α cd3 α cd28 immobilized on beads

Splenic CD8 T cells were magnetically enriched using an AutoMACS pro (Miltenyi Biotec) to >95% purity per manufacturer's instructions. The cells were fluorescein-labeled with 5 μM carboxyfluorescein succinimidyl ester (CFSE) at 37° for 10', washed 3x with RPMI-1640 medium supplemented with 2-mercaptoethanol, L-glutamine, pyruvate, non-essential amino acids, and 10% fetal bovine serum (abbreviated as RP10 in the text). The cells were cultured at 2 × 10⁵ cells/ml at a 1:1 ratio with α-CD3/α-CD28 immobilized on beads (Miltenyi), in the presence of combinations of 10 U/ml rIL-2, 10 ng/ml rIL-12 (Biolegend), and 10 ng/ml rIL-18 (Biolegend), as indicated, for up to 3 days. On each day of analysis, cells were stained for viability (LIVE/DEAD reagent, Invitrogen), as well as with surface and/or intracellular antibodies.

Naïve splenic CD8⁺ T cells were magnetically enriched using an AutoMACS pro (Miltenyi Biotec) using the CD8a T cell isolation kit supplemented with anti-CD44-biotin (eBioscience). The cells were cultured at 2 × 10⁵ cells/ml in RPMI-1640 with L-glutamine (Lonza, Basel, Switzerland) + 10% FCS 1:1 with α-CD3/α-CD28 immobilized on beads (Miltenyi), 10 U/ml rmIL-2 (eBioscience). The purity of isolated naïve T cells was assessed by flow cytometry and was >95% in all experiments. Lymph node (LN) stromal cells were obtained by dissociating lymph nodes in Liberase TL enzyme mixture (Roche, Basel, Switzerland) using GentleMACS tissue dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). When lymph nodes were fully digested lymph node stromal cells were negatively enriched using anti CD45 and anti Ter119 magnetic beads (Miltenyi Biotec). Stromal cells were passaged 5–10 times in alpha-modified MEM (Sigmaaldrich, St.Louis, Mo) with 20% FCS. Naïve T cells were cultured with lymph node stromal cells in 1:10 ratio in 96 well-round plates.