自己免疫に対する幹細胞由来の抗原特異的制御性T細胞の開発
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
自己免疫疾患は、免疫学的自己寛容の損失に起因し発生します。制御性T細胞（Treg）免疫学的自己寛容の重要なメディエーターです。末梢血中を循環するものTregの2％ -約1で、マウスおよびヒトにおける成熟したCD4 + T細胞亜集団の10％ – Tregは約5を表します。人工多能性幹細胞（iPS細胞）は、自己免疫疾患の細胞ベースの治療のために使用される可能性を持っている機能のTregへ分化させることができます。ここでは、iPS細胞からの抗原（Ag）から固有のTreg（ すなわち 、IPSC-のTreg）を開発するための方法を提示します。この方法は、iPS細胞への転写因子のFoxP3及びAg特異的T細胞受容体（TCR）を組み込み、次いでノッチを発現OP9間質細胞上で分化に基づいてデルタ様（DL）1とDL4のリガンド。 in vitroでの分化に続いて、IPSC-Tregは、CD4、CD8、CD3、CD25、FoxP3の、およびAg特異的TCRを発現し、Agの刺激に応答することができます。この方法は、正常マウスモデルにおいて自己免疫性関節炎の細胞ベースの治療に適用されています。マウス-bearingのAg誘導関節炎（AIA）にこれらのAg特異的IPSC – Tregの養子移入は、関節の炎症および腫脹を軽減し、骨損失を防止する能力を有します。
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
Representative Results
Discussion
このプロトコルでは、重要なステップは、TCR / FoxP3の遺伝子形質iPS細胞のインビトロ分化である。 インビトロで Notchシグナル伝達は、T細胞系統に向けて開発を誘導します。 CD4 + FoxP3の+ TregのにiPS細胞を区別するために、我々は非常にMHC II（IA b）の分子を発現するOP9-DL1 / DL4 / IA B細胞を、使用していました。 iPS細胞の大部分は、CD4 +細胞?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
このプロジェクトは、健康（R01AI121180、R21AI109239とK18CA151798）、米国糖尿病協会の国立研究所からの助成金の下で、一部には、資金を供給された（1月16日 – IBS-281）、および健康のペンシルベニア州省（たばこ和解基金）へ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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