פיתוח תאי גזע שמקורם אנטיגן ספציפי תקינים בתאי T נגד אוטואימוניות
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
מחלות אוטואימוניות להתעורר עקב אובדן סובלנות עצמית אימונולוגית. בתאי T רגולטוריים (Tregs) הם מתווכים חשובים של סובלנות עצמית אימונולוגית. Tregs מייצג כ 5 – 10% של תת-אוכלוסיית תא CD4 + T הבוגר בעכברים ובבני אדם, עם כ 1 – 2% מאלה Tregs במחזור הדם ההיקפי. תאי גזע מושרים (iPSCs) יכול להיות מובחן לתוך Tregs תפקודית, אשר יש פוטנציאל לשמש עבור טיפולים מבוססי תאים של מחלות אוטואימוניות. כאן, אנו מציגים שיטה לפתח אנטיגן (Ag) Tregs -specific מ iPSCs (כלומר, iPSC-Tregs). השיטה מבוססת על שילוב גורם שעתוק FoxP3 ו קולטן תא Ag-T ספציפי (TCR) לתוך iPSCs ולאחר מכן הבחנה על תאי סטרומה OP9 להביע Notch הליגנדים דלתא דמוית (DL) 1 ו DL4. בעקבות הבידול חוץ גופית, iPSC-Tregs להביע CD4, CD8, CD3, CD25, FoxP3, ו Ag ספציפי TCR והם מסוגלים להגיב לגירוי Ag.שיטה זו יושמה בהצלחה לטיפול מבוסס תאים של דלקת אוטואימונית מודל בעכברים. העברת המאמצת של Ag ספציפי iPSC-Tregs אלה לתוך מפרקים הנגרמת Ag (AIA) עכברים -bearing יש את היכולת להפחית דלקת מפרקים ונפיחות וכדי למנוע איבוד מסת העצם.
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
בפרוטוקול זה, צעד קריטי הוא בידול במבחנה של iPSCs TCR / FoxP3-transduced הגן. איתות Notch חוץ גופית גורם להתפתחות כלפי השושלת תא T. כדי לבדל iPSCs לתוך CD4 + FoxP3 + Tregs, השתמשנו תאים ב OP9-DL1 / DL4 / IA, אשר מאוד מפורשת MHC II מולקולות (IA ב). רוב iPSCs להתמיין CD4 + תאים. …
Divulgations
The authors have nothing to disclose.
Acknowledgements
פרויקט זה מומן בחלקו תחת מענקים מהמוסד הלאומי לבריאות (R01AI121180, R21AI109239 ו K18CA151798), ה- American Diabetes Association (1-16-IBS-281), ואת מחלקת פנסילבניה הבריאות (קופות להתיישבות טבק) כדי 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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