Figure 4. DLE activates DCs to stimulate memory Tc17 cells.

Splenocytes were harvested from OVA-naïve mice and then sorted by flow cytometry before being placed into coculture with DC^OVA in 1 ml total media. 2e6 ceq of OVA:DLE was added to the media. (A) 1e6 CD3⁺ splenocytes sorted by CD4 and CD8 positivity cocultured for 72 h with 4e5 DC^OVA. Differences (Δ) in IL-17A from media control without DLE are shown. (B) IL-17A levels at 72 h from 2e5 CD3/8⁺ cells subsorted by CD62L and CD44 expression before coculture with 4e4 DC^OVA in the presence of 2e6 ceq of OVA:DLE. (C) 2e6 CD3⁺/CD4⁺ cells were cultured under Th17 conditions, with and without 2e6 ceq of OVA:DLE. IL-17A at 72 h is shown. (D–F) 2e6 whole splenocytes were cultured with 4e5 DC^OVA separated by a semipermeable Transwell membrane. 1e6–5e6 ceq of OVA:DLE was added to either the DC (above the transwell membrane) or splenocyte compartment (below the membrane). Controls included cocultures without the Transwell separator and DC^OVA cultured in isolation (DC, no spleens). Change vs. diluent control for IL-17A after 72 h (D) and IL-6 at 24 h (E) are shown (dotted line in E represents no change from diluent control). (F) Antibodies against IL-6 and/or IL-1β were added to the DC compartment before coculture with whole splenocytes and OVA:DLE; change in 72 h values of IL-17A versus media control without DLE are shown. (G and H) IL-17 (G) and IL-6 (H) induced by OVA:DLE (0, 1e4, 1e5, and 1e6 ceq) from cocultures of whole splenocytes and DC^e-OVA. (I) IL-6 production from 5e5 DC^OVA derived from C57BL/6 wild-type (WT) mice or TLR4^−/− mice was incubated for 18 h with 1e6 ceq of OVA:DLE or diluent control. Antigen-naïve splenocytes were assayed independently from at least 3 mice/group. Data shown are representative of 3–4 independent experiments and displayed as means ± sem. Significance shown versus media control baseline or as indicated. ****P < 0.0001, ***P < 0.001, **P < 0.01, as determined by ANOVA.