Figure 1. Ebi3^−/−Il10^−/− T_regs are suppressive in vitro and in vivo.

(A) Wild type or knock out T_reg purified by FACS were titrated in a standard T_reg assay with T_conv cells and anti-CD3 and anti-CD28 coated latex beads. Proliferation of T_conv responder cells was determined by [³H]-thymidine incorporation (p-value: wild type T_regs compared to Ebi3^−/−Il10^−/−, and Il12a^−/−Il10^−/− T_regs, Not significant (NS)). (B) Wild type or knock out T_reg were cultured with anti-CD3 and anti-CD28 coated latex beads and T_conv cells in the inserts of a Transwell™ culture plate. Third party, wild type responder T_conv was activated in the bottom chamber of the plate. Proliferation of responder cells was determined by [³H]-thymidine incorporation. Proliferation ranged from 30,000–60,000 cpm. p-value: *: <0.05, NS: Not significant. (C) Congenically marked wild type T_conv cells, wild type or knock out T_regs purified by FACS were injected at 4:1 ratio into Rag1^−/− mice. CD4⁺ cell numbers in the spleen were analyzed after 7 days by flow cytometry. p-value: * <0.05. (D) Wild type T_conv cells (0.5×10⁶) were injected into Rag1^−/− mice. The weight of the mice was monitored weekly for weight loss. Once the mice had lost 5% of its body weight wild type or knock out T_regs were injected. Mice were monitored for percent weight change calculated based on the weight at the time of T_reg injection. p-value: * <0.05 and NS: Not significant. (E) Colonic tissue sections stained with H & E stain were scored in a blinded manner. Representative images of sections from 3 independent experiments are shown with histological score in parentheses. Data represent the mean ± SEM of (A) 3, (B) 3–5, (C) 4–9 mice per group, (D&E) 3 independent experiments.