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.
Autoimmuna sjukdomar uppstår på grund av förlusten av immunologisk självtolerans. Regulatoriska T-celler (tregs) är viktiga mediatorer för immunologisk självtolerans. Tregs representerar omkring 5 – 10% av den mogna CD4 + T-cell subpopulation i möss och människor, med ca 1 – 2% av dessa tregs som cirkulerar i det perifera blodet. Inducerade pluripotenta stamceller (iPSCs) kan delas in i funktionella tregs, som har en potential att användas för cellbaserade terapier för autoimmuna sjukdomar. Här presenterar vi en metod för att utveckla antigen (Ag) specifika tregs från iPSCs (dvs IPSC-tregs). Metoden är baserad på införlivande av den transkriptionsfaktorn Foxp3 och en Ag-specifik T-cellreceptor (TCR) i iPSCs och därefter differentiera på OP9 stromala celler som uttrycker Notch-ligander deltaliknande (DL) 1 och DL4. Efter in vitro-differentiering, IPSC-tregs uttrycka CD4, CD8, CD3, CD25, foxp3, och Ag-specifik TCR och kan svara på Ag stimulering.Denna metod har med framgång tillämpas på cellbaserad terapi av autoimmun artrit i en murin modell. Adoptiv överföring av dessa Ag-specifik iPSC-tregs i Ag-inducerad artrit (AIA) -haltig möss har förmågan att minska ledinflammation och svullnad och för att förhindra benförlust.
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.
I detta protokoll är ett kritiskt steg differentieringen av TCR / foxp3 genen-omvandlade iPSCs in vitro. In vitro Notch-signalering inducerar utveckling mot cell härstamning T. För att skilja iPSCs i CD4 + FOXP3 + tregs använde vi OP9-DL1 / DL4 / IA B-celler, som i hög grad express MHC II (IA B) molekyler. De flesta av de iPSCs differentiera till CD4 + celler. Men efter ytan TCR uttryck, många differentierade pre-T-celler förlorar förmågan…
The authors have nothing to disclose.
Detta projekt har finansierats delvis under anslag från National Institutes of Health (R01AI121180, R21AI109239 och K18CA151798), American Diabetes Association (1-16-IBS-281), och Pennsylvania Department of Health (Tobaksavvecklingsfonder) till JS
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 |