Figure 5.

Apoptotic T_reg cells release ATP and generate high levels of adenosine. (a) Kinetics of ATP release by apoptotic T_reg cells. Mouse T_reg cells and conventional T cells (T_conv) were treated with anti-Fas mAb or isotype IgG. ATP in the supernatant was measured by colorimetric assay. Mean ± s.d., n = 6. *P < 0.05, Student’s t-test between T_reg cells and conventional T cells at each time point. (b,c) ATP released by mouse apoptotic T_reg cells under serum starvation (b) or after irradiation (c) in the presence of ectonuclease inhibitor ARL67156. The ATP concentration in apoptotic T_reg supernatants was measured at 16 h. Mean and s.d., n = 5. *P < 0.05, Student’s t-test. (d) Kinetics of adenosine generation by apoptotic T_reg cells. Mouse T_reg cells and conventional T cells were treated with anti-Fas mAb or isotype IgG in the presence of the adenosine deaminase inhibitor EHNA. Adenosine was measured in the supernatant. Mean ± s.d., n = 6. *P < 0.05, Student’s t-test between T_reg cells and conventional T cells at each time point. (e,f) Adenosine generated from exogenous ADP and ATP by apoptotic T_reg cells. Exogenous ADP or ATP (5 μM) was added to fresh medium with apoptotic T_reg cells for 6 h. Adenosine was measured in the culture supernatants (e). Supernatants were used for immunosuppression assays (f). Mean and s.d., n = 5. *P < 0.05 versus control, Wilcoxon test. (g) The effect of ARL67165 or AMP-CP on ATP release by apoptotic T_reg cells. Mouse T_reg cells were treated for 8 h with anti-Fas mAb with or without ARL67165 or AMP-CP. ATP was measured in the supernatants. Mean ± s.d., n = 5. *P < 0.05 versus the untreated control, Wilcoxon test. (h) The effect of ARL67165 or AMP-CP on apoptotic T_reg cell–mediated immunosuppression. Mouse T_reg cells were cultured for 8 h with anti-Fas mAb with or without ARL67165 or AMP-CP. The culture supernatants were used for T cell immunosuppression. IL-2 was measured on day 3 by ELISA. Mean and s.d., n = 8. *P < 0.05 versus control, ANOVA with Dunett’s post-test. (i,j) Immunosuppression mediated by wild-type (WT) and Nt5e^−/− apoptotic T_reg cells. Apoptotic T_reg cells from wild-type and Nt5e^−/− mice were used for T cell immunosuppressive assays. TNF expression was measured by flow cytometry (i), and IL-2 was detected by ELISA (j). Mean and s.d., n = 5. *P < 0.05 versus control, Wilcoxon test. (k) The effect of Nt5e^−/− apoptotic T_reg cells on tumor growth in vivo. MC38 tumors were inoculated subcutaneously with wild-type or Nt5e^−/− apoptotic T_reg cells. The images show representative tumors at the endpoint of the experiment. Mean and s.e.m., n = 10. Student’s t-test, *P < 0.05 versus control. (l,m) The effects of CD39 and A_2A inhibitors on tumor growth. MC38-bearing mice were treated with the CD39 inhibitor ARL67165 (5 mg/kg, 5 d per week) or the A_2A inhibitor ZM241385 (1 mg/kg, 5 d per week) with anti-PD-L1 in the presence of apoptotic T_reg cells. Mean and s.e.m., n = 10. *P < 0.05 compared with isotype control in the presence of the apoptotic T_reg cell group, ANOVA with Dunnett’s post hoc test. All data in a–j were generated in single experiments; n values for those panels indicate the number of animals used for cell isolation. Data in k–m are from 1 experiment with 10 animals per group.