이 프로젝트는 건강 (R01AI121180, R21AI109239 및 K18CA151798), 미국 당뇨병 협회 (American Diabetes Association)의 국립 연구소에서 보조금에서 부분적으로 투자되었다 (16-IBS-281), 건강의 펜실베니아 부 (담배 결제 자금)에 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.

이 프로토콜에서 중요한 단계는 TCR / Foxp3의 유전자 형질 iPSCs의 체외 분화된다. 시험관 노치 신호는 T 세포 계통으로의 개발을 유도한다. CD4 + Foxp3의 + Tregs에 iPSCs를 구별하기 위해, 우리는 OP9-DL1 / DL4 / IA의 B 세포, 매우 빠른 MHC II (IA 나) 분자를 사용했다. iPSCs 대부분은 CD4 + 세포로 분화. 그러나, 표면 TCR 발현 후 많은 분화 이전 T 세포 분화 결국 죽게 할 수있는 능력을 잃는다. 그 결과, IPSC 유래 기능성 Tregs의 셀 수가 대폭 체외 분화 4 주 후에 감소시킨다. 이를 방지하기 위해, IL-2의 첨가는 그 시점에서 세포 생존을 향상시킬 수있다. 다른 방법은 마우스에 주 동안 시험 관내에서 분화 한 프리 Tregs을 전송 AG-특정 사전 Tregs의 성숙 및 생존을 구동하도록되어있다. 이 사전 Tregs 공동 수ntinue 차별화 된 또 다른 3 주 동안 생체 내에서 성숙. 이 방법을 사용하는 기능 AG-특정 TREG 인구 nTregs 형상이며 뮤린 모델에서자가 면역 관절염을 억제하는 기능이 iPSCs로부터 생성 될 수있다. AG-특정 IPSC-Tregs의 생체 현상의 효과를 향상시키기 위해, 다른 화합물 (예를 들어, 레티노 산, gelectin) 13, 14 또는 억제 사이토 카인 (예를 들면, TGF-β, IL-10)은 대체 전송 후에 사용될 수있다 사전 Tregs의. 세포 생존을 증가시키는 것 이외에,이 결합 방법은 Foxp3를 발현 14,15 유지 및 AG-특정 IPSC-Tregs의 품질을 향상시킬 수있다. 생체 TREG 개발에 발생할 수있는 잠재적 문제점은 감염 또는 후속 감량시 합병증 발생 전반적인 면역이다. VI에서 개발 AG-특정 Tregs의 많은 수의VO는 감염을 악화시킬 수 있습니다. 이 경우, 자살 유전자의 유도 카스파 제 9 (iCasp9) 15,16은 TCR / Foxp3의 벡터 내로 혼입 될 수있다. 이 방법은 이러한 잠재적 인 문제를 극복 줄기 세포 유래 Tregs의 생성을 &quot;차단&quot;할 에이전트 (AP1903)을 이량 생체 불활성 작은 분자의 주사에 의한 줄기 세포 유래 Tregs의 제거를 허용한다. 자체 AG의 개별자가 ​​면역 질환을 앓고있는 호스트의 면역 시스템에 의해 인식 전형적인 단백질이다. 이 자동 AG는 면역계의 대상이며, T 세포 상호 삭제하지 않는다. 자기 AG에 특정 Tregs를 생성하기 위해, TCR 전달의 사용은 좋은 선택이다. 셀프 AG (예, 열 충격 단백질) 특정 TCR 말초 혈액 단핵 세포 (PBMC)로부터 성숙한 CD4 + CD25 + Tregs로 형질 도입 될 수 있고, 이러한 접근은 임상에서 이용되고있다. 또한,로 데스, 세포 유래 Tregs 줄기 노치 신호와 유전자 자체의 Ag의 전달 Foxp3를 가진 특정 TCR 자극을 사용하여이 프로토콜 cribed는, 자기의 Ag 특정 Tregs 수 있습니다. 설계 Tregs를 이용하여자가 면역 질환에 대한 세포 기반 치료는 17 유용 할 수 있습니다. T 세포의 TCR 전달이 가능한 금고 및 임상에 적용 가능한 것으로 판명되었지만, 건강한 조직에 의한 18 교차 반응에 의해 야기되는자가 면역에 중요한 안전성 문제는 여전히 존재한다. 또한, 현재의 방법을 이용하여 유전자 조작 Tregs는 일반적으로 생체 내에서 19 단기 지속성을 갖는다. 대안 적으로, 모노클 AG-특정 Tregs 다수의 개발을위한 유망한 공급원은 줄기 세포이다. 배아 줄기 세포 (는 ESC)가 가장 능성 자기 갱신해야하지만, 환자들을 입수 가능하지 않다. 쉽게 말초 혈액으로부터 분리되지만, 조혈 줄기 세포 (조혈 모세포)가있는 멀티강력한 줄기 세포와는 ESC (20)와 마찬가지로 세포 배양 물에서 확장 될 수있다. 본 IPSC 기술은 다양한 전사 인자의 형질 도입에 의해 환자의 체세포에서 PSC의 더 효율적인 생성에 진출하고있다. 방법론 개선 다수 최대한 이러한 면역 원성 및 발암 등의 잠재적 인 위험을 감소시킴으로써 iPSCs를 생성하는 최근에 개발되었다. 는 ESC에 비해 iPSCs는 동일 능성과 자기 갱신을해야합니다. 그 결과, IPSC 기술은 환자 - 및 / 또는 질병 특정한 PSC를 개발하는 이점을 제공 할 수있다. iPSCs의 사용은 AG-특정 Tregs는자가 면역 질환 10, 11에 대한 세포 기반 치료법의 필드를 진행할 수 개발.

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

자가 면역 질환으로 인해 면역 자기 관용의 손실이 발생한다. 규제 T 세포 (Tregs)는 면역 자기 관용의 중요한 매개체이다. 말초 혈액 순환 그 Tregs 2 % - 1과, 마우스 및 인간 성숙 CD4 + T 세포 개체군의 10 % - 약 5 Tregs를 나타낸다. 유도 된 다 능성 줄기 세포 (iPSCs)은 전위가자가 면역 질환의 세포 기반 치료에 사용할 수있는 기능 Tregs로 분화 될 수있다. 여기서는 iPSCs에서 항원은 (Ag) 특이 Tregs (즉, IPSC-Tregs)를 개발하기위한 방법을 제시한다. 상기 방법은 iPSCs으로 전사 인자 Foxp3의 및 AG-특정한 T 세포 수용체 (TCR)를 포함하고 노치 발현 OP9 간질 세포 분화에 기초하여 델타 형 (DL) 1 DL4 리간드. 체외 분화 후, IPSC-Tregs는 CD4, CD8, CD3, CD25, Foxp3를하고, AG-특정 TCR을 표현하고의 Ag 자극에 응답 할 수 있습니다.이 방법은 성공적 뮤린 모델에서자가 면역 관절염 세포 기반 치료에 적용되고있다. AG-유도 관절염 (AIA) -bearing 마우스로 이러한 AG-특정 IPSC - Tregs의 입양 전송은 관절 염증을 감소하고 붓기와 뼈 손실을 방지 할 수있는 능력을 가지고 있습니다.

28 일에, 다음과 같이),은 (Ag 특정 Tregs는 실질적으로 CD3과 AG-특정 TCR,이 T 세포 마커를 표명했다. CD3 + TCRVβ5 + 인구는 CD4를 표명했다. 일반적으로 자연적으로 T의 REGS (nTregs)를 발생 및 ectopically Foxp3를 표현 T 세포에서 높은 수준으로 발현된다 또한 CD25, CD127, 및 CTLA-4를 표현 CD3 + TCRVβ5 + CD4 + 세포의 대부분. 유동 세포 계측법 (도 1)에 의해 분석하여 세포 내 염색으로 검출되는 IPSC 유래 세포 Foxp3를 발현 노치 리간드 관내 자극 후에도 장기간 지속되었다. IPSC-Tregs 잠재적 억제 활동했다 나타내는 AG-펄스 비장 세포 (도 2)와 함께 생체 외에서 자극 된 경우뿐만 아니라, AG-특정 IPSC-Tregs는, 예컨대 TGF-β와 IL-10 억제 사이토 카인을 생산했다. 형광 현미경 밝혀<html> 더 Foxp3를 + 세포에 존재하는 OVA-처리 된 PBS 처리 된 무릎보다; 을 DsRed + 벡터 - 형질 iPSCs를받은 쥐의 무릎에 존재하는 어떤 Foxp3를 + 세포는 없었다. 더 많은 CD4 +를 MiDR-Foxp3를보다 MiDR-TCR-Foxp3를 형질 도입 iPSCs를받은 쥐의 무릎에 제시 Foxp3의 + TCRVβ5 + 세포 (그림 3). 이러한 관찰은 AG-특정 IPSC-Tregs받는 사람 마우스로 입양 전송 후 AIA 무릎으로 마이그레이션하는 것이 좋습니다. 전송 된 IPSC 파생 된 세포는 실질적으로 OVA가 존재하는 경우 부종 염증 무릎을 감소하지만 쥐 모델에서 MBSA와 주입 제어 무릎 (그림 4)에 영향을 미치지 아니합니다; 고해상도 마이크로 CT 시스템 (도 5)에 의해 가시화 셀 전송 무릎 뼈 손실 감소. tp_upload / 54720 / 54720fig1.jpg &quot;/&gt; 그림 1 :. AG-특정 IPSC - Tregs의 차별화 쥐 iPSCs는 구조로 형질 도입했다 : MiDR-TCRα-2A-TCRβ-2A-Foxp3의는 OVA 특정 TCR 및 Foxp3의 유전자를 포함. 유전자 형질 도입 세포 (을 DsRed +)는 mFlt3L 및 MIL-7의 존재 OP9-DL1-DL4-IA의 B 세포에 공동 - 배양 하였다. (A)는 T의 형태학은 0 일, 7, 14에 대한 세포 분화를 REGS 하루 28 CD3에 IPSC 파생 된 세포의 단백질 발현을위한 22 (B) 유세포 분석 + TCRVβ5 + 표시 (R1)로 게이트와 CD4와 CD8의 발현을 분석, CD25, CD127, CTLA-와 한 세포 CD4 + CD8으로 게이트 셀 표시 4, Foxp3의 - 세포 (R2). (진한 라인, 음영 영역은 이소 타입 컨트롤을 나타냅니다) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig1large.jpg" target="_blank">larg의를 보려면 여기를 클릭하십시오</a>이 그림의 어 버전입니다. <img alt="그림 2" src="/files/ftp_upload/54720/54720fig2.jpg" /> 그림 2 : I n은 체외 차별화 된 AG-특정 Tregs의 기능 분석 쥐 IPSC-Tregs는 비장 세포에 의해 자극했다. (장갑차, T의 REGS / 장갑차 = 1 : 4), OVA 323-339 펩타이드와 펄스. 세포 내 사이토 카인 생산 (TGF-β와 IL-10) 세포 계측법 라이브 CD4 + CD25 + 세포에 게이팅 후 흐름에 의해 분석 하였다. (진한 라인, 음영 영역은 이소 타입 컨트롤을 표시) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 3" src="/files/ftp_upload/54720/54720fig3.jpg" /> 그림 3 :g-고유의 무릎 관절에 IPSC-Tregs에 침투. AG-특정 IPSC-Tregs는 적응 적 C57BL / 6 마우스로 옮겼다. 얼마 관절염 유도 (일 7-14) 후, 무릎을 제거하고 면역 조직학을 위해 염색 (스케일 바 : 20 μm의). OVA 특정 IPSC - Tregs를 수신 마우스가 무릎에 TCRVβ5 + 세포의 큰 숫자가있다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 4" src="/files/ftp_upload/54720/54720fig4.jpg" /> 그림 4 : AG-특정 IPSC - Tregs의 입양 전송 쥐에 AIA를 개선한다 쥐 iPSCs는 레트로 바이러스 구조 MiDR, MiDR-Foxp3를, 또는 MiDR-TCR-Foxp3를하고 있었다 OP9-DL1에 공동 배양 / DL4 / 형질 도입 하였다. IA의 B 세포. 7 일에, 유전자 형질 세포 (3 × 10 6 / 마우스)이었던doptively 2 주 셀 전송 후에 AIA로 유도 된 암컷 C57BL / 6 마우스에 옮겼다. 하루 다음 관절염 유도에서 관절염의 심각도는. 무릎 직경의 측정에 의해 모니터링 된 무릎 직경 (AC) % 증가. 무릎 직경의 증가는 눈을 멀게 방식으로 두 무릎을 검토하여 평가 일 0 관절염 점수에 주입하기 전에 각 마우스 preinjection 무릎 직경을 기준으로 계산 하였다 각 무릎 점수 할당 (0 : 눈에 보이는 부종이나 변색을 1 : 또는 변색없이 붓기 볼 2 : 변색과 붓기 보통 3 : 심한 변색과 붓기). 각 그룹에서, 다섯 마우스를 사용하고, 데이터는 세개의 독립된 실험의 대표 값이다. 데이터는 평균 ± 표준 편차로 표시됩니다. (D)의 평균 다섯 생쥐에서 모두 무릎을 위해 하루 7 득점. 데이터는 3 개의 독립적 인 실험 (에서 SD 평균 ±로 표시됩니다 ** p &lt;0.01, *** p &lt;0.001, 양방향 ANOVA). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 5" src="/files/ftp_upload/54720/54720fig5.jpg" /> 그림 5 :. 난자 주조 무릎에 IPSC-Tregs 마이그레이션 AG-특정과 뼈 손실을 줄일 수는 IPSC-Tregs 적응 적 C57BL / 6 마우스로 옮겼다. 이십일일 후 관절염 유도 내에 마우스를 마취시키고, 마우스의 무릎 주위의 뼈 구조는 마이크로 CT 장치에 의해 촬영 하였다. 수신 마우스 OT-II TCR / Foxp3의 유전자 형질 도입 벡터 형질 iPSCs 수용 생쥐 제어 비해 IPSC-Tregs는 골 손실의 상당한 감소를 나타낸다 쥐. (A)을 스캔이 2.2 mm 길이로 수행하고, 이미지 후에 캡처 된 상기 화상 처리부 (볼륨 렌더링 및 변환;스케일 바 :. (스케일 바 1mm) (B) 무릎 관절 주위의 뼈 볼륨 마이크로 CT 이미지의 3 차원 재구성을 사용하여 평가하고, 관심의 볼륨 내부 뼈의 볼륨을 계산 하였다. 1mm) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54720/54720fig5large.jpg" target="_blank">여기를 클릭하십시오 이 그림의 더 큰 버전을 볼 수 있습니다.</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.

자가 면역 대해 줄기 세포 유래의 항원 특이 적 조절 T 세포의 발달

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 조회수.  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. 

비디오: 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.

기본 인적 나이브 및 메모리 T의 세포로부터 유도된 규제 T 세포의 생성

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

연구

면역학 및 감염병학

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.

새로운 도구는 HIV-1에 감염된 개인으로부터 규제 T 세포를 확장하려면

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.

효율적인위한 킬러 인공 항원 제시 세포 (KaAPC)<em&gt; 체외</em&gt; 인간의 항원 특이 T 세포의 고갈

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.

생성<em&gt; 드 노보</em&gt; 중심 Hemichain의 레트로 바이러스 형질 도입에 의해 항원 특이 적 인간 T 세포 수용체

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.

TGF-β 함유 프로토콜을 사용하여 나이브 CD4 + T 세포에서 인간의 CD4 + Foxp3의 + 유도 규제 T 세포 (iTregs)의 체외 분화

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.

카르복시플루아세인 수치니미딜 에스테르를 이용한 증식 인간 항원 특이적 CD4+ T 세포의 정량화

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.

자가 면역 당뇨병 모델에서 기능 테스트를 위한 항원 특이적 원발성 원발성 T 세포의 고효율 생성

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.

줄기 세포 유래 바이러스 Ag 특이적 T 림프구는 마우스에서 HBV 복제를 억제

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.

내장 호밍 규제 T 세포 유도의 생체 내 확대

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 유도만능줄기세포 유래 뇌내피세포의 감염

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