Figure 6.

Oxidative stress induces T_reg cell apoptosis in the tumor environment. (a) The effect of ovarian cancer ascites on human T_reg cell apoptosis. Mouse T_reg cells and conventional T cells (T_conv) were cocultured with 50% human ovarian cancer ascites or hydrogen peroxide for 24 h. Additional cultures were treated with N-acetyl-cysteine (NAC) as a free radical scavenger. Annexin V⁺ T_reg cells and T_conv cells were analyzed by flow cytometry. n = 5. (b,c) The effect of ovarian cancer ascites on proapoptotic and antiapoptotic gene transcripts in T_reg cells. T_reg cells were exposed to ascites or ascites plus NAC after 24 h. Proapoptotic (b) and antiapoptotic (c) transcripts were quantified by real-time PCR. n = 3. (d,e) The effect of hydrogen peroxide on proapoptotic and antiapoptotic transcripts in T_reg cells. T_reg cells were exposed to hydrogen peroxide or hydrogen peroxide plus NAC after 24 h. Proapoptotic (d) and antiapoptotic (e) mouse gene transcripts were quantified by real-time PCR. n = 3. (f,g) Expression of mouse NRF2 and NRF2-associated gene transcripts (f) and proteins (g) in T cell subsets as determined by real-time PCR and immunoblotting, respectively. n = 5. (h) The expression of intracellular ROS in T_reg cells. T_reg cells and conventional T cells were cultured overnight. Amounts of intracellular ROS were measured by flow cytometry. n = 5. MFI, mean fluorescence intensity. (i) The effect of NRF2 inducers on T_reg cell apoptosis in vitro. Mouse T_reg cells were cultured for 24 h with 50% ascites or medium containing H₂O₂. NRF2 inducers diethylmaleate (DEM; 100 μM) and sulforaphane (10 μM) were added to the culture for 24 h. Controls were treated with vehicle (DMSO). Apoptosis was measured by flow cytometry with annexin V. n = 4. (j–l) The effect of sulforaphane on tumor immunity. MC38-bearing mice were treated with sulforaphane (25 mg/kg, 5 d per week) or vehicle (DMSO). Tumor T_reg cell apoptosis (j) and CD8⁺ T cell polyfunctional cytokine expression (k) were analyzed by flow cytometry. Tumor volume was monitored (l). n = 10. All data are shown as the mean and s.d. and are from 1 experiment with 10 animals per group (i–l) or from single experiments with cells isolated from individual animal for each data point (a–h). *P < 0.05 versus control, Wilcoxon test (b–e), paired Student’s t-test (f,g), Student’s t-test (i–l), or Mann–Whitney U-test (a,h).