Differences in cytokine secretion levels between IgM⁺IgD⁺CD27⁺ and switched B cells in response to different TLR agonists. Switched (IgG⁺ and IgA⁺) CD27⁺ cells and IgD⁺CD27⁺ were sorted and activated with different agonists, and cytokine secretion in culture supernatants after 4 days was evaluated by multiplex assays (see “Methods”). CpG (TRL9 agonist), R848 (TLR7 and TLR8 agonist), and Pam₂CSK4 (Pam2, TLR2/6 agonist) did induce a reproducible secretion of IL-6, IL-8, MIP-1-α, and MIP-1-β (and IL-2R and IL-1RA for CpG stimulation). Because of a high variability of the amount of cytokines secreted by B-cell subsets from one splenic sample to another, the mean ratio of cytokine secretion ± SD between IgD⁺CD27⁺ and switched cells from the same sample is represented. The data were obtained from 3 or 4 independent experiments. The dashed line indicates a ratio equal to 1.