Differentiation of Human CD4+ T Cells

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)].