Figure 3. In vivo programming of Ag-specific iPSC-T_regs.

The iPSCs transduced with the MiDR-TCR-FoxP3 were co-cultured with the OP9-DL1/DL4/I-A^b cells in the presence of murine rFlt3L and rIL-7. On day 7, the DsRed⁺ cells were sorted were adoptively transferred into Thy1.1 congenic mice and in the following days, mice were i.p. injected with 0.25 mg agonistic α-Notch2 Ab, 5 μg mouse rIL-7 and 10 μg mouse rFlt3L or a mouse IgG/PBS control twice a week. (a) After 2 weeks, CD4⁺ TCRVβ5⁺ cells from the pooled lymph nodes and spleen were analyzed by flow cytometry, after gating on Thy1.2⁺ cell population. (b) The expression of CD25 and FoxP3 was analyzed by flow cytometry, after gating on CD4⁺ Thy1.2⁺ TCRVβ5⁺ T cells from the pooled lymph nodes and spleen (dark lines; shaded areas indicate isotype controls). Data are representative of three independent experiments. (c) IL-10 and TGF-β production (dark lines; shaded areas indicate isotype controls). The pooled lymph nodes and spleen were stimulated with OVA_323-339 peptide and analyzed by intracellular IL-10 or surface TGF-β (LAP) staining, after gating on Thy1.2⁺ TCRVβ5⁺ cells. Data are representative of three independent experiments.