Figure 8. DLE contains S100 peptides and binds target antigen.

(A and B) OVA:DLE or naïve DLE (0 and 1e2–1e8 ceq) was incubated in 96-well immunoplates overnight to replace the capture antibody in a standard sandwich ELISA. OVA or BSA was then added, and signal for bound OVA (A) and BSA (B) is shown versus expected value for the added concentration (dotted, horizontal lines). (C) 1e5 DC^OVA were subsequently cultured with diluent; 1e6 OVA:DLE; or 1, 10, or 100 μg e-OVA (no DLE), and IL-6 concentrations were measured after 18–24 h. (D) 1e5 DC^OVA cultured with 1e6 ceq of OVA:DLE extracted via ultrafiltration, dialysis, or both. (E) 1e6 DC^SIINFEKL, DC^OVA, or DC^HKCA from C57BL/6 mice were incubated for 24 h with 1e6 ceq of DLE from OVA-immunized mice or unimmunized OT-I or OT-II mice. Change (Δ) in IL-6 compared with diluent condition is shown. (F) Ninety-six-well plates were coated with 1e5 ceq of OT-I, single OVA immunized (OVA:DLE), repeat OVA immunized (Boosted), or naïve:DLE. An ELISA was performed using anti-TCR-α, TCR-β, S100a9, or isotype control as primary antibodies. Absorbance at 405 nm for TCR chains and S100a9 minus the absorbance for isotype control are shown with significance versus naïve:DLE or as indicated. (G) 1e5 DC^OVA were incubated with 1e6 ceq of OTI:DLE that had undergone antibody precipitation with antibodies to the TCR α chain (α)TCR-α, TCR-β, and/or S100a9. IL-6 production at 20 h is shown with significance versus the isotype condition (dotted, horizontal line). (H) IL-6 production from 1e6 DC^OVA from C57BL/6 wild-type (WT) or TLR4 knockout (TLR4^−/−) mice cultured with 5e4 ceq of DLE from CD8⁺/OVA tetramer-positive (Tet⁺) or tetramer-negative (Tet⁻) in the presence of anti-MHC-I or isotype control (Iso). Data shown are representative of 3 or more independent experiments, each using at least 3 mice/group, and displayed as means ± sem. Significance shown versus media control baseline or as indicated. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, as determined by ANOVA.