Fig. 2: CD4⁺ T cell activation in vitro required USP12.

A–E Purified naïve CD4⁺ T cells isolated from WT and Usp12^−/− mice were either not treated (0) or stimulated with plate-bound anti-CD3 (1 μg/ml or indicated concentrations) and anti-CD28 (1 μg/ml) for 48 h (A, C, E) or 72 h (D) or indicated amounts of time (B). A The intracellular production of IFN-γ, TNF-α, and IL-2 by CD4⁺ T cells was determined. Pooled data are presented in the right panel. B Cytokine production was measured by ELISA. C The incorporation of thymidine was measured during the final 8 h. D Isolated purified naïve CD4⁺ T cells were labeled with CFSE, stimulated and determined by flow cytometry. E Purified WT and Usp12^−/− naive CD4⁺ T cells first stimulated with anti-CD3 and CD28 and then cultured with various concentrations of IL-2. The incorporation of thymidine was measured for 8 h. F OVA (323–339) coated DCs from WT and Usp12^−/− mice were cocultured with OT-II CD4⁺ T cells from WT OT-II or Usp123 OT-II mice for indicated amounts of time. IFN-γ and TNF-α production was measured by ELISA+/+: WT; −/−: Usp12^−/−. Data (n = 5 in A–F) shown are the mean ± SD. **P < 0.01 and ***P < 0.001 by an unpaired t test (in A) or two-way ANOVA (in B, C, E, and F). Data are representative of three independent experiments with similar results. ND no detected.