Influence of the level of viral replication and the magnitude of the CD8⁺ T-cell response on the expansion of Treg during FV infection. (A) Mice were infected with 4 different doses of FV (100; 1000; 10 000; 25 000 SFFU) to investigate the role of the viral inoculation dose on the expansion of Treg. Flow cytometry was used to detect numbers of CD4⁺ Foxp3⁺ Treg in lymph nodes, spleen, and bone marrow at 10 dpi. Each column represents mean numbers + SEM of CD4⁺ Foxp3⁺ Treg per 1 million nucleated cells for a group of 6 mice. Data were pooled from 2 independent experiments with similar results. (B) Numbers of CD4⁺ Foxp3⁺ Treg were plotted against numbers of CD8⁺ CD43⁺ T cells in the bone marrow of mice at 10 dpi with different doses of FV. Each symbol represents an individual mouse. Pearson 1-grouping variable tables correlation analysis was performed for the numbers of CD4⁺ Foxp3⁺ Treg and CD8⁺ CD43⁺ T cells for individual mice (r = 0.7886; P = .0039). A representative experiment is shown. The experiment was repeated twice with similar results. (C) Mice were infected with 25 000 SFFU of FV and depleted for their CD8⁺ T ells by injecting monoclonal antibody 169.4. At 10 dpi, numbers of CD4⁺ Foxp3⁺ Treg were determined by flow cytometry in the spleen and bone marrow. Each column represents mean numbers + SEM of CD4⁺ Foxp3⁺ Treg per 1 million nucleated cells for a group of 5 to 7 mice. Data were pooled from 2 independent experiments with similar results. Differences between the 2 groups were analyzed using the Mann-Whitney test. Statistically significant differences between the groups are indicated in the figure. (D) Noninfected mice (□) and mice at day 7 post-FV infection (■) were exposed to BrdU in drinking water. At 10 dpi, cells from lymph nodes, spleen, and bone marrow were stained for CD4, CD25, Foxp3, and incorporated BrdU. The figure shows mean numbers + SEM of CD4⁺ Foxp3⁺ Treg per 1 million nucleated cells positive for BrdU in the 3 different organs of naive and FV-infected mice. Six mice per group were analyzed. The experiment was repeated twice with similar results. Differences between naive mice and mice infected for 10 days with FV were analyzed by the unpaired Student t test. Statistically significant differences between the groups are indicated in the figure.