Figure 7
Devising eAPCs efficiently presenting whole allergens.
(a) Uptake transport to lysosomal compartments of Bet v 1 coupled to pHrodo® visualized by fluorescence microscopy. Sections of microscopic pictures representative for multiple experiments are shown. (b) Effect of HLA-DM on the capacity of eAPCs to present allergenic peptides and to process whole allergen for MHCII presentation. Bet v 1 specific T cell reporters were cocultured in presence of control eAPC and eAPC expressing HLA-DM. Allergenic peptide Bet v 1142–153, Bet v 1 or birch pollen (BP) extract was added at the indicated concentrations. Mean fold induction is shown for duplicate values and median fold induction of multiple experiments is indicated as line. Statistics by two-way ANOVA, followed by Bonferroni post-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns not significant). (c) Comparison of the stimulatory capacity of eAPCs and EBV cell lines using Bet v 1 specific T cell reporters (antigen source as indicated in (b)). Mean fold induction ± SD is shown for duplicate values. (d) Schematic illustration of a fusion protein of LAMP1 (lysosomal-associated membrane protein 1) and the whole Bet v 1 allergen; (TM … transmembrane domain). (e) Effect of HLA-DM on the reporter activation using stimulator cells harboring the Bet v 1-LAMP1 fusion protein. eAPC expressing the Bet v 1-LAMP1 fusion protein and eAPC coexpressing the Bet v 1-LAMP1 fusion protein and HLA-DM were used to stimulate Bet v 1 specific T cell reporter cells. Mean fold induction is shown for duplicate values and median fold induction of multiple experiments is indicated as line. Statistics by Mann-Whitney test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns not significant). (f) A CFSE-labeled Bet v 1-specific T cell line was cocultured with eAPC expressing HLA-DRB1*01 and CD80. eAPC expressing the Bet v 1-LAMP1 fusion protein or unloaded eAPC were used. Following 5 days of coculture, cells were harvested, stained for CD4 expression and analyzed by flow cytometry.
