In Vitro T-Reg Cell Suppression Assay

For analysis of T_reg-cell suppression in vitro, CD4⁺CD25^hi T_reg cells or CD4⁺Foxp3-YFP⁺ T_reg cells, isolated from the lymphoid organs of the respective Cd4^Cre- or Foxp3^Cre-expressing mice, were co-cultured with naive CD4⁺ T cells and irradiated splenocytes as antigen presenting cells as previously described^{30 (link)}. For suppression assays using in vitro activated T_reg cells, CD25^hi T_reg cells were sorted from the lymphoid organs of C57BL/6 mice, resuspended in complete Click’s medium containing IL-2 (200 U ml⁻¹), and activated using anti-CD3 (10 μg ml⁻¹) and anti-CD28 (10 μg ml⁻¹) antibodies for 3 days in the presence of PP242 (500 nM, Tocris Bioscience) or vehicle control. The live cells were then isolated using Lymphocyte Separation medium and co-cultured with naive CD4⁺ T cells and irradiated splenocytes for 3 days, and the incorporation of [³H]-thymidine was assessed as described^{30 (link)}.