Total splenocytes were stimulated for 5 h with 2 µg/mL of the MHC class I restricted LCMV-NP396–404 peptide (>99% pure; Synpep, Dublin, CA, USA) in the presence of 50 U/mL recombinant murine IL-2 (R&D Systems, Minneapolis, MN, USA) and 1 mg/mL brefeldin A (Sigma, St. Louis, MO, USA). The absolute number of NP396–404-specific CD8 T cells was determined by multiplying the frequency of IFNγ+ cells by the total number of splenocytes.

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