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.

les cellules mononucléaires du sang périphérique humain (CMSP) ont été fraîchement isolés de donneurs sains buffy coats anonymisées achetés à l&#39;hôpital universitaire Karolinska, en Suède. permis éthique pour les expériences a été obtenue auprès du Conseil régional d&#39;éthique Revue à Stockholm (Regionala etikprövningsnämnden i Stockholm), Suède (numéro d&#39;agrément: 2013 / 1458-1431 / 1). 1. T cellulaire Isolement du sang périphérique <ol><li> PBMC Isolation ...

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Le protocole décrit permet l&#39;induction robuste de CD4 humaine + FOXP3 + iTregs de cellules naïves humaines T CD4 +. Elle comprend un nouveau protocole que nous avons récemment décrit, en utilisant une combinaison de TGF-β, l&#39; ATRA et de la rapamycine, pour l&#39; induction de iTregs in vitro avec fonction supérieure de suppression 22. Par rapport aux autres protocoles publiés, un autre avantage réside dans l&#39;induction de diverses populations iTreg en parallèle par diff...

Des cellules T régulatrices CD4 + CD25 + FOXP3 + (Treg) suppriment d&#39; autres cellules immunitaires et sont des médiateurs essentiels de la tolérance périphérique, la prévention de l&#39; auto - immunité et l&#39; inflammation excessive 1. L&#39;importance de Tregs est illustré par la maladie humaine syndrome immunodysregulation polyendocrinopathie entéropathie liée à l&#39;X (IPEX), dans laquelle la perte de Tregs due à des mutations dans le `master&#39; Treg facteur de transcription forkhead box P3 (FOXP3) conduit à une maladie auto-immune systémique sévère, létale à un âge précoce. Cependant, Tregs agir comme une épée à double tranchant dans le système immunitaire , car ils peuvent aussi nuire à l&#39; immunité anti-tumorale dans certains paramètres 2. manipulation thérapeutique du nombre et de la fonction Treg est donc soumis à de nombreuses investigations cliniques. Dans le cancer, l&#39; épuisement des Tregs peut être souhaitable et un certain succès des approches cliniques encourage la poursuite de la recherche 3. Dans les maladies auto-immunes et inflammatoires, en plus des effets thérapeutiques des Treg à plusiedes modèles de maladies de la souris relles, récents essais en homme de transfert Treg adoptive pour prévenir la maladie du greffon contre -host (GvHD) 4-7 et d&#39;évaluer la sécurité dans le traitement du diabète de type 1 8 a montré des résultats très prometteurs. D&#39; origine naturelle Tregs (nTregs) comprennent tTregs thymiques dérivées et pTregs périphériquement induites, avec des fonctions essentielles non redondantes dans le maintien de la santé 9 - 11. Cependant, les numéros nTreg sont limitées, en encourageant l&#39;approche complémentaire induisant Treg (iTregs) in vitro à partir de précurseurs naïfs des lymphocytes T 12. La stabilité morte de iTregs, probablement en raison d&#39; un manque de déméthylation dans ce qu&#39;on appelle la région déméthylé Treg spécifique (TSDR) dans le locus du gène FOXP3 13, reste une préoccupation et plusieurs études indiquent que in vivo induite par Tregs sont plus stables 14. À ce jour, FOXP3 reste la meilleure protéine marker pour Tregs mais il est absolument pas spécifique parce que les cellules conventionnelles humaines CD4 + CD25 T expriment transitoirement des niveaux intermédiaires de FOXP3 lors de l&#39; activation 15,16. Bien que des progrès significatifs ont été accomplis dans l&#39;élucidation de la régulation de l&#39;expression FOXP3, il reste encore beaucoup à découvrir en ce qui concerne l&#39;induction, la stabilité et la fonction de FOXP3 en particulier dans les cellules humaines. En dépit des différences à nTregs, in vitro induite FOXP3 + cellules T CD4 + peuvent être utilisés comme un système modèle pour étudier les mécanismes moléculaires de FOXP3 induction et comme un point de départ pour élaborer des protocoles à l&#39;avenir qui permettent de génération de iTregs qui sont plus semblables à vivo généré Tregs, qui pourrait être applicable pour les stratégies de transfert adoptif à l&#39;avenir. Il n&#39;y a pas `protocole standard&#39; d&#39;or pour induire iTregs humains, et les protocoles actuels ont été développés sur la base de conditions mimant Treg induisant in vivo: l&#39; interleukine 2 (IL-2) Et la transformation β du facteur de croissance (TGF-β) de signalisation sont cruciaux pour FOXP3 induction in vivo 17, et tout-trans de l&#39; acide rétinoïque (ATRA) - qui est produit in vivo par les cellules dendritiques associés à l&#39; intestin - est fréquemment utilisé pour améliorer FOXP3 induction in vitro 18-21. Nous avons développé des protocoles humains supplémentaires Treg induisant en utilisant butyrate 22, un acide gras à chaîne courte microbiote intestinal dérivé qui a été récemment montré pour augmenter murin Treg induction 23,24. Récemment , nous avons établi un nouveau protocole pour la production de iTregs ayant une fonction suppressive supérieure in vitro en utilisant une combinaison de TGF-β, l&#39; ATRA et la rapamycine 22, cette dernière étant une cible mammalienne cliniquement approuvé de la rapamycine (mTOR) , un inhibiteur qui est connu pour promouvoir entretien FOXP3 lors de l&#39; expansion humaine Treg 25,26. Cette méthode décrit le reproductiblela génération in vitro de cellules CD4 + humaines FOXP3 + iTregs en utilisant un ensemble de conditions différentes, ainsi que leur phénotypage ultérieure par cytométrie en flux et la réaction inverse quantitative en chaîne par polymérase (qRT-PCR) pour révéler les profils d&#39;expression de FOXP3 et d&#39; autres signatures des molécules Treg telles spécifiques au protocole comme CD25, CTLA-4, EOS, ainsi que la répression de l&#39; IFN-γ et l&#39; expression SATB1 22. Les populations cellulaires générées peuvent être utilisées pour des analyses fonctionnelles relatives à l&#39;activité suppressive ou pour des études moléculaires, que ce soit en ce qui concerne les régulateurs généraux FOXP3 ou pour étudier les effets spécifiques à certains composés tels que le butyrate ou la rapamycine. Une meilleure compréhension des mécanismes moléculaires de conduite différenciation Treg est très pertinente pour les futures approches thérapeutiques de l&#39;auto-immunité ou d&#39;un cancer de cibler spécifiquement les molécules impliquées dans la production et la fonction Treg.

<table border="0" cellpadding="0" cellspacing="0" width="1689">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<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>
		</tr>
		<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" />
			<table cellpadding="0" cellspacing="0">
				<tbody>
					<tr>
						<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" />
			<table cellpadding="0" cellspacing="0">
				<tbody>
					<tr>
						<td height="20" style="height:21px;width:176px;">Miltenyi Biotec</td>
					</tr>
				</tbody>
			</table>
			</td>
			<td>130-094-131</td>
			<td></td>
		</tr>
		<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.

La figure 1 montre un schéma du dispositif expérimental. La figure 2 montre une coloration de contrôle de pureté représentant pour les cellules T CD4 + naïves magnétiquement isolées et nTregs. F igure 3A montre la cytométrie en flux stratégie de déclenchement et la figure 3B montre FOXP3 et CD25 représentant de cytométrie de flux Colora...

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 NaÃƒÆ’Ã†'Ãƒâ€šÃ‚Â¯ve CD4+ T Cells Using a TGF-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-containing Protocol

Ce protocole décrit la génération reproductible et phénotypage des cellules T régulatrices humaines induites (les iTregs) à partir de cellules T CD4 + naïves in vitro. Différents protocoles pour FOXP3 induction permettent l&#39;étude des phénotypes iTreg spécifiques obtenus avec les protocoles respectifs.

In Vitro Differentiation of Human CD4 + FOXP3 cellules + Induced T régulatrices (de iTregs) de Naïf cellules T CD4 + en utilisant un protocole TGF-β contenant

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

Retroviral Transduction of T-cell Receptors in Mouse T-cells

Transduction rétrovirale des récepteurs de cellules T dans les lymphocytes T de souris

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 ...

Recherche

Immunologie et infection

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

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

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

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Différenciation dirigée des cellules souches pluripotentes induites à l&#39;égard des lymphocytes T

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

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piggyBac Transposon System Modification of Primary Human T Cells

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

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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 ...

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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 ...

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Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

L&#39;évaluation de l&#39;Innate détection du VIH-1 CD4 infectés<sup&gt; +</sup&gt; Cellules T par les cellules dendritiques plasmacytoïdes Utilisation d&#39;un<em&gt; Ex vivo</em&gt; Système de co-culture.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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