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

This project was funded, in part, under grants from the National Institutes of Health (R01AI121180, R21AI109239 and K18CA151798), the American Diabetes Association (1-16-IBS-281), and the Pennsylvania Department of Health (Tobacco Settlement Funds) to J.S.

All animal experiments are approved by the Pennsylvania State University College of Medicine Animal Care Committee (IACUC protocol #45470) and are conducted in compliance with the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care.
1. Stem Cell Culture
<ol>
	<li>Incubate a 10 cm dish with 10 ml of 0.1% gelatin for at least 30 min at 37 &#176;C (incubator) in order to coat the plate.</li>
	<li>Remove gelatin from the dish and plate 3 x 106 ...

<ol>
	<li>Firestein, G. 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. J., 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=Regulatory+T+cells+that+recognize+a+ubiquitous+stress-inducible+self-antigen+are+long-lived+suppressors+of+autoimmune+arthritis.">Regulatory T cells that recognize a ubiquitous stress-inducible self-antigen are long-lived suppressors of autoimmune arthritis.</a> Proc Natl Acad Sci U S A. 109, 14134-14139 (2012).</li><li>Wright, G. P., 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=Adoptive+therapy+with+redirected+primary+regulatory+T+cells+results+in+antigen-specific+suppression+of+arthritis.">Adoptive therapy with redirected primary regulatory T cells results in antigen-specific suppression of arthritis.</a> Proc Natl Acad Sci U S A. 106, 19078-19083 (2009).</li><li>Schmitt, T. M., 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=Induction+of+T+cell+development+and+establishment+of+T+cell+competence+from+embryonic+stem+cells+differentiated+in+vitro.">Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro.</a> Nat Immunol. 5, 410-417 (2004).</li><li>La Motte-Mohs, R. N., Herer, E., Zuniga-Pflucker, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+T-cell+development+from+human+cord+blood+hematopoietic+stem+cells+by+Delta-like+1+in+vitro.">Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro.</a> Blood. 105, 1431-1439 (2005).</li><li>Lei, F., Haque, R., Weiler, L., Vrana, K. E., Song, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+lineage+differentiation+from+induced+pluripotent+stem+cells.">T lineage differentiation from induced pluripotent stem cells.</a> Cell Immunol. 260, 1-5 (2009).</li><li>Lei, F., Haque, R., Xiong, X., Song, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+differentiation+of+induced+pluripotent+stem+cells+towards+T+lymphocytes.">Directed differentiation of induced pluripotent stem cells towards T lymphocytes.</a> J Vis Exp. , e3986 (2012).</li><li>Lei, F., 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+vivo+programming+of+tumor+antigen-specific+T+lymphocytes+from+pluripotent+stem+cells+to+promote+cancer+immunosurveillance.">In vivo programming of tumor antigen-specific T lymphocytes from pluripotent stem cells to promote cancer immunosurveillance.</a> Cancer Res. 71, 4742-4747 (2011).</li><li>Haque, R., 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=Programming+of+regulatory+T+cells+from+pluripotent+stem+cells+and+prevention+of+autoimmunity.">Programming of regulatory T cells from pluripotent stem cells and prevention of autoimmunity.</a> J Immunol. 189, 1228-1236 (2012).</li><li>Chi, V., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry:+paraffin+sections+using+the+Vectastain+ABC+kit+from+vector+labs.">Immunohistochemistry: paraffin sections using the Vectastain ABC kit from vector labs.</a> J Vis Exp. , (2007).</li><li>Lu, L., 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=Critical+role+of+all-trans+retinoic+acid+in+stabilizing+human+natural+regulatory+T+cells+under+inflammatory+conditions.">Critical role of all-trans retinoic acid in stabilizing human natural regulatory T cells under inflammatory conditions.</a> Proc Natl Acad Sci U S A. 111, E3432-E3440 (2014).</li><li>Wu, C., 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=Galectin-9-CD44+interaction+enhances+stability+and+function+of+adaptive+regulatory+T+cells.">Galectin-9-CD44 interaction enhances stability and function of adaptive regulatory T cells.</a> Immunity. 41, 270-282 (2014).</li><li>Di Stasi, 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=Inducible+apoptosis+as+a+safety+switch+for+adoptive+cell+therapy.">Inducible apoptosis as a safety switch for adoptive cell therapy.</a> N Engl J Med. 365, 1673-1683 (2011).</li><li>Ramos, C. 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=An+inducible+caspase+9+suicide+gene+to+improve+the+safety+of+mesenchymal+stromal+cell+therapies.">An inducible caspase 9 suicide gene to improve the safety of mesenchymal stromal cell therapies.</a> Stem Cells. 28, 1107-1115 (2010).</li><li>Haque, R., Lei, F., Xiong, X., Wu, Y., Song, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxP3+and+Bcl-xL+cooperatively+promote+regulatory+T+cell+persistence+and+prevention+of+arthritis+development.">FoxP3 and Bcl-xL cooperatively promote regulatory T cell persistence and prevention of arthritis development.</a> Arthritis Res Ther. 12, R66 (2010).</li><li>van Loenen, M. M., 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=Mixed+T+cell+receptor+dimers+harbor+potentially+harmful+neoreactivity.">Mixed T cell receptor dimers harbor potentially harmful neoreactivity.</a> Proc Natl Acad Sci U S A. 107, 10972-10977 (2010).</li><li>Kim, Y. C., 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=Engineered+antigen-specific+human+regulatory+T+cells:+immunosuppression+of+FVIII-specific+T-+and+B-cell+responses.">Engineered antigen-specific human regulatory T cells: immunosuppression of FVIII-specific T- and B-cell responses.</a> Blood. 125, 1107-1115 (2015).</li><li>Himburg, H. 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=Pleiotrophin+regulates+the+expansion+and+regeneration+of+hematopoietic+stem+cells.">Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells.</a> Nat Med. 16, 475-482 (2010).</li></ol>

In this protocol, a critical step is the in vitro differentiation of TCR/FoxP3 gene-transduced iPSCs. In vitro Notch signaling induces development towards the T cell lineage. To differentiate iPSCs into CD4+FoxP3+ Tregs, we used the OP9-DL1/DL4/I-Ab cells, which highly express MHC II (I-Ab) molecules. Most of the iPSCs differentiate into CD4+ cells. However, after the surface TCR expression, many differentiated pre-T cells lose the ability to differ...

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.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<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 diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

As shown here, on day 28, Ag-specific Tregs substantially expressed CD3 and Ag-specific TCR, two T cell markers. The CD3+TCRV&#946;5+ population expressed CD4. Most of the CD3+TCRV&#946;5+CD4+ cells also expressed CD25, CD127, and CTLA-4, which are typically expressed at elevated levels in naturally occurring Tregs (nTregs) and in T cells expressing FoxP3 ectopically. FoxP3 expression in iPSC-derived cells persisted even af...

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.

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

Research

JoVE Journal

Immunology and Infection

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

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

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

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

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

Killer Artificial Antigen Presenting Cells (KaAPC) for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Killer Artificial Antigen Presenting Cells KaAPC for Efficient In Vitro Depletion of Human Antigen-specific T Cells

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied ...

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

Derivation of T Cells In Vitro from Mouse Embryonic Stem Cells

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

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

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

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

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

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

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