Name: development of stem cell-derived antigen-specific regulatory t cells against autoimmunity
Uploaded: 2016-11-08T15:00:00
Description: Watch this Scientific Journal Video about Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity at JoVE.com

Dette projekt blev finansieret, delvis under tilskud fra National Institutes of Health (R01AI121180, R21AI109239 og K18CA151798), American Diabetes Association (1-16-IBS-281), og Pennsylvania Department of Health (Tobacco Settlement Funds) til JS

Alle dyreforsøg godkendes af Pennsylvania State University College of Medicine Animal Care udvalg (IACUC protokol # 45470) og gennemføres i overensstemmelse med retningslinjerne fra foreningen for vurdering og akkreditering af Laboratory Animal Care. 1. Stem Cell Culture <ol><li> Inkubér en 10 cm skål med 10 ml 0,1% gelatine i mindst 30 min ved 37 ° C (inkubator) med henblik på at belægge pladen. </li><li> Fjern gelatine fra skålen og pladen 3 x 10 6 bestrålet SNL76 / 7...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolving+concepts+of+rheumatoid+arthritis.">Evolving concepts of rheumatoid arthritis.</a> Nature. 423, 356-361 (2003).</li><li>Ferraro, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interindividual+variation+in+human+T+regulatory+cells.">Interindividual variation in human T regulatory cells.</a> Proc Natl Acad Sci U S A. 111, E1111-E1120 (2014).</li><li>Tang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro-expanded+antigen-specific+regulatory+T+cells+suppress+autoimmune+diabetes.">In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes.</a> J Exp Med. 199, 1455-1465 (2004).</li><li>van Herwijnen, M. 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I denne protokol, et kritisk trin er in vitro differentiering af TCR / Foxp3 gen-transducerede iPSCs. In vitro Notch signalering inducerer udvikling mod T-celle afstamning. For at differentiere iPSCs i CD4 + foxp3 + tregs, vi brugte OP9-DL1 / DL4 / IA B-celler, som meget udtrykkelige MHC II (IA b) molekyler. De fleste af de iPSCs differentiere til CD4 + celler. Men efter at overfladen TCR ekspression mange differentierede præ-T-celler taber, evnen...

Autoimmune arthritis is a systemic disease characterized by hyperplasia of synovial tissue and progressive destruction of articular cartilage, bone, and ligaments1. The defective generation or function of Tregs in autoimmune arthritis contributes to chronic inflammation and tissue injury because Tregs play a crucial role in preventing the development of auto-reactive immune cells. 
Manipulation of Tregs is an ideal strategy for the development of therapies to suppress inflammation in an Ag-dependent manner. For Treg-based immunotherapy, the specificity of the transferred Tregs is important for the treatment of ongoing autoimmunity2. To exhibit the suppressive activity, Tregs need to migrate and be retained at the afflicted region, which can be directed by the specificity of the TCR for the Ag at that location3. Although polyclonal Tregs may contain a small population containing this Ag specificity from their TCRs, the numbers of these Ag-specific Tregs are usually low. Consequently, cell-based therapies using polyclonal Tregs against autoimmune disorders require adoptive transfers of a large number of Tregs4,5. Because pluripotent stem cells (PSCs) have the ability to develop into any type of cell, Ag-specific PSC-Tregs may prove to be good candidates for Treg-based immunotherapy. Previous studies have shown the successful development of PSC-derived T cells, including Tregs6-8. 
Here, we describe a protocol to develop Ag-specific iPSC-Tregs. We further describe a cell-based therapy of autoimmune arthritis in a murine model using such Tregs. This method is based upon genetically modifying murine iPSCs with Ag-specific TCRs and the transcriptional factor FoxP3. The engineered iPSCs then differentiate into Ag-specific Tregs on the OP9 stromal cells expressing Notch ligands DL1, DL4, and MHC-II (I-Ab) molecules in the presence of cytokines mFlt3L and mIL-7. These Ag-specific iPSC-Tregs can produce suppressive cytokines, such as TGF-&#946; and IL-10, when stimulated with the Ag, and adoptive transfer of such Tregs has the ability to suppress AIA development in a murine model. The described protocol can be used to develop stem cell-derived Ag-specific Tregs for potential therapeutic interventions.

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	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">C57BL/6j mice</td>
			<td style="width:71px;">Jackson Laboratory</td>
			<td style="width:119px;">664</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">B6.129S7 Rag1tm1Mom/J</td>
			<td style="width:71px;">Jackson Laboratory</td>
			<td style="width:119px;">2216</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Anti-CD3 (2C11) antibody</td>
			<td style="width:71px;">BD Pharmingen</td>
			<td style="width:119px;">553058</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Anti-CD28 (37.51) antibody</td>
			<td style="width:71px;">BD Pharmingen</td>
			<td style="width:119px;">553295</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-CD4 (GK1.5) antibody</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">100417</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-CD8 (53&#8211;6.7) antibody</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">100714</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-CD25 (3C7) antibody</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">101912</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-TCR-&#946; (H57597) antibody</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">109220</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-IL10</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">505010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-TGF&#946;</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">141402</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">DMEM</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">ABCD1234</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">&#945;-MEM</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">A10490-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">FBS</td>
			<td style="width:71px;">Hyclone</td>
			<td style="width:119px;">SH3007.01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Brefeldin A</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">B7651</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Polybrene</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">107689</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Genejammer</td>
			<td style="width:71px;">Integrated science</td>
			<td style="width:119px;">204130</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">ACK Lysis buffer</td>
			<td style="width:71px;">Lonza</td>
			<td style="width:119px;">10-548E</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">mFlt-3L</td>
			<td style="width:71px;">peprotech</td>
			<td style="width:119px;">250-31L</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">mIL-7</td>
			<td style="width:71px;">peprotech</td>
			<td style="width:119px;">217-17</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Gelatin</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">G9391</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Paraformaldehyde</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P6148-500G</td>
			<td style="width:167px;">Caution: Allergenic, Carcenogenic, Toxic</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Permeabilization buffer</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">421002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">mBSA</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A7906</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Ova albumin</td>
			<td style="width:71px;">Avantor</td>
			<td style="width:119px;">0440-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">CFA</td>
			<td style="width:71px;">Difco</td>
			<td style="width:119px;">2017014</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Tailveiner restrainer</td>
			<td style="width:71px;">Braintree scientific</td>
			<td style="width:119px;">RTV 150-STD</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

development of stem cell-derived antigen-specific regulatory t cells against autoimmunity

Autoimmune sygdomme opstår på grund af tabet af immunologisk selv-tolerance. Regulatoriske T-celler (tregs) er vigtige mediatorer af immunologisk selv-tolerance. Tregs for ca. 5 - 10% af det modne CD4 + T-celle subpopulationen i mus og mennesker, med ca. 1 - 2% af disse tregs cirkulerer i det perifere blod. Inducerede pluripotente stamceller (iPSCs) kan differentieres til funktionel tregs, som har potentiale til at blive anvendt til cellebaserede behandlingsformer på autoimmune sygdomme. Her præsenteres en metode til at udvikle antigen (Ag) -specifikke tregs fra iPSCs (dvs. IPSC-tregs). Fremgangsmåden er baseret på inkorporering af transskriptionsfaktoren foxp3 og en Ag-specifik T-celle receptor (TCR) i iPSCs og derefter differentiere på OP9 stromaceller udtrykker Notch-ligander delta-lignende (DL) 1 og DL4. Efter in vitro differentiering, IPSC-tregs udtrykke CD4, CD8, CD3, CD25, foxp3, og Ag-specifik TCR og er i stand til at reagere på Ag-stimulering.Denne metode er blevet anvendt med succes til celle-baseret behandling af autoimmun arthritis i en murin model. Adoptiv overførsel af disse Ag-specifik iPSC-tregs i Ag-induceret arthritis (AIA) -fyldt mus har evnen til at reducere ledbetændelse og hævelse og forebygge knogletab.

Som vist her, på dag 28, Ag-specifik tregs udtrykte væsentlige CD3 og Ag-specifik TCR, to T-celle-markører. CD3 + TCRVβ5 + population udtrykt CD4. De fleste af de CD3 + TCRVβ5 + CD4 + celler udtrykte også CD25, CD127, og CTLA-4, der typisk er udtrykt på et højt niveau i naturligt forekommende T Fo (nTregs) og i T-celler, der udtrykker foxp3 ektopisk. Foxp3 ekspression i iPSC-afledte celler fortsatte selv efter lang...

Watch this Scientific Journal Video about Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity at JoVE.com

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Udvikling af Stem Cell-afledte antigen-specifikke regulatoriske T celler mod Autoimmunitet

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

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Regulatory T cells (Tregs) are an integral part of peripheral tolerance, suppressing immune reactions against self-structures and thus preventing ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Kvantificering af prolifererende humane antigen-specifikke CD4+ T-celler ved hjælp af Carboxyfluorescein Succinimidyl ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Højeffektiv generation af antigen-specifikke primære mus cytotoksiske T-celler til funktionel testning i en autoimmun diabetes model

Type 1 Diabetes (T1D) is characterized by islet-specific autoimmunity leading to beta cell destruction and absolute loss of insulin production. In the ...