Figure 3.

Naive and infection-experienced T_regs have comparable phenotypes in systemic, homologous challenges. CD4+GFP+ T_regs were sorted from naive, LCMV- (A–C) or VV- (D–F) experienced CD45.1 + Foxp3-GFP reporter mice >30 days after the primary infection and 500’000 of each population were adoptively transferred into separate groups of CD45.2+ recipient mice followed by acute LCMV (A–C) or VV (D–F) infection. (A–C) T_reg recipients were sacrificed 10 days post LCMV infection and the transferred T_regs characterized in the spleen. (A) Frequencies of transferred naive or LCMV-experienced T_regs within the total T_reg population (top) and absolute numbers of recovered, transferred T_regs (bottom). (B) Naive or LCMV-experienced transferred T_regs were phenotypically characterized by flow cytometry. Representative plots (top) and cumulative data (bottom) depicting frequencies of marker positive cells among the transferred T_regs. (C) Splenocytes of recipient mice were re-stimulated with LCMV peptides (gp33 and gp61) for 4 h followed by intracellular cytokine staining for IFN-γ. (Mean ± SD; biological replicates: naive n = 4–14, LCMV-experienced n = 3–10; 3 independent experiments). (D–F) T_reg recipients were sacrificed 7 days post VV infection and the transferred T_regs characterized in the spleen (D). Frequencies of transferred naive or VV-experienced T_regs within the total T_reg population (top) and absolute numbers of recovered, transferred T_regs (bottom). (E) Naive or VV-experienced transferred T_regs were phenotypically characterized by flow cytometry. Representative plots (top) and cumulative data (bottom) depicting frequencies of marker positive cells among the transferred T_regs. (F) Splenocytes of recipient mice were re-stimulated with VV peptide for 4 h followed by intracellular cytokine staining for IFN-γ. (Mean ± SD; biological replicates: naive n = 6, VV-experienced n = 8; 2 independent experiments) (t Test, *p < 0.05, **p < 0.01, ***p < 0.001).