Figure 4.

Impaired conversion of naive Arrb2^−/− T cells into Foxp3⁺CD4⁺ inducible regulatory T (Treg) cells. Naive (CD25⁻ CD4⁺) T cells purified from spleens of Arrb2^−/− or Arrb2^+/+ mice were cultured with anti-CD3 and anti-CD28. For T helper type 1 (Th1) differentiation, cells received interleukin-12 (IL-12; 10 ng/ml; PeproTech). For Th2 differentiation, cells received anti-interferon-γ (IFN-γ; 5 mg/ml; XMG1.2; BD Pharmingen) and IL-4 (40 ng/ml; PeproTech). For Th17 differentiation, cells received anti-IFN-γ, transforming growth factor-β₁ (TGF-β₁) (3 ng/ml; PeproTech), IL-6 (30 ng/ml; eBioscience). For inducible Treg cells, cells received TGF-β₁ (1 ng/ml), and anti-IFN-γ. (a) Representative flow cytometry graphs show the generation of IFN-γ⁺ IL-17⁺ Foxp3⁺ cells in Arrb2^−/− or Arrb2^+/+ cell cultures. Numbers adjacent to outlined areas indicate frequency of cells within the CD4⁺ gate. (b) Quantification of results in (a). Data were shown as mean ± SEM from three independent experiments. (c) Flow cytometry analysis of GFP expression (indicative of endogenous Foxp3 transcription) by naive T cells purified from either Arrb2^−/− Foxp3^egfp or Arrb2^+/+ Foxp3^egfp mice (n = 3 for each group) and cultured under iTreg conditions (TGF-β₁; 1 ng/ml) for 5 days. (d) and (e), quantification of results in (c).