Programming of regulatory T cells from pluripotent stem cells and prevention of autoimmunity

Abstract

Regulatory T (Treg) cells are being used to treat autoimmunity and prevent organ rejection; however, Treg cell-based therapies have been hampered by the technical limitation in obtaining a high number of functional Treg cells. In this study, we show how to generate functional Treg cells from induced pluripotent stem (iPS) cells and to determine the potential role of such cells for Treg cell-based immunotherapy against autoimmunity in a therapeutic setting. Ligation of a Notch ligand and transduction of the gene Foxp3 induce iPS cells to differentiate into Treg cells. Expression of Foxp3 and coculture on Notch ligand-expressing stromal cells augment expression of CD3, TCR, CD4, CD25, and CTLA-4 on iPS cell-differentiated Treg cells, which are able to secrete TGF-β and IL-10 both in vivo and in vitro. Importantly, adoptive transfer of iPS cell-derived Treg cells expressing large amounts of Foxp3 and Bcl-x(L) significantly suppresses host immune responses and reduces arthritis development within murine models. These data suggest that Notch signaling and Foxp3 regulate the development and function of Treg cells derived from iPS cells. Our results provide a novel approach for generating potentially therapeutic Treg cells for the treatment of autoimmune diseases.

Figure 1. iPS cells differentiate into Treg cells

Murine iPS cells were transduced with the FoxP3 gene (a-c) and co-cultured with adult thymus organ (d-e) or on OP9-DL1 cells (f-g). (a) FoxP3-transduced iPS cells were visualized by fluorescence microscopy. (b) GFP⁺ iPS cells (left) were transduced with the retroviral construct, and GFP⁺ DsRed⁺ iPS cells (middle) were analyzed by flow cytometry and sorted by a high-speed cell sorter (right). (c) GFP⁺ DsRed⁺ iPS cells were analyzed for by Western blot (left) and flow cytometry, after gating on DsRed⁺ cells (right). (d) FoxP3-transduced iPS cells were co-cultured on OP9-DL1 cells in the presence of murine recombinant Flt3L and IL-7 for 7 days then with adult thymus organ from Rag1-/- mice for additional 8 days, Treg cell differentiation was analyzed by flow cytometry (dark lines; shaded areas indicate isotype controls). (e) iPS cell-derived cells from the culture were stimulated with anti-CD3 plus anti-CD28 mAbs, intracellular TGF-β and IL-10 was analyzed by flow cytometry, after gating on CD4⁺ CD25⁺ cells. (f) Gene-transduced iPS cells were co-cultured on OP9-DL1 cells for 7 days, and adoptively transferred into thymectomized Rag1-/- mice. 4 weeks later, spleen and LN were analyzed for the expression of CD4 and FoxP3 by flow cytometry shown for cells gated as CD4⁺ cell. (g) Purified CD4⁺ T cells from pooled LN and spleen were stimulated with anti-CD3 plus anti-CD28 mAbs, intracellular TGF-β and IL-10 was analyzed by flow cytometry, after gating on CD4⁺ CD25⁺ cells. All data are representative of three experiments.

Figure 2. Characterization of iPS cell-derived Treg cells

FoxP3-transduced iPS cells were co-cultured on OP9-DL1 cells in the presence of murine recombinant Flt3L and IL-7. (a) Morphology of Treg cell differentiation on days 0, 7, 14, and 30. Data are representative of three independent experiments. (b) Flow cytometric analysis for the protein expression of iPS cell-derived cells on day 30. CD3⁺ TCRαβ⁺ cells were gated as indicated, and analyzed for the expression of CD4 and CD8, with CD25, CD127, CTLA-4, and FoxP3 expression shown for cells gated as CD4⁺ cells (R1), CD4⁺CD8⁺ cells (R2) and CD8⁺ cells (R3) (dark lines; shaded areas indicate isotype controls). Data are representative of four experiments. (c) Western blots for the expression of FoxP3 and β-actin on day 30. Data are representative three experiments.

Figure 3. Functional analyses of in vitro differentiated Treg cells

Murine iPS cell-derived Treg cells were stimulated with plate-coated anti-CD3/anti-CD28 mAbs (a-b) or co-cultured with naive CD4⁺ T cells from C57BL/6 mice (Tregs/T cells = 1:1) in the presence of anti-CD3/anti-CD28 mAbs for 2 days (c-d). For suppressive assays, a group of CD4⁺ T cells stimulated with CD4⁺ CD25⁺ Treg cells from C57BL/6 mice as the positive control and a group of CD4⁺ T cells stimulated without Treg cells as the negative control. Intracellular cytokine production was analyzed by flow cytometry after gating on live CD4⁺ CD25⁺ cells (a-b) (dark lines; shaded areas indicate isotype controls) or by ELISA (c), and proliferation was determined by [³H] thymidine incorporation. (a) Intracellular TGF-β and IL-10. (b) Intracellular IL-2 and IFN-γ. (c) IL-2 and IFN-γ. Data are representative of three experiments. (d) Thymidine incorporation during the last 12 hrs. Data are representative of three experiments. *P <0.05, one-way ANOVA tests.

Figure 4. Adoptive transfer of iPS cell-derived Treg cells suppresses arthritis development

Murine iPS cells were transduced with retroviral constructs: vector (MiDR), FoxP3 (MiDR-FoxP3), or FoxP3 with Bcl-xL (MiDR-Bcl-xL-2A-FoxP3), and co-cultured on OP9-DL1 cells. On day 7, DsRed-positive (DsRed⁺) T cells were sorted and prepared for adoptive cell transfer. Collagen-induced arthritis (CIA) was induced in male C57BL/6J mice by one (day 0) intradermal immunization at two sites in the base and slightly above of the tail with chicken type II collagen in complete Freund's adjuvant. On day 15 after the immunization, mice received transduced DsRed⁺ cells (2.5 × 10⁶/mouse). (a-b) In the following days, the arthritis incidence (a) and clinical score (b) were evaluated by examining the paws. Values are the mean ± standard error of the mean of data obtained in three experiments, and in each experiment, ten mice per group were used. Significance was tested by one-way ANOVA with Bonferroni multiple comparison test with ***, ** and * as p < 0.001, p < 0.01 and p < 0.05. (c) On day 60 of immunization, sera were separated from all samples and the levels of serum total IgG and anti-chicken type II collagen Abs were determined by ELISA. Values are the mean ± standard error of the mean (n = 6). (d-g) On day 60 of immunization, hind foot paws were amputated, fixed, and decalcified. The tissues were embedded in paraffin, sectioned, and stained for Hematoxylin and eosin (HE) staining (d), HE semiquantitative score (e), Safranin O-fast green staining (f), and Safranin O-fast green semiquantitative score (g). Infiltrations of polymorphonuclear (PMN) cells () in HE staining and massive destruction of cartilage () in Safranin O-fast green staining are indicated. Data are representative of three similar experiments (*P <0.05, one-way ANOVA tests).

Figure 5. iPS cell-derived Treg cells infiltrate into joints

On day 60 of immunization as described in Fig.4, joints from hind foot paws were examined for Treg cell infiltration. There were iPS cell-derived Treg cells (red) infiltrating in joints from mice receiving iPS cell-derived Treg cells, but not in tissues from mice receiving vector control-transduced iPS cells or without cell transfer (Green: CD4 or CD25, Red: FoxP3, Blue: DAPI). Data are representative of three independent experiments.

Figure 6. Adoptive transfer of iPS cell-derived Treg cells overcomes strain limitation and suppresses arthritis development

Adoptive transfer of Treg cells derived from C57BL/6 iPS cells as described in Fig. 4 suppressed CIA-induced arthritis in DBA/1 mice. (a) Arthritis incidence. (b) Clinical score. Significance was tested by one-way ANOVA with Bonferroni multiple comparison test with *** and ** as p < 0.001 and p < 0.01. (c) Serum total IgG and anti-chicken type II collagen antibodies. (d-e) HE staining and semiquantitative score. (f-g) Safranin O-fast green staining and semiquantitative score. Infiltrations of polymorphonuclear (PMN) cells () in HE staining and massive destruction of cartilage () in Safranin O-fast green staining are indicated. Data are representative of three independent experiments.

Figure 7. Allogeneic Treg cells derived from iPS cells infiltrate into joints

On day 60 of immunization as described in Fig. 6, joints from hind foot paws were examined for Treg cell infiltration. There were iPS cell-derived Treg cells (red) infiltrating in joints from mice receiving allogeneic Treg cells derived from iPS cells. Data are representative of three independent experiments.