Human cd4 t cells

Human CD4+ T cells (STEMCELL Technologies) were cultured in RMPI 1640 (Glutamax Supplement, Gibco) supplemented with 5 mM HEPES, pH 8.0 (Gibco), 50 µg/mL penicillin/streptomycin (Gibco), 50 µM 2-mercaptoethanol (Sigma-Aldrich), 5 mM MEM nonessential amino acids (Gibco), 5 mM sodium pyruvate (Gibco), and 10% FBS (Seradigm). Cells were activated for 5–7 days post-thaw by plating every 2 days on dishes coated with 10 µg/mL of anti-CD3 (UCHT1, eBioscience, Invitrogen) and anti-CD28 (CD28.2, eBioscience, Invitrogen) monoclonal antibodies.

Purified human CD4+ T cells from peripheral blood (1.5 × 10⁷ cells (Stemcell Technologies)) were resuspended in complete RPMI (10% FBS, 1% l-Glut, 1% Pen/Strep) at a concentration of 5 × 10⁵ cells per mL (30 mL). Human T-Activator CD3/CD28 Dynabeads™ (Gibco) were resuspended in the vial by vortexing and the desired volume was transferred to an Eppendorf tube. Beads were rinsed with PBS + 0.1% BSA (1 mL) by vortexing for at least 30 s. Beads were isolated using a magnet and resuspended in the same volume of RPMI culture medium as the initial volume of beads taken from vial. Beads were then added to T cell culture at a ratio of 1 : 1 (375 μL beads for 1.5 × 10⁷ cells). Recombinant human IL-2 was added to T cell culture at a concentration of 30 U per mL. The culture was incubated in 5% CO₂ at 37 °C for ten days. Cell density was measured daily. When cells reached 2.5 × 10⁶ cells per mL the culture was split to 5 × 10⁵ cells per mL with fresh complete RPMI with 30 U per mL recombinant human IL-2.