FIGURE 2.

Effect of GSK-3β inhibition, using SB216763, on Treg cell suppression. Performing a classical suppression assay, CD4⁺CD25⁻ T cells and CD4⁺CD25⁺ Treg cells were isolated using magnetic separation and co-cultured in a 1:1 ratio with anti-CD3/CD28 microbeads with and without SB216763. Cells were pulsed with tritiated thymidine after 18–20 h, and proliferation was measured. Using this assay, we show that SB216763 potentiates Treg cell function in vitro as judged by increased suppression of T effector CD4⁺CD25⁻ cell proliferation when T effector CD4⁺CD25⁻ cells were incubated at a 1:1 ratio with CD4⁺CD25⁺ Treg cells in 5 μm SB216763 (86% inhibition versus 65% without the drug (DMSO vehicle control)). Moreover, cells incubated in a 1:8 ratio, respectively, with Treg cells again in 5 μm SB216763 demonstrate 95% inhibition versus 78% without the drug, suggesting that GSK-3β inhibition can significantly enhance Treg cell suppression. Data are represented as means ± S.E. of triplicate samples. All data are representative of at least two independently performed experiments.