Fig. 4: Ubc9 deficiency impairs the homeostatic maintenance of Treg cells.

A Representative photograph of mouse size and spleen from WT (left) and Treg-KO (right) 5-week-old mice. B Treg cells were isolated and cocultured with Tcon cells at different ratio (Treg/Tcon = 0:1, 1:4, 1:2, 1:1, and 2:1). Proliferation of Tcon cells was indicated by the intensity of [3][H] incorporation, which was negatively correlated with Treg suppressive function (n = 3, representative of two experiments). C, D CD4⁺CD25⁺ or CD4⁺CD25⁻ T cells were isolated and stimulated by anti-CD3 (10ug/ml) for 3 days, supernatants were collected for the detection of cytokine concentration of IL-10 (WT: 4727 ± 108.7 pg/ml vs. Treg-KO: 2167 ± 31.51 pg/ml, p < 0.001), IL-2 (WT: 67.80 ± 4.73 pg/ml vs. Treg-KO: 79.32 ± 3.84 pg/ml, p = 0.13), IFN-γ (WT: 735.7 ± 29.36 pg/ml vs. Treg-KO: 1,152 ± 36.57 pg/ml, p < 0.001), IL-4 (WT: 45.28 ± 4.04 pg/ml vs. Treg-KO: 121.0 ± 7.35 pg/ml, p < 0.001), and IL-17A (WT: 48.71 ± 3.06 pg/ml vs. Treg-KO: 447.5 ± 17.76 pg/ml, p < 0.001) (n = 3). E, F Time-dependent increase of activated T cells and decrease of Treg cells are shown. G Proportion (WT: 11.64 ± 0.69% vs. Treg-KO: 7.03 ± 0.72%, p < 0.01) and absolute number (WT: 11.67 ± 0.63 × 10⁵ vs. Treg-KO: 8.78 ± 0.61 × 10⁵, p < 0.05) of Treg cells in 4–5-week-old WT and Treg-KO mice (n = 4). H Percentage of Ki67⁺ proliferative Treg cells (WT: 21.78 ± 0.91% vs. Treg-KO: 14.08 ± 1.36%, p < 0.01) in 4–5-week-old WT and Treg-KO mice (n = 4). The p-value was determined by Student’s unpaired t-test.