Summary

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

Published: November 08, 2016
doi:

Summary

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.

Abstract

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.

Introduction

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-β 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.

Protocol

모든 동물 실험은 의학 동물 관리위원회의 펜실베니아 주립 대학 (IACUC 프로토콜 # 45470)에 의해 승인 및 실험 동물 관리의 평가 및 인증을위한 협회의 가이드 라인을 준수하여 실시하고 있습니다. 1. 줄기 세포 배양 코트하기 위해서는 37 ° C (인큐베이터)에서 최소 30 분 동안 0.1 % 젤라틴 10 ml의 플레이트와 10cm 접시를 품어. 접시와 접시 3 × 106에서 젤라?…

Representative Results

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 <st…

Discussion

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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

Materials

C57BL/6j mice Jackson Laboratory 664
B6.129S7 Rag1tm1Mom/J Jackson Laboratory 2216
Anti-CD3 (2C11) antibody BD Pharmingen 553058
Anti-CD28 (37.51) antibody BD Pharmingen 553295
Anti-CD4 (GK1.5) antibody Biolegend 100417
Anti-CD8 (53–6.7) antibody Biolegend 100714
Anti-CD25 (3C7) antibody Biolegend 101912
Anti-TCR-β (H57597) antibody Biolegend 109220
Anti-IL10 Biolegend 505010
Anti-TGFβ Biolegend 141402
DMEM Invitrogen ABCD1234
α-MEM Invitrogen A10490-01
FBS Hyclone SH3007.01
Brefeldin A Sigma B7651
Polybrene Sigma 107689
Genejammer Integrated science 204130
ACK Lysis buffer Lonza 10-548E
mFlt-3L peprotech 250-31L
mIL-7 peprotech 217-17
Gelatin Sigma G9391
Paraformaldehyde Sigma P6148-500G Caution: Allergenic, Carcenogenic, Toxic
Permeabilization buffer Biolegend 421002
mBSA Sigma A7906
Ova albumin Avantor 0440-01
CFA Difco 2017014
Tailveiner restrainer Braintree scientific RTV 150-STD

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Cite This Article
Haque, M., Fino, K., Sandhu, P., Song, J. Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity. J. Vis. Exp. (117), e54720, doi:10.3791/54720 (2016).

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