Single-Cell Analysis of Tfh ICOS and IL-21 Variation Links Local Immune Activation to Anti-Thyroid Antibodies in Graves’ Disease

Graves' disease (GD) is an autoimmune cause of hyperthyroidism in which T cell and B cell interactions drive thyroid autoantibody production.To test whether intrathyroid T follicular helper (Tfh) signatures relate to downstream immune activation, we analyzed single-cell RNA sequencing (scRNA-seq) data from GD and healthy thyroid tissue.Data were processed in Scanpy with filtering, normalization to 10,000 reads per cell and log-transformation, PCA (principal component analysis, summarizing gene expression into components that capture variation across cells), and Leiden clustering.Batch effects were addressed using BBKNN (Batch Balanced K-Nearest Neighbors), which builds a batch-balanced neighbor graph by selecting neighbors from each sample or batch.We examined Tfh-cell ICOS and IL21 because ICOS co-stimulation supports Tfh survival and IL-21 secretion, and IL-21 promotes B-cell activation and differentiation.ICOS expression was quantified as normalized ICOS transcript abundance in Tfh cells and summarized per donor.We integrated these signals with a per-sample B-cell activation index and serum anti-thyroglobulin (anti-TG) and anti-thyroid peroxidase (anti-TPO) titers.At the donor level, mean Tfh ICOS expression was higher in GD than in controls but variable and did not separate groups (Welch's t-test p = 0.302; healthy n = 4, GD n = 5).IL21 expression was low overall and similarly did not separate groups (p = 0.427).We therefore stratified GD donors into Tfh ICOS⁺ and Tfh ICOS⁻ subgroups.B-cell activation differed across Healthy, Tfh ICOS⁻, and Tfh ICOS⁺ groups (ANOVA p = 0.0013), with higher activation in Tfh ICOS⁺ donors.Serum anti-TG (ANOVA p = 0.0011) and anti-TPO (ANOVA p = 0.0289) were highest in the Tfh ICOS⁺ subgroup.Together, these results suggest that heterogeneity in Tfh ICOS expression may mark a subset of GD donors with stronger B-cell activation and higher anti-thyroid antibodies, while highlighting power limits from small donor counts for donor-level comparisons.