Figure 5
Factors influencing peptide loading - MHC loading enhancers and peptide processing machinery.
(a + b) Effect of MHCII loading enhancers (MLE) on exogenous (a) or endogenous (b) peptide loading. Art v 1 (left) or Bet v 1 (right) specific T cell reporters were stimulated with eAPC exogenously loaded with peptide or endogenously loaded with immunodominant peptides expressed as invariant chain fusion proteins without MLE or in presence of MLE. (c) Influence of molecules involved in MHCII antigen processing on exogenous peptide loading. Art v 1 (left) or Bet v 1 (right) specific T cell reporters were stimulated with allergenic peptides in presence of eAPC overexpressing the indicated molecules. (d) Influence of molecules involved in MHCII antigen processing on endogenous loading. Art v 1 specific T cell reporters were stimulated with eAPC expressing Art v 1 peptides as invariant chain fusion protein alone or in combination with the indicated molecules. (e) Evaluation of the synergy between HLA-DM and non-peptide MLEs. Art v 1 specific reporter cells were stimulated with Art v 1 peptide in the presence of control eAPC or eAPC overexpressing HLA-DM. MLE were added as indicated. Statistics by two-way ANOVA, followed by Bonferroni post-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns not significant). Art v 123–36 and Bet v 1142–153 peptides were used at 5 and 0.5 μg/ml for exogenous loading, respectively. The non-peptide MLEs 2-(1-adamantyl) ethanol (short AdEtOH) and p-chlorophenol (short p-CP) were used at concentrations of 100 μM and 1000 μM, respectively. The peptide-MLEs AE206 (NH2-LRLKLPK-COOH) and Di-Peptide (Ac-NH-FR-CONH2) were evaluated at a concentration of 150 μM. Mean fold induction is shown for duplicate values and median fold induction of multiple experiments is indicated as line. Statistics (a–d) by one-way ANOVA, followed by Tukey’s multiple comparison post-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns not significant).
