Lo sviluppo di cellule staminali derivate antigene-specifiche cellule T regolatorie contro autoimmunità
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
Malattie autoimmuni insorgono a causa della perdita di immunologica auto-tolleranza. cellule T regolatorie (Tregs) sono importanti mediatori di immunologica auto-tolleranza. Tregs rappresentano circa il 5 – 10% del maturo sottopopolazione di cellule T CD4 + in topi e nell'uomo, con circa 1 – 2% di tali Tregs circolanti nel sangue periferico. Le cellule staminali pluripotenti indotte (iPSCs) possono essere differenziati in Tregs funzionale, che hanno un potenziale da utilizzare per le terapie a base di cellule di malattie autoimmuni. Qui, vi presentiamo un metodo per sviluppare antigene (Ag) Treg specifico d'da iPSCs (vale a dire, IPSC-Tregs). Il metodo si basa sulla incorpora il fattore di trascrizione FoxP3 e un recettore delle cellule T Ag-specifica (TCR) in iPSCs e differenziazione di cellule stromali OP9 esprimenti Notch ligandi delta-simili (DL) 1 e DL4. A seguito di differenziazione in vitro, l'IPSC-Treg esprimono CD4, CD8, CD3, CD25, FoxP3, e Ag-specifici TCR e sono in grado di rispondere alla stimolazione Ag.Questo metodo è stato applicato con successo a terapia cellulare di artrite autoimmune in un modello murino. trasferimento adottivo di questi Ag-specifici iPSC-Tregs in artrite Ag-indotta (AIA) topi -bearing ha la capacità di ridurre l'infiammazione articolare e gonfiore e per prevenire la perdita ossea.
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 questo protocollo, un passaggio fondamentale è la differenziazione in vitro di TCR / FOXP3 iPSCs gene-trasdotte. In vitro Notch induce lo sviluppo verso la linea cellulare T. Per differenziare iPSCs in CD4 + FoxP3 + Tregs, abbiamo utilizzato le cellule B OP9-DL1 / DL4 / Ia, altamente espressa molecole MHC II (IA b). La maggior parte dei iPSCs differenziano in cellule CD4 +. Tuttavia, dopo l'espressione del TCR di superficie, molte cellule p…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Questo progetto è stato finanziato, in parte, sotto sovvenzioni dal National Institutes of Health (R01AI121180, R21AI109239 e K18CA151798), l'American Diabetes Association (1-16-IBS-281), e il Dipartimento della Salute della Pennsylvania (tabacco Settlement Funds) 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 |
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