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.

İnsan periferal kan tek-çekirdekli hücreleri (PBMC), taze Karolinska Üniversite Hastanesi, İsveç satın anonim sağlıklı donor buffy katlarından izole edilmiştir. deneyler için etik izin Stockholm Bölgesel Etik Değerlendirme Kurulu (Regionala etikprövningsnämnden i Stockholm) elde edilmiştir, İsveç (onay numarası: 2013 / 1458-1431 / 1). Periferal kandan, 1&#39;dir, T Hücre İzolasyonu <ol><li> PBMC İzolasyon <ol><li> 15 ml yoğunluğu santrifüj ortamı 50 ml tüpler (buff...

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Açıklanan protokol, insan CD4 + FoxP3 + insan naif CD4 + T hücrelerinden iTregs sağlam indüksiyon sağlar. Biz üstün in vitro baskılayıcı fonksiyonu 22 iTregs uyarılması için, TGF-p, ATRA ve rapamisin bir arada kullanarak, son zamanlarda açıklanan yeni bir protokol içerir. Diğer yayınlanmış protokollere göre, bir başka avantajı, tek başına IL-2 varlığında, aktive edilmiş olan kontrol hücreleri ile birlikte, belirli bir iTreg uyaran faktörlerin etkileri doğru...

CD4 + CD25 + FoxP3 + düzenleyici T hücreleri (Tregs) diğer bağışıklık hücrelerini bastırmak ve periferik tolerans kritik arabulucular, önlenmesi otoimmünite ve aşırı enflamasyon 1 bulunmaktadır. Tregs önemi, ciddi sistemik otoimmün hastalık yol açar Tregs kaybı nedeniyle `master&#39; Treg transkripsiyon faktör forkhead kutusu P3 mutasyonlar insan hastalık immunodysregulation poliendokrinopati enteropatisi X&#39;e bağlı sendromu (IPEX), (FoxP3) tarafından örneklenmiştir erken yaşta ölümcül. Ancak, Treg&#39;ler onlar da bazı ayarlarda 2 anti-tümör bağışıklığını engel gibi bağışıklık sisteminde bir iki ucu keskin kılıç olarak hareket ederler. Treg sayısı ve fonksiyonu Terapötik manipülasyon birçok klinik araştırmalarda bu nedenle tabidir. Kanserinde Tregs tükenmesi arzu edilebilir ve klinik yaklaşımlar bazı başarı daha fazla araştırma 3 teşvik eder. Pek çok dil içinde Tregs terapötik etkilerine ek olarak, otoimmün ve enflamatuar hastalıkların, bölgesindekiral fare hastalık modelleri, evlatlık Treg transferi son ilk-man denemeleri greftleme karşı -host hastalığı (GVHH) 4 önlemek için - 7 ve çok umut verici sonuçlar vermiştir tip 1 diyabet 8 tedavisinde güvenliğini değerlendirmek için. 11 - Doğal Tregs (nTregs üretme) SAĞLIK 9 korunmasında yedeksiz temel fonksiyonları ile timik türevli tTregs ve periferik kaynaklı pTregs içermektedir meydana. Bununla birlikte, nTreg numaraları naif T hücresi öncüleri 12 in vitro olarak uyarılması Tregs (iTregs) tamamlayıcı bir yaklaşım teşvik sınırlıdır. Bunun nedeni, muhtemelen FoxP3 gen lokusu 13 olarak adlandırılan regülatör özgü demetile bölge (TSDR) &#39;de demetilasyon eksikliği iTregs Hala stabilite, bir sorun olmaya devam etmektedir ve birçok çalışma, Tregs bağlı in vivo 14 daha kararlı olduğunu göstermektedir. Bugüne kadar, FoxP3 en iyi protein m kalırinsan geleneksel CD4 + CD25 T hücreleri geçici aktivasyon 15,16 üzerine FOXP3 orta düzeyde ifade çünkü Tregs için arker ama kesinlikle özel değildir. önemli bir ilerleme FoxP3 ekspresyonunun düzenlenmesi açıklık yapılmış olmasına rağmen, daha özel olarak, insan hücrelerinde FOXP3 indüklenmesi, kararlılık ve işlevi ile ilgili tespit edilmesi gerekmektedir. NTregs için farklılıklara rağmen, in vitro FoxP3 neden + CD4 + T hücreleri FoxP3 indüksiyon moleküler mekanizmaları incelemek için bir model sistem olarak da kullanılabilir ve daha benzer iTregs üretilmesine izin gelecek protokolleri geliştirmek için bir başlangıç noktası olarak gelecekte adaptif transfer stratejileri için geçerli olabilir oluşturulan Tregs, vivo. Orada insan iTregs ikna etmek için hiçbir `altın standard&#39; protokolü ve mevcut protokoller in vivo regülatör uyaran koşulları taklit dayalı geliştirilen edilmiştir: 2 (IL-2, interlökin) Ve transforme edici büyüme faktörü β (TGF-β) sinyal vivo 17 FoxP3 indüksiyonu için çok önemli olan, ve all-trans retinoik asit (ATRA) - bağırsak ile ilişkili dendritik hücreler tarafından in vivo üretilen - sıkça FoxP3 arttırılması için kullanılır 21 - 18, in vitro olarak. Bu bütirat 22 en son sıçangil regülatör indüksiyon 23,24 artırdığı gösterilmiştir bağırsak Mikrobiyota türetilen kısa zincirli yağ asit kullanılarak ek insan regülatör T indükleyici protokolleri geliştirmiştir. Ayrıca son zamanlarda bu teşvik ettiği bilinir önleyicisi ikinci rapamisin (mTOR) klinik olarak kabul memeli hedefi olarak, TGF-p, ATRA bir kombinasyonu kullanılarak ve 22 rapamisin ile, in vitro olarak, üstün bastırma fonksiyonu ile iTregs üretimi için yeni bir protokol kurulan insan Treg genişleme 25,26 sırasında FoxP3 bakım. Bu yöntem tekrarlanabilir açıklarinsan CD4 + FoxP3 + farklı olan bir dizi şartı ile iTregs ve akış sitometrisi ve kantitatif ters transkripsiyon polimeraz zincir reaksiyon ile daha sonra fenotipleme nitro nesil (qRT-PCR) FOXP3 sentezlenmesi ve diğer regülatör imza molekülleri, protokole özel desenler ortaya çıkarmak için CD25, CTLA-4, EOS, hem de IFN-y ve SATB1 ifade 22 baskı olarak. oluşturulan hücre popülasyonları, genel FoxP3 düzenleyicileri ile ilgili ya da bu tür butirat veya rapamisin gibi bazı bileşiklerin özel etkilerini incelemek için, ya baskılayıcı aktivitesi ile ilgili olarak, işlevsel tayinler için veya moleküler çalışmalar için de kullanılabilir. Treg farklılaşma sürüş moleküler mekanizmaların daha iyi anlaşılması, özellikle Treg nesil ve fonksiyonunda yer alan molekülleri hedef otoimmünite veya kanser gelecekteki terapötik yaklaşımlara için büyük önem taşımaktadır.

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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>
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		<tr height="20">
			<td height="20" style="height:21px;">anti-human CD25 microbeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-983</td>
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			<td height="20" style="height:21px;">anti-human CD25-PE</td>
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			<td>130-091-024</td>
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			<td height="20" style="height:21px;">anti-human CD28 antibody, LEAF Purified&#160;</td>
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			<td>302914</td>
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			<td height="20" style="height:21px;">anti-human CD3 Antibody, LEAF Purified&#160;</td>
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			<td height="20" style="height:21px;width:501px;">anti-human CD45RA , FITC</td>
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			<td height="20" style="height:21px;">anti-human CD45RO PE clone UCHL1</td>
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			<td height="20" style="height:21px;">anti-human CD4-PerCP clone SK3; mIgG1</td>
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			<td>345770</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>
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			<td>562743</td>
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			<td height="20" style="height:21px;">anti-human IFN-g FITC clone 4S.B3; mIgG1k</td>
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			<td>11-7319-81&#160;</td>
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			<td height="20" style="height:21px;width:501px;">Brefeldin A-containing solution: GolgiPlug</td>
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			<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>
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			<td height="20" style="height:21px;width:501px;">Density centrifugation medium: Ficoll-Paque</td>
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			<td style="width:248px;">17-1440-03</td>
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			<td height="21" style="height:22px;">DMSO 99,7%</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;">human naive CD4 T cell isolation kit II</td>
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			<td>130-094-131</td>
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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 height="20" style="height:21px;width:176px;">Miltenyi Biotec</td>
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			<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>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>
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			<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>
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			<td>563464</td>
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			<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>
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			<td>240-B-010/CF</td>
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			<td height="20" style="height:21px;">RNA isolation kit: RNAqueous-Micro Kit</td>
			<td style="width:176px;">Ambion</td>
			<td>AM1931&#160;&#160;</td>
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		<tr height="20">
			<td height="20" style="height:21px;">RPMI 1640 Medium&#160;</td>
			<td>Invitrogen</td>
			<td>72400-054&#160;</td>
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		<tr height="20">
			<td height="20" style="height:21px;">Sodium butyrate</td>
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			<td>B5887-250MG&#160;</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>
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			<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.

Şekil 1 deney düzeneği bir şemasını göstermektedir. Şekil 2, manyetik izole saf CD4 + T hücreleri ve nTregs için temsili bir saflıkta kontrol boyamasını göstermektedir. F ŞEKIL 3A yolluk stratejisi sitometri akışını gösterir ve Şekil 3B temsilcisi FoxP3 ve CD25 belirtilen iTreg veya kontrol koşulları altında kültür 6. günd...

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

Bu protokol, in vitro olarak naif CD4 + T hücrelerinden üretilebilir üretimi ve insan kaynaklı düzenleyici T hücrelerinin (iTregs) fenotipleme tarif eder. FoxP3 indüksiyonu için farklı protokoller ilgili protokolleri ile elde edilen spesifik iTreg fenotipleri çalışması için izin verir.

Bir TGF-β-içeren Protokolü kullanılarak naif CD4 + T hücrelerinden insan CD4 + FoxP3 + Bağlı regülatör T hücreleri (iTregs) in vitro ayrımında

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

Fare T-hücrelerinin, T-hücre reseptörleri retroviral İletimi

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

İlköğretim İnsan naif ve Bellek T Hücreleri gelen İsteyerek Düzenleyici T Hücreleri Üretimi

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 Lenfositler doğru İsteyerek Pluripotent Kök Hücrelerinin Yönetmen Farklılaşma

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

Primer insan T hücrelerinin piggyBac Transposon Sistem Modifikasyon

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

Yeni Araçlar HIV-1 ile enfekte bireyler gelen Düzenleyici T hücreleri büyütmek için

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

Naif CD4 adenoviral İletimi Treg Farklılaşma Eğitim Hücreler T

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

İzolasyon ve naif CD4 T Lenfositler Th17 Farklılaşma

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 Enfekte CD4 Doğuştan Algılama Değerlendirilmesi<sup&gt; +</sup&gt; Bir kullanılması plazmositoid dendritik hücreler tarafından T Hücreleri<em&gt; Ex vivo</em&gt; Co-kültür sistemi.

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 hücrelerinin türetilmesi<em&gt; İn Vitro</em&gt; Fare Embriyonik Kök Hücreleri

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

Periferik Kan İnsan alloantijen-özgü T hücrelerinin üretilmesi

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