Rheumatoid Arthritis (RA) is a chronic inflammatory disorder affecting 1% of the world population and results in the eventual destruction of bone and joints. A two amino acid Shared Epitope (SE) LA 67,74 in HLA-DRB1 is highly associated with RA and is also positively correlated with high levels of anti-citrullinated protein antibodies. In contrast, a single amino acid SE D 70 in HLA-DRB1 is associated with resistance in a resistance dominance fashion. We have previously demonstrated through assessment of peptide binding to 91 HLA class II alleles on Luminex beads that citrullinated vimentin binds significantly over native to HLA-DRB1 alleles containing the LA 67,74 epitope. In this study, we assess the importance of these HLA SE in binding of citrullinated peptides to alleles containing SE mutations.We measured citrullinated and native vimentin binding by flow cytometry on T2 cells transduced with either DRB1∗04:01 Wild Type (WT), DRB1∗04:02 WT, or DRB1∗04:01 containing the single mutations L67I and Q70D in addition to DRB1∗04:01 containing both mutations.DRB1∗04:01 WT bound citrullinated vimentin significantly over native. DRB1∗04:02 bound both citrullinated and native vimentin equally. Mutagenesis of DRB1∗04:01 L67I resulted in a modest reduction of binding of citrullinated vimentin, but excluded native vimentin binding. A Q70D mutation however allowed both citrullinated and native vimentin binding. DRB1∗04:01 containing both epitope mutations showed binding comparable to DRB1∗04:02.As observed in our Luminex peptide binding assay, we found that LA 67,74 displays a preference for citrullinated peptides. In contrast, the D 70 epitope confers no preference for citrullinated peptides. Our results suggest that the presence of LA 67,74 in the HLA cannot accommodate arginine in the binding pocket, whereas D 70 alleles allow both arginine and citrulline binding. This provides rationale for LA 67,74 associated susceptibility to RA in addition to D 70 associated resistance to RA.