Isolation and Stimulation of T Cells with Vitamin D and TGF-β

Human peripheral blood mononuclear cells (PBMCs) were isolated, as previously described Xystrakis et al. (31 (link)). CD4+ and CD8+ T cells were isolated using Dynabeads CD4 or CD8 positive selection kits (Invitrogen, Paisley, UK). Cells (1 × 10⁶/ml) were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, and 50 μg/ml gentamicin, and stimulated with plate-bound anti-CD3 (1 μg/ml; OKT3) plus 50 IU/ml recombinant human IL-2 (Eurocetus, UK) in the presence or absence of 1,25(OH)₂D3 (BIOMOL research labs, UK), TGF-β1 (R&D Systems, UK), TGF-β2 (R&D Systems) or anti-TGF-β (R&D Systems) at indicated concentrations for 7 days. NIST SRM1648a Urban Particulate Matter (National Institute of Standards & Technology, USA) is an urban total particulate matter reference material with mean particle diameter 5.85 μm, collected in the USA. NIST was prepared as previously described by Pfeffer et al. (32 (link)).