Fig. 5.

Effect of IL-12 on the expression of CD40L on CD4⁺ T cells stimulated with immobilized MoAb to CD3: effect of B cells. CD4⁺ T cells (2 × 10⁵/well) were cultured in wells with immobilized anti-CD3 (64.1, 100 ng/well) in the presence or absence of a metalloproteinase inhibitor KB8301 (10 μm). B cells (2.5 × 10⁴/well) and IL-12 (10 ng/ml) were added from the initiation of cultures where indicated. After 96 h of incubation, the supernatants were assayed for IFN-γ content by ELISA, and the cells were harvested and stained with PE-conjugated anti-CD4 MoAb and FITC-conjugated anti-CD40L MoAb (solid line), or control MoAb (dotted line), followed by fixation with paraformaldehyde (PFA) (a). Alternatively, the cells were fixed with ethanol cooled to −20°C prior to staining (b). The cells were then analysed by flow cytometry. The staining for CD40L or control MoAb on cells gated for positive CD4 staining is displayed. Percent positive as well as mean fluorescence intensity (MFI) for specific CD40L staining were calculated as described in Materials and Methods. The data are representative of three different experiments.