Production of cytokines in response to IL-12 and IFN-γ stimulation was evaluated using ELISA assays specific for IFN-γ (LEGEND MAX human IFN-γ kit, BioLegend), TNF (Human TNF Platinum ELISA kit, eBioScience) and IL-12p40 (Human IL-12/23 p40 Platinum ELISA kit, eBioScience) according to the manufacturer’s instructions.
Using heparinized blood samples from patients and healthy controls, peripheral blood mononuclear cells (PBMC) were separated by centrifugation on a Ficoll-Hypaque density gradient as previously described [20 (link)]. The PBMCs were cultured (106 cell/well) in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 UI/mL penicillin and 100 μg/mL streptomycin (GibcoBRL). For generation of phytohemagglutinin (PHA)-activated T cells, PBMCs were activated with 20 μg PHA for 72 hours. IL-12Rβ1 expression on the surface of PHA-activated T cells was detected using PE-anti-IL-12Rβ1 mAh (BD) and anti-CD-3-FITC mAh (BD). FITC-anti-mouse IgG2a,κ and PE-anti-mouse IgG1,κ were used as isotype controls. IL-12Rβ1 expression was assessed on CD3+ PHA-activated T cells on a FACS-Calibur flow cytometer using CellQuest Pro software (BD Biosciences).
Using heparinized blood samples from patients and healthy controls, peripheral blood mononuclear cells (PBMC) were separated by centrifugation on a Ficoll-Hypaque density gradient as previously described [20 (link)]. The PBMCs were cultured (106 cell/well) in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 UI/mL penicillin and 100 μg/mL streptomycin (GibcoBRL). For generation of phytohemagglutinin (PHA)-activated T cells, PBMCs were activated with 20 μg PHA for 72 hours. IL-12Rβ1 expression on the surface of PHA-activated T cells was detected using PE-anti-IL-12Rβ1 mAh (BD) and anti-CD-3-FITC mAh (BD). FITC-anti-mouse IgG2a,κ and PE-anti-mouse IgG1,κ were used as isotype controls. IL-12Rβ1 expression was assessed on CD3+ PHA-activated T cells on a FACS-Calibur flow cytometer using CellQuest Pro software (BD Biosciences).