Treg suppression of the MLR-IL-2 response requires dendritic cell IL-2.

<p>Panels A–F show Treg suppression of IL-2-secreting cells in primary MLR cultures. MLR cultures of DO11.10 CD4⁺CD25⁻ cells and allogeneic DCs from WT or IL-2^−/− KO littermates were incubated overnight with or without DO11.10 Tregs, washed, and transferred to IL-2-ELISPOT plates to measure IL-2-secreting cells as per materials and methods. <b>A&B:</b> Tregs fail to suppress MLR-IL-2 response in cultures containing IL-2^−/− KO DCs. Bars are mean ± SE IL-2 producing cells/culture from N = 5 independent experiments with splenic DCs (A) and N = 3–4 independent experiments with BMDCs (B). * indicates <i>p</i><.001 (A) and <i>p</i><.005 (B) compared with all other conditions, Holm-Sidak method of multiple pairwise comparison. <b>C&D:</b> Anti-CD25 blocks Treg suppression of MLR-IL-2 response. MLR cultures of DO11.10 CD4⁺CD25⁻ cells and allogeneic WT splenic DCs (C) or BMDCs (D), with or without Treg, were treated with anti-CD25 or isotype control antibody, washed, then transferred to IL-2-ELISPOT plates as in material and methods. C: Symbols are mean ± SE IL-2 producing cells/culture from N = 6 independent experiments. * indicates <i>p</i><.001, anti-CD25 versus isotype control, Holm-Sidak method of multiple pairwise comparison. D: Symbols are mean ± SE IL-2-producing cells of triplicate cultures from one of four representative experiments. * indicates <i>p</i><.001, Treg treatment groups anti-CD25 versus isotype control, Tukey's test for all pairwise multiple comparison. <b>E&F:</b> Exogenous IL-2 restores Treg suppression of MLR cultures containing IL-2KO DCs. MLR cultures of DO11.10 CD4⁺CD25⁻ cells and allogeneic IL-2^−/− KO splenic DCs (E) or BMDCs (F), with or without Treg, were treated with exogenous IL-2, washed, then transferred to IL-2 ELISPOT plates as in material and methods. Symbols are mean ± SE IL-2 producing cells of triplicate cultures from one of three representative experiments. * indicates <i>p</i><.001, IL-2 treatment DC/CD4eff + Treg versus DC/CD4eff control, Tukey's test for all pairwise multiple comparison.