In vitro inhibition assay of GPI-induced TNF-α and IFN-γ production using anti-cytokine mAbs or anti-co-stimulators. High amounts of tumor necrosis factor (TNF)-α and IFN-γ were produced by splenocytes cultured with glucose-6-phosphate isomerase (GPI). Thus, we used anti-TNF-α mAb (10 μg/ml), anti-IFN-γ mAb (1 μg/ml), and anti-IL-12 mAb (0.3 μg/ml) to neutralize these cytokines in the in vitro cytometric bead array system. Inhibition study was conducted by adding the above concentrations at commencement of culture. These concentrations were calculated to produce more than 80% blockade of these cytokines. The percentage inhibition rate is calculated by cytokine production with this system: 100 – ([cytokine mAb – control antibody]/control antibody). The inhibition rate of (a) TNFα and (b) IFN-γ are shown. Cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA-4Ig; 1 μg/ml), anti-inducible co-stimulator (ICOS) mAb (0.5 μg/ml), and anti-CD40L mAb (1 μg/ml) were also used to block co-stimulatory pathways, and the inhibition rate of (c) TNF-α and (d) IFN-γ are shown. Three independent experiments were performed. Data are expressed as mean ± standard error of the mean.