Figure 4. IL15-DCs primed with TLR3 or TLR4 agonists preferentially induce Th17 responses compared with IL4-DCs.

(A) IL4-DCs and IL15-DCs were primed with the panel of TLR agonists described in Fig. 3 and washed with PBS to remove cytokines and TLR agonists. CD4⁺ T cells were purified from autologous lymphocytes by negative selection and cultured with unprimed or TLR-primed DC populations in fresh cRPMI for 72 h. Indicated cytokines were measured in cell-free culture fluids by ELISA kits. Each symbol is the mean of duplicate ELISA values obtained from the different individuals tested after subtracting out levels from cocultures with unprimed IL4-DCs and IL-15DCs. Horizontal bars represent the means in pg/ml from five independent experiments. *P < 0.05, as determined by the paired two-tailed Student's t test. (B) Analysis of intracellular cytokine production by purified CD4⁺ T cells cultured with unprimed or TLR3-primed, autologous IL15-DCs in fresh cRPMI for 48 h following stimulation with PMA and ionomycin for the last 6 h; in the presence of monensin, for the last 4 h. Two hundred thousand CD4⁺CD3⁺ cells were gated on, and intracellular detection of FITC-IFN-γ and PE-IL-17 was determined by FACS. Numbers are percent-positive cells, as determined by appropriate intracellular Ig isotype controls. Density plots are from one of five different donors tested in A.