Macs separation

The spleens were extracted from Balb/c mice and naive CD4⁺ T cells were purified by using a magnetic cell sorting system (MACS® separation, Miltenyi Biotech, Bergisch Gladbach, Germany). The cells were cultured in RPMI 1640 culture media with 10% heat-inactivated FBS (Cellgro, Herndon, VA, USA), 100 U/mL penicillin (Cellgro), 0.1 mg/mL streptomycin (Cellgro), 2 mM L-glutamine (Cellgro), and 0.05 μM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, USA) at 37°C in a 5% CO₂-humidified incubator. Plate-bound anti-CD3 antibody (1 μg/mL, ebioscience) and anti-CD28 antibody (1 μg/mL, ebioscience) were used to stimulate T cells. Recombinant mouse IL-6 (25 ng/mL, BD, San Jose, CA, USA) and TGF-β (2.5 ng/mL, BD) were used for Th17 differentiation, and IL-2 (10 ng/mL, BD), IL-12 (5 ng/mL, Biosource, Camerillo, CA, USA), and anti-IL-4 (5 μg/mL) were added to induce differentiation into Th1 cells.

Human blood was obtained from healthy donors with informed consent and ethical approval in the form of leukocyte cones purchased from the NHS Blood and Transplant service. Leukocyte cones contain waste leukocytes isolated from individuals donating platelets via apheresis and consist of a small volume (~10 ml) of packed leukocytes with few red blood cells or platelets. For monocyte isolation, blood was diluted with 1:2 with PBS followed by separation using a Histopaque gradient and centrifugation as previously described (Purvis et al., 2020 (link)). Following human peripheral blood mononuclear cell (PBMC) isolation and washing, approximately 1 × 10⁸ PBMCs were labelled and negatively selected using the pan human monocytes isolation kit and MACS separation (Miltenyi Biotec, Bisley, Surrey, UK), typically yielding around 5 × 10⁶ monocytes defined as CD11b⁺CD114⁺CD16⁻. Monocytes were resuspended at 4 × 10⁶ cells·ml⁻¹ in chemotaxis buffer (RPMI 1640, 25‐mM HEPES, 0.5% [w/v] BSA) and left on ice until required.