Supplementary Material for: RPS26 Expression as a Predictive Biomarker and Functional Modulator in Sublingual Immunotherapy for Japanese Cedar Pollinosis

Introduction: Japanese cedar pollinosis (JCP) is highly prevalent in Japan, and sublingual immunotherapy (SLIT) remains one of the few effective disease-modifying treatments. While many patients have significant benefits, biomarkers predicting SLIT responsiveness are lacking. Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) identified the ribosomal protein gene RPS26 as a candidate associated with treatment response. This study aimed to determine whether RPS26 expression contributes to SLIT efficacy and to define its involvement in T-cell dynamics, cytokine production, and epigenetic regulatory pathways. Methods: PBMCs from 35 patients with JCP were analysed before SLIT, and paired samples from seven patients were evaluated after 1 year of SLIT. scRNA-seq was performed in four responders and three non-responders. Changes in T-cell subsets and interleukin (IL)-5 and IL-10 secretion were evaluated during SLIT. Functional relevance was examined through small interfering RNA-mediated RPS26 knockdown followed by allergen stimulation. The relationship between RPS26 and the ten-eleven translocation family (TET2 and TET3) expression was assessed by quantitative PCR. Results: scRNA-seq revealed significant RPS26 upregulation in responders. Although bulk RPS26 expression did not differ between responders and non-responders, all subjects with high RPS26 expression (≥100 relative units) were responders. SLIT increased follicular regulatory and type 1 regulatory T-cells in responders, whereas IL-10 elevation occurred exclusively in high-RPS26 responders. RPS26 knockdown enhanced early apoptosis and cell death, and reduced IL-10 expression following allergen challenge. RPS26 expression was strongly correlated with the epigenetic regulators TET2 and TET3, both essential for regulatory T-cell stability. Conclusion: RPS26 appears to function as both a biomarker and a contributor to immune tolerance during SLIT. Its association with IL-10 induction, regulatory T-cell expansion, apoptosis resistance, and TET2/TET3 expression suggests a translational–epigenetic axis supporting allergen desensitization. These findings highlight ribosomal specialization as a determinant of immunotherapy responsiveness. Larger studies are needed to validate RPS26 and further define its mechanistic role in allergen-specific immunotherapy.