Hp91 uptake by DCs is dose, time, and sequence dependent . (A) Immature human DCs were pre-incubated on ice for 30 min, then incubated with biotinylated-Hp91 (0, 10, or 100 μg/ml) for 30 min at 37°C. Cells were permeabilized with Cytofix/Cytoperm, stained with Streptavidin-Alexa 488, and analyzed by flow cytometry. The results shown is one representative experiment (N = 6). (B) Immature human DCs were incubated with 100 μg/ml biotinylated Hp91 for 10 or 30 minutes, permeabilized, stained, and analyzed as above. Results shown are mean (±SEM) of N = 4. (C) Pre-cooled immature human DCs (iDCs) were incubated on ice for 30 min with 200 μg/ml biotinylated Hp91 or Hp121 to allow peptide binding, washed, then incubated for 15, or 30 additional min at 37°C. Cells were cytospun, fixed, permeabilized, and stained with Streptavidin-Alexa 488 to visualize biotinylated peptides (Green) and Hoechst DNA stain (Blue). Cells were imaged on a Zeiss LSM confocal microscope. Data shown is one representative experiment of N = 3. (D-E) Immature human DCs were pre-cooled on ice for 30 min, then incubated with media only, biotinylated-Hp91, biotinylated-Hp121, or biotinylated-scrambled Hp91 (“Scramble”) at 200 μg/ml for 30 min at 37°C. Cells were permeabilized, stained, and analyzed by flow cytometry as above. (D) is mean (±SEM) for N = 3 and (E) is one representative experiment. *p < 0.05 compared to medium; Student’s t-test. (F, G) Mice were immunized with OVA-I peptide in PBS with Hp91 or scrambled Hp91 peptide (250 μg). (F) Freshly isolated splenocytes from the immunized mice were examined in an OVA IFN-γ ELISpot assay. (G) Supernatants were collected and analyzed for IL-2 secretion by ELISA. The data shown is mean (±SEM) for 5–10 mice/group. *p < 0.05 between groups; Student’s t-test.