI3, mIgG2ak, Brilliant violet 421</td>
			<td>BD Biosciences</td>
			<td>562743</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">anti-human IFN-g FITC clone 4S.B3; mIgG1k</td>
			<td>eBioscience</td>
			<td>11-7319-81&#160;</td>
			<td style="width:764px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">Brefeldin A-containing solution: GolgiPlug</td>
			<td>BD Biosciences</td>
			<td>555029</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">cDNA synthesis kit: SuperScript VILO(Reverse transcriptase) cDNA Synthesis Kit</td>
			<td style="width:176px;">Invitrogen</td>
			<td style="width:248px;">11754-250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">Density centrifugation medium: Ficoll-Paque</td>
			<td style="width:176px;">GE healthcare</td>
			<td style="width:248px;">17-1440-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;">DMSO 99,7%</td>
			<td>Sigma Aldrich</td>
			<td>D2650-5X5ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS, heat inactivated</td>
			<td>Invitrogen</td>
			<td>10082-147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Fixable Viability Dye, eFluor 780&#160;&#160;</td>
			<td>eBioscience</td>
			<td>65-0865-14 or 65-0865-18&#160;</td>
			<td style="width:764px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">Foxp3 Staining Buffer Set</td>
			<td>eBioscience</td>
			<td>00-5523-00&#160;</td>
			<td style="width:764px;">Caution, contains Paraformaldehyde 
			Can be also bought in combined kit with antibody; 77-5774-40 Anti-Human Foxp3 Staining Set APC Clone: 236A/E7 Set&#160;</td>
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		<tr height="20">
			<td height="20" style="height:21px;">GlutaMAX (200 mM L-alanyl-L-glutamine)</td>
			<td>Invitrogen</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">human naive CD4 T cell isolation kit II</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-131</td>
			<td></td>
		</tr>
		<tr height="20">
			<td align="left" height="20" style="height:21px;"><img alt="nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT" height="1" src="file:///C:/Users/DAVIDK~1/AppData/Local/Temp/msohtmlclip1/01/clip_image002.png" width="2" />
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						<td height="20" style="height:21px;width:501px;">human naive CD4 T cell isolation kit II</td>
					</tr>
				</tbody>
			</table>
			</td>
			<td align="left"><img alt="nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT,nojavascript&amp;WT" height="1" src="file:///C:/Users/DAVIDK~1/AppData/Local/Temp/msohtmlclip1/01/clip_image004.png" width="1" />
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				<tbody>
					<tr>
						<td height="20" style="height:21px;width:176px;">Miltenyi Biotec</td>
					</tr>
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			</td>
			<td>130-094-131</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:21px;">Human serum albumin 50 g/l</td>
			<td>Baxter</td>
			<td>1501057</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Ionomycin from Streptomyces conglobatus &#62;98%</td>
			<td style="width:176px;">Sigma Aldrich</td>
			<td>I9657-1MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">MACS LS-columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">mouse IgG1 K Isotype Control APC Clone: P3.6.2.8.1&#160;</td>
			<td>eBioscience</td>
			<td>17-4714-42&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">mouse IgG1 K Isotype Control FITC 50 ug&#160;</td>
			<td>eBioscience</td>
			<td>11-4714-81&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">mouse IgG2a isotype control, Brilliant violet 421, clone MOPC-173</td>
			<td>BD Biosciences</td>
			<td>563464</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Pasteur pipet plastic, individually packed</td>
			<td>Sarstedt</td>
			<td>86.1172.001&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">PMA PHORBOL 12-MYRISTATE 13-ACETATE</td>
			<td>Sigma Aldrich</td>
			<td>P1585-1MG&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Rapamycin</td>
			<td>EMD (Merck)</td>
			<td>553210-100UG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Recombinant Human IL-2, CF</td>
			<td>R&#38;D</td>
			<td>202-IL-050/CF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:501px;">Recombinant Human TGF-beta 1, CF</td>
			<td>RnD</td>
			<td>240-B-010/CF</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">RNA isolation kit: RNAqueous-Micro Kit</td>
			<td style="width:176px;">Ambion</td>
			<td>AM1931&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">RPMI 1640 Medium&#160;</td>
			<td>Invitrogen</td>
			<td>72400-054&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Sodium butyrate</td>
			<td>Sigma Aldrich</td>
			<td>B5887-250MG&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">T cell culture medium: X-Vivo 15 medium, with gentamicin+phenolred</td>
			<td>Lonza</td>
			<td>04-418Q</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">TaqMan Gene Expression Assay, FOXP3 (Best Coverage)&#160;</td>
			<td style="width:176px;">Applied Biosystems</td>
			<td style="width:248px;">4331182; assay ID: Hs01085834_m1</td>
			<td>Caution, contains Paraformaldehyde</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">TaqMan Gene Expression Assay, RPL13A (Best Coverage)</td>
			<td style="width:176px;">Applied Biosystems</td>
			<td>4351370; assay ID: Hs04194366_g1&#160;&#160;</td>
			<td>Caution, contains Paraformaldehyde</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:501px;">TaqMan Gene Expression Master mix</td>
			<td style="width:176px;">Applied Biosystems</td>
			<td style="width:248px;">4369514</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing autoimmune diseases. Clinical approaches to adoptively transfer Tregs, or to deplete Tregs in cancer, are underway with promising first outcomes. 
Because the number of naturally occurring Tregs (nTregs) is very limited, studying certain Treg features using in vitro induced Tregs (iTregs) can be advantageous. To date, the best although not absolutely specific protein marker to delineate Tregs is the transcription factor FOXP3. Despite the importance of Tregs including non-redundant roles of peripherally induced Tregs, the protocols to generate iTregs are currently controversial, particularly for human cells. This protocol therefore describes the in vitro differentiation of human CD4+FOXP3+ iTregs from human na&#239;ve T cells using a range of Treg-inducing factors (TGF-&#946; plus IL-2 only, or their combination with retinoic acid, rapamycin or butyrate) in parallel. It also describes the phenotyping of these cells by flow cytometry and qRT-PCR. 
These protocols result in reproducible expression of FOXP3 and other Treg signature genes and enable the study of general FOXP3-regulatory mechanisms as well as protocol-specific effects to delineate the impact of certain factors. iTregs can be utilized to study various phenotypic aspects as well as molecular mechanisms of Treg induction. Detailed molecular studies are facilitated by relatively large cell numbers that can be obtained. 
A limitation for the application of iTregs is the relative instability of FOXP3 expression in these cells compared to nTregs. iTregs generated by these protocols can also be used for functional assays such as studying their suppressive function, in which iTregs induced by TGF-&#946; plus retinoic acid and rapamycin display superior suppressive activity. However, the suppressive capacity of iTregs can differ from nTregs and the use of appropriate controls is crucial.

Figur 1 viser en ordning av den eksperimentelle oppsettet. Figur 2 viser en representativ styre renhet farging for magnetisk isolerte naive CD4 + T-celler og nTregs. F igur 3A viser flowcytometri gating strategi og Figur 3B viser representative foxp3 og CD25 flowcytometri stainings på dag 6 av kultur i henhold til de angitte iTreg eller kontrollfor...

Watch this Scientific Journal Video about In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells iTregs from NaÃƒÂ¯ve CD4+ T Cells Using a TGF-ÃŽÂ²-containing Protocol at JoVE.com

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells iTregs from NaÃƒÂ¯ve CD4+ T Cells Using a TGF-ÃŽÂ²-containing Protocol (Video) | JoVE

Denne protokollen beskriver reproduserbar generering og fenotyping av menneskeskapte regulatoriske T-celler (iTregs) fra naive CD4 + T-celler in vitro. Forskjellige protokoller for foxp3 induksjons tillater studiet av spesifikke fenotyper iTreg oppnådd med respektive protokoller.

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 ...

Research

JoVE Journal

Immunology and Infection

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

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