Effects of LPS, sCD14 and autologous serum on perforin production by CD4+ T cells. Fresh peripheral blood mononuclear cells of patients with ankylosing spondylitis were incubated with medium (as a negative control), soluble CD14 (sCD14) or 10 μg/ml lipopolysaccharide (LPS) alone or with LPS in combination with 25 μg/ml sCD14 and 5% autologous serum for 16 hours. After staining with fluorescence-marked monoclonal antibodies directed against perforin, CD4 and CD28, cells were counted by flow cytometry. (a) Histograms show perforin production by CD4+CD28+ (upper row) and CD4+CD28null T cells (lower row) in response to medium, sCD14, LPS, LPS + sCD14 and LPS + serum as indicated (red curves). Black lines represent isotype control staining. Values indicate the mean fluorescence intensity. Gates for CD4+CD28null and CD4+CD28+ T cells were set as shown in Figure 3a. (b) Box blots show percentages of perforin-producing CD4+CD28null T cells from seven independent experiments. Differences were tested for significance using the Wilcoxon test. ***P ≤ 0.001.