CB6F1 mice were immunized once with MCMV.env, or with a non transgene-encoding MCMV as control, and infected with 5 000 SFFU FV six weeks later. Spleens were collected from mice either five weeks after immunization, or two weeks after FV infection, and restimulated in vitro with Env-derived peptide pools. (A) Spleen cells were depleted of CD4+ cells by magnetic cells sorting, and subjected to restimulation with 15 pools of overlapping peptides spanning the whole Env protein in an ELISpot assay; stimulation with a pool of CMV-derived peptides, or incubation without any peptide stimulation, served as controls. (B) Whole spleen cells were restimulated in vitro with pools of Env-derived peptides, and production of the indicated cytokines was analysed by flow cytometry after intracellular cytokine staining. Data were acquired in one experiment, data shown in (A) are means of two replicates. Each dot indicates an individual mouse (A) or mean values (B). Data were analysed for statistically significant differences by One Way ANOVA on Ranks with Dunn’s post test. * indicates statistically significant differences compared to unstimulated control (P < 0.05).