Association of phenotypic and functional characteristics of regulatory T cells from minor salivary glands with expression of the delta 7 FoxP3 splice variant in patients with Sjögren's disease.

Background: Sjögren's disease (SjD) is a systemic autoimmune disorder characterized by lymphoplasmacytic infiltration of exocrine glands, including minor salivary glands (MSG), with potential formation of ectopic germinal centers. Regulatory T cells (Tregs) are critical suppressors of autoimmune responses, and their dysfunction has been implicated in SjD pathogenesis. FoxP3, the master transcription factor of Tregs, undergoes alternative splicing to produce four variants: full-length (FL), Δ2, Δ7, and Δ2Δ7. While FoxP3 splice variants are known to influence Treg proliferation and suppressive activity, their profile and functional significance in Tregs from SjD patients remain poorly characterized. Methods: Peripheral blood was collected from 20 SjD patients or 20 healthy donors (HD). MSG biopsies were obtained from 10 SjD patients. Tregs were isolated from blood and biopsy tissue by immunomagnetic separation and expanded ex vivo. FoxP3 splice variant mRNA levels were quantified by real-time RT-PCR. Treg phenotype (CD4, CD25, CD127, CD152, Helios), suppressive molecules (CD39, CD223, granzymes A/B, perforin), was assessed by flow cytometry. The concentrations of suppressive cytokines IL-10 andIL-35 were accessed by Bio-Plex assay. Proliferation was evaluated by cell counting. Suppressive activity was determined by mixed lymphocyte reaction. Splice-switching oligonucleotides (SSOs) were used to induce Δ7 FoxP3 expression to confirm direct functional effects. Results: Peripheral blood Tregs from SjD patients showed significantly decreased increased Δ7 FoxP3 splice variant compared to HD. MSG-derived Tregs from SjD patients also predominantly expressed the Δ7 FoxP3 variant. High-expressing Δ7 FoxP3 Tregs exhibited reduced proliferative activity, division frequency and decreased suppressive activity compared to low-expressing Δ7 FoxP3 Tregs. High-expressing Δ7 FoxP3 Tregs showed significantly reduced CD127low and Helios expression, decreased CD39, CD223, granzyme, and granzyme B. IL-10 and IL-35 production was also reduced. SSO-mediated induction of Δ7 FoxP3 MSG-derived Tregs confirmed these phenotypic and functional changes. Conclusions: This study demonstrates that elevated Δ7 FoxP3 splice variant expression in MSG-derived Tregs from SjD patients is associated with impaired proliferative and suppressive function, reduced expression of suppressive molecules and cytokines, and a phenotype suggestive of a transitional or immature Treg state. The results highlight the functional consequences of altered FoxP3 alternative splicing in SjD pathogenesis and suggest a possible involvement of Tregs in the biology of ectopic germinal centers in SjD.