Data Sheet 1_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.docx

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.</p>Results<p>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.</p>Conclusions<p>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.</p>