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This study aims to screen and authenticate immune cell-associated biomarkers in AS. Two Gene Expression Omnibus datasets (GSE25101 and GSE41038) were combined as the discovery dataset, with candidate biomarkers screened via differential expression analysis, immune cell infiltration analysis, and weighted gene co-expression network analysis (WGCNA). Immune cell-related biomarkers were further identified and validated by receiver operating characteristic (ROC) analysis using the confirmatory dataset GSE73754, and potential diagnostic biomarkers were finally confirmed by reverse transcription quantitative polymerase chain reaction (RT-qPCR), immunofluorescence staining, and single-cell RNA sequencing (scRNA-seq) analysis (GSE194315). Thirty-two differentially expressed genes between the AS and control samples were identified. The ratio of M2 macrophages was significantly different between the AS and control samples. Seven candidate biomarkers associated with immune cells in AS were identified by WGCNA and Venn diagram. Then, three genes (SBK1, HNRPR, and CX3CR1) were authenticated as immune cell-associated biomarkers in AS by ROC curves, indicating a possible diagnostic value in clinical settings. The results of RT-qPCR, immunofluorescence staining, and scRNA-seq analysis all confirmed that CX3CR1 was down-regulated in AS, which was in line with bioinformatics study findings. Dysregulation of the CX3CR1 and M2-type macrophage ratio are key factors in AS, which lay the groundwork for exploring illness pathophysiology and yielding fresh perspectives on AS diagnosis and therapy.<\/jats:p>","DOI":"10.1063\/5.0252297","type":"journal-article","created":{"date-parts":[[2025,9,9]],"date-time":"2025-09-09T14:35:47Z","timestamp":1757428547000},"update-policy":"https:\/\/doi.org\/10.1063\/aip-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Analysis of immune cell infiltration landscape and identification of diagnostic biomarkers in ankylosing spondylitis"],"prefix":"10.1063","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-3004-5882","authenticated-orcid":false,"given":"Meng","family":"Chen","sequence":"first","affiliation":[{"name":"Department of Nephrology, Henan Clinical Medical Research Center for Nephropathy, Henan Provincial Key Laboratory of Kidney Disease and Immunology, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Henan University People's Hospital 1 , Zhengzhou,","place":["China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shanbang","family":"Zhu","sequence":"additional","affiliation":[{"name":"Department of Orthopedics, Affiliated Jinling Hospital, Medical School of Nanjing University 2 , Nanjing,","place":["China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yue","family":"Gu","sequence":"additional","affiliation":[{"name":"Department of Nephrology, Henan Clinical Medical Research Center for Nephropathy, Henan Provincial Key Laboratory of Kidney Disease and Immunology, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Henan University People's Hospital 1 , Zhengzhou,","place":["China"]},{"name":"Department of Nephrology, Fuwai Central China Cardiovascular Hospital 3 , Zhengzhou,","place":["China"]}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7072-095X","authenticated-orcid":false,"given":"Xinzhe","family":"Feng","sequence":"additional","affiliation":[{"name":"Department of Joint Bone Disease Surgery, Changhai Hospital, Naval Medical University 4 , Shanghai,","place":["China"]}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"317","published-online":{"date-parts":[[2025,9,9]]},"reference":[{"key":"2025090910354310200_c1","doi-asserted-by":"publisher","first-page":"996103","DOI":"10.3389\/fimmu.2022.996103","article-title":"Joint together: The etiology and pathogenesis of ankylosing spondylitis","volume":"13","year":"2022","journal-title":"Front. 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