Roswell park memorial institute (rpmi)

Blood samples were collected in heparinised tubes and processed on the day of vaccination and then at 7, 14, and 28 days after the booster dose. To obtain peripheral blood mononuclear cells (PBMCs), the samples were centrifuged to separate the blood plasma, and the plasma was maintained at −80 °C. The remaining blood samples were diluted with RPMI (Gibco, Waltham, MA, USA) and placed into SepMATE tubes containing Lymphoprep (STEMCELL Technologies, Vancouver, Canada), according to the manufacturer’s protocol. The cell pellet was resuspended in 3 mL of R10 media (RPMI-1640 supplemented with 1% penicillin–streptomycin, 2 mM L-glutamine, and 10% foetal calf serum (FCS, Labtech, Heathfield, UK). The cells were adjusted to a concentration of 3 × 10⁶ PBMCs per mL before cryopreservation in FCS containing 10% DMSO. The cell suspensions were aliquoted and stored in CoolCell (Corning, Glendale, NY, USA) for freezing at −80 °C overnight. The tubes were stored in liquid nitrogen until further analysis.

Memory CD4⁺ T cells were cultured in complete RPMI (RPMI (Labtech) supplemented with 10% FCS (Sigma Aldrich), 2 mM L-glutamine with 1% penicillin/streptomycin (Sigma Aldrich) with irradiated antigen-presenting cells (irrAPC), anti-CD3 (eBioscience) and, where indicated, in the presence of dichloroacetate (DCA) (10 mM, Sigma Aldrich), rapamycin (20 nM, Sigma Aldrich), 5-(Tetradecyloxy)-2-furoic acid (TOFA) (1.2 μg/ml, Sigma Aldrich), C-75 (0.6 μg/ml, Sigma Aldrich), cerulenin (3.1 μM, Sigma Aldrich), or palmitate (25 μM, Seahorse Biosciences) for 5 days. Isolated CD161⁺, CD161⁻, and Tconv cells were cultured with irradiated antigen presenting cells (irrAPC), anti-CD3 and IL-2 (20 u/ml, eBioscience) for 6 days or in some cases with PMA/ionomycin for 18 h prior to Seahorse analysis. Isolated Treg cells were cultured using the Treg cell expansion kit (Miltenyi Biotec) in the presence of IL-2 (500 u/ml) for 6 days prior to Seahorse analysis. During glucose deprivation, memory CD4⁺ T cells were cultured in glucose-free complete RPMI (RPMI (Biosciences) supplemented with 10% dialysed FCS (Sigma Aldrich), 1% vitamin cocktail (InvivoGen), and 1% selenium/insulin cocktail (InvivoGen) supplemented with either 10 mM glucose (Sigma Aldrich) or 10 mM galactose (Sigma Aldrich) for 5 days.