Figure 1.

Characteristics of polyclonally iTreg cells. (A) Naïve CD4⁺ T cells from DBA/2 or C57BL/6 Foxp3^gfp knock-in mice were stimulated with anti-CD3/28 beads and rmIL-2 with (iTreg) or without TGFβ (CD4_con) for 3–4 days. CD25 and Foxp3 (or GFP) expression was determined by flow cytometry. Numbers in the panel represents for the frequency of the quadrants. (B) iTreg or CD4_con cells generated as before were cultured with CFSE-labeled CD25⁺-depleted T cells (1:5 ratio) in the presence of anti-CD3 and irradiated APC for 3 days. The proliferation (CFSE dilution) of responder T cells was analyzed by flow cytometry. Cells frequency showed in panel was gated on CD8⁺ cells. Data were representative of three independent experiments. (C) iTreg cells generated with IL-2 and TGFβ were cultured with CD25⁺-depleted T cells (1:5 ratio) in the presence of anti-CD3 and syngeneic APC for 3 days. Anti-TGFβ, ALK5i, anti-IL-10R, and isotype IgG (all 10 μg/ml) were added to the culture system separately. Transwell was also used to determine the cell-contact effect. [³H]thymidine was added to cultures for the last 18 h and incorporation was measured. Values were mean ± SEM of three independent experiments. ***P <0.001, iTreg vs. baseline. (D) iTreg or CD4_con cells were added to CD25⁺-depleted T cells in the presence of allogeneic APC for 3 days. [³H]thymidine was used to measure the incorporation by proliferating T cells. Values were mean ± SEM of three independent experiments. P =0.02, iTreg vs. baseline.