Figure 6. Ebi3^−/−Il10^−/− T_reg mediated suppression is TRAIL-dependent.

Wild type or knock out T_regs purified by FACS (A) were titrated in a T_reg assay with wild type or DR5^−/− T_conv cells and stimulated with anti-CD3 and anti-CD28 coated latex beads or (B) were cultured with wild type T_conv cells in the insert of a Transwell™ culture plate. Wild type or DR5^−/− T_conv cells were activated in the bottom chamber of the plate with anti-CD3 and anti-CD28 coated latex beads. Proliferation of responder wild type or DR5^−/− T_conv cells was determined by [³H]-thymidine incorporation. CPM ranged between 30,000–65,000. Results shown here are average of 4–5 independent experiments. (C) Wild type and Ebi3^−/−Il10^−/− T_regs were stimulated with anti-CD3 and anti-CD28 coated latex beads in the presence of T_conv cells in the insert of a Transwell™ culture plate. Freshly purified wild type responder T_conv cells were activated in the bottom wells in the presence of a titrated amount of DR5-Fc. Data is average of 2–3 independent experiments, One way ANCOVA p-value *= 0.01 (D) Congenically marked wild type T_conv cells and wild type or knock out T_regs were injected at 4:1 ratio in to Rag^−/− mice. On days 1 and 3 TRAIL neutralizing mAb or isotype control were injected by i.p. CD4, Thy1.1 and Thy 1.2 T cell numbers in the spleen were analyzed after 7 days by flow cytometry. Data includes 3–6 mice per group from 3 independent experiments. (E) Wild type littermate control T_conv cells or (F) DR5^−/− T_conv cells (0.5 × 10⁶ cells) were injected into Rag1^−/− mice. The weight of the mice was monitored weekly for weight loss. Percent weight change was calculated based on the weight at the time of T_reg injection. (G) Wild type or knock out T_regs purified by FACS were cultured with wild type T_conv cells in the insert of a Transwell™ culture plate. Wild type T_conv cells were activated in the bottom chamber of the plate with anti-CD3 and anti-CD28-coated latex beads. Proliferation of responder T_conv cells was determined by [³H]-thymidine incorporation. CPM ranged between 30,000–70,000. Results shown here are mean ± SEM of 3 independent experiments. p-value - A–G; *: < 0.05, ** < 0.005, NS: Not significant.