Fig 2: SOD3 suppress the activation-induced downstream signaling pathways. CD4+T cells were first pre-treated with 200 U/mL of SOD3 for 1 h and then treated with (A) anti-CD3/CD28 (3 µg/mL) or (B) PMA (100 ng/mL) and Ionomycin (300 ng/ml) for indicated time periods. CD4+T cells isolated from wild type and SOD3 knock out mice were first pre-treated with 200 U/mL of SOD3 and DETCA (10 µM) for 1 h and then treated with (C) anti-CD3/CD28 (3 µg/mL) or (D) PMA (100 ng/mL) and Ionomycin (300 ng/ml) for 30 min. Activation of p38, ERK1/2 and NF-?B were analyzed by western blot. All experiments were performed in triplicate. Band densities of blot are shown in Supplementary Figures 5A–D .

Fig 3: SOD3 suppressed CD69 expression and IL-2 production. CD4+T cells were isolated from SOD3 knock out and wild type mice first pre-treated with 200 U/mL of SOD3 and DETCA (10 µM) for 1 h and then treated with anti-CD3/CD28 (3 µg/mL) or PMA (100 ng/mL) and Ionomycin (300 ng/ml) for 24 h. (A) Expression levels of surface proteins were analyzed by FACS. (B) Absolute cell numbers of CD69 positive T cells. (C) IL-2 productions were determined from cell free supernatant by ELISA. All experiments were performed in triplicate. The data shown represent one of three independent experiments. Data are expressed as mean ± standard deviation. #p<0.05, ##p<0.01, ###p<0.001 (Control group vs. anti-CD3/CD28 or PMA/ION-treated group); *p<0.05, **p<0.01, ***p<0.001 (anti-CD3/CD28 or PMA/ION-treated group vs. SOD3 and anti-CD3/CD28 or PMA/ION-treated group); @p<0.05, @@p<0.01, @@@p<0.001 (wild-type vs SOD3 Knock out).

Fig 4: Effects of SOD3 on CD4+T cell proliferation. CD4+T cells were first pre-treated with 100, 200, and 300 U/mL of SOD3 for 1 h and then treated with (A) anti-CD3/CD28 (3 µg/mL) or (B) PMA (100 ng/mL) and Ionomycin (300 ng/ml) for 3 days. CD4+T cells isolated from wild type and SOD3 knock out mice were first pre-treated with 200 U/mL of SOD3 and DETCA (10 µM) for 1 h and then treated with (C) anti-CD3/CD28 (3 µg/mL) or (D) PMA (100 ng/mL) and Ionomycin (300 ng/ml) for 3 days. Cell proliferation was measured with BrdU cell proliferation assay kit. All experiments were performed in triplicate. Data are expressed as mean ± standard deviation. #p<0.05, ##p<0.01, ###p<0.001 (Control group vs. anti-CD3/CD28 or PMA/ION-treated group); *p<0.05, **p<0.01, ***p<0.001 (anti-CD3/CD28 or PMA/ION-treated group vs. SOD3 and anti-CD3/CD28 or PMA/ION-treated group); @p<0.05, @@p<0.01, @@@p<0.001 (wild-type vs SOD3 knock out).

Fig 5: Effect of SOD3 on surface activation markers. (A) CD4+T cells isolated from wild type and SOD3 knock out mice were first pre-treated with 200 U/mL of SOD3 and DETCA (10 µM) for 1 h and then treated with anti-CD3/CD28 (3 µg/mL) or PMA (100 ng/mL) and Ionomycin (300 ng/ml) for 24 h. Expression levels of surface proteins were determined by FACS. (B) Absolute cell numbers of CD25 positive T cells. (C) IL-2 productions were determined from cell free supernatant by ELISA. All experiments were performed in triplicate. The data shown represent one of three independent experiments. Data are expressed as mean ± standard deviation. #p<0.05, ##p<0.01, ###p<0.001 (Control group vs. anti-CD3/CD28 or PMA/ION-treated group); *p<0.05, **p<0.01, ***p<0.001 (anti-CD3/CD28 or PMA/ION-treated group vs. SOD3 and anti-CD3/CD28 or PMA/ION-treated group); @p<0.05, @@p<0.01, @@@p<0.001 (wild-type vs SOD3 Knock out).