Immune profiling of anti-SSA-positive at-risk individuals reveals increased type I IFN activity preceding lymphocyte abnormalities in Sjögren's disease.

Objectives: Identifying immune features that distinguish asymptomatic autoantibody-positive at-risk individuals from patients with established autoimmune disease is key to understanding the evolution from autoreactivity to established autoimmune disease. This study investigated immune characteristics of anti-SSA-positive at-risk individuals, including pregnant women, compared with patients with Sjögren's disease (SjD) and healthy donors (HDs). Methods: Peripheral blood samples were obtained from asymptomatic anti-SSA-positive women (at-risk individuals; n = 7, including two ongoing pregnancies with congenital heart block [CHB] and one woman with a prior CHB pregnancy), patients with SjD (n = 10), and age- and sex-matched HDs (n = 21; median age 35.1 years; 100% female). T- and B-cell subsets and IFN signaling molecules, including STAT1, phosphorylated STAT1 (pSTAT1), STAT2, IRF1, and IRF9, were analyzed by flow cytometry. In addition, circulating IFN subtypes were quantified in available plasma samples using the NULISA platform. Results: At-risk individuals showed lower ANA and rheumatoid factor titers and lacked anti-SSB autoantibodies compared with patients with SjD. At-risk individuals had B-cell distributions similar to HDs, whereas patients with SjD showed a typical increase in naïve B cells and reduced pre-switched and switched memory B-cell frequencies, together with increased CD8+ T-cell frequencies and a non-significant trend toward reduced double-negative T-cell frequencies. At-risk individuals also showed significantly increased IFN activity, reflected by Siglec-1 (CD169) expression on CD14+ monocytes, compared with HDs, while levels tended to be lower than in patients with SjD. Two of three women with a current or previous CHB pregnancy had the highest Siglec-1 levels within the at-risk group. Cytokine profiling revealed a stage-associated IFN pattern, with IFN-α1 and IFN-ω already increased in at-risk individuals, whereas IFN-γ, IFN-β1, and type III IFNs showed a trend toward higher levels in established SjD. Intracellular STAT1 expression was elevated across CD19+ B-cell subsets in at-risk individuals compared with HDs, but was generally less pronounced than in SjD. IRF9 was also increased in B-cell subsets from at-risk individuals and patients with SjD compared with HDs. Similar findings were observed in CD3+ T-cell subsets, where STAT1 and IRF9 expression were elevated in at-risk individuals compared with HDs and were largely comparable to levels observed in patients with SjD. Conclusions: Our data suggest that increased type I IFN activity and IFN-associated signaling in T- and B-cell subsets are already present in anti-SSA-positive at-risk individuals. In contrast, altered B- and T-cell homeostasis was restricted to established SjD, indicating progression from autoreactivity toward overt autoimmune disease.