Fig 1.
Depletion of Tregs during FV infection.
Gating strategy for Tregs (A-C). B6-FOXP3-DTR mice were injected with 20,000 SFFU i.v. FV and left untreated (B) or injected with DT on day 0, 3, and 6 relative to infection (C). Spleens were harvested at 1 (C) or 2 wpi (B, C) and cells were analyzed by flow cytometry compared to naïve mice. (B) Splenic Tregs from non-depleted mice were analyzed for CD43, CD62L and Ki-67 expression, represented as a percentage of Tregs. (C) The total number of Tregs at 1 wpi or 2 wpi with or without Treg depletion was compared to the levels in naïve mice. Each dot represents an individual mouse pooled from experiments with 2–6 mice per group. Lines indicate the mean of each group. * P < .05, **P < .01, ****P < .0001 as determined by ordinary one-way ANOVA with Tukey post-test for multiple comparisons or an unpaired two-tailed t test for comparisons between only two groups.
Fig 2.
Transient Treg depletion enhances B cell activation in FV-infected mice.
B6.FOXP3-DTR mice were injected with 20,000 SFFU i.v. and left untreated or injected with 0.5 μg DT i.p. at 0, 3, and 6 dpi. Spleens were harvested at 2 wpi and total splenocytes (A, B) or CD19+CD4- B cells (C, D, E) were analyzed by flow cytometry. (B) Absolute numbers of CD19+ cells per spleen. (C) Representative histograms of CD86 expression on B cells; Mean MFI of Naïve: 221, FV: 304.8, FV + DT: 506.5, P < .001. (D) %Ki-67+CD86+ B cells in the spleen. (E) Number of Ki-67+ CD86+ B cells/spleen. Each dot in (B, C, D, E) represents an individual mouse pooled from experiments with 2–6 mice per group. Lines indicate the mean of each group. *P < .05, **P < .01, ***P < .001, ****P < .0001 as determined by ordinary one-way ANOVA with Tukey post-test for multiple comparisons.
Fig 3.
Transient Treg depletion enhances GC B cell and plasma cell responses in FV-infected mice.
B6.FOXP3-DTR mice were injected with 20,000 SFFU i.v. and left untreated or injected with 0.5 μg DT i.p. at 0, 3, and 6 dpi. Spleens were harvested at 2wpi and CD19+CD4- B cells (A–C, G–I), CD38-Gl7+ germinal center B cells (D–F), or CD38+CD138+ plasma cells (J–L) were analyzed by flow cytometry. Representative FACS plots showing GC B cell (A) or plasma cell gates (G). (D, J) Representative histograms of CD86 expression on GC B cells; (D) Mean MFI of Naïve: 611, No DT: 800.0, DT: 944.3, P < .05, or plasma cells (J) Mean MFI of Naïve: 476.5, No DT: 542.2, DT: 1340.7, P < .001. Quantification of %Ki-67+CD86+ (E, K), and total number of Ki-67+CD86+ (F, L) GC B cells or plasma cells as indicated. Each dot represents an individual mouse pooled from experiments with 2–6 mice per group. Lines indicate the mean of each group. * P < .05, **P < .01, ***P < .001, ****P < .0001 as determined by ordinary one-way ANOVA with Tukey post-test for multiple comparisons.
Fig 4.
Transient Treg depletion enhances B cell class switching in FV-infected mice.
B6.FOXP3-DTR mice were injected with 20,000 SFFU i.v. and left untreated or injected with 0.5 μg DT i.p. at 0, 3, and 6 dpi. Mice were bled and spleens were harvested at 2wpi or 3 wpi and total CD19+CD4- B cells were analyzed for IgM and IgD expression by flow cytometry. Each dot represents an individual mouse pooled from experiments with 2–6 mice per group. Lines indicate the mean of each group. ****P < .0001 as determined by ordinary one-way ANOVA with Tukey post-test for multiple comparisons.
Fig 5.
Transient Treg depletion enhances antibody responses against FV.
B6.FOXP3-DTR mice were injected with 20,000 SFFU i.v. and left untreated or injected with 0.5 μg DT i.p. at 0, 3, and 6 dpi. Mice were bled at 1, 2, and 3 wpi and plasma was analyzed for FV-binding IgG by ELISA (A), IgM (1:10 dilution) by ELISA (B), or FV-neutralizing antibody by neutralizing antibody assay (C). Each dot represents plasma from an individual mouse. Lines indicate the mean of each group. ***P < .001, as determined by ordinary one-way ANOVA with Tukey post-test for multiple comparisons.
Fig 6.
Transient Treg depletion enhances antibody responses against FV.
B6.FOXP3-DTR mice were injected with 20,000 SFFU i.v. and left untreated or injected with 0.5 μg DT i.p. at 0, 3, and 6 dpi. Mice were bled at 1, 2, and 3 wpi and infectious units/mL blood were determined by viremia assay (A). Spleens were harvested at 2 and 3 wpi (B, C). Infectious centers per spleen were determined by infectious center assay and %Ter119+ cells were analyzed by flow cytometry. Each dot represents an individual mouse from experiments with 2–6 mice per group. Lines indicate the mean of each group. * P < .05, **P < .01, ****P < .0001, as determined by ordinary one-way ANOVA with Tukey post-test for multiple comparisons.