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.
Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 – 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 – 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.
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.
All animal experiments are approved by the Pennsylvania State University College of Medicine Animal Care Committee (IACUC protocol #45470) and are conducted in compliance with the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care.
1. Stem Cell Culture
2. In Vitro Differentiation of Ag-specific iPSC-Tregs
3. Evaluation of In Vitro Treg Differentiation and Maturation
4. In Vivo Maturation and Suppression of Autoimmune Arthritis
5. Measurement of Bone Loss in the Knees with the High-resolution Micro-computed Tomography (micro-CT) System
As shown here, on day 28, Ag-specific Tregs substantially expressed CD3 and Ag-specific TCR, two T cell markers. The CD3+TCRVβ5+ population expressed CD4. Most of the CD3+TCRVβ5+CD4+ cells also expressed CD25, CD127, and CTLA-4, which are typically expressed at elevated levels in naturally occurring Tregs (nTregs) and in T cells expressing FoxP3 ectopically. FoxP3 expression in iPSC-derived cells persisted even after long-term in vitro stimulation with the Notch ligand, as detected by intracellular staining analyzed by flow cytometry (Figure 1). In addition, the Ag-specific iPSC-Tregs produced suppressive cytokines, such as TGF-β and IL-10, when stimulated in vitro with Ag-pulsed splenocytes (Figure 2), indicating the iPSC-Tregs had potential suppressive activities. Fluorescence microscopy revealed that more FoxP3+ cells were present in the OVA-treated than the PBS-treated knees; there were no FoxP3+ cells existing in the knees in mice receiving the DsRed+ vector-transduced iPSCs. Many more CD4+FoxP3+ TCRVβ5+ cells presented in the knees of mice receiving iPSCs transduced with the MiDR-TCR-FoxP3 than the MiDR-FoxP3 (Figure 3). These observations suggest that Ag-specific iPSC-Tregs migrate to the AIA knee after the adoptive transfer into recipient mice. The transferred iPSC-derived cells substantially decreased the inflammatory knee swelling when OVA was present, but had no effect on the control knee that was only injected with mBSA in the murine model (Figure 4); reduced bone loss in the cell transfer knee visualized by the high-resolution micro-CT system (Figure 5).
Figure 1: Differentiation of Ag-specific iPSC-Tregs. Murine iPSCs were transduced with a construct: MiDR-TCRα-2A-TCRβ-2A-FoxP3 containing genes of OVA-specific TCR and FoxP3. The gene-transduced cells (DsRed+) were co-cultured on OP9-DL1-DL4-I-Ab cells in the presence of mFlt3L and mIL-7. (A) Morphology of Tregs cell differentiation on day 0, 7, 14 and 22. (B) Flow cytometric analysis for the protein expression of iPSC-derived cells on day 28. CD3+ TCRVβ5+ cells were gated as indicated (R1) and analyzed for the expression of CD4 and CD8, with CD25, CD127, CTLA-4, and FoxP3 shown for cells gated as CD4+CD8– cells (R2) (dark lines; shaded areas indicate isotype controls). Please click here to view a larger version of this figure.
Figure 2: Functional Analyses of In Vitro Differentiated Ag-specific Tregs. Murine iPSC-Tregs were stimulated by splenocytes (APCs; Tregs/APCs = 1:4) and pulsed with OVA323-339 peptide. Intracellular cytokine production (TGF-β and IL-10) was analyzed by flow cytometry after gating on live CD4+ CD25+ cells (dark lines; shaded areas indicate isotype controls). Please click here to view a larger version of this figure.
Figure 3: Ag-specific iPSC-Tregs Infiltrate into the Knee Joints. Ag-specific iPSC-Tregs were adoptively transferred into C57BL/6 mice. Shortly after arthritis induction (days 7 – 14), the knees were removed and stained for immunohistology (scale bars: 20 μm). Mice receiving OVA-specific iPSC-Tregs have large numbers of TCRVβ5+ cells in the knees. Please click here to view a larger version of this figure.
Figure 4: Adoptive Transfer of Ag-specific iPSC-Tregs Ameliorates AIA in Mice. Murine iPSCs were transduced with the retroviral construct MiDR, MiDR-FoxP3, or MiDR-TCR-FoxP3 and were co-cultured on the OP9-DL1/DL4/I-Ab cells. On day 7, the gene-transduced cells (3 × 106/mouse) were adoptively transferred into female C57BL/6 mice that were induced with AIA two weeks after the cell transfer. On the day following arthritis induction, the arthritis severity was monitored by measurement of the knee diameter. (A-C) Percent increase in knee diameter. Increase in knee diameter was calculated based on preinjection knee diameter for each mouse before injection on day 0. Arthritis score was evaluated by examining both knees in a blinded manner; each knee was assigned a score (0: no visible swelling or discoloration; 1: visible swelling with or without discoloration; 2: moderate swelling with discoloration; 3: severe swelling with discoloration). In each group, five mice were used, and data are representative of three independent experiments. Data are represented as the mean ± SD. (D) The mean scoring on day 7 for both knees from five mice. Data are represented as the mean ± SD from three independent experiments (** p< 0.01, *** p< 0.001, two-way ANOVA). Please click here to view a larger version of this figure.
Figure 5: Ag-specific iPSC-Tregs Migrate to the OVA Injected Knee and Reduce the Bone Loss. iPSC-Tregs were adoptively transferred into C57BL/6 mice. Within 21 days post-arthritis induction, mice were anaesthetized and the bone architecture around the mouse knees was imaged by the micro-CT system. Mice receiving OT-II TCR/FoxP3 gene-transduced murine iPSC-Tregs exhibit significant reduction of bone loss as compared to control mice receiving vector-transduced iPSCs. (A) The scans were performed with a 2.2 mm length, and images were captured after further image processing (volume rendering and transformation; scale bars: 1 mm). (B) Bone volume around knee joint was evaluated using three-dimensional reconstruction of micro-CT images, and bone volume inside the volume of interest was calculated (scale bars: 1 mm). Please click here to view a larger version of this figure.
In this protocol, a critical step is the in vitro differentiation of TCR/FoxP3 gene-transduced iPSCs. In vitro Notch signaling induces development towards the T cell lineage. To differentiate iPSCs into CD4+FoxP3+ Tregs, we used the OP9-DL1/DL4/I-Ab cells, which highly express MHC II (I-Ab) molecules. Most of the iPSCs differentiate into CD4+ cells. However, after the surface TCR expression, many differentiated pre-T cells lose the ability to differentiate and eventually die. As a result, the cell number of the iPSC-derived functional Tregs dramatically reduces after four weeks of in vitro differentiation. To avoid this, addition of IL-2 can improve cell survival at those time points. An alternative method to drive the maturation and survival of Ag-specific pre-Tregs is to transfer the pre-Tregs that have differentiated in vitro for a week into mice. These pre-Tregs can continue differentiating and maturing in vivo for another three weeks. Using this method, a functional Ag-specific Treg population can be generated from iPSCs, which are nTregs-like and have the ability to suppress autoimmune arthritis in the murine model.
To improve the efficacy of in vivo development of Ag-specific iPSC-Tregs, different compounds (e.g., retinoic acid, gelectin)13,14 or suppressive cytokines (e.g., TGF-β, IL-10) can be used after the adoptive transfer of the pre-Tregs. In addition to increasing the cell survival, this combined approach can maintain FoxP3 expression14,15 and enhance the quality of the Ag-specific iPSC-Tregs.
A potential problem that could arise for in vivo Treg development is overall immunosuppression, resulting in complications during infections or subsequent weight loss. A large number of Ag-specific Tregs developing in vivo may worsen infections. In this case, a suicide gene, the inducible caspase 9 (iCasp9)15,16, can be incorporated into the TCR/FoxP3 vector. This approach allows the removal of the stem cell-derived Tregs by the injection of a bioinert small-molecule dimerizing agent (AP1903) to "shut off" the generation of stem cell-derived Tregs, which will overcome this potential issue.
A self-Ag is a typical protein recognized by the immune system of hosts suffering from an individual autoimmune disorder. This self-Ag is the target of the immune system, and the correlated T cells are not deleted. To generate self-Ag specific Tregs, the use of TCR transduction is a good choice. A self-Ag (e.g., heat shock protein) specific TCR can be transduced into mature CD4+ CD25+ Tregs from peripheral blood mononuclear cells (PBMCs), and this approach has been utilized in clinical trials. Alternatively, as described in this protocol, using the gene transduction of self-Ag specific TCR with FoxP3 and stimulation with Notch signaling, stem cell-derived Tregs can be self-Ag specific Tregs.
Cell-based therapies for autoimmune diseases utilizing engineered Tregs may be useful17. Although TCR transduction in T cells has proved to be safe, feasible, and applicable in clinical trials, there are still major safety concerns due to the autoimmunity caused by cross-reactivity with healthy tissues18. Furthermore, using current methods, genetically modified Tregs usually have a short-term persistence in vivo19. Alternatively, a promising source for developing large numbers of monoclonal Ag-specific Tregs is stem cells. Embryonic stem cells (ESCs) have the best pluripotency and self-renewal, but it is not feasible to obtain them from patients. Although easily isolated from the peripheral blood, hematopoietic stem cells (HSCs) are multi-potent stem cells and can be expanded in cell culture similarly to ESCs20. Present iPSC technology has advanced to a more efficient generation of PSC from patients' somatic cells by transduction of different transcription factors. A number of methodological improvements have been developed in recent years to generate iPSCs by maximally reducing potential hazards such as immunogenicity and tumorigenicity. Compared to ESCs, iPSCs have identical pluripotency and self-renewal. As a result, iPSC technology can provide an advantage in developing patient- and/or disease-specific PSCs. The use of iPSCs to develop Ag-specific Tregs may advance the field of cell-based therapies for autoimmune diseases10,11.
The authors have nothing to disclose.
This project was funded, in part, under grants from the National Institutes of Health (R01AI121180, R21AI109239 and K18CA151798), the American Diabetes Association (1-16-IBS-281), and the Pennsylvania Department of Health (Tobacco Settlement Funds) to J.S.
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 |