Deep Immunophenotyping Unravels the Double Negative T cells and PD-1-Mediated Inflammatory Exhaustion in Ankylosing Spondylitis

Ankylosing spondylitis (AS) is a chronic, immune-mediated inflammatory disorder primarily affecting the axial skeleton. It is characterized by bony fusion and syndesmophyte formation of the spine, leading to compromised mobility, reduced quality of life and increased mortality. However, the underlying immune dysregulation in AS remains poorly understood. We comprehensively profiled the peripheral immune landscape of 30 patients with active AS, all of whom were TNF-α blocker-naïve, capturing the immune profile during the active disease phase without biologic interference. Using advanced 11-color flow cytometry and FlowSOM clustering, 584 immunophenotypes were analyzed in AS and 30 age-and sex-matched healthy controls, with emphasis on both cellular composition and functional characteristics. AS patients displayed a distinctive functional “activated-exhausted” immune phenotype, dominated by CD4 + CD28 + CD38 + PD-1 + T cells and HLA-DR + CD38 + CD4 − CD8 − double-negative (DN) T cells. DN T cells were significantly enriched in HLA-B27-positive patients, suggesting potential as a biomarker. Network analysis revealed seven central immune subsets associated with active disease, while functional assessment demonstrated impaired CD95 + CD8 + T cell apoptosis and disrupted B-cell maturation. These findings provide a detailed functional immune map of biologic-naïve active AS, offering novel mechanistic insights and identifying candidate biomarkers for precision intervention.