Activation and Expansion of Murine CD8+ T Cells

Spleens were harvested from 6 to 8-week-old mice. Naïve CD8⁺ T cells (CD44^lo CD62L^hi) T cells were purified (>95% purity) by negative selection (Dynabeads™ Untouched™ Mouse CD8 Cells Kit, Invitrogen) from RBC-lysed single-cell suspensions from spleen followed by cell sorting. For stimulation, purified CD8⁺ T cells were plated at 10⁶ cells/ml in 24-well plates precoated with 0,3 mg/mL goat anti-hamster and coated with anti-CD3 (clone 2C11) and anti-CD28 (clone 37.51) (1 μg/mL), as previously described (30 (link), 31 (link)). After, 48 h, cells were removed from the TCR signal and re-cultured by diluting 1:2 in media (DMEM (11995-065, Gibco) supplemented with 10% FBS (26140079, Gibco), 1x Penicillin/Streptomycin (15140-122, Gibco), 1x L-Glutamine (25030-081, Gibco), 1x MEM Vitamins (11120-052, Gibco), 1x Sodium Pyruvate (11360-070, Gibco), 1x Essential Amino Acids (M5550, Sigma),1x Non-Essential Amino Acids (11140-050, Gibco), 10 mM HEPES (15630-080, Gibco) and 50 μM β-Mercaptoethanol (M3148, Sigma), containing 200 U/ml final concentration of recombinant murine IL-2 (Peprotech). Every 24 h, cells were diluted 1:2 with fresh media containing IL-2. On day 5, cells were stained with anti-CD25-PE, anti-CD44-FITC, anti-CD62L-APC and anti-CD127-PEcy7 antibodies (Biolegend) for phenotypic analysis and were assessed by flow cytometry.