Induction of DC maturation and cytokines secretion by recombinant Hsp70L1 protein. (A) Phenotypic maturation of DCs induced by recombinant Hsp70L1 protein. DCs were treated with Hsp70L1, Hsp70, LPS, or His-CK for 48 hours then collected for FACS analysis of CD40, CD80, CD83, CD86, and HLA-DR expression. Gray histograms indicate autofluorescence; open thin line histograms, His-CK–treated DCs; and open thick line histograms, HSP- or LPS-treated DCs. (B) Functional DC maturation, assessed by MLR. DCs pretreated with the indicated stimuli, as described for panel A, were irradiated and used as stimulators, with T cells from a different donor as responders. T-cell proliferation was measured by thymidine incorporation. Experiments were repeated 3 times, and data are displayed as means ± SEM. (C) Cytokine production of DCs stimulated with recombinant Hsp70L1. DCs were cultured with Hsp70L1, Hsp70, His-CK, or LPS proteins in 3 formats; native protein (▪), native protein in the presence of 50 μg/mL PMB (▪), or denatured protein (100°C, 20 minutes) for 24 hours (□). The levels of IL-12p70, TNF-α, and IL-1β in supernatants were measured by ELISA. Data are displayed as mean cytokine concentration (pg/mL) ± SEM. P < .05 for IL-12p70 production between Hsp70L1 and human Hsp70.