Recombinant human tgf β

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)].

Where indicated, CD45RA⁺RO⁻ naïve 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) and recombinant human TGF-β (5 ng/μl; PAN™-Biotech GmbH, Aidenbach, Germany), IL-1β (12.5 ng/μl), IL-6 (25 ng/μl; both Miltenyi Biotec), and IL-23 (25 ng/μl; PeproTech, Rocky Hill, USA). Medium and cytokines were refreshed after 3 days.

Naïve CD4⁺ T cells were cultured in X-Vivo 15 media (Lonza, Cologne, Germany), which combines with 1% human serum and 1% penicillin–streptomycin (Both from Sigma-Aldrich, Saint Louis, USA). For the activation of the T cell receptor (TCR), cells were incubated with the antibodies from T cell Activation/Expansion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). For the induction of Th17 cells, recombinant human IL-1β (12.5 ng/μl), IL-6 (25 ng/ml; Both from Miltenyi Biotec, Bergisch Gladbach, Germany), recombinant human IL-23 (25 ng/ml; PeproTech, Rocky Hill, USA), and recombinant human TGF-β (25 ng/μl; PAN™-Biotech GmbH, Aidenbach, Germany) were added to cell culture for 96 h as described previously [29 (link)]. To ensure a high induction quality, we changed the medium and cytokines after 72 h of incubation. The induced Th17 cells were collected for quantitative real-time PCR.