Background/Purpose: Extracellular Vesicles (EVs) are double lipid bilayer-enclosed membranous vesicles, produced and discharged from almost all cells. EVs are known for inter-cellular communication by releasing complex chemical messages and have the capacity of inducing functional responses in recipient cells. EVs can also serve as circulating markers of diseases. Our hypothesis is that circulating EVs could be a platform of autoantigen recognition to induce the secretion of autoantibodies known in
Sjögren’s syndrome (SjS). SjS is the second most frequent chronic systemic autoimmune disease with a prevalence ranging between 0,1% and 0,6% in the overall adult population.
Presence of autoantibodies (anti-Ro52/TRIM21, anti-Ro60/Trove2 and anti-La/SSB), rheumatoid factor and other conventional diagnostic methods are commonly used for SjS diagnosis, but these markers are lacking in one third of the patients and their pathogenic role is still debated.

Methods: SjS patients fulfilling the AECG 2002 and/or ACR/EULAR 2016 classification criteria from the “I GET DRY” study
(
NCT03003572
)
were included and divided in 2 subgroups
based on their ESSDAI score: High Activity (HA, ESSDAI ≥ 5; n = 17) and Low Activity (LA, ESSDAI < 5; n = 31), along with non-SjS sicca syndrome patients (S; n = 14) and healthy blood donors (H; n=20). To explore the size of EVs, serum samples were characterized with Dynamic Light Scatter (Malvern). To evaluate the EV’s cell origin, several antibodies against lymphoid, platelet, epithelial and myeloid markers were used in combination with anti-La/SSB, Ro52 and Ro60 antibodies via  spectral flow cytometry (Cytek Northern Light). Validation of EV detection was performed with specific treatment such as Triton, centrifugation, and EDTA for flow cytometry. Characterization of EV content was performed using Western Blot analysis.

Results: In serum samples from all groups, large EVs were predominant and their concentration were significantly modulated through the disease state. Annexin V+ CD3+CD4+CD8+ or CD45+, CD14+, CD15+, CD41+ EV concentrations were significantly higher in LA and HA groups compared to Healthy donors (H). Interestingly, CD15+, CD19+, and CD45+ EVs expressed Ro52, Ro60 and La/SSB, and their concentration were significantly increased in LA and HA patients’ sera. These flow cytometry data were analyzed with unbiased manner, with a SPADE algorithm. We obtained similar data. Then we performed a correlation study between serum concentrations of anti-La/SSB, anti-Ro52 and anti-Ro60 autoantibodies that highlighted several significative correlations between level of expression of La/SSB, Ro52 and Ro60 autoantigens within different types of EVs. In parallel,  Principal Component Analysis, t-SNE analysis revealed the multiple possibility of expression of autoantigens on EVs with the complexity of the EVs (origin, composition etc.).

Conclusion: EVs from patient suffering from SjS, carry special markers of the disease that prompted us to use EV detection methods in clinics as an additional and complementary method to better understand their role and possible pathogenic mechanisms.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/extracellular-vesicles-from-lymphocyte-b-and-neutrophil-presents-ro52-ro60-and-la-autoantigens-in-patient-suffering-from-sjogren-syndrome/