FIGURE 1.

DCs loaded with anti-hDectin-1-HA1 can elicit HA1-specific Th₁, Th₂, and Th₁₇ responses. (A) Reduced SDS-PAGE analysis of anti-hDectin-1 mAb (lane 1), anti-hDectin-1-HA1 (lane 2) and IgG4-HA1 (lane 3). (B and C) Binding of anti-hDectin-1-HA1 and IgG4-HA1 to 293F cells transfected with hDectin-1 (B) and IFNDCs (C). (D) Total CD4⁺ T cell proliferation induced by IFNDCs alone or IFNDCs loaded with 1 μg/ml anti-hDectin-1-HA1, IgG4-HA1, anti-hDectin-1, or combination of IgG4-HA1 (1 μg/ml) and anti-hDectin-1 (1 μg/ml). (E) CFSE-labeled total CD4⁺ T cells were co-cultured for 7 days with 1 μg/ml anti-hDectin-1-HA1-loaded IFNDCs or blood mDCs. T cells were restimulated with 1 μM of the clusters (upper panels, IFNDCs) or individual peptides from cluster 8 (middle panels, IFNDCs and lower panels, mDCs). (F) CD4⁺ T cells were restimulated for 48h in the presence or absence of 1 μM peptides indicated. Cytokines in the supernatants were assessed. In (B-F), three independent experiments showed similar results. (G) CFSE-labeled total CD4⁺ T cells were co-cultured for 7 days with 1 μg/ml anti-hDectin-1-HA1-loaded IFNDCs. T cells were restimulated with 1 μM of the indicated peptides and IL-17 and IFNγ expression were analyzed by flow cytometry. Two independent experiments using cells from donors #1 and #2 showed similar results. One representative data generated with donor #1 are presented. (H) 1×10⁵ IFNDCs were incubated overnight with 1 μg/ml HA1 fusion proteins. The amount of IL-23 in the supernatants was assessed. (I) Total CD4⁺ T cells were co-cultured with 1 μg/ml anti-hDectin-1-HA1-loaded IFNDCs for 7 days in the presence of 5 μg/ml anti-IL-23p19 or control antibodies. CD4⁺ T cells were then restimulated with 1 μM peptides for 48h. Cytokines in culture supernatants were assessed. (J and K) Experiments in (H and I) were performed with blood mDCs. In (H-K), Error bars indicate mean±SD of triplicate assay.