Mtor inhibitor rapamycin

Naïve CD4⁺ T cells were isolated from fresh human blood samples with a negative selection isolation kit from Miltenyi Biotec as per protocol. The purity of naïve CD4⁺ T cells was evaluated with staining of anti-CD3 (Clone UCHT1, BioLegend, San Diego, USA), anti-CD4 (clone RPA-T4, BioLegend, San Diego, USA), and anti-CD45RA (HI100, BioLegend, San Diego, USA). Isolated cell purities were over 95% (Supplementary Figure S1). Cells were cultured in RPMI 1640 media (GE Health Care Life Sciences, Marlborough, USA) supplemented with 10% FBS (Life Technologies, Carlsbad, USA). Cells were stimulated overnight with anti-CD3 (10 μg/mL, pre-coated on plate, Clone UCHT1, BD Biosciences, San Jose, USA) and anti-CD28 (2.5 μg/mL, Clone CD28.2, BD Biosciences, San Jose, USA), with and without glycolysis inhibitor 2-deoxy-d-glucose (2-DG, 2 mg/mL, Acros Organics, New Jersey, USA), mTOR inhibitor rapamycin (100 Nm, Alfa Aesar, Ward Hill, USA) or H₂O₂ (50 μM, Sigma-Aldrich, St. Louis, USA). The next day the antibodies were removed and fresh media (RPMI and 10% FBS) were added with and without 2-DG, rapamycin, or H₂O₂. The cells were cultured for a total of 3 days before harvesting.