Figure 3
Tumor necrosis factor (TNF)-like cytokine 1A (TL1A) directly induces TNFα and interferon (IFN)γ in interleukin (IL)12 plus IL18-stimulated whole blood. (a) Peripheral blood was treated with IL12 and IL18 plus increasing concentrations of TL1A or TNFα for 24 h. Supernatants were analyzed for IFNγ expression by AlphaLISA. (b) Whole blood was treated with 50 ng ml−1 TL1A as in a, but increasing concentrations of anti-TL1A-0011 antibody was added. IFNγ was measured as above. (c) Whole blood was treated as in a, but TNF-α was analyzed instead by AlphaLISA. (d) Whole blood was treated with 50 ng ml−1 of TL1A or TNFα, plus 15 μg ml−1 anti-TL1A-0011 or anti-TNFα, and IFNγ was analyzed. (e) Human peripheral blood mononuclear cells (hPBMCs) were treated with IL12 and IL18 plus TL1A, anti-TL1A-0011, or isotype (iso) control, and the total amount of TNFα was measured in the supernatant after 24 h. (f) hPBMCs were cultured with IL12, IL18, and increasing amounts of membrane-bound TL1A (MbTL1A) Chinese hamster ovary (CHO) cells and IFNγ was measured. Untransfected CHO cells did not induce IFNγ (data not shown). (g) A total of 5 × 104 CHO-MbTL1A cells were cultured with hPBMCs, IL12, IL18, plus increasing concentrations of anti-TL1A-0011, anti-TNFα, or iso control. IFN-γ was measured as above after 24 h. (h, i) hPBMCs were treated with IL12 and IL18 plus 50 ng ml−1 TL1A. Cells were then gated on CD3+ T cells, CD3+CD56− natural killer (NK) cells, CD3+CD56+ NK T (NKT) cells, or CD3−CD56− lymphocytes, and (g) the percentage of IFNγ-producing cells or (h) the mean fluorescence intensity (MFI) of IFNγ was analyzed by flow cytometry. All samples were run with a minimum of three replicates per sample. IgG, immunoglobulin G; NT, not receiving any treatment.
