T cell synergy is mediated by a soluble factor.

Cells were flow sorted, stimulated with anti-CD3ε antibody for 3 days, and treated as indicated. ELISA measurements of IL-10 concentration in culture supernatant from FoxP3+ Tregs co-cultured with IL-10-knockout RbLoTem (A), fixed RbLoTem or RbHiTn cells (B) or the change in IL-10 in the supernatant from cultured FoxP3+ Tregs supplemented with conditioned media from RbLoTem, RbHiTn or FoxP3+ Tregs stimulated previously for 3 days as before (C). Conditioned media from activated RbLoTem or RbHiTn cells were administered to HDM-challenged IL-10-GFP reporter mice intranasally and compared to resting and HDM-challenged mice without conditioned media. Mice were sacrificed and the BALf was analyzed by flow cytometry, showing percent IL-10+ among CD4+ cells in representative histograms (D) as well as replicates and the total CD4+ cell counts (E). ELISA measurements of IFNγ concentration in culture supernatants from FoxP3+ Tregs, WT RbLoTem, or both cells in co-culture with anti-CD3ε stimulation for 4 days (F). * = p0.05. P value calculated from Student’s T-Test. Error bars show mean with SEM. For A-C, n = 3 to 6 experiments. For E-F, n = 3 experiments.