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. 2012 Jul 6;4(6):409–419. doi: 10.1093/jmcb/mjs040

Figure 1.

Figure 1

Characteristics of polyclonally iTreg cells. (A) Naïve CD4+ T cells from DBA/2 or C57BL/6 Foxp3gfp knock-in mice were stimulated with anti-CD3/28 beads and rmIL-2 with (iTreg) or without TGFβ (CD4con) 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 CD4con 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 CD4con 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.