Background/Purpose: Carriage of HLA-DRB1 shared epitope (SE) alleles and a history of smoking, have been identified as the most prominent risk factors for development of autoantibody positive rheumatoid arthritis (RA). The hallmark autoantibodies, anti-citrullinated protein antibodies (ACPA) can target various antigens, resulting in heterogeneous ACPA profiles among patients. Their exact nature and etiological significance remain unknown. ACPA are multi-reactive and bind citrulline-amino acid motifs where “cit-gly” is a common target. We investigated the co-occurrence of RA-specific autoantibodies, and ACPA-patterns by recognized citrulline (cit)-motifs in relation to HLA-DRB1 and smoking.

Methods: We measured rheumatoid factor (RF) IgM IgG and IgA, anti-cyclic citrullinated peptide (CCP2) IgG and IgA, and IgG to 15 anti-cit- peptides elected to cover different cit-motifs and 4 Carb/Acet-peptides, in 6,907 patients from five Scandinavian RA cohorts [1]. Analyses were performed centrally using fluoroenzyme immunoassay and custom-made multiplex solid-phase microarray. Cutoffs was set based on 400 healthy controls and high signals were 3x cutoff. HLA-DRB1 SE alleles were imputed from SNP genotyping data.

Results: Compared to using only CCP2-IgG and/or RF-IgM, seropositivity increased from 76% to 84% when all autoantibody tests were included. Single-cit peptides derived from four multi-cit peptides (Cit Fiba_36-50, Cit Fibb_60-74, Cit TNC5, Cit Vim_60-75) showed differential binding patterns, supporting recognition of cit-motifs rather than long peptides. Four cit-peptides (Cit Fibb_36-52, Cit Fibb_60-74-Cit3, Cit Fil_307-324, Cit Vim_60-75-Cit1) (table 1) captured 97% of IgG anti-CCP2+ patients. Different ACPA overlapped, but when dichotomizing patients based on high reactivity ( >3x cutoff) to peptide cit-motifs, non-glycine but not glycine-motif ACPA associated with HLA-SE alleles (figure 1). 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, yielding an odds ratio (OR) of 4.5 (95% CI: 1.9-11, Fisher’s exact test). Smoking status did not correlate with fine-specificity subsets or IgG anti-Carb/Acet but independently associated with IgA/IgM RF and IgA/IgG anti-CCP2 double positivity.

Conclusion: While glycine-motifs are commonly citrullinated and prevalent ACPA-targets, our data in this large cohort of routine care treated patients with RA reveals that HLA-SE alleles are primarily associated with ACPA to non-glycine cit-motif, providing new insight in ACPA epitope spreading. The association between IgA and smoking supports a chronic mucosal response. Our results advance our understanding of citrulline responses in relation to key environmental and genetic RA risk factors.

Reference:

1. Westerlind et al RMD Open 2023 Sep;9(3):e003027

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/refined-autoantibody-profiles-in-ra-reveal-that-primarily-acpas-binding-non-glycine-citrulline-motifs-are-associated-with-shared-epitope-alleles/