Développement de cellules souches dérivées de cellules T régulatrices spécifiques de l'antigène contre Autoimmunité
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
Maladies auto-immunes surviennent en raison de la perte de l'auto-tolérance immunologique. Les lymphocytes T régulateurs (Treg) sont des médiateurs importants de l'auto-tolérance immunologique. Treg représentent environ 5 – 10% de la sous – population de lymphocytes T CD4 + matures chez les souris et les humains, avec environ 1 – 2% de ces Treg circulant dans le sang périphérique. Les cellules souches pluripotentes induites (CISP) peuvent être différenciées en Tregs fonctionnelle, qui ont un potentiel d'être utilisé pour les thérapies à base de cellules de maladies auto-immunes. Ici, nous présentons une méthode pour développer l' antigène (Ag) Tregs de CSPi spécifique de (c. -à- iPSC-Treg). La méthode est basée sur l'intégration du facteur de transcription FoxP3 et un récepteur Ag-spécifique des cellules T (TCR) en CSPi puis différenciation sur les cellules OP9 stromales exprimant Notch ligands delta-like (DL) 1 et DL4. Après la différenciation in vitro, les iPSC-Tregs expriment CD4, CD8, CD3, CD25, FoxP3 et Ag-TCR spécifique et sont capables de répondre à Ag stimulation.Cette méthode a été appliquée avec succès à la thérapie à base de cellules de l'arthrite auto-immune chez un modèle murin. Le transfert adoptif de ces Ag spécifique iPSC-Tregs dans l'arthrite Ag-induite (AIA) souris -bearing a la capacité de réduire l'inflammation des articulations et de l'enflure et de prévenir la perte osseuse.
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
Dans ce protocole, une étape critique est la différenciation in vitro de TCR / FoxP3 CSPi géniques-transduction. In vitro signalisation Notch induit le développement vers la lignée cellulaire T. Pour différencier CSPi en CD4 + Foxp3 + Tregs, nous avons utilisé les cellules b OP9-DL1 / DL4 / IA, qui expriment des molécules hautement CMH II (IA) b. La plupart des CSPi se différencient en cellules CD4 +. Cependant, après l'expression du …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Ce projet a été financé, en partie, au titre des subventions des Instituts nationaux de la santé (R01AI121180, R21AI109239 et K18CA151798), l'American Diabetes Association (1-16-IBS-281), et le ministère de la Santé en Pennsylvanie (Fonds de règlement du tabac) à 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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