Fig. 2

CHD8 deficiency does not affect Treg homeostasis but causes Treg activation. A Freshly isolated splenocytes were subjected to surface staining of CD4 and intracellular staining of Foxp3 (versus isotype control) and then analyzed by FACS. Representative dot plots of Foxp3 staining gated from CD4⁺ cells are shown. The graph panels show the proportions and absolute numbers of Tregs. B Treg proliferation. BrdU was injected into WT and Chd8^−/− mice 16 h before splenocytes were harvested for surface staining of CD4 and intracellular staining of Foxp3 and BrdU (versus isotype control). Representative dot plots of BrdU staining gated on CD4⁺Foxp3⁺ cells are shown. The percentages of CD4⁺Foxp3⁺ Tregs incorporated with BrdU are shown in a bar graph. C Treg apoptosis. Splenocytes were subjected to surface staining of CD4 and intracellular staining of Foxp3 and active caspase 3 (versus isotype control). Representative dot plots of active caspase3 staining gated on CD4⁺Foxp3⁺ cells are shown. The percentages of CD4⁺Foxp3⁺ Tregs expressing active caspase 3 are shown in a bar graph. D Splenocytes were subjected to surface staining of CD4, CD62L, and CD44 and intracellular staining of Foxp3. Representative dot plots of CD62L and CD44 staining gated on CD4⁺Foxp3⁺ cells are shown. The percentages and mean fluorescence intensity (MFI) of CD44⁺ cells are shown in bar graphs. E–G Splenocytes were subjected to surface staining of CD4 E–G, GITR E, ICOS F, and PD-1 G, and intracellular staining of Foxp3 E–G. Representative dot plots of GITR, ICOS, and PD-1 staining gated on CD4⁺Foxp3⁺ cells are shown. The percentages and MFI of the positively stained cells are shown in bar graphs. Results in bar graphs represent mean plus SD (WT: five males + five females; Chd8^−/−: three males + three females). *P < 0.05, **P < 0.01 versus WT control group