Este projecto foi financiado, em parte, sob bolsas de Institutos Nacionais de Saúde (R01AI121180, R21AI109239 e K18CA151798), a American Diabetes Association (1-16-IBS-281), e do Departamento de Pensilvânia da Saúde (tabaco fundos de liquidação) para JS

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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. 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The authors have nothing to disclose.

Neste protocolo, um passo crítico é a diferenciação in vitro de iPSCs transduzidas com genes / FoxP3 TCR. In vitro sinalização Notch induz o desenvolvimento para a linhagem de células T. Para diferenciar iPSCs em células CD4 + Foxp3 + Tregs, foram utilizadas as células B OP9-DL1 / DL4 / IA, que moléculas altamente expressa MHC II (IA b). A maioria dos iPSCs diferenciar-se em células T CD4 +. No entanto, após a expressão de TCR da superfície, muitas células pré-T diferenciadas perdem a capacidade de diferenciar e eventualmente morrem. Como resultado, o número de células das Tregs funcionais iPSC derivados reduz dramaticamente após quatro semanas de diferenciação in vitro. Para o evitar, a adição de IL-2 pode melhorar a sobrevivência de células nos pontos de tempo. Um método alternativo de conduzir a maturação e sobrevivência de Ag-específicas pré-Tregs é para transferir os pré-Tregs que diferenciadas in vitro durante uma semana em ratinhos. Estes pré-Tregs podem continue diferenciação e maturação in vivo por mais três semanas. Usando este método, uma população específica Treg Ag-funcional pode ser gerada a partir de iPSCs, que são nTregs-like e têm a capacidade de suprimir a artrite auto-imune no modelo murino. Para melhorar a eficácia de no desenvolvimento in vivo de Ag-specific iPSC-Tregs, compostos diferentes (por exemplo, ácido retinóico, 13,14 gelectin) ou supressores de citocinas (por exemplo, TGF-β, IL-10) pode ser usado após a transferência adoptiva do pré-Tregs. Além de aumentar a sobrevivência de células, esta abordagem combinada pode manter a expressão FoxP3 14,15 e melhorar a qualidade do específico do Ag iPSC-Tregs. Um problema potencial que pode surgir para o desenvolvimento in vivo Treg é geral imunossupressão, resultando em complicações durante infecções ou subsequente perda de peso. Um grande número de Ag-específica Tregs em desenvolvimento VIvo pode piorar infecções. Neste caso, um gene suicida, a caspase 9 indutível (ICasp9) 15,16, podem ser incorporadas no vector de TCR / FoxP3. Esta abordagem permite a remoção das Tregs derivadas de células estaminais através da injecção de uma pequena molécula bio-inerte dimerização agente (AP1903) para &quot;desligar&quot; a geração de Tregs derivadas de células estaminais, a qual irá ultrapassar este problema potencial. Um auto-Ag é uma proteína típica reconhecido pelo sistema imune de hospedeiros que sofrem de uma desordem auto-imune indivíduo. Esta auto-Ag é o objectivo do sistema imunitário, e as células T não são eliminadas correlacionados. Para gerar a auto-Ag Tregs específicos, a utilização de transdução de TCR é uma boa escolha. Um auto-Ag (por exemplo, proteína de choque térmico) de TCR específica pode ser transduzido para madura CD4 + CD25 + a partir de células mononucleares do sangue periférico (PBMCs), e esta abordagem tem sido utilizada em ensaios clínicos. Em alternativa, como descrita neste protocolo, utilizando a transdução de genes de auto-Ag TCR específico com FoxP3 e estimulação com a sinalização Notch,-tronco derivadas de células Tregs pode ser auto-Ag Tregs específico. Terapias à base de células para doenças auto-imunes que utilizam Tregs manipuladas podem ser úteis 17. Embora TCR transdução em células T provou ser seguro, viável e aplicável em ensaios clínicos, ainda há grandes preocupações de segurança, devido à auto-imunidade causada por reação cruzada com os tecidos saudáveis 18. Além disso, usando os métodos atuais, geneticamente modificado Tregs geralmente têm uma persistência de curto prazo in vivo 19. Em alternativa, uma fonte promissora para o desenvolvimento de um grande número de anticorpos monoclonais específicos de Ag-Tregs é células estaminais. As células estaminais embrionárias (CES) tem o melhor pluripotência e auto-renovação, mas não é possível obter-los a partir de pacientes. Embora facilmente isolados a partir do sangue periférico, células estaminais hematopoiéticas (HSCs) são multi-As células estaminais e potentes podem ser expandidas em cultura de células de forma semelhante à CES 20. tecnologia iPSC presente avançou a uma geração mais eficiente de CPS a partir de células somáticas de doentes por transdução de diferentes factores de transcrição. Um certo número de aperfeiçoamentos metodológicos têm sido desenvolvidos nos últimos anos para gerar iPSCs reduzindo ao máximo os riscos potenciais, tais como a imunogenicidade e a tumorigenicidade. Em comparação com os CES, iPSCs possuem pluripotência idênticos e auto-renovação. Como resultado, a tecnologia iPSC pode proporcionar uma vantagem no desenvolvimento de pacientes-unidades e / ou de doenças específicas. O uso de iPSCs para desenvolver Ag-específica Tregs podem avançar no campo de terapias à base de células para doenças auto-imunes 10,11.

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

As doenças autoimunes surgem devido à perda da auto-tolerância imunológica. As células T reguladoras (Tregs) são importantes mediadores da auto-tolerância imunológica. Tregs representam cerca de 5 - 10% da subpopulação maduro de células T CD4 + em ratinhos e humanos, com cerca de 1 - 2% dos Tregs que circulam no sangue periférico. Induzidas células estaminais pluripotentes (IPSCs) podem ser diferenciados em Tregs funcional, que tem um potencial para ser utilizados para terapias baseadas em células de doenças auto-imunes. Aqui, apresentamos um método para desenvolver antigénio (Ag) Tregs especï¿½icos de iPSCs (ou seja, IPSC-Tregs). O método baseia-se na incorporação do factor de transcrição Foxp3 e um receptor de células T específico de Ag-(TCR) em iPSCs e, em seguida, a diferenciação em células do estroma expressando Notch OP9 ligandos semelhante a delta (DL) e uma DL4. Seguindo diferenciação in vitro, o CPSP-Tregs expressam CD4, CD8, CD3, CD25, FoxP3, e Ag-TCR específico e são capazes de responder à estimulação Ag.Este método tem sido aplicado com sucesso para a terapia à base de células de artrite auto-imune num modelo murino. A transferência adoptiva destes específica iPSC-Ag-Tregs em artrite induzida por Ag (AIA) -bearing ratinhos tem a capacidade para reduzir a inflamação das articulações e inchaço e para prevenir a perda óssea.

Como mostrado aqui, no dia 28, específico do Ag Tregs expressa substancialmente CD3 e TCR específico do Ag, dois marcadores de células T. A população de células CD3 + + TCRVβ5 expressa CD4. A maioria das células CD3 +, CD4 + TCRVβ5 + também expressa CD25, CD127, e CTLA-4, que são normalmente expressas em níveis elevados em que ocorre naturalmente (regs T nTregs) e em células T que expressam ectopicamente FoxP3. FoxP3 expressão em células-derivadas iPSC persistiu mesmo depois de longo prazo na estimulação in vitro com o ligando do entalhe, tal como detectado por coloração intracelular analisadas por citometria de fluxo (Figura 1). Além disso, a Ag-specific iPSC-Tregs produzido citoquinas supressoras, tais como TGF-β e IL-10, quando estimulada in vitro com esplenócitos Ag-pulsados (Figura 2), indicando a iPSC-Tregs tinham potenciais actividades supressivas. A microscopia de fluorescência revelou<html> que mais células FoxP3 + estavam presentes na que os joelhos tratados com PBS-OVA tratadas; não havia células FoxP3 + existentes nos joelhos em camundongos que receberam as iPSCs transduzidas por vetores DsRed +. Muitos mais células CD4 + FoxP3 + + TCRVβ5 células apresentados nos joelhos de ratos que receberam iPSCs transduzidas com o TCR-Midr-Foxp3 que o Midr-FoxP3 (Figura 3). Estas observações sugerem que específicas IPSC-Ag-Tregs migrar para o joelho AIA após a transferência adoptiva para ratinhos receptores. As células-derivadas iPSC transferidos diminuiu substancialmente o joelho inchaço inflamatório quando OVA estava presente, mas não teve nenhum efeito sobre o controlo do joelho que apenas foi injectada com mBSA no modelo de murino (Figura 4); reduziu a perda óssea no joelho transferência de células visualizados pelo sistema de micro-TC de alta resolução (Figura 5). tp_upload / 54720 / 54720fig1.jpg &quot;/&gt; Figura 1:. Diferenciação de Ag-specific iPSC-Tregs Murino iPSCs foram transduzidas com um constructo: Midr-TCRα-2A-2A-TCRβ-FoxP3 contendo genes de OVA-específica de TCR e FoxP3. As células transduzidas com genes (DsRed +) foram co-cultivados em células B OP9-DL1-DL4-IA na presença de mFlt3L e mIL-7. (A) Morfologia de T regs diferenciação de células no dia 0, 7, 14 e 22. análise por citometria de (B) de fluxo para a expressão da proteína das células-derivadas iPSC no dia 28. CD3 + + TCRVβ5 células foram bloqueadas conforme indicado (R1) e analisados para a expressão de CD4 e CD8, com CD25, CD127, CTLA- 4 e FoxP3 mostrado para células fechadas como CD4 + CD8 - células (R2). (linhas escuras; áreas sombreadas indicam controlos isotípicos) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig1large.jpg" target="_blank">por favor clique aqui para ver uma larg</a>versão er desta figura. <img alt="Figura 2" src="/files/ftp_upload/54720/54720fig2.jpg" /> Figura 2: As análises funcionais de I n Tregs Vitro Diferenciada Ag específicos de murino IPSC-Tregs foram estimuladas por esplenócitos.;: E pulsadas com OVA 323-339 peptídeo (APCs regs T / APC = 1 4). Produção de citocinas intracelular (TGF-β e IL-10) foi analisada por citometria de fluxo após gating em directo de CD4 + CD25 +. (Linhas escuras; áreas sombreadas indicam controlos isotípicos) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/54720/54720fig3.jpg" /> Figura 3: UmiPSC-Tregs infiltrar-se as articulações do joelho específico do g. Ag-específicos IPSC-Tregs foram transferidos adoptivamente para ratinhos C57BL / 6. Pouco tempo após a indução da artrite (dias 7 - 14), os joelhos foram removidas e coradas para a imuno-histologia (barras de escala: 20 mm). Os ratinhos que receberam OVA específica do iPSC-Tregs têm um grande número de células TCRVβ5 + nos joelhos. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 4" src="/files/ftp_upload/54720/54720fig4.jpg" /> Figura 4: Transferência adoptiva de específicas de Ag iPSC-Tregs melhora AIA em Ratos iPSCs de ratinho foram transduzidas com a construção retroviral Midr, Midr-FoxP3, ou Midr-TCR-Foxp3 e foram co-cultivados sobre o OP9-DL1 / DL4 /. As células B IA. No dia 7, as células transduzidas com o gene (3 x 10 6 / rato) foram umdoptively transferidos para fêmeas C57BL / 6 ratos que foram induzidos com AIA duas semanas após a transferência de células. No dia seguinte a indução da artrite, a gravidade da artrite foi monitorizada por medição do diâmetro do joelho. (AC) por cento de aumento no diâmetro do joelho. Aumento do diâmetro do joelho foi calculada com base no diâmetro de pré-injecção do joelho para cada rato antes da injecção no dia 0. A pontuação de artrite foi avaliada por análise de ambos os joelhos de uma maneira cega; cada joelho foi atribuída uma pontuação (0: nenhum inchaço visível ou descoloração; 1: visível inchaço com ou sem manchas; 2: moderada inchaço com descoloração; 3: inchaço grave com descoloração). Em cada grupo foram utilizados cinco ratos, e os dados são representativos de três experiências independentes. Os dados estão representados como a média ± DP. (D) A média marcando no dia 7 para ambos os joelhos a partir de cinco murganhos. Os dados estão representados como a média ± DP de três experiências independentes (** p &lt;0,01, *** p &lt;0,001, two-way ANOVA). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 5" src="/files/ftp_upload/54720/54720fig5.jpg" /> Figura 5: Ag. Específica iPSC-Tregs migrar para o OVA injetado no joelho e reduzir a perda óssea IPSC-Tregs foram adoptively transferidos para camundongos C57BL / 6. Dentro de 21 dias de indução de pós-artrite, os ratos foram anestesiados e a arquitetura óssea ao redor dos joelhos rato foi fotografada pelo sistema de micro-CT. Os ratinhos que receberam OT-II TCR / FoxP3 gene transduzido murino IPSC-Tregs exibem redução significativa da perda óssea, em comparação com ratinhos de controlo que receberam iPSCs transduzidas por vectores. (A) Os exames foram realizados com um comprimento de 2,2 mm, e as imagens foram capturadas depois posterior processamento de imagem (renderização de volume e de transformação;barras de escala:. 1 mm) (B) de volume ósseo ao redor da articulação do joelho foi avaliada usando reconstrução tridimensional de imagens micro-CT, e foi calculado o volume ósseo no interior do volume de juros (barras de escala:. 1 mm) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig5large.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</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.

Desenvolvimento de Células-Tronco de derivados de células T regulatórias antígeno específico da Contra Auto-imunidade

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 visualizações.  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. 

Vídeo: 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.

Geração de Células T reguladoras induzidas a partir de células T humanas primárias Naïve e Memória

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

Imunologia e Infecção

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.

Novas ferramentas para expandir as células T reguladoras de indivíduos HIV-1-infected

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.

Assassino Artificial células apresentadoras de antígenos (KaAPC) para a eficiente<em&gt; In Vitro</em&gt; Depleção de células T humanas antígeno específico da

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.

Gerando<em&gt; De Novo</em&gt; Antígeno-específica T Humano receptores celulares por Transdução retroviral de 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

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.

Na diferenciação in vitro de células T CD4 + humano FOXP3 + Induzida T reguladoras (iTregs) a partir de células naive T CD4 + usando um protocolo de TGF-β contendo

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.

Quantificação de células T antígeno-específicas humanas proliferating de CD4+ usando o éster do Succinimidyl do carboxyfluorescein

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.

Geração de alta eficiência de células T Cytotoxic do rato preliminar antígeno-específico para o teste funcional em um modelo auto-imune do diabetes

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.

Células-tronco derivadas virais AG-specific T da haste suprimir a replicação de HBV em ratos

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.

In Vivo Aumento da Indução de CélulaS T Reguladoras de Gut-Homing

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.

Neisseria meningitidis Infecção de Células-Tronco Pluripotentes Induzidas Derivadas do Endotélio Cerebral

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