干细胞衍生的抗原特异性调节性T细胞对自身免疫的发展
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
自身免疫疾病的出现是由于免疫自身耐受丧失。调节性T细胞（Treg细胞）是免疫自身耐受的重要介质。调节性T细胞代表约5 -在小鼠和人类成熟的CD4 + T细胞亚群的10％，与约1 -这些Treg细胞在外周血中循环的2％。诱导多能干细胞（iPS细胞）可以分化成官能的Treg，其具有用于自身免疫性疾病的基于细胞的疗法的电位。这里，我们目前开发iPS细胞从抗原（银）特异性调节性T细胞（ 即 iPSC的调节性T细胞）的方法。该方法是基于结合转录因子FoxP3的和Ag特异性T细胞受体（TCR）插入的iPSC，然后分化上表达的Notch OP9基质细胞配体三角状（DL）1和DL4。 下列体外分化，所述iPSC的的Treg表达CD4，CD8，CD3，CD25，FoxP3的，和Ag-特异性TCR并能够对于Ag刺激作出响应。该方法已成功地应用于自身免疫性关节炎的基于细胞的疗法在小鼠模型。这些特定的Ag-iPSC的调节性T细胞的过继转移到银 – 诱导的关节炎（AIA）的-bearing小鼠具有降低关节发炎和肿胀，并防止骨损失的能力。
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
在这个协议中，一个关键的步骤是TCR / FoxP3基因转导的iPSC的体外分化。 在体外 Notch信号传导诱导发展朝向T细胞谱系。分化的iPSC成CD4 + FOXP3 + Treg细胞，我们使用了OP9-DL1 / DL4 / IA 的B细胞，其中高表达MHC II类分子（ⅠAB）的分子。大部分的iPSC的分化成CD4 +细胞。然而，表面TCR表达后，许多分化的前T细胞失去分化并最终死亡的能力。其结果是，在?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
该项目资助，一部分，根据卫生（R01AI121180，R21AI109239和K18CA151798），美国糖尿病协会全国学院助学金（1-16 IBS-281），和健康的宾夕法尼亚部门（烟草和解基金） 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 |
References
- Firestein, G. S. Evolving concepts of rheumatoid arthritis. Nature. 423, 356-361 (2003).
- Ferraro, A., et al. Interindividual variation in human T regulatory cells. Proc Natl Acad Sci U S A. 111, E1111-E1120 (2014).
- Tang, Q., et al. In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J Exp Med. 199, 1455-1465 (2004).
- van Herwijnen, M. J., et al. Regulatory T cells that recognize a ubiquitous stress-inducible self-antigen are long-lived suppressors of autoimmune arthritis. Proc Natl Acad Sci U S A. 109, 14134-14139 (2012).
- Wright, G. P., et al. Adoptive therapy with redirected primary regulatory T cells results in antigen-specific suppression of arthritis. Proc Natl Acad Sci U S A. 106, 19078-19083 (2009).
- Schmitt, T. M., et al. Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat Immunol. 5, 410-417 (2004).
- La Motte-Mohs, R. N., Herer, E., Zuniga-Pflucker, J. C. Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro. Blood. 105, 1431-1439 (2005).
- Lei, F., Haque, R., Weiler, L., Vrana, K. E., Song, J. T lineage differentiation from induced pluripotent stem cells. Cell Immunol. 260, 1-5 (2009).
- Lei, F., Haque, R., Xiong, X., Song, J. Directed differentiation of induced pluripotent stem cells towards T lymphocytes. J Vis Exp. , e3986 (2012).
- Lei, F., et al. In vivo programming of tumor antigen-specific T lymphocytes from pluripotent stem cells to promote cancer immunosurveillance. Cancer Res. 71, 4742-4747 (2011).
- Haque, R., et al. Programming of regulatory T cells from pluripotent stem cells and prevention of autoimmunity. J Immunol. 189, 1228-1236 (2012).
- Chi, V., Chandy, K. G. Immunohistochemistry: paraffin sections using the Vectastain ABC kit from vector labs. J Vis Exp. , (2007).
- Lu, L., et al. Critical role of all-trans retinoic acid in stabilizing human natural regulatory T cells under inflammatory conditions. Proc Natl Acad Sci U S A. 111, E3432-E3440 (2014).
- Wu, C., et al. Galectin-9-CD44 interaction enhances stability and function of adaptive regulatory T cells. Immunity. 41, 270-282 (2014).
- Di Stasi, A., et al. Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med. 365, 1673-1683 (2011).
- Ramos, C. A., et al. An inducible caspase 9 suicide gene to improve the safety of mesenchymal stromal cell therapies. Stem Cells. 28, 1107-1115 (2010).
- Haque, R., Lei, F., Xiong, X., Wu, Y., Song, J. FoxP3 and Bcl-xL cooperatively promote regulatory T cell persistence and prevention of arthritis development. Arthritis Res Ther. 12, R66 (2010).
- van Loenen, M. M., et al. Mixed T cell receptor dimers harbor potentially harmful neoreactivity. Proc Natl Acad Sci U S A. 107, 10972-10977 (2010).
- Kim, Y. C., et al. Engineered antigen-specific human regulatory T cells: immunosuppression of FVIII-specific T- and B-cell responses. Blood. 125, 1107-1115 (2015).
- Himburg, H. A., et al. Pleiotrophin regulates the expansion and regeneration of hematopoietic stem cells. Nat Med. 16, 475-482 (2010).
