Figure 3

Immune focusing of T cell response towards PSA. Mice were treated as described for Figure 1. Pooled splenocytes were assayed for vaccinia virus (VV)-specific (A and C panels on left) or PSA-specific (A and C panels on right) cytotoxic activity by flow cytometry (% CD107+ IFNγ + CD8 T cells) 14 days after the last treatment. Splenocytes were restimulated overnight with vaccinia E3L and F2L peptides or with PSA OPL (or PSA peptide HPQKVTKFML, see Additional file 1: Figure SD4) in the presence of anti-CD107 antibody. The following day, cells were stained intracellularly for IFNγ and with the surface markers CD127 and KLRG1.% antigen-specific cytotoxic SLEC and DPEC were determined by gating on (CD8 + CD127-KLRG1+) and (CD8 + CD127 + KLRG1+) cells, respectively. (B, D) The PSA:VV ratio was determined by dividing % CD107+ IFNγ + PSA-specific DPECs or SLECs by % CD107+ IFNγ + VV-specific DPECs or SLECs. Graphs show representative data of two independently performed experiments. Similar responses were observed when splenocytes were restimulated with PSA peptide HPQKVTKFML instead of a PSA OPL (Additional file 1: Figure SD4). The gating strategy for these experiments is outlined in Additional file 1: Figure SD3).