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.

Human peripheral blood mononuclear cells (PBMCs) were freshly isolated from anonymized healthy donor buffy coats purchased from the Karolinska University Hospital, Sweden. Ethical permit for the experiments was obtained from the Regional Ethical Review Board in Stockholm (Regionala etikpr&#246;vningsn&#228;mnden i Stockholm), Sweden (approval number: 2013/1458-31/1).
1. T Cell Isolation from Peripheral Blood
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	<li>PBMC Isolation
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		<li>Pre-lay 15 ml density centrifugation medium (such a...

<ol>
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The described protocol enables the robust induction of human CD4+FOXP3+ iTregs from human na&#239;ve CD4+ T cells. It includes a new protocol that we described recently, using a combination of TGF-&#946;, ATRA and rapamycin, for induction of iTregs with superior in vitro suppressive function22. Compared to other published protocols, another advantage is the induction of different iTreg populations in parallel by different protocols, which enables the direct comparison of effects of certain ...

CD4+CD25+FOXP3+ regulatory T cells (Tregs) suppress other immune cells and are critical mediators of peripheral tolerance, preventing autoimmunity and excessive inflammation1. The importance of Tregs is exemplified by the human disease immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX), in which loss of Tregs due to mutations in the `master&#180; Treg transcription factor forkhead box P3 (FOXP3) leads to severe systemic autoimmune disease, lethal at an early age. However, Tregs act as a double-edged sword in the immune system as they can also hamper anti-tumor immunity in certain settings2. Therapeutic manipulation of Treg number and function is therefore subject to numerous clinical investigations. In cancer, depletion of Tregs can be desirable and some success of clinical approaches encourages further research3. In autoimmune and inflammatory diseases, in addition to therapeutic effects of Tregs in several mouse disease models, recent first in-man trials of adoptive Treg transfer to prevent graft-versus-host disease (GvHD)4-7 and to assess safety in treating type 1 diabetes8 showed very promising outcomes.
Naturally occurring Tregs (nTregs) comprise thymic-derived tTregs and peripherally induced pTregs, with non-redundant essential functions in maintaining health9-11. However, nTreg numbers are limited, encouraging the complementary approach of inducing Tregs (iTregs) in vitro from na&#239;ve T cell precursors12. Still stability of iTregs, presumably due to lack of demethylation in the so-called Treg-specific demethylated region (TSDR) in the FOXP3 gene locus13, remains a concern and several studies indicate that in vivo induced Tregs are more stable14.
To date, FOXP3 remains the best protein marker for Tregs but it is not absolutely specific because human conventional CD4+CD25- T cells transiently express intermediate levels of FOXP3 upon activation15,16. Although significant progress has been made in elucidating the regulation of FOXP3 expression, much remains to be discovered regarding the induction, stability and function of FOXP3 particularly in human cells. Despite differences to nTregs, in vitro induced FOXP3+ CD4+ T cells can be used as a model system to study molecular mechanisms of FOXP3 induction and as a starting point to develop protocols in the future that allow for generation of iTregs that are more similar to in vivo generated Tregs, which could be applicable for adoptive transfer strategies in the future.
There is no `gold standard&#180; protocol to induce human iTregs, and current protocols have been developed based on mimicking Treg-inducing conditions in vivo: interleukin 2 (IL-2) and transforming growth factor &#946; (TGF-&#946;) signaling are crucial for FOXP3 induction in vivo17, and all-trans retinoic acid (ATRA) &#8212; which is produced in vivo by gut-associated dendritic cells &#8212; is frequently used to enhance FOXP3 induction in vitro18-21. We have developed additional human Treg-inducing protocols using butyrate22, a gut microbiota-derived short-chain fatty acid that was recently shown to augment murine Treg induction23,24. We also recently established a new protocol for generation of iTregs with superior suppressive function in vitro by using a combination of TGF-&#946;, ATRA and rapamycin22, the latter being a clinically approved mammalian target of rapamycin (mTOR) inhibitor that is known to promote FOXP3 maintenance during human Treg expansion25,26.
This method describes the reproducible in vitro generation of human CD4+FOXP3+ iTregs using a set of different conditions, and their subsequent phenotyping by flow cytometry and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to reveal protocol-specific patterns of expression of FOXP3 and other Treg signature molecules such as CD25, CTLA-4, EOS, as well as repression of IFN-&#947; and SATB1 expression22. The generated cell populations can be used for functional assays regarding suppressive activity or for molecular studies, either concerning general FOXP3 regulators or to study effects specific to certain compounds such as butyrate or rapamycin. Further understanding of molecular mechanisms driving Treg differentiation is highly relevant for future therapeutic approaches in autoimmunity or cancer to specifically target molecules involved in Treg generation and function.

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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>
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			<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>
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		<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>
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			<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.

Figure 1 shows a scheme of the experimental setup. Figure 2 shows a representative purity control staining for magnetically isolated na&#239;ve CD4+ T cells and nTregs.
Figure 3A shows the flow cytometry gating strategy and Figure 3B shows representative FOXP3 and CD25 flow cytometry stainings on day 6 of culture under the indicated iTreg or control c...

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

This protocol describes the reproducible generation and phenotyping of human induced regulatory T cells (iTregs) from na&#239;ve CD4+ T cells in vitro. Different protocols for FOXP3 induction allow for the study of specific iTreg phenotypes obtained with respective protocols.

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 ...

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