T cell proliferation and HLA-DQ2 binding capacity of DQ2-Glia-α variants.

Variants of the DQ2-Glia-α1 and DQ2-Glia-α2 epitopes as encoded by the α-gliadin transcriptome were synthesized as deamidated 14- to 17-mer peptides (column 1, underlined: DQ2-Glia-α1/α2 epitope region) and tested for stimulation of DQ2-Glia-α1 and DQ2-Glia-α2 specific T cell clones in a proliferation assay. ± = 100 times reduced T cell stimulation compared to the ‘canonical’ epitope; - = 1000 times reduced T cell stimulation compared to the ‘canonical’ epitope. For each epitope shorter versions of the peptide variant, including the putative epitope core flanked by at least two amino acids, were tested in a cell free in vitro peptide binding assay for binding to HLA-DQ2 antigen presenting cells (lysates from HLA-DR3/DQ2 positive EBV-transfored B-cells). IC50 DQ2-Glia-α1/α2 = mean value of the half maximal inhibitory concentration (IC50) returned by the binding assays for respectively DQ2-Glia-α1 and DQ2-Glia-α2 epitope variants. IC50 values were calculated based on the observed competition between the tested peptides and biotin-labelled indicator peptides and indicate the concentration of the tested peptide required for half maximal inhibition of the binding of the indicator peptide.