Ia ie 2g9

CD8 T cells were purified from the spleens of adult P14 mice via negative selection by antibody depletion of B220 (RA3-6B2, BD Pharmingen), CD11c (HL3, BD Pharmingen), CD11b (M1/70, BD Pharmingen), CD16/32 (2.4G2, BD Pharmingen), IA/IE (2G9, BD Pharmingen) and CD4 (L3T4 RM4-5, BD Pharmingen) with magnetic bead separation (anti-rat IgG Dynal beads, Invitrogen). CD4 T cells were purified from the spleens of adult Smarta mice via negative selection by antibody depletion of B220 (RA3-6B2, BD Pharmingen), CD11b (M1/70, BD Pharmingen), CD16/32 (2.4G2, BD Pharmingen), IA/IE (2G9, BD Pharmingen), CD8α (Clone 53–6.7, BD Pharmingen) with magnetic bead separation (anti-rat IgG Dynal beads, Invitrogen). Cells were counted and labeled with CellTrace^TM Violet proliferation dye (Invitrogen) according to manufacturers instructions. FACS sorted DCs were plated at 1×10⁴ cells/well of a round bottom 96 well plate together with 1×10⁵ cells/well of purified CD8 T cells or 3×10⁴ CD4 T cells in RPMI 1640 (Invitrogen) supplemented with 5 mM Hepes, 10% FBS (HyClone), 1% Penicillin Streptomycin (Life Technologies), 1% Glutamax (Life Technologies) and 50μM ≤β-mercaptoethanol (Life Technologies). As positive control for T cell proliferation, T cells were co-cultured with GP33 or GP61 (Abgent) peptide pulsed naïve CD8α^neg DCs.