Anti tgf β

PBMCs were isolated from peripheral blood using Ficoll density-gradient centrifugation. Treg cells were obtained as CD4⁺CD25⁻T cell subsets, and naïve CD4⁺ T cells were isolated and purified using a naïve CD4⁺ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Then, CD4⁺CD25⁻ T cells (1 × 10⁶/well) were cultured with soluble anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) antibodies, with the addition of recombinant human TGF-β1 (10 ng/ml; R&D Systems, USA) and IL-2 (100 U/ml; Peprotech, USA) to induce Treg cell conversion. After culturing for 5–6 days, the cells were collected for the measurement of CD4⁺CD25⁺ percentages by flow cytometry.
Tfh cells were obtained as naive CD4⁺ T cells (1 × 10⁶/well) and stimulated with soluble anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) antibodies, with the addition of recombinant human IL-2 (100 U/ml; Peprotech, USA), IL-6 (20 ng/ml; Peprotech, USA), anti-IL-4 (10 μg/ml; R&D Systems, USA), anti-IFN-γ (10 μg/ml; R&D Systems, USA) and anti-TGF-β (10 μg/ml; R&D Systems, USA). After 5–6 days, the cells were collected for the measurement of CXCR5⁺⁺PD-1⁺⁺CD4⁺ T cell or CXCR5^{+ +}PD-1⁺⁺ Foxp3⁺CD4⁺ T cell percentages by flow cytometry.

Spleen and lymph nodes from mice were harvested, mechanically disrupted, and passed through a 70-μm filter. Naive CD4⁺ T cells from the sample were purified by magnetic bead separation using the CD4⁺CD62L⁺ isolation kit (Miltenyi Biotec). Cells were plated in 96-well plates coated with anti-CD3 antibody (5 μg/mL, BD, clone 145-2C11) at a density of 3 × 10⁶ cells/mL. Cells were plated in Tfh-conditioning media (RPMI complete media with 10% fetal bovine serum), 10 μg/mL anti-IL4 (BD Biosciences, clone 11B11), 10 μg/mL anti-IFNγ (BD Biosciences, clone XMG1.2), 10 μg/mL anti-TGFβ (R&D Systems, clone 1D11), 30 ng/mL IL6 (Shenandoah Biotechnology), and 50 ng/mL IL21 (Shenandoah Biotechnology) in the presence of soluble anti-CD28 (2 μg/mL, BD, clone 37.51). Polarized cells were harvested for Tfh phenotyping by flow cytometric analysis 4 days after plating.
For surface phenotype and transcription factor analysis, harvested cells were stained with Live/Dead Fixable Aqua dead cell stain (Thermo Scientific Fisher) to exclude dead cells. Cells were subsequently stained for surface markers (CD4, TCRβ, PD1, CXCR5) and then fixed and permeabilized using the Intracellular Fixation and Permeabilization Buffer Set (eBioscience). Fixed cells were stained with antibody to BLC6 (clone 7D1) or isotype control.

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)).