Recognition of glycine versus non‐glycine citrulline motifs dictates the HLA class II association of anti‐citrullinated protein antibodies ‐ insights from autoantibody profiling of 6900 Scandinavian rheumatoid arthritis patients

Rheumatoid arthritis (RA) associated anti-citrullinated protein antibodies (ACPA) target various antigens by binding short citrulline-amino acid motifs, resulting in heterogeneous ACPA-profiles among patients. Here we analyzed ACPA-patterns by recognized citrulline-motifs in relation to the RA risk factors HLA-DRB1 shared epitope (SE) alleles and history of smoking. Rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP2) isotypes, 15 anti-Cit- and four anti-Carb/Acet-peptide-IgG, were centrally measured in 6,907 patients from five Scandinavian RA cohorts using fluoroenzyme immunoassay and a custom-made multiplex solid-phase microarray. HLA-DRB1 SE alleles were imputed from SNP genotyping data. Single-citrulline peptides derived from four multi-citrulline peptides (Cit Fibα36-50, Cit Fibβ60-74, Cit TNC5, Cit Vim60-75) showed differential binding patterns, supporting recognition of citrulline-motifs rather than long peptides. Four citrulline-peptides (Cit Fibβ36-52, Cit Fibβ60-74-Cit3, Cit Fil307-324, Cit Vim60-75-Cit1) captured 97% of IgG anti-CCP2+ patients. Patient subsets based on ACPA, anti-Carb/Acet, and RF displayed differences in ACPA composition and disease activity, but not comorbidities. Different ACPA overlapped, but when dichotomizing patients based on high reactivity to peptide citrulline-motifs, only ACPA to non-glycine citrulline-motif associated with HLA-SE alleles. In IgG anti-CCP2+ patients, 90% of those with only high non-glycine ACPA were HLA-SE allele carriers, compared to 67% in the group with glycine-motif only ACPA (OR=4.5). Smoking status associated with IgA and glycine-motif ACPA. While citrulline-glycine-motifs are prevalent ACPA-targets, our data reveals that HLA-SE alleles are primarily associated with ACPA to non-glycine citrulline-motifs, providing insight in ACPA T-cell dependance. Yet, the etiological significance of ACPA targeting different protein structures remains unknown.