Restoration of the anti-FrCas^E immune response in infected/nontreated mice on Treg depletion. (A) Experimental design. Mice were infected perinatally. At 8 weeks of age, they were rechallenged to facilitate immune response analysis, which was followed by Treg depletion by repeated injections of an anti-CD25 mAb. Anti-FrCas^E IgGs and D^b-GagL⁺CD8⁺ T cells were assayed 10 days after challenge. To assay the in vivo anti-FrCas^E CTL activity, a mixture of splenocytes loaded with the GagL peptide or a control peptide was administered 9 days after challenge and death of GagL-loaded cells was assayed on the following day, as described in “Methods.” (B) Depletion of Tregs. Mice were treated as described in panel A. CD25^highCD4⁺ T cells were assayed by flow cytometry analysis for FoxP3 positivity at the moment of immune response analysis. (C) Anti-FrCas^E Ig concentrations. Antiviral IgG concentrations were assayed by ELISA in the sera of mice 10 days after rechallenge, as described in panel A. Three independent experiments with 5 mice per group were conducted. Data are presented as means ± SEM. The statistical significance between groups was established using the Student t test. *P < .05. (D-E) Flow cytometry assay of D^b-GagL tetramer⁺CD8⁺ T cells in Treg-depleted and Treg-nondepleted infected/nontreated mice. Analyses were carried out 10 days after challenge as described in panel A. (D) Representative flow cytometry analysis of CD8⁺ splenocytes of an individual mouse. (E) Means ± SEM of 3 independent experiments with 5 mice per group. The statistical significance between groups was established using the Student t test. *P < .05. (F-G) In vivo assay of anti-FrCas^E–infected cell CTL responses. FrCas^E-infected splenocytes were labeled with the CFSE vital dye at a 10× concentration and splenocytes recovered from naive mice were labeled with CFSE at a 1× concentration. Both types of splenocytes were mixed in an approximately 1:1 ratio and the mixture was administered to Treg-depleted and Treg-nondepleted infected/nontreated mice on day 9 after challenge. Cell death was monitored on the following day by CFSE flow cytometry assay of splenocytes recovered from killed mice. (F) Individual mouse analysis. Left peaks correspond to noninfected splenocytes and right peaks to infected splenocytes. The decrease of the right peak in the presence of the anti-CD25 mAb is indicative of CTL activity against FrCas^E-infected cells. (G) Means ± SEM of 3 independent experiments with 5 mice per group. The statistical significance between groups was established using the Student t test. **P < .01.