eGFP⁺Foxp3⁻ T cells lack suppressive activity.

<p>FACS-sorted CD4⁺eGFP⁻, CD4⁺eGFP⁺CD25^lo and CD4⁺eGFP⁺CD25^hi populations were added at varying ratios to responder T cells (Tresp) with simultaneous anti-CD3 stimulation. <i>Ex vivo</i> isolated CD4⁺CD25⁺eGFP⁺ cells (nTregs) from DEREG mice were used as control. (A) Dot plots demonstrate the purity of various FACS-sorted iTreg populations. Sorting was performed on the basis of eGFP and CD25 expression. (B) Comparison of Foxp3 expression on sorted iTreg sub-populations. Dotted line represents Foxp3 expression on live CD4⁺eGFP⁻ T cells, solid gray line represent Foxp3 expression on live CD4⁺eGFP⁺CD25^lo and solid black line represent Foxp3 expression on CD4⁺eGFP⁺CD25^hi T cells. (C) Representative histograms for dilution of proliferation dye on gated live CD4⁺ Tresp cells (left panel). Quantification of proliferated Tresp cells under various conditions (right panel). Stimulated and non-stimulated Tresp cells served as positive (Pos) and negative (Neg) controls, respectively. Error bars designate SD of triplicates from one representative of three individual experiments.</p

Culture conditions demonstrating eGFP and Foxp3 expression dichotomy amongst in vitro generated iTregs from DEREG mice.

<p>(A) Dot plots display eGFP and Foxp3 expression among the live CD4⁺ gated DEREG cells. Shown are <i>ex vivo</i> stained T cells (left), <i>in vitro</i> differentiated iTregs using soluble anti-CD3, TGF-β, RA and GM-CS- or Flt3L-derived BMDC (second and third panels respectively) and iTregs using plate-bound anti-CD3, anti-CD28, TGF-β and RA (right). (B) Differential expression of various surface antigens on eGFP⁺Foxp3⁻ (dotted line, upper panel) and eGFP⁺Foxp3⁺ (solid line, upper panel) iTregs and eGFP⁻Foxp3⁻ (dotted line, lower panel) and eGFP⁻Foxp3⁺ (solid line, lower panel) cells differentiated in the presence of TGF-β, RA, soluble anti-CD3 and DC. Gray histograms represent isotype controls. Graphs shown are representative of four individual experiments.</p

Removal of Tregs after <i>Mtb</i> aerosol infection in DEREG mice does not influence pathogen control.

<p>DEREG mice were infected with approx. 100<i>Mtb</i> 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 ESAT6_1–20-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).</p

Limited impact of Treg depletion on inflammatory cytokine production after <i>M. bovis</i> BCG infection.

<p>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 <i>M. bovis</i> 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. <i>*p<0.05; **p<0.01; and ***p<0.001</i>.</p

Preferential expansion of Teff cells over Tregs during acute BCG infection.

<p>WT mice were infected i.v. with 2×10⁶ CFU <i>M. bovis</i> 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. <i>*p<0.05; **p<0.01; and ***p<0.001</i>.</p

eGFP⁻ diTregs rapidly replenish the pool of Tregs in <i>M. bovis</i> BCG infected DEREG mice.

<p>DEREG mice were infected i.v. with 2×10⁶ CFU <i>M. bovis</i> 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. <i>*p<0.05; **p<0.01; and ***p<0.001</i>.</p