PMC

F-MuLV load in DEREG mice infected with F-MuLV and depleted for Tregs. F-MuLV-infected DEREG mice were treated with DT to deplete Foxp3⁺ Treg during the first 10 days of infection (white columns). Control DEREG mice were infected but received PBS instead of DT (black columns). Viral loads were measured in the spleen of the 10 days infected mice. Each column represents mean numbers plus SEM per one million nucleated cells. Data was pooled from two independent experiments with similar results. Differences between the groups were analyzed by an unpaired t-test. A statistically significant difference between the groups was not found (P = 0.19).

Expansion of Treg after infection with F-MuLV. C57BL/6 mice were infected with F-MuLV and sacrificed 8 or 10 days after infection. Then, suspensions of spleen cells were prepared. Flow cytometry was used to quantify the numbers of CD8⁺ T cells specific for the H-2D^b-restricted F-MuLV glycosylated gag epitope (MHC I Tetramer⁺), and CD4⁺ T cells specific for the A^b-restricted FV-H19-Env epitope (MHC II Tetramer⁺) in the spleen. A. Numbers of CD8⁺ MHC I Tetramer⁺ T cells per one million spleen cells. B. Numbers of CD4⁺ MHC II Tetramer⁺ T cells per one million spleen cells. C. Numbers of CD4⁺ Foxp3⁺ T cells per one million spleen cells. D. Numbers of CD4⁺Foxp3⁺ T cells expressing Ki-67 per one million spleen cells. E. A representative dot plot shows the gating-strategy to identify CD4⁺ Foxp3⁺ that were Npn-1⁺ or Npn-1^-. Upper graph, numbers of CD4⁺Foxp3⁺Npn-1⁺ cells per one million nucleated cells. Lower panel, numbers of CD4⁺Foxp3⁺Npn-1^-cells per one million nucleated cells. F. A representative dot plot shows the expression of Helios and Ki67 on gated CD4⁺Foxp3⁺Npn-1⁺ nTregs (upper panel) and on gated CD4⁺Foxp3⁺Npn-1^-iTregs (lower panel). The numbers in the upper quadrants represent the percentages of Helios⁺ cells. Data was pooled from three independent experiments with similar results. Differences between naïve mice and mice infected for 10 days with F-MuLV were analyzed by an unpaired t-test. Statistically significant differences between the groups are indicated in the figure.

CD8+ T cell responses in F-MuLV infected Treg-depleted DEREG mice. F-MuLV-infected DEREG mice were treated with diphtheria toxin (DT) to deplete Foxp3⁺ Tregs during the first 10 days of infection (white columns). Control mice were infected but received PBS instead of DT (black columns). Flow cytometry was used to determine numbers of CD8⁺ T cells reactive with FV-D^bgagL class I tetramers (A), or numbers of CD43⁺CD8⁺ T cells expressing granzyme A (GzmA) or granzyme B (GzmB) (B). Each column represents mean numbers plus SEM per one million nucleated cells for a group of 5-7 mice. Data was pooled from two independent experiments with similar results. Differences between the groups were analyzed by an unpaired t-test. Statistically significant differences between the groups are indicated in the figure. C. Splenocytes from naïve mice were loaded with FV-DbGagL peptides and labelled with CFSE to perform an in vivo CTL assay. As negative control, equal numbers of cells without FV peptide were used from CD45.1 B6 mice. Target cells were injected intravenously into naïve DEREG mice, DEREG mice infected for 10 days or F-MuLV-infected DEREG mice treated with DT. A histogram of CD45.1⁺ CFSE^- (transferred cells without peptide) and CFSE⁺ cells (peptide loaded) from the spleen of representative mice for each group (one from a group of 5 animals) is presented. The figure shows the percentage of target cell killing in the spleen. D. Flow cytometry was used to determine the mean fluorescence intensity (MFI) of Eomesodermin (Eomes) in CD8⁺ T cells from naïve mice and CD8⁺ T cells reactive with FV-DbgagL class I tetramers from 10 days F-MuLV infected mice. Differences between the groups were analyzed by an unpaired t-test. Statistically significant differences between the groups are indicated in the figure.