Figure 4.

CD4+ Teff cell suppression assay using Tregs cells and MCs. (A) A standard coculture system was designed using varying ratios of CD4 Teffs and CD4+ Tregs derived from LNs of mice draining sites of MPO immunization. Coculture of CD4+foxp3− LN cells from MPO-immunized mice with CD4+foxp3+ cells of a ratio of 32:1 reduced Teff cell proliferation responses to MPO by 18%. All cells were cultured in the presence of antigen-presenting cells, and results are representative of two independent ex vivo experiments. (B) Coculturing Teffs with MCs does not increase MPO proliferative responses. Minimal baseline proliferation from MCs cultured with MPO was observed (dotted line). Results are expressed as an average from four independent experiments. (C) The capacity of MCs to enhance Treg immunosuppression was assessed in the presence or absence of either wild-type or IL-10−/− MCs in a ratio of 32 Teffs to 1 Treg to 1 MC. Wild-type MCs significantly enhanced MPO-specific Treg suppression shown by a 40% reduction in T effector cell proliferation, whereas the addition of IL-10−/− MCs had no effect. Results are expressed as an average from four individual experiments. (D–F) There is a decrease in production of proinflammatory cytokines IFNγ, IL-17A, and IL-6 by Teffs when cultured in the presence of Tregs and wild-type MCs. Results are representative of three independent experiments. Error bars represent mean ± SEM with statistical analysis by one-way ANOVA. **P<0.01.