Figure 3.

Determination of MHC-II binding affinities of VP1 epitopes and antigen-specificity of T cell responses by MHC class II dextramer staining. (a) MHC-binding affinity. The reaction mixtures containing thrombin-cleaved IA^k monomers and competitor peptides VP1 681–700, VP1 721–740 and VP1 771–790, and biotinylated reference peptide, HEL 46–61, were individually prepared and added in duplicates to fluorescence plates coated with anti-IA^k. After washing and the addition of europium-labeled streptavidin and DELFIA-enhancer, fluorescence intensities were measured at excitation/emission wavelengths of 340/615 nm, and IC₅₀ values were then calculated. Representative data from two individual experiments, each involving two replicates are shown. (b) Antigen-specificity. Lymphocytes prepared from animals immunized with individual VP1 peptides (VP1 681–700, VP1 721–740 or VP1 771–790) were stimulated with the corresponding peptides and were maintained in medium supplemented with IL-2 for a week. Cells were harvested between days 7–10 post-stimulation and stained with indicated IA^k/dextramers, anti-CD4 and 7-AAD. After acquiring the cells by flow cytometry, dextramer positive cells were analyzed in the live (7-AAD¯) CD4 subset using FlowJo software. Representative flow cytometric dot-plots for specific and control dextramers are shown in the left panel. Data sets obtained from three individual experiments, each involving three to four mice are shown in the right panel. RNase 43–56, control. ** p ≤ 0.01, and **** p ≤ 0.0001.