Testing A2.1-restricted VACV T cell epitopes in A2.1 Tg mice using purified CD8⁺ T cells and individual peptides. (A) Splenic CD8⁺ cells were purified from VACV-infected A2.1 Tg mice and incubated with peptide-pulsed Jurkat-A2.1/K^b cells, after which number of IFN-γ-producing cells was enumerated in an ELISPOT assay. Shown are the average net SFC/10⁶ CD8⁺ T cells in response to each peptide; black bars, epitopes identified by Oseroff et al. [9] in A2.1 positive donors and recognized in Tg mice upon re-screening; white bars, peptides identified by Pasquetto et al. [5] in Tg mice; grey bars, epitopes identified in both studies. (B) The specific T cell response to three different epitopes (B14R_327–335, M1L_374–383, and A14L_51–59) was assessed 7 days after VACV infection in A2.1 Tg mice. Shown is the stimulation index for the epitopes tested either as individual peptides (black bars) or in pools of ten peptides (white bars). (C) Shown is the stimulation index for the epitope-specific responses by using either CD8⁺ T cells (black bars) or splenocytes (white bars). (D) Shown are the average net SFC/10⁶ CD8⁺ T cells in response to groups of epitopes recognized in Tg mice upon re-screening (black bar) and identified in Tg mice in the original study by Pasquetto et al. [5] (white bar). The unpaired t test with Welch's correction was used to determine if differences were significant. For all panels, average data are shown from at least two independent experiments. For each individual experiment samples are tested in triplicate. Error bars indicate SEM.