Figure 1.
Preferential expansion of Teff cells over Tregs during acute BCG infection.
WT mice were infected i.v. with 2×106 CFU M. bovis BCG (black squares), or not (black dots), and the (A) frequency and (B) total cell number of FoxP3+ Tregs within the live CD4+ T cell gate was determined in spleen (left) and lungs (right) at day 20 p.i.. Data are pooled from three independent experiments and represent the mean ± SD of 11–12 mice per group. Each symbol represents an individual mouse. N = 3. Statistical analysis: Mann-Whitney-U-Test. *p<0.05; **p<0.01; and ***p<0.001.
Figure 2.
M. bovis BCG infection induces Treg activation but does not affect their suppressive capacity.
Mice were infected i.v. with 2×106 CFU M. bovis BCG, or not, and the phenotype and suppressive capacity of Tregs was evaluated at day 20 p.i. (A) Expression of CTLA-4, GITR, CD103, ICOS, PD-1 and CD25 within the FoxP3+CD4+ Treg population in the blood (upper panel) and spleen (lower panel) of M. bovis BCG-infected (black line) vs non-infected (grey shaded area) WT mice. Results show representative FACS plots of 3 mice per group. N = 2. (B) Suppressive capacity of Tregs isolated from naïve (white bars) or day 20 p.i. (black bars) M. bovis BCG-infected DEREG mice determined by an in vitro suppression assay. Proliferation of CD25−CD4+ Thy1.1+ effector T cells was assessed using different ratios of Treg:Teffector cells in the presence of 1 µg/mL soluble αCD3. For the negative control, αCD3 was omitted. Means of three wells ± SD are depicted. N = 3.
Figure 3.
Minor effect of Treg depletion on M. bovis BCG bacterial burden.
(A) DEREG mice were infected i.v. with 2×106 CFU M. bovis BCG and Tregs were depleted by DT administration on days 2/3, 9/10 or 14/15 (black dots) in a single-depletion round or left untreated (white dots). CFU were determined in lungs, spleen and liver 20 days p.i.. Each symbol represents an individual mouse. Data represent mean ± SD of 3 mice per group. N = 1. (B) DEREG mice were infected i.v. with 2×106 CFU M. bovis BCG and Tregs were depleted by DT administration on days 7/8 and 14/15 (black dots) in two rounds of depletion or were left untreated (white dots). CFU were assessed in lungs, spleen and liver on day 20 p.i.. Each symbol represents an individual mouse. Data represent mean ± SD of 3–5 mice per group. N = 3.
Figure 4.
Limited impact of Treg depletion on inflammatory cytokine production after M. bovis BCG infection.
Intracellular cytokine production by FoxP3−CD4+ T cells was analysed in the spleen of day 7/8 and 14/15 double-depleted (black bars) or untreated DEREG mice (white bars) on day 20 after i.v. infection with 2×106 CFU M. bovis BCG. Percentages of (A) IFN-γ, (B) IL-17A and (C) IL-10 production within live FoxP3−CD4+ T cells after PMA/ionomycin restimulation are shown. Bar graphs represent mean ± SD of 3–5 mice per group. N = 3. Statistical analysis: Mann-Whitney-U-Test. *p<0.05; **p<0.01; and ***p<0.001.
Figure 5.
eGFP− diTregs rapidly replenish the pool of Tregs in M. bovis BCG infected DEREG mice.
DEREG mice were infected i.v. with 2×106 CFU M. bovis BCG and treated, or not, with DT on days 7/8 and 14/15 p.i. and (A) the percentage of eGFP+ (grey)- and eGFP− (white) Foxp3+CD4+ Tregs cells in the spleen (left) and lungs (right) or (B) the expression of the thymic Treg markers Helios (left) and Nrp-1 (right) in the eGFP+ (grey) and eGFP− (white) Treg population were analysed in the spleen at day 20 p.i. in DT-treated mice. Bar graphs represent mean ± SD of 3–5 mice per group. N = 3. Statistical analysis: Mann-Whitney-U-Test. *p<0.05; **p<0.01; and ***p<0.001.
Figure 6.
Removal of Tregs after Mtb aerosol infection in DEREG mice does not influence pathogen control.
DEREG mice were infected with approx. 100Mtb via the aerosol route. Tregs were depleted by DT administration on days 11/12, 18/19 and 25/26 (black dots/bars) or not (white dots/bars). (A) Experimental scheme. (B) Mycobacterial colony enumeration assays were performed in lungs (left), spleen (middle) and liver (right) on day 20, 28 and 42 p.i.. Data represent mean ± SD of 5 mice per group. (C) The frequency of ESAT61–20-specific IFN-γ- and IL-17A-producing CD4+ cells per 105 total lung cells was determined by ELISPOT assay at different time points after infection. Bar graphs represent the mean ± SD of 5 mice per group. N = 3 (day 20), N = 1(day 28 and 42).