Isolation of Naive CD8+ T Cells

This was performed as in previous reports^{22 (link)}; briefly, inguinal, axillary, brachial, cervical, and mesenteric lymph nodes (LNs) were harvested from WT OT-I or B6 mice, pooled, and homogenized to obtain single cell suspensions. CD8⁺CD44^lo cells were enriched by negative selection using MACS magnetic beads (Milteny Biotec, Auburn, CA) wherein cells were coated with FITC-labeled antibodies specific for CD4, B220, I-A^b, and CD44 (Biolegend San Diego, CA). Anti-FITC magnetic MicroBeads (Miltenyi Biotech) were added, and the suspension passed through separation columns attached to a MACS magnet. Cells that did not bind were collected, and were >95% CD8⁺ and <0.5% CD44^hi.