Ontwikkeling van stamcel-afgeleide Antigeen-specifieke regulatoire T-cellen tegen auto-immuniteit
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
Auto-immuunziekten worden veroorzaakt door het verlies van immunologische zelftolerantie. Regulatoire T-cellen (Tregs) zijn belangrijke mediatoren van immunologische zelf-tolerantie. Tregs voor ongeveer 5-10% van de volwassen CD4 + T-cel subpopulaties in muis en mens, met ongeveer 1-2% van de Tregs circuleren in het perifere bloed. Geïnduceerde pluripotente stamcellen (iPSC) kunnen worden gedifferentieerd in functionele Tregs, die een potentieel om te worden gebruikt voor celtherapie van auto-immuunziekten. Hier presenteren we een methode voor antigeen (Ag) -specifieke Tregs van iPSCs (dwz iPSC-Tregs) ontwikkelen. De werkwijze is gebaseerd op het opnemen van de transcriptiefactor Foxp3 en een Ag-specifieke T-celreceptor (TCR) in iPSCs en vervolgens differentiëren naar OP9 stromale cellen die Notch liganden delta-achtige (DL) en 1 DL4. Naar aanleiding van in vitro differentiatie, de iPSC-Tregs uiten CD4, CD8, CD3, CD25, FoxP3 en Ag-specifieke TCR en zijn in staat om te reageren op Ag stimulatie.Deze werkwijze is met succes toegepast op cellen gebaseerde behandeling van autoimmune artritis in een muizenmodel. Adoptieve overdracht van deze Ag-specifieke iPSC-Tregs in Ag-geïnduceerde artritis (AIA) dragende muizen heeft het vermogen om gewrichtsontsteking te verminderen en zwelling en botverlies voorkomen.
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
In dit protocol, een cruciale stap is in vitro differentiatie van TCR / Foxp3-gen getransduceerd iPSCs. In vitro Notch signalering induceert ontwikkeling is in de T-cellijn. Om iPSCs differentiëren in CD4 + Foxp3 + Tregs, gebruikten we de OP9-DL1 / DL4 / IA B-cellen, die zeer express MHC II (IA b) moleculen. De meeste iPSCs differentiëren in CD4 + cellen. Na de oppervlak TCR-expressie vele gedifferentieerde pre-T-cellen verliezen het vermogen om …
Divulgations
The authors have nothing to disclose.
Acknowledgements
Dit project werd gefinancierd, voor een deel, in het kader van subsidies van de National Institutes of Health (R01AI121180, R21AI109239 en K18CA151798), de American Diabetes Association (1-16-IBS-281), en de Pennsylvania Department of Health (Tobacco Settlement Funds) aan 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).
