Anti human ifnγ mab

Where indicated, CD4⁺ T cells were cultured in X-Vivo 15 (Lonza, Cologne, Germany) media supplemented with 1% human serum and 1% penicillin-streptomycin (both Sigma-Aldrich, Saint Louis, USA) Cells were cultured for 4 days with the T cell Activation/Expansion Kit (Miltenyi Biotec). For Th0 cell induction recombinant human IL-2 (20 U; Miltenyi Biotech) was added. For Th1 cell induction, recombinant human IL-12 (10 ng/ml; Miltenyi Biotec) and anti-human IL-4 mAb (10 μg/ml; BioLegend) were added. For Th2 cell induction, recombinant human IL-4 (10 ng/ml; PeproTech, Rocky Hill, USA) and anti-human IFNγ mAb (10 μg/ml; BioLegend) were added. For Th9 cell induction, recombinant human IL-4 (10 ng/ml; PeproTech, Rocky Hill, USA), recombinant human TGF-β (5 ng/μl; PAN™-Biotech GmbH, Aidenbach, Germany), and anti-human IFNγ mAb (10 μg/ml; BioLegend) were added. For iT_reg cell induction, recombinant human TGF-β (5 ng/μl; PAN™-Biotech GmbH, Aidenbach, Germany) and recombinant human IL-2 (20 U; Miltenyi Biotech) were added as described previously [30 (link)].

T_N (5,000–10,000) or T_CM (20,000) were added to 24-well plates coated with anti-CD3 mAb (clone OKT3, purchased from the Walter and Eliza Hall Institute, Parkville, Australia) at 1 µg/mL and anti-CD11a mAb (clone MEM-83, generous gift from Vaclav Horejsi, Institute of Molecular Genetics, Prague, Czech Republic) at 19 µg/mL unless otherwise indicated. Cells were incubated in culture medium (RPMI supplemented with 9% FBS, 2-mM L-glutamine, 1-mM MEM sodium pyruvate, 100-U/mL penicillin/streptomycin, 100-µM MEM non-essential amino acids, 5-mM HEPES buffer, and 55-µM 2-mercaptoethanol). rHuman IL-2 (Roche 11 147 528 001) was added at 25 U/mL for neutral and T2 conditions. T2 cultures also contained 10-ng/mL rHuman IL-4 (Shenandoah Cat. #100-09) and 1-µg/mL antihuman IFN-γ mAb (Biolegend Cat. #502404). Antihuman IL-12p70 mAb clone QS12p70 (U-Cy Tech biosciences Cat. #CT276) was added to T2 media at 1 µg/mL in one experiment. Cells were recovered from stimulation plates after 5 days and assessed directly or rested in fresh plates for 3 days in culture medium containing IL-2.