scFvMTBHsp70 induces DC maturation and promotes antigen presentation and cross-presentation. A, CD11c+ BMDCs isolated form FVB/NJ mice were incubated for 24 h with 2 μg/ml scFvMTBHsp70, 1.3 μg/ml MTBHsp70, 1 μg/ml LPS as positive control, or 0.1 ng/ml LPS as contamination control (thick lines), or medium only (solid), stained for CD11c, CD40, CD80, CD86, and MHC II, and analyzed by flow cytometry. Histograms were gated on CD11c+ DCs. Data are representative of three independent experiments in duplicate wells. B, Median fluorescence intensity (MFI) of LPS- or protein-stimulated BMDCs normalized to MFI of BMDCs maintained in medium. Data are expressed as means ± SEM in arbitrary units. P values were determined using One-Way ANOVA followed by Dunnett’s multiple comparison tests. C, BMDCs cultured from FVB/NJ mice were pulsed with BR5FVB1 cells alone (Column a), or BR5FVB1 cells pre-complexed with MTBHsp70 (Column b) or scFvMTBHsp70 (Column c), and then incubated with BR5FVB1 tumor cell-primed T cells. Intracellular granzyme B and IFNγ expressions in CD3+CD4+ and CD3+CD8+ T cells were analyzed by flow cytometry. Data from three independent experiments in duplicate wells are pooled and analyzed using One-Way ANOVA followed by Turkey’s multiple comparison tests. Data are presented as mean ± SEM. D, Representative flow data are presented. E, scFvMTBHsp70 enhanced tumor cell immunogenicity in vivo. Results are reported as the difference between nonstimulated (media alone) and stimulated cells and expressed as the frequency of parent CD3+CD4+ or CD3+CD8+ cells. P values were determined using One-Way ANOVA followed by Turkey’s multiple comparison tests. *,p < 0.05; **,p < 0.01; ***,p < 0.001; ****,p < 0.0001.