Figure 3. Female Ndfip1^fl/flFoxp3-Cre^+/− mice show similar T_reg cell changes as males.

(a–l) Lung homogenates, from 8- to 16-week-old hemizygous female Ndfip1^fl/fl Foxp3-Cre^+/− mice and Ndfip1^+/+ Foxp3-Cre^+/− controls, were analysed ex vivo by flow cytometry. (a) Representative flow plots showing the gating of T_reg cells (previously gated as live CD3⁺CD4⁺ cells). This gating was used to determine the overall frequencies of T_reg cells (b) in mice from the indicated genotypes. (c) Representative flow plots showing the gating of T_reg cells from a that are eT_reg or cT_reg cells. This gating was used to determine the (d) frequencies of eT_reg cells. (e) Representative flow plot of T_reg cells as gated in a and analysed for frequencies of YFP⁺ and YFP⁻ cells. (f) Ratio of YFP⁺ to YFP⁻ T_reg cells. (g,h) Percentages of (g) eT_reg cells or (h) Ki-67⁺ eT_reg cells, shown as a ratio of YFP⁺:YFP⁻ cells within each mouse to normalize for a Cre effect. (i–l) mean fluorescence intensity (MFI) of (i) ICOS, (j) GITR, (k) CD25 and (l) PD-1 expression on eT_reg cells, again shown as a ratio of YFP⁺:YFP⁻. P values determined by Student's t-test, with correction for unequal variances as appropriate. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Each dot shows data acquired from a single female mouse. Graphs show mean±s.e.m. All experiments were performed on at least two independent occasions.