Figure 3.

Apoptotic T_reg cells mediate immunosuppression via soluble factor(s). (a–c) The effect of macrophage depletion on apoptotic T_reg cell–mediated suppression. Mice bearing MC38 and apoptotic T_reg cells were treated with clodronate-loaded liposomes. The plots show tumor volume (a) and numbers of tumor-infiltrating effector T cells (b,c). Data in a and c are the mean and s.d. of n = 10 mice per group. *P < 0.05 versus the control, Student’s t-test. (d) The expression of immunosuppression-associated molecules in live and apoptotic T cell subsets. The indicated mouse T cell subsets were immunoblotted with antibodies to the indicated proteins. (e) The effect of PD-L1 or PD-1 blockade on apoptotic T_reg cell–mediated T cell suppression. Apoptotic T_reg cells and apoptotic conventional T cells were induced by irradiation. Apoptotic T_reg cells and apoptotic conventional T cells were cultured with normal T cells at a ratio of 1:2 in the presence of anti-CD3. We measured the expression of T cell TNF and IL-2 by flow cytometry. (f,g) Immunosuppression mediated by PD-L1⁻, PD-1⁻, and wild-type (WT) apoptotic T_reg cells. Apoptotic T_reg cells from Cd274^−/− (f), Pdcd1^−/− (g), and wild-type mice were cultured with normal T cells at a ratio of 1:2 in the presence of anti-CD3. We measured the expression of T cell TNF and IL-2 by flow cytometry. Data shown are the mean and s.e.m. (n = 5 mice). *P < 0.05 compared with controls, Wilcoxon test. (h) Immunosuppression mediated by low-molecular-weight components in apoptotic T_reg cell–derived supernatants. T_reg cells were treated with anti-Fas mAb for 6 h. T_reg cell supernatants were passed through a 100-kDa cutoff filter and divided into two fractions with a 3-kDa cutoff filter. The two fractions were collected for T cell immunosuppression assays. T cell IL-2 was detected on day 3 by ELISA. The data shown are the mean and s.d. (n = 5 mice). *P < 0.05 versus the control, ANOVA with Dunett’s post-test. (i,j) Immunosuppression mediated by nonprotein elements in apoptotic T_reg cell supernatants. Apoptotic T_reg cell supernatants (apo) were treated with proteinase K (i) or trypsin (j) and subsequently used for immunosuppression assays. T cell IL-2 was detected on day 3 by ELISA. The data shown are the mean and s.d. (n = 5 mice). ANOVA with Dunett’s post-test, *P < 0.05. (k) The effect of the removal of small lipid-like factors on immunosuppression mediated by apoptotic T_reg cell supernatants. Apoptotic T_reg cell supernatants were mixed with charcoal-treated dextran to remove small lipid-like molecules and were subsequently used for immunosuppression assays. T cell IL-2 was detected on day 3 by ELISA. The data shown are the mean and s.d. (n = 5 mice). Wilcoxon test, *P < 0.05 versus the control. Data are representative of 10 (d) or 5 (e) experiments. In b and e, numbers in the upper right show the percentage of cells in the indicated quadrant.