Deconvolution of immune responses to PR3. Flow analyses of ICC assays analyzing responses of rVV-EGFP-PR3–immunized HHDII mice to a PR3 peptide library. Splenocytes from immunized mice were expanded for 1 week in culture by stimulation with the whole PR3 library (A) and aliquots of these cultures tested, first by restimulation with peptide pools (D-G), and then in a separate experiment, with single peptides (B-C, H-K) followed by labeling with Abs to CD8 and IFN-γ. The robust response to the PR3 library was shown to be because of 2 peptides (64 and 65) within peptide subpool D (H-I). The minimal cytotoxic epitope common to these 2 peptides was shown to be a 10-mer peptide termed PR2 (K). This deconvolution of PR3 responses to the PR2 epitope was performed in 2 repeat experiments, each using at least 3 mice.