Desarrollo de derivados de células madre Las células T reguladoras específicas de antígeno contra la autoinmunidad
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
Las enfermedades autoinmunes se presentan debido a la pérdida de inmunológica auto-tolerancia. Las células T reguladoras (Tregs) son importantes mediadores de la auto-inmunológica tolerancia. Tregs representan aproximadamente el 5 – 10% de la subpoblación de células T CD4 maduro + en ratones y humanos, con aproximadamente 1 – 2% de esas células T reguladoras que circulan en la sangre periférica. células madre pluripotentes inducidas (iPSCs) pueden diferenciarse en células T reguladoras funcional, que tienen un potencial para ser utilizado para terapias basadas en células de enfermedades autoinmunes. A continuación, presentamos un método para desarrollar antígeno (Ag) Tregs específicos de células iPS a partir de (es decir, IPSC-Tregs). El método se basa en la incorporación del factor de transcripción FoxP3 y un receptor de células T Ag-específico (TCR) en iPSCs y luego diferenciar en células que expresan Notch OP9 estromales ligandos delta-como (DL) 1 y DL4. Después de la diferenciación in vitro, las células T reguladoras IPSC-expresan CD4, CD8, CD3, CD25, FoxP3, y Ag-TCR específico y son capaces de responder a la estimulación Ag.Este método se ha aplicado con éxito a la terapia basada en células de la artritis autoinmune en un modelo murino. La transferencia adoptiva de estas Ag-específica IPSC-Tregs en la artritis inducida por Ag (AIA) ratones llevando los tiene la capacidad de reducir la inflamación articular y la inflamación y para prevenir la pérdida de hueso.
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
En este protocolo, un paso crítico es la diferenciación in vitro de TCR / FoxP3 iPSCs de genes transducidos. In vitro de señalización Notch induce el desarrollo hacia el linaje de células T. Para diferenciar células iPS en CD4 + FoxP3 + células T reguladoras, hemos utilizado las células B OP9-DL1 / DL4 / IA, que MHC II (IA b) moléculas altamente exprés. La mayoría de las iPSCs se diferencian en células CD4 +. Sin embargo, después de la…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Este proyecto fue financiado, en parte, en virtud de subvenciones de los Institutos Nacionales de Salud (R01AI121180, R21AI109239 y K18CA151798), la Asociación Americana de la Diabetes (1-16-IBS-281), y el Departamento de Salud de Pensilvania (Fondos del Acuerdo de Tabaco) a 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).
