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.

ヒト末梢血単核細胞（PBMC）を新たにカロリンスカ大学病院（スウェーデン）から購入した匿名の健常ドナーのバフィーコートから単離しました。実験のための倫理的許可は、ストックホルムの地域倫理審査委員会（RegionalaetikprövningsnämndenIストックホルム）から入手した、スウェーデン（承認番号：2013/1458から31/1）。 末梢血から1 T細胞の単離<ol><li> PBMCの単離 <ol><li> 15ミ?...

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記載されているプロトコルは、人間のナイーブCD4 + T細胞からのヒトCD4 + FOXP3 + iTregsの堅牢な誘導を可能にします。それは我々が優れたin vitroでの抑制機能22とiTregsの誘導のために、TGF-β、ATRA及びラパマイシンの組み合わせを使用して、最近記載新しいプロトコルが含まれています。他の公表されたプロトコルに比べ、他の利点は、単独で、IL-2の存在下で活性化されたコ...

CD4 + CD25 + FOXP3 +調節性T細胞（Tregは）他の免疫細胞を抑制し、末梢寛容の重要なメディエーターであり、自己免疫及び過度の炎症1を防止します 。 Tregの重要性は、重度の全身性の自己免疫疾患につながるTregの損失による `master&#39;のTreg転写因子フォークヘッドボックスP3における変異にヒトの疾患immunodysregulationの多発性内分泌腺の腸のX連鎖症候群（IPEX）、（FOXP3）が例示されます早い年齢で致死。しかし、Tregは、彼らはまた、特定の設定2での抗腫瘍免疫を妨げることができますように免疫系における両刃の剣として作用します。 Treg細胞の数および機能の治療的操作は、したがって、多数の臨床研究の対象です。癌では、Tregの枯渇が望ましい可能性があると臨床的アプローチのいくつかの成功は、さらなる研究3を奨励しています。自己免疫および炎症性疾患において、セベにおけるTregの治療効果に加えてRALマウス疾患モデル、-host病（GVHD）4 対移植片を防ぐために、養子のTreg転送の最近の最初の人の試験- 7と1型糖尿病8は非常に有望な結果を示した治療の安全性を評価すること。 11 -当然のTreg（nTregs）を発生することは健康に9を維持する上で非冗長本質的な機能で、胸腺由来tTregsおよび末梢誘導さpTregsを備えます。しかし、nTreg番号は、ナイーブT細胞前駆体12からのin vitroで誘導したTreg（iTregs）の補完的なアプローチを奨励し、限られています。まだiTregsの安定性は、おそらくFOXP3遺伝子座13におけるいわゆるTreg細胞に特異的脱メチル化領域（TSDR）における脱メチル化の欠如のため、懸念のままであり、いくつかの研究は、 生体内でのTregを誘導することを示している14以上の安定しています。 現在までに、FOXP3は、最良のタンパク質メートルのままTregのためのarker人間従来のCD4 + CD25-T細胞は一過性の活性化15,16時FOXP3の中間レベルで発現するので、それは絶対に固有のものではありません。重要な進歩は、FOXP3発現の調節を明らかにしたが、多くは、特にヒト細胞におけるFOXP3の誘導、安定性及び機能に関する発見されていません。 nTregsの違いにもかかわらず、in vitroでの FOXP3誘導さ+ CD4 + T細胞はFoxp3誘発の分子機構を研究するためのモデル系として使用することができ、 中に、より類似しているiTregsの生成を可能にする将来のプロトコルを開発するための出発点として将来的に養子移入戦略の適用可能性が生成されたTregを、 生体内 。 そこに人間iTregsを誘導する全く`金standard&#39;プロトコルはなく、現在のプロトコルは、 生体内でのTreg誘導条件を模倣することに基づいて開発されている：2（IL-2、インターロイキン）およびトランスフォーミング増殖因子β（TGF-β）シグナル伝達は、 インビボ 17 における Foxp3誘発のために重要であり、全トランスレチノイン酸（ATRA） -腸管関連樹状細胞によってインビボで産生される-頻繁Foxp3誘発を増強するために使用されます21 - 試験管 18インチ我々は、酪酸22、最近マウスのTreg誘導23,24を増強することが示された腸内細菌叢由来の短鎖脂肪酸を使用して、追加のヒトTregの誘導プロトコールを開発しました。我々はまた、最近、それが促進することが知られている阻害剤、後者はラパマイシン（mTORの）の臨床的に承認された哺乳類の対象となる、TGF-β、ATRAの組み合わせを使用し、22ラパマイシン することにより、in vitroでの優れた抑制機能付きiTregsの世代のための新しいプロトコルを確立しました人間のTreg拡張25,26の間にFOXP3のメンテナンス。 この方法は、再現性の中での説明しますFOXP3の発現のプロトコル固有のパターンや、その他のTreg署名分子を明らかにするために、フローサイトメトリーおよび定量的逆転写ポリメラーゼ連鎖反応（QRT-PCR）により、異なる条件のセット、およびその後続の表現型を用いて、ヒトCD4 + FOXP3 + iTregsのインビトロでの生成CD25、CTLA-4、EOS、ならびにIFN-γおよびSATB1 式 22の抑制など。生成された細胞集団は、一般的なFOXP3レギュレータに関するあるいは酪酸またはラパマイシンなどの特定の化合物に特有の効果を研究するか、抑制活性に関する機能アッセイまたは分子的研究のために使用することができます。 Treg細胞の分化を駆動する分子メカニズムのさらなる理解は、具体的Treg細胞の発生と機能に関与する分子を標的とする自己免疫または癌における今後の治療的アプローチのために非常に関連性があります。

<table border="0" cellpadding="0" cellspacing="0" width="1689">
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		<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>
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		<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>
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		<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>
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			<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>
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			<td height="20" style="height:21px;">anti-human CD45RO PE clone UCHL1</td>
			<td>BD Biosciences</td>
			<td>555493</td>
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		<tr height="20">
			<td height="20" style="height:21px;">anti-human CD4-PerCP clone SK3; mIgG1</td>
			<td>BD Biosciences</td>
			<td>345770</td>
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			<td height="20" style="height:21px;">anti-human CD8-eFluor 450 (clone OKT8), mIgG2a</td>
			<td>eBioscience</td>
			<td>48-0086-42&#160;</td>
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			<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>
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			<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>
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			<td height="20" style="height:21px;width:501px;">Brefeldin A-containing solution: GolgiPlug</td>
			<td>BD Biosciences</td>
			<td>555029</td>
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			<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>
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			<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>
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			<td height="21" style="height:22px;">DMSO 99,7%</td>
			<td>Sigma Aldrich</td>
			<td>D2650-5X5ML</td>
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			<td height="20" style="height:20px;">FBS, heat inactivated</td>
			<td>Invitrogen</td>
			<td>10082-147</td>
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			<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>
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			<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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			<td height="20" style="height:21px;">GlutaMAX (200 mM L-alanyl-L-glutamine)</td>
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			<td>35050-061</td>
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			<td height="20" style="height:21px;">human naive CD4 T cell isolation kit II</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-131</td>
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			<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">
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						<td height="20" style="height:21px;width:501px;">human naive CD4 T cell isolation kit II</td>
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			<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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						<td height="20" style="height:21px;width:176px;">Miltenyi Biotec</td>
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			</td>
			<td>130-094-131</td>
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			<td height="20" style="height:21px;">Human serum albumin 50 g/l</td>
			<td>Baxter</td>
			<td>1501057</td>
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			<td height="20" style="height:21px;">Ionomycin from Streptomyces conglobatus &#62;98%</td>
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			<td>I9657-1MG</td>
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			<td height="20" style="height:21px;">MACS LS-columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
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			<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>
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			<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>
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			<td height="20" style="height:21px;width:501px;">mouse IgG2a isotype control, Brilliant violet 421, clone MOPC-173</td>
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			<td>563464</td>
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			<td height="20" style="height:21px;">Pasteur pipet plastic, individually packed</td>
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			<td>86.1172.001&#160;&#160;</td>
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			<td height="20" style="height:21px;">PMA PHORBOL 12-MYRISTATE 13-ACETATE</td>
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			<td>P1585-1MG&#160;</td>
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			<td height="20" style="height:21px;">Rapamycin</td>
			<td>EMD (Merck)</td>
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			<td height="20" style="height:21px;">Recombinant Human IL-2, CF</td>
			<td>R&#38;D</td>
			<td>202-IL-050/CF</td>
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			<td height="20" style="height:21px;width:501px;">Recombinant Human TGF-beta 1, CF</td>
			<td>RnD</td>
			<td>240-B-010/CF</td>
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		<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>
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		<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.

図1は、実験のスキームを示します。 図2は、磁気的に分離されたナイーブCD4 + T細胞およびnTregsのための代表的な純度制御染色を示します。 Fのigure 3Aはゲート戦略フローサイトメトリーを示し、 図3Bは 、代表FOXP3及びCD25が示さiTregまたは制御条件下での培養6日目...

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

このプロトコルは、in vitroでのナイーブCD4 + T細胞から人為的な制御性T細胞（iTregs）の再現性の生成と表現型を説明しています。 Foxp3誘発のための異なるプロトコルは、それぞれのプロトコルを用いて得られた特定のiTregの表現型の研究を可能にします。

TGF-βを含むプロトコルを使用したナイーブCD4 + T細胞からのヒトCD4 + FOXP3 +誘導された調節性T細胞（iTregs）のインビトロ分化における

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

マウスT細胞におけるT細胞受容体のレトロウイルス導入

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 ...

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

初代ヒトナイーブおよびメモリーT細胞から誘導した調節性T細胞の生成

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

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Tリンパ球に向かって誘導多能性幹細胞の分化の指示

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

プライマリーヒトT細胞のpiggyBacトランスポゾンシステムの変更

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

HIV-1に感染した個体からの制御性T細胞を増殖するための新しいツール

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

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

ナイーブCD4のアデノウイルス形質導入Tregの分化を研究するためにT細胞

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 ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Isolation and Th17 Differentiation of NaÃƒÆ’Ã‚Â¯ve CD4 T Lymphocytes

離とナイーブCD4 Tリンパ球のTh17分化

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 ...

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

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

HIV-1感染CD4の生得センシングを評価<sup&gt; +</sup&gt;使用して形質細胞様樹状細胞によるT細胞<em&gt;エクスビボ</em&gt;共培養系。

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

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

T細胞の導出<em&gt;体外</em&gt;マウス胚性幹細胞からの

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

末梢血からのヒトアロ抗原特異的T細胞の生成

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...