Fig. 1: Comprehensive analysis of single-cell profiles of PBMCs in TNFi-treated patients with AS.

a Schematic of the study design, showing blood sample collection from 167 patients with ankylosing spondylitis (AS) before and after TNFi treatment. This includes a discovery cohort (n = 32), of which 22 patients (responders: R_Pre, R_Post, n = 12; non-responders: NR_Pre, NR_Post, n = 10) underwent scRNA-seq analysis, as well as validation cohort 1 (n = 18) and validation cohort 2 (n = 117). Figure created with BioRender. Created in BioRender. https://BioRender.com/q70l192. b UMAP of single-cell RNA sequencing data from PBMCs (n = 199,926), revealing 10 major cell clusters colored according to cell type. c Comparisons between pre- and post-treatment within the same group (R, NR) were performed using the Wilcoxon matched-pairs signed rank test or a paired t-test, while comparisons between different groups (R_Pre vs. NR_Pre, R_Post vs. NR_Post) used a two-sided Mann-Whitney U test or an unpaired t-test, based on normality. In the cluster names, “CD14 M” and “CD16 M” refer to CD14⁺ and CD16⁺ monocytes, respectively, and “prolif” indicates proliferating cells. Bar plots indicate means, and error bars represent standard deviation (SD). Exact p-values for CD14 Monocyte and NK cell comparisons within responders are 0.00012 and 0.00029, respectively (Wilcoxon matched-pairs signed rank test, two-sided). d Milo analysis comparing PBMC neighborhoods (k = 45) between NR and R baselines. Red indicates neighborhoods more abundant in the NR group, and blue indicates neighborhoods more abundant in the R group. Nhood size corresponds to circle size, and overlap size corresponds to line thickness. e Neighborhood function that uses community detection to partition neighborhoods, showing automatic grouping of 15 Nhood groups in different colors. f Visualization of differential abundance (DA) fold changes in different neighborhoods, focusing on the distribution in PBMCs: cytotoxic (CTX), proliferating (PRF), central memory (CM), and effector memory (EM). Statistical testing for differential abundance utilized the quasi-likelihood F-test implemented in edgeR (two-sided), with spatial FDR controlled using a weighted Benjamini–Hochberg method. g Pearson correlation coefficient analysis of TNFi-induced changes. Responder pairs (n = 12), non-responder pairs (n = 10); error bars indicate standard error. Statistical significance was tested using the Wilcoxon matched-pairs signed rank test (two-sided). Source data are provided as a Source Data file.