Background/Purpose: Sjögren’s Disease (SjD) is characterized by aberrant autoimmune reactions in lacrimal and salivary glands (SG) leading to severe dryness. Evidence for B cell help in situ in SjD SG predicts recognition of common antigens by interacting T and B cells. We identified 149 candidate autoantigens by screening SG plasmablast-derived recombinant monoclonal antibodies (mAbs), stimulated parotid saliva, and plasma from SjD cases, on human proteome arrays. Our objectives were to determine whether the peptides eluted from SjD SG HLA-DR match candidate autoantigens discovered with proteome arrays and to detect any differences in SG tissue-enriched HLA-DR peptide presentation between SjD and non-SjD (nSjD) sicca subjects.

Methods: HuProtä arrays (CDI Labs) were screened with recombinant mAb pools (n=14 pools from 7 cases), citric acid-stimulated parotid saliva, or plasma from SjD cases (n=30) and healthy controls (HC; n=15)¹. Candidate antigens were prioritized by the presence of autoantibodies in ³3 cases and/or tissue enrichment in SG. HLA DR molecules were immunoprecipitated from SG biopsies following solubilization in a mild detergent solution using LB 3.1 immunoaffinity chromatography and a microscale HLA isolation protocol. Bound peptide ligands were analyzed using a Bruker timsTOF Pro 2 mass spectrometer after separation using an EvoSep One LC coupled to an IonOpticks Aurora Elite nanoflow column using 20 SPD methodology. Incidence and abundance of peptides were evaluated using Fisher’s Exact and Mann-Whitney U tests at a=0.05.

Results: Peptides eluted from SG HLA-DR of SjD (n=8) and nSjD (n=22) subjects mapped to 35/149 candidate autoantigens and 88 SG tissue-enriched proteins (Protein Atlas). The number of peptides detected per subject varied from 1-100, and the average peptide length was 15.5 amino acids. Of the 35 candidate autoantigens from which peptides were detected, peptides from 13 were found in SjD only, 7 in nSjD only, and 15 in both. Peptides encoded by HNRNPAB and TPI1 were detected more frequently among SjD vs. nSjD (3/8 SjD; 0/22 nSjD, p=0.014), while greater quantities of peptides encoded by RALY (p=0.036) and TPI1 (p=0.014) were detected in SjD vs. nSjD. Of the 88 SG tissue-enriched proteins from which peptides were detected, peptides from 17 were found in SjD only, 18 in nSjD only, and 53 in both. Peptides encoded by ELAPOR1, MGAM2, and SLC12A2 were detected more frequently (p=0.016, p=0.029, and p=0.032, respectively) and in greater in abundance (p=0.003, p=0.006, and p=0.043, respectively) in SjD vs. nSjD. Peptides from APCDD1L (3/8 SjD; 0/22 nSjD, p=0.014) and FAM3D (7/8 pSjD; 9/22 nSjD, p=0.040) were detected more frequently, while peptides from NDRG2 were detected in greater abundance (p=0.039) in SjD vs. nSjD.

Conclusion: This is the first study reporting HLA-eluted peptides from SG tissue in SjD. Further studies will i) assess peptides bound by HLA-DQ, -DP, -A, -B, and -C, ii) look for overall differences in presentation between SjD and nSjD groups, iii) incorporate HLA haplotypes, and iv) test SG TCRs for recognition of peptides from individuals harboring antibodies to the same protein.

¹Longobardi, et al. Ann Rheum Dis. 82:1181-1190, 2023.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/peptides-from-candidate-sjogrens-disease-autoantigens-and-salivary-gland-tissue-enriched-proteins-eluted-from-human-salivary-gland-hla-dr/