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Our aim was to assess the phenotype of T-cell subsets in patients with ankylosing spondylitis (AS), a chronic inflammatory rheumatic disease. In addition, we also tested short-term T-cell activation characteristics. Measurements were done in 13 AS patients before and during the intravenous therapy with anti-TNF agent infliximab (IFX). Flow cytometry was used to determine T-cell subsets in peripheral blood and their intracellular signaling during activation. The prevalence of Th2 and Th17 cells responsible for the regulation of adaptive immunity was higher in AS than in 9 healthy controls. Although IFX therapy improved patients' condition, immune phenotype did not normalize. Cytoplasmic and mitochondrial calcium responses of CD4+ and CD8+ cells to a specific activation were delayed, while NO generation was increased in AS. NO generation normalized sooner upon IFX than calcium response. These results suggest an abnormal immune phenotype with functional disturbances of CD4+ and CD8+ cells in AS.