Fig. 4

Enforced IκB kinase (IKK) activation cannot rescue the Bcl10 deficiency in regulatory T cells (Tregs). a PhosFlow experiment to detect the median fluorescence intensity (MFI) of phospho-nuclear factor-κB (p-NF-κB) p65 at serine 536 in the viable CD4⁺Foxp3⁺ Treg population in spleen samples of Rosa26^LSL-IKK2-CA;FIC female mice with or without (w/o) PMA/ionomycin stimulation. Due to random X inactivation, female mice were mosaic with GFP⁺ Tregs expressing the IKK2-CA variant and GFP^– non-IKK2-CA-expressing Tregs. Statistical significances between the GFP^– and GFP⁺ populations were assessed by a two-tailed paired t test. b Quantified analysis of CD4⁺Foxp3^–CD44^hiCD62L^lo-gated splenic CD4 T effector (T_eff) cells (left graph), CD8⁺CD44^hiCD62L^lo-gated splenic CD8 T_eff cells (middle graph), and the median fluorescence intensity (MFI) of cell surface CD86 on CD19⁺B220⁺-gated splenic B cells (right graph) of either wild-type control (Ctrl) or diseased Bcl10^fl/flRosa26^LSL-IKK2-CA;FIC male mice. A statistical significance between genotypes was assessed by a two-tailed unpaired Student’s t test. c Survival curves of Bcl10^+/+Rosa26^LSL-IKK2-CA;FIC control (gray line, n = 4), Bcl10^fl/flRosa26^LSL-IKK2-CA;FIC (red line, n = 3), and Bcl10^fl/fl;FIC male mice (black line, n = 14). Survival equals the day the animal had to be sacrificed to avoid severe burden. Statistical significances between the survival curves were calculated by a log-rank (Mantel–Cox) test; (ns) not significant. d In vitro Treg-suppressor assay assessing the suppressor activity of Tregs of FIC and Rosa26^{LSL-CARD11-CA};FIC mice. Sorted splenic CD4⁺CD25^–CD45RB^hi naive conventional T cells of FIC mice were labeled with Cell Proliferation Dye eFluor 450 and cultivated for 3 days without any Tregs (lower panel), with sorted splenic CD4⁺CD25⁺CD45RB^lo Tregs of FIC mice (upper panel) or with sorted splenic CD4⁺CD25⁺CD45RB^loGFP⁺ Tregs of Rosa26^{LSL-CARD11-CA};FIC mice (middle panel) in the presence of irradiated splenocytes and soluble anti-CD3. FACS plots show the proliferation profiles of Cell Prolif Dye 450⁺-gated cells cultivated with different ratios of Tregs. Bars in a, b represent the mean ± D. Data in a, b are cumulative from two independent experiments and data in d are representative of three independent experiments. Source data are provided as a Source Data File