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.

引言：日本雪松花粉症（Japanese cedar pollinosis, JCP）在日本患病率极高，舌下免疫疗法（sublingual immunotherapy, SLIT）仍是为数不多的有效疾病修饰治疗手段之一。尽管多数患者可从中获得显著获益，但目前仍缺乏可预测SLIT治疗应答的生物标志物。既往通过对外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）开展单细胞RNA测序（single-cell RNA sequencing, scRNA-seq），已将核糖体蛋白基因RPS26鉴定为与治疗应答相关的候选标志物。本研究旨在明确RPS26的表达是否对SLIT的治疗效能存在影响，并阐明其在T细胞动态、细胞因子产生及表观遗传调控通路中的作用。
方法：本研究对35例JCP患者的PBMCs进行了SLIT治疗前的检测，并对其中7例患者接受1年SLIT治疗后的配对样本进行了评估。我们对4例治疗应答者与3例无应答者开展了scRNA-seq分析。同时评估了SLIT治疗期间T细胞亚群的变化，以及白细胞介素（interleukin, IL）-5与IL-10的分泌水平。通过小干扰RNA介导的RPS26基因敲低联合过敏原刺激实验，验证了RPS26的功能相关性。此外，通过定量PCR（quantitative PCR）分析了RPS26与十十一易位家族（ten-eleven translocation family, TET2和TET3）表达水平的关联。
结果：scRNA-seq分析显示，治疗应答者体内RPS26的表达显著上调。尽管整体水平的RPS26表达在应答者与无应答者间并无显著差异，但所有RPS26表达量≥100相对单位的受试者均为治疗应答者。SLIT治疗可使应答者体内的滤泡调节性T细胞与1型调节性T细胞水平升高，而IL-10水平升高仅见于高表达RPS26的应答者。敲低RPS26可增强过敏原刺激后的早期细胞凋亡与死亡，并降低IL-10的表达水平。RPS26的表达与表观遗传调控因子TET2、TET3的表达呈显著正相关，而二者均为维持调节性T细胞稳定性所必需的蛋白。
结论：RPS26可作为SLIT治疗期间免疫耐受的生物标志物，同时也是该治疗过程中免疫耐受的促成因子。其与IL-10诱导、调节性T细胞扩增、凋亡抵抗及TET2/TET3表达的关联，提示存在一条支持过敏原脱敏的翻译-表观遗传轴。本研究结果凸显了核糖体特异性作为免疫治疗应答决定因素的重要性。未来需开展更大规模的研究以验证RPS26的临床价值，并进一步阐明其在过敏原特异性免疫治疗中的具体机制。