Induction of Antigen-Specific CD8+ T Cells

Bone marrow derived dendritic cells (BMDC) from AAD mice were prepared as described.5 23 (link) BMDC were pulsed with 10 µg/mL phosphopeptide and 10 µg/mL β2-microglobulin for 2–3 hours, irradiated (3000 cGy), washed and injected intravenous (1.5–2.5×10⁵) into AAD mice. Mice received intravenous booster immunizations 6 days later with phosphopeptide, CpG (type C, ODN 2395, InvivoGen (San Diego, California, USA)), and antimouse CD40 (FGK45, gift of Stephen Schoenberger, La Jolla Institute of Allergy and Immunology). CD8α⁺ T-cells were enriched from spleens and lymph nodes 5 d later using magnetic beads (Miltenyi) and incubated for 5 hours with peptide-pulsed stimulators (C1R-AAD) in medium containing 0.5 µg/mL anti-CD107a, 5 µg/mL Brefelden A (Sigma-Aldrich) and 1 µM monensin (eBioscience). Cells were stained for CD8α (eBioscience), permeabilized and fixed with Cytoperm/Cytofix (BD Bioscience), and stained for intracellular interferon-γ (IFNγ) (eBioscience). Samples were analyzed on a FACSCanto I or II (BD Bioscience) using FlowJo software (Tree Star, Ashland, Oregon, USA).