Fig. 6

MALT1 protease activity in regulatory T cells (Tregs) is critical for immune suppression. a Immunoblot analysis to detect full-length (FL) and cleaved (CL) Regnase-1, and FL Roquin in sorted CD4⁺EYFP^–CD25^–CD45RB^hi naive conventional T cells (Tconv) and CD4⁺EYFP⁺ Tregs of diseased male Malt1^fl/PM;Rosa26^LSL-EYFP;FIC mice minus (−) or plus (+) phorbol myristate acetate (PMA)/ionomycin. Phospho (p)-Erk served as a stimulation control, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control. b Image stream analysis detecting nuclear translocation of p65 (upper two images) and c-Rel (lower two images) in splenic CD4⁺Foxp3⁺ Tregs of male Malt1^fl/PM;FIC and Malt1^fl/+;FIC mice upon PMA/ionomycin. 4′,6-Diamidino-2-phenylindole (DAPI) was used as a nuclear stain. Black bars indicate 10 μm. (BF) brightfield. c Histograms of the similarity score of p65 (upper panel) or c-Rel (lower panel) and the DAPI stain. Black (Malt1^fl/+;FIC) and red (Malt1^fl/PM;FIC) histograms represent the similarity score in splenic CD4⁺Foxp3⁺ Tregs upon PMA/ionomycin. The gray histogram indicates unstimulated splenic CD4⁺Foxp3⁺ Tregs (Malt1^fl/PM;FIC). d Frequencies of splenic CD44^hiCD62L^lo effector Tregs in the viable CD4⁺Foxp3⁺ cell gate of male Malt1^fl/PM and Malt1^fl/PM;FIC mice e Frequencies of EYFP^–- and EYFP⁺-gated CD44^hiCD62L^lo effector Tregs in the viable CD4⁺Foxp3⁺ cell gate of female Malt1^fl/+;Rosa26^LSL-EYFP;FIC and Malt1^fl/PM;Rosa26^LSL-EYFP;FIC mice. Statistical significances were assessed by ordinary one-way analysis of variance (ANOVA) combined with Tukey’s multiple comparisons test. f, g Quantified median fluorescence intensities (MFIs) of f PD-1 and g CTLA4 on viable CD4⁺Foxp3⁺CD44^hiCD62L^lo-gated splenic effector (eff) Tregs in male Malt1^fl/PM;FIC and Malt1^fl/PM mice. h Size of spleens (left) and lymph nodes (right) in male Malt1^fl/PM;FIC (n = 4) and Malt1^fl/PM (n = 4) mice, aged 40 days. Scale bars represent 1 cm. i FACS plots of CD44^hiCD62L^loCD4⁺Foxp3^–-gated splenic cells of Malt1^fl/PM (n = 4) and Malt1^fl/PM;FIC (n = 4) mice. j Quantified serum concentrations of anti-cardiolipin (left) and anti-nuclear immunoglobulin (right) in male Malt1^fl/PM;FIC and Malt1^fl/PM mice. k Survival curves of male Malt1^fl/PM;FIC (red line, n = 4) and Malt1^fl/PM control (black line, n = 4) mice. Statistical significance was calculated by a log-rank (Mantel–Cox) test. l In vitro Treg-suppressor assay with sorted and Cell Proliferation Dye 450-labeled naive Tconv cells cultivated at a 2:1 ratio without any Tregs (left panel), with sorted splenic CD4⁺EYFP⁺ Tregs of either Malt1^fl/+;Rosa26^LSL-EYFP;FIC/FIC (middle panel) or Malt1^fl/PM;Rosa26^LSL-EYFP;FIC/FIC mice (right panel) in the presence of irradiated splenocytes and anti-CD3. FACS plots show the proliferation of viable cells. Bars in d–g and j represent the mean ± SD. Data are representative of a–c, l two or cumulative from d–g two independent experiments with d, f, g n = 2 or b, c, l n = 3 mice per genotype. Statistical significances in d, f, g, j were assessed by a two-tailed unpaired Student’s t test. Source data are provided as a Source Data File