Развитие стволовых клеток, полученных из антиген-специфических регуляторных Т-клеток против аутоиммунитета
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
Аутоиммунные заболевания возникают из-за потери иммунологической аутотолерантности. Регуляторные Т-клетки (Tregs) являются важными медиаторами иммунологической аутотолерантности. Tregs составляют около 5 – 10% от зрелого субпопуляции CD4 + Т – клеток у мышей и людей, с примерно 1 – 2% от тех Tregs , циркулирующих в периферической крови. Индуцированные плюрипотентные стволовые клетки (ИПСК) можно дифференцировать в функциональную Tregs, которые имеют потенциал для использования в клеточной терапии аутоиммунных заболеваний. Здесь мы представляем метод для разработки антигена (Ag) Tregs из – специфических ИПСК (т.е. IPSC-Tregs). Метод основан на включении фактор транскрипции FoxP3 и антиген-специфические Т-клеточного рецептора (TCR) в ИПСК, а затем дифференцированием по OP9 стромальных клеток, экспрессирующих Notch лигандов дельта-подобный (DL), 1 и DL4. После дифференцировки в пробирке, в IPSC-Tregs экспрессировать CD4, CD8, CD3, CD25, FoxP3 и АГ-специфических TCR и способны реагировать на стимуляцию Ag.Этот метод был успешно применен к клеточной терапии аутоиммунного артрита в мышиной модели. Приемные передачи этих Ag-специфической иПСК-Tregs в Ag-индуцированного артрита (AIA) водоносного мышей обладает способностью снижать воспаление суставов и отек и предотвратить потерю костной массы.
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
В этом протоколе, важным шагом является дифференциация в пробирке TCR / FOXP3 ген-трансдуцированных ИПСК. В пробирке передача сигналов Notch индуцирует развитие к клеточной линии Т. Чтобы дифференцировать ИПСК в CD4 + FoxP3 + Tregs, мы использовали клетки б OP9-DL1 / DL4 / IA, котор…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Этот проект был профинансирован, в частности, в рамках грантов от Национального института здоровья (R01AI121180, R21AI109239 и K18CA151798), Американской ассоциации диабета (1-16-IBS-281) и Пенсильвании Департамент здравоохранения (Табак Расчетно фонды) в 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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