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.

<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>

The authors have nothing to disclose.

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 differentiate and eventually die. As a result, the cell number of the iPSC-derived functional Tregs dramatically reduces after four weeks of in vitro differentiation. To avoid this, addition of IL-2 can improve cell survival at those time points. An alternative method to drive the maturation and survival of Ag-specific pre-Tregs is to transfer the pre-Tregs that have differentiated in vitro for a week into mice. These pre-Tregs can continue differentiating and maturing in vivo for another three weeks. Using this method, a functional Ag-specific Treg population can be generated from iPSCs, which are nTregs-like and have the ability to suppress autoimmune arthritis in the murine model.
To improve the efficacy of in vivo development of Ag-specific iPSC-Tregs, different compounds (e.g., retinoic acid, gelectin)13,14 or suppressive cytokines (e.g., TGF-&#946;, IL-10) can be used after the adoptive transfer of the pre-Tregs. In addition to increasing the cell survival, this combined approach can maintain FoxP3 expression14,15 and enhance the quality of the Ag-specific iPSC-Tregs.
A potential problem that could arise for in vivo Treg development is overall immunosuppression, resulting in complications during infections or subsequent weight loss. A large number of Ag-specific Tregs developing in vivo may worsen infections. In this case, a suicide gene, the inducible caspase 9 (iCasp9)15,16, can be incorporated into the TCR/FoxP3 vector. This approach allows the removal of the stem cell-derived Tregs by the injection of a bioinert small-molecule dimerizing agent (AP1903) to &#34;shut off&#34; the generation of stem cell-derived Tregs, which will overcome this potential issue.
A self-Ag is a typical protein recognized by the immune system of hosts suffering from an individual autoimmune disorder. This self-Ag is the target of the immune system, and the correlated T cells are not deleted. To generate self-Ag specific Tregs, the use of TCR transduction is a good choice. A self-Ag (e.g., heat shock protein) specific TCR can be transduced into mature CD4+ CD25+ Tregs from peripheral blood mononuclear cells (PBMCs), and this approach has been utilized in clinical trials. Alternatively, as described in this protocol, using the gene transduction of self-Ag specific TCR with FoxP3 and stimulation with Notch signaling, stem cell-derived Tregs can be self-Ag specific Tregs.
Cell-based therapies for autoimmune diseases utilizing engineered Tregs may be useful17. Although TCR transduction in T cells has proved to be safe, feasible, and applicable in clinical trials, there are still major safety concerns due to the autoimmunity caused by cross-reactivity with healthy tissues18. Furthermore, using current methods, genetically modified Tregs usually have a short-term persistence in vivo19. Alternatively, a promising source for developing large numbers of monoclonal Ag-specific Tregs is stem cells. Embryonic stem cells (ESCs) have the best pluripotency and self-renewal, but it is not feasible to obtain them from patients. Although easily isolated from the peripheral blood, hematopoietic stem cells (HSCs) are multi-potent stem cells and can be expanded in cell culture similarly to ESCs20. Present iPSC technology has advanced to a more efficient generation of PSC from patients&#39; somatic cells by transduction of different transcription factors. A number of methodological improvements have been developed in recent years to generate iPSCs by maximally reducing potential hazards such as immunogenicity and tumorigenicity. Compared to ESCs, iPSCs have identical pluripotency and self-renewal. As a result, iPSC technology can provide an advantage in developing patient- and/or disease-specific PSCs. The use of iPSCs to develop Ag-specific Tregs may advance the field of cell-based therapies for autoimmune diseases10,11.

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 after long-term in vitro stimulation with the Notch ligand, as detected by intracellular staining analyzed by flow cytometry (Figure 1). In addition, the Ag-specific iPSC-Tregs produced suppressive cytokines, such as TGF-&#946; and IL-10, when stimulated in vitro with Ag-pulsed splenocytes (Figure 2), indicating the iPSC-Tregs had potential suppressive activities. Fluorescence microscopy revealed that more FoxP3+ cells were present in the OVA-treated than the PBS-treated knees; there were no FoxP3+ cells existing in the knees in mice receiving the DsRed+ vector-transduced iPSCs. Many more CD4+FoxP3+ TCRV&#946;5+ cells presented in the knees of mice receiving iPSCs transduced with the MiDR-TCR-FoxP3 than the MiDR-FoxP3 (Figure 3). These observations suggest that Ag-specific iPSC-Tregs migrate to the AIA knee after the adoptive transfer into recipient mice. The transferred iPSC-derived cells substantially decreased the inflammatory knee swelling when OVA was present, but had no effect on the control knee that was only injected with mBSA in the murine model (Figure 4); reduced bone loss in the cell transfer knee visualized by the high-resolution micro-CT system (Figure 5).
<img alt="Figure 1" src="/files/ftp_upload/54720/54720fig1.jpg" /> 
Figure 1: Differentiation of Ag-specific iPSC-Tregs. Murine iPSCs were transduced with a construct: MiDR-TCR&#945;-2A-TCR&#946;-2A-FoxP3 containing genes of OVA-specific TCR and FoxP3. The gene-transduced cells (DsRed+) were co-cultured on OP9-DL1-DL4-I-Ab cells in the presence of mFlt3L and mIL-7. (A) Morphology of Tregs cell differentiation on day 0, 7, 14 and 22. (B) Flow cytometric analysis for the protein expression of iPSC-derived cells on day 28. CD3+ TCRV&#946;5+ cells were gated as indicated (R1) and analyzed for the expression of CD4 and CD8, with CD25, CD127, CTLA-4, and FoxP3 shown for cells gated as CD4+CD8- cells (R2) (dark lines; shaded areas indicate isotype controls). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54720/54720fig2.jpg" /> 
Figure 2: Functional Analyses of In Vitro Differentiated Ag-specific Tregs. Murine iPSC-Tregs were stimulated by splenocytes (APCs; Tregs/APCs = 1:4) and pulsed with OVA323-339 peptide. Intracellular cytokine production (TGF-&#946; and IL-10) was analyzed by flow cytometry after gating on live CD4+ CD25+ cells (dark lines; shaded areas indicate isotype controls). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54720/54720fig3.jpg" /> 
Figure 3: Ag-specific iPSC-Tregs Infiltrate into the Knee Joints. Ag-specific iPSC-Tregs were adoptively transferred into C57BL/6 mice. Shortly after arthritis induction (days 7 - 14), the knees were removed and stained for immunohistology (scale bars: 20 &#956;m). Mice receiving OVA-specific iPSC-Tregs have large numbers of TCRV&#946;5+ cells in the knees. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54720/54720fig4.jpg" /> 
Figure 4: Adoptive Transfer of Ag-specific iPSC-Tregs Ameliorates AIA in Mice. Murine iPSCs were transduced with the retroviral construct MiDR, MiDR-FoxP3, or MiDR-TCR-FoxP3 and were co-cultured on the OP9-DL1/DL4/I-Ab cells. On day 7, the gene-transduced cells (3 &#215; 106/mouse) were adoptively transferred into female C57BL/6 mice that were induced with AIA two weeks after the cell transfer. On the day following arthritis induction, the arthritis severity was monitored by measurement of the knee diameter. (A-C) Percent increase in knee diameter. Increase in knee diameter was calculated based on preinjection knee diameter for each mouse before injection on day 0. Arthritis score was evaluated by examining both knees in a blinded manner; each knee was assigned a score (0: no visible swelling or discoloration; 1: visible swelling with or without discoloration; 2: moderate swelling with discoloration; 3: severe swelling with discoloration). In each group, five mice were used, and data are representative of three independent experiments. Data are represented as the mean &#177; SD. (D) The mean scoring on day 7 for both knees from five mice. Data are represented as the mean &#177; SD from three independent experiments (** p&#60; 0.01, *** p&#60; 0.001, two-way ANOVA). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54720/54720fig5.jpg" /> 
Figure 5: Ag-specific iPSC-Tregs Migrate to the OVA Injected Knee and Reduce the Bone Loss. iPSC-Tregs were adoptively transferred into C57BL/6 mice. Within 21 days post-arthritis induction, mice were anaesthetized and the bone architecture around the mouse knees was imaged by the micro-CT system. Mice receiving OT-II TCR/FoxP3 gene-transduced murine iPSC-Tregs exhibit significant reduction of bone loss as compared to control mice receiving vector-transduced iPSCs. (A) The scans were performed with a 2.2 mm length, and images were captured after further image processing (volume rendering and transformation; scale bars: 1 mm). (B) Bone volume around knee joint was evaluated using three-dimensional reconstruction of micro-CT images, and bone volume inside the volume of interest was calculated (scale bars: 1 mm). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

development-stem-cell-derived-antigen-specific-regulatory-t-cells

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.6K Views.  The Pennsylvania State University College of Medicine. 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. 

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

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;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 immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

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 measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood 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

Current treatment of T cell mediated autoimmune diseases relies mostly on strategies of global immunosuppression, which, in the long term, is accompanied by adverse side effects such as a reduced ability to control infections or malignancies. Therefore, new approaches need to be developed that target only the disease mediating cells and leave the remaining immune system intact. Over the past decade a variety of cell based immunotherapy strategies to modulate T cell mediated immune responses have been developed. Most of these approaches rely on tolerance-inducing antigen presenting cells (APC). However, in addition to being technically difficult and cumbersome, such cell-based approaches are highly sensitive to cytotoxic T cell responses, which limits their therapeutic capacity. Here we present a protocol for the generation of non-cellular killer artificial antigen presenting cells (KaAPC), which allows for the depletion of pathologic T cells while leaving the remaining immune system untouched and functional. KaAPC is an alternative solution to cellular immunotherapy which has potential for treating autoimmune diseases and allograft rejections by regulating undesirable T cell responses in an antigen specific fashion.

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

Guidelines are presented for the generation of killer artificial antigen presenting cells, KaAPC, an efficient tool for in vitro depletion of human antigen-specific T cells and an alternative solution to cellular immunotherapy for the treatment of T cell-mediated autoimmune diseases in an antigen-specific fashion without compromising the remaining immune system.

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

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, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

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

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

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

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.

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.

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

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

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

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

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 spontaneous non-obese diabetes (NOD) mouse model, insulin is the primary target, and genetic manipulation of these animals to remove a single key insulin epitope prevents disease. Thus, selective elimination of professional antigen presenting cells (APCs) bearing this pathogenic epitope is an approach to inhibit the unwanted insulin-specific autoimmune responses, and likely has greater translational potential.
Chimeric antigen receptors (CARs) can redirect T cells to selectively target disease-causing antigens. This technique is fundamental to recent attempts to use cellular engineering for adoptive cell therapy to treat multiple cancers. In this protocol, we describe an optimized T-cell retrovirus (RV) transduction and in vitro expansion protocol that generates high numbers of functional antigen-specific CD8 CAR-T cells starting from a low number of naive cells. Previously multiple CAR-T cell protocols have been described, but typically with relatively low transduction efficiency and cell viability following transduction. In contrast, our protocol provides up to 90% transduction efficiency, and the cells generated can survive more than two weeks in vivo and significantly delay disease onset following a single infusion. We provide a detailed description of the cell maintenance and transduction protocol, so that the critical steps can be easily followed. The whole procedure from primary cell isolation to CAR expression can be performed within 14 days. The general method may be applied to any mouse disease model in which the target is known. Similarly, the specific application (targeting a pathogenic peptide/MHC class II complex) is applicable to any other autoimmune disease model for which a key complex has been identified.

This article describes a protocol for the generation of antigen-specific CD8 T cells, and their expansion in vitro, with the aim of yielding high numbers of functional T cells for use in vitro and in vivo.

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

Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of liver cancer. This is a major concern, especially in developing countries. Failure of the immune system to mount an effective response against HBV leads to chronic infection. Although HBV vaccine is present and novel antiviral medicines are being created, eradication of virus-reservoir cells remains a major health topic. Described here is a method for the generation of viral antigen (Ag) -specific CD8+ cytotoxic T lymphocytes (CTLs) derived from induced pluripotent stem cells (iPSCs) (i.e., iPSC-CTLs), which have the ability to suppress HBV replication. HBV replication is efficiently induced in mice through hydrodynamic injection of an HBV expression plasmid, pAAV/HBV1.2, into the liver. Then, HBV surface Ag-specific mouse iPSC-CTLs are adoptively transferred, which greatly suppresses HBV replication in the liver and blood as well as prevents HBV surface Ag expression in hepatocytes. This method demonstrates HBV replication in mice after hydrodynamic injection and that stem cell-derived viral Ag-specific CTLs can suppress HBV replication. This protocol provides a useful method for HBV immunotherapy.

Presented here is a protocol for the effective suppression of hepatitis B virus (HBV) replication in mice by utilizing adoptive cell transfer (ACT) of stem cell-derived viral antigen (Ag)-specific T lymphocytes. This procedure may be adapted for potential ACT-based immunotherapy of HBV infection.

Hepatitis B virus (HBV) infection is a global health issue. With over 350 million people affected worldwide, HBV infection remains the leading cause of ...

In Vivo Augmentation of Gut-Homing Regulatory T Cell Induction

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 million IBD patients. It is generally accepted that a dysregulated immune response to gut bacteria initiates the disease and disrupts the mucosal epithelial barrier. We recently show that gut-homing regulatory T (Treg) cells are a promising therapy for IBD. Accordingly, this article presents a protocol for in vivo augmentation of gut-homing Treg cell induction. In this protocol, dendritic cells are engineered to produce locally high concentrations of two molecules de novo, active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and active vitamin A (retinoic acid or RA). We chose 1,25(OH)2D and RA based on previous findings showing that 1,25(OH)2D can induce the expression of regulatory molecules (e.g., forkhead box P3 and interleukin-10) and that RA can stimulate the expression of gut-homing receptors in T cells. To generate such engineered dendritic cells, we use a lentiviral vector to transduce dendritic cells to overexpress two genes. One gene is the cytochrome P450 family 27 subfamily B member 1 that encodes 25-hydroxyvitamin D 1&#945;-hydroxylase, which physiologically catalyzes the synthesis of 1,25(OH)2D. The other gene is the aldehyde dehydrogenase 1 family member A2 that encodes retinaldehyde dehydrogenase 2, which physiologically catalyzes the synthesis of RA. This protocol can be used for future investigation of gut-homing Treg cells in vivo.

Here we present a protocol for in vivo augmentation of gut-homing regulatory T cell induction. In this protocol, dendritic cells are engineered to locally produce high concentrations of the active vitamin D (1,25-dihydroxyvitamin D or 1,25[OH]2D) and the active vitamin A (retinoic acid or RA) de novo.

Inflammatory bowel disease (IBD) is an inflammatory chronic disease in the gastrointestinal tract (GUT). In the United States, there are approximately 1.4 ...

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central nervous system (CNS) by penetrating highly specialized brain endothelial cells (BECs). As Nm is a human-specific pathogen, the lack of robust in vivo model systems makes study of the host-pathogen interactions between Nm and BECs challenging and establishes a need for a human based model that mimics native BECs. BECs possess tighter barrier properties when compared to peripheral endothelial cells characterized by complex tight junctions and elevated trans-endothelial electrical resistance (TEER). However, many in vitro models, such as primary BECs and immortalized BECs, either lack or rapidly lose their barrier properties after removal from the native neural microenvironment. Recent advances in human stem-cell technologies have developed methods for deriving brain-like endothelial cells from induced pluripotent stem-cells (iPSCs) that better phenocopy BECs when compared to other in vitro human models. The use of iPSC-derived BECs (iPSC-BECs) to model Nm-BEC interaction has the benefit of using human cells that possess BEC barrier properties, and can be used to examine barrier destruction, innate immune activation, and bacterial interaction. Here we demonstrate how to derive iPSC-BECs from iPSCs in addition to bacterial preparation, infection, and sample collection for analysis.

Neisseria meningitidis Infection of Induced Pluripotent Stem-Cell Derived Brain Endothelial Cells

The protocol described here highlights the major steps in the differentiating induced pluripotent stem-cell derived brain-like endothelial cells, the preparation of Neisseria meningitidis for infection, and sample collection for other molecular analyses.

Meningococcal meningitis is a life-threatening infection that occurs when Neisseria meningitidis (meningococcus, Nm) can gain access to the central ...