Fig. 5

BCL10 and MALT1 proteolytic activity control regulatory T cell (Treg) suppression. a Heat map of a subcluster of differentially expressed genes in sorted CD4⁺EYFP⁺CD44^hiCD62L^lo splenic effector Tregs (eTregs) of Bcl10^+/+;Rosa26^LSL-EYFP;FIC (+/+) and Bcl10^fl/fl;Rosa26^LSL-EYFP;FIC (fl/fl) female mice. The blue color indicates a low abundance of messenger RNAs (mRNAs), whereas the red color depicts high mRNA expression. b Quantified median fluorescence intensities (MFIs) of CTLA4, OX40, PD-1, and TIGIT on splenic CD4⁺Foxp3⁺CD44^hiCD62L^lo-gated eTregs in Bcl10^+/+;Rosa26^LSL-EYFP;FIC and Bcl10^fl/fl;Rosa26^LSL-EYFP;FIC female mice. c MFI of CTLA4 in sorted CD4⁺EYFP⁺CD44^loCD62L^hi resting Tregs (rTregs) of ≥2 pooled female Bcl10^fl/fl;Rosa26^LSL-EYFP;FIC and Bcl10^+/fl;Rosa26^LSL-EYFP;FIC control mice following 3 days of differentiation with anti-CD3/CD28. Data are cumulative from four independent experiments; statistical significance was assessed by a two-tailed ratio-paired t test. d MFI of CTLA4 on viable EYFP^– or EYFP⁺ CD4⁺Foxp3⁺CD44^hiCD62L^lo-gated eTregs of female Bcl10^+/+;Rosa26^{LSL-EYFP/LSL-IKK2-CA};FIC controls, Bcl10^fl/fl;Rosa26^{LSL-EYFP/LSL-IKK2-CA};FIC and Bcl10^fl/fl;Rosa26^LSL-EYFP;FIC mice. Statistical significances were calculated by ordinary one-way analysis of variance (ANOVA) combined with Tukey’s multiple comparisons test; (ns) not significant. e MFIs of CTLA4 on viable splenic CD4⁺Foxp3⁺CD44^hiCD62L^lo-gated eTregs in 16-day-old Bcl10^fl/fl and Bcl10^fl/fl;FIC mice. Data are cumulative from two independent experiments with ≥2 mice each. f, g CTLA4 expression on in vitro differentiated, sorted CD4⁺EYFP⁺CD44^loCD62L^hi rTregs of ≥2 pooled female Bcl10^+/+;Rosa26^LSL-EYFP;FIC or Bcl10^+/fl;Rosa26^LSL-EYFP;FIC control mice after anti-CD3/CD28 stimulation in the presence of the MALT1 protease inhibitor mepazine (5 μM) f alone or g with added tumor necrosis factor (TNF (20 ng mL⁻¹) and interleukin-1β (IL-1β (20 ng mL⁻¹). Dimethyl sulfoxide (DMSO) was used as a control. The graphs show the MFI of viable CD4⁺EYFP⁺-gated cells on day 3 from f six and g two independent experiments. Statistical significance was assessed by a two-tailed ratio-paired t test. h Western immunoassay (WES) immunoblot analysis of Regnase-1 expression in sorted CD4⁺EYFP⁺ Tregs of male Bcl10^fl/fl;FIC or Bcl10^+/+;FIC control mice. β-Actin served as a loading control. The bands shown are from one WES immunoblotting run. i Normalized Regnase-1 to β-actin peak area of different biological replicates analyzed for Regnase-1 and β-actin expression using the WES immunoblotting system. Each dot represents one mouse. Statistical significances (b, e, i) were assessed by a two-tailed unpaired Student’s t test. Bars in b–g and in i represent the mean ± SD. Source data are provided as a Source Data File