تطوير المستمدة الخلية الجذعية خلايا T التنظيمية مستضد محددة ضد المناعة الذاتية
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
أمراض المناعة الذاتية تنشأ بسبب فقدان المناعة التسامح مع الذات. الخلايا التائية التنظيمية (Tregs) هي سطاء مهمة من المناعية التسامح مع الذات. تمثل Tregs حوالي 5 – 10٪ من نضجا حيوانية خلايا CD4 + T في الفئران والبشر، مع حوالي 1-2٪ من تلك Tregs في الدم المحيطي. الناجم عن الخلايا الجذعية المحفزة (iPSCs) يمكن أن تكون متباينة في Tregs وظيفية، والتي لديها القدرة على استخدامها في العلاجات القائمة على خلية من أمراض المناعة الذاتية. هنا، فإننا نقدم وسيلة لتطوير المستضد (حج) Tregs -specific من iPSCs (أي IPSC-Tregs). وتستند هذه الطريقة على دمج عامل النسخ FoxP3 ومستقبلات الخلايا التائية حج محددة (TCR) في iPSCs ثم التمييز على خلايا OP9 انسجة التعبير عن الشق بروابط-دلتا مثل (DL) (1) وDL4. التالية في المختبر التمايز، وIPSC-Tregs التعبير عن CD4، CD8، CD3، CD25، FoxP3، وحج محددة TCR وتكون قادرة على الاستجابة لتحفيز حج.وقد تم تطبيق هذه الطريقة بنجاح لعلاج خلية القاعدة من التهاب المفاصل المناعة الذاتية في نموذج الفئران. نقل بالتبني من هذه حج محددة IPSC-Tregs إلى التهاب المفاصل الناجم عن حج (AIA) الفئران -bearing لديه القدرة على تقليل التهاب المفاصل والانتفاخ ولمنع فقدان العظام.
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
في هذا البروتوكول، خطوة حاسمة هي التمايز في المختبر من / FoxP3 iPSCs-transduced الجينات TCR. في المختبر الشق يشير تحرض التطور نحو الخلية النسب تي. للتمييز iPSCs في CD4 + FoxP3 + Tregs، استخدمنا OP9-DL1 / DL4 / IA خلايا ب، التي صريحة للغاية MHC II (IA ب) الجزيئات. معظم iPSCs …
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
وقد تم تمويل هذا المشروع، في جزء منه، في إطار المنح المقدمة من المعاهد الوطنية للصحة (R01AI121180، R21AI109239 وK18CA151798)، والجمعية الأمريكية للسكري (1-16-IBS-281)، ووزارة الصحة ولاية بنسلفانيا (صناديق تسوية التبغ) ل 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 |
Referencias
- 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).
