(a) Spontaneous apoptosis, and activation-induced intracellular production of (b) IFN-γ and (c) perforin in CD28+ and CD28- CD4+ T cells from patients with ankylosing spondylitis (AS). For determination of subdiploidy, peripheral blood mononuclear cells of healthy control individuals and AS patients were surface stained with monoclonal antibodies directed against CD4 and CD28, and then intracellularly stained with 7-aminoactinomycin D. Peripheral blood mononuclear cells were stimulated with phorpbol 12-myristate 13-acetate and ionomycin in the presence of brefeldin A. Cells were stained with fluorescence-labelled monoclonal antibodies (mAb) directed against CD4, CD28 and either IFN-γ or perforin, and counted by flow cytometry. The number of positive cells were compared between CD28+ and CD28-CD4+ T cells using the two-sided paired t-test. P ≤ 0.05 was considered statistically significant. PE, phycoerythrin; PerCP, peridinin chlorophyll protein.