Figure 1. Citrulline residues are required in cit-TNC17 and cit-TNC56 peptides for HLA-DRB1*0401 binding and immunogenicity.

(A) The binding of cit-TNC and their counterpart arginine peptides (arg-TNC) to DRB1*04:01 was tested using a peptide competition assay; each peptide was tested in triplicate. Europium-conjugated streptavidin was used to label residual biotinylated peptide bound to the HLA-DR protein. Binding curves were fitted by nonlinear regression with a sigmoidal dose response curve model, and EC₅₀ values were calculated as the peptide concentration needed to displace 50% of the reference peptide. The HA306–318 peptide was used as a positive control. The EC₅₀ cutoff for measurable binding was 50 μM. Results indicated that citrullination at 1 specific residue was required for cit-TNC17 and cit-TNC56 binding. X stands for 1 citrullinated amino acid in cit-TNC; R stands for 1 arginine amino acid in arg-TNC. (B) Citrullination of a distinct T cell contact residue was associated with cit-TNC17 and cit-TNC56 immunogenicity. Representative plots showing representative CD4⁺ T cell response to TNC peptide citrullinated at either both residues (TNC17XX and TNC56XX) or only 1 residue (TNC17RX and TNC56XR); n = 14 RA subjects tested for all peptides.