Figure 3

Retrovirus-mediated transduction of FoxP3 and Bcl-xL enhances suppressive activity of regulatory T cells (Tregs) in vitro. Naive CD4⁺ T cells from C57BL/6J mice were stimulated with anti-CD3 plus anti-CD28 antibodies (Abs). On day 2/3, T cells were transduced with retroviral vectors expressing green fluorescent protein (GFP), GFP with FoxP3, or GFP with Bcl-xL and FoxP3. On day 6 of primary culture, GFP⁺ CD4⁺ Tregs were sorted and re-stimulated with anti-CD3 and anti-CD28 Abs (a) or co-cultured with naive CD4⁺ T cells from C57BL/6J mice (Tregs/T cells = 1:1) stimulated with anti-CD3 and anti-CD28 Abs for various periods (b, c). (a) Recall survival of Tregs, based on recovery of GFP⁺ CD4⁺ T cells over time. Cell numbers present on day 0 were assigned a value of 100%, and cell numbers surviving on day 2 to day 8 were used to calculate the percentage recovery. Data represent the mean percentage change ± standard deviation from three separate experiments. (b) Interleukin-2 (IL-2) production and interferon-gamma (IFN-γ) production were measured by enzyme-linked immunosorbent assay at 48 hours. Data are representative of three independent experiments. Proliferation on day 2 or 4 was analyzed by flow cytometry for 5-bromo-2-deoxyuridine (BrdU) incorporation (c) and thymidine incorporation during the last 12 hours (d). Data are representative of three experiments. *P <0.05, Student unpaired t test.