Fig. 4

Follicular Treg (Tfr) cell differentiation and prevention of autoantibody production depend on STIM1/STIM2. a Volcano plot of DEG in unstimulated WT and Stim1/2-deficient Treg cells overlaid with upregulated (red) or downregulated (blue) genes of the ‘Tfr cell signature’⁸. b Analysis of Tfr cells in the spleen and LNs of male WT and Stim1/2^Foxp3 mice; means ± SEM of four mice. c Representative immunofluorescence of PNA⁺ germinal centers (GCs) in submandibular LNs of untreated male WT and Stim1/2^Foxp3 mice; scale bar: 200 µm. d Analysis of GC B cells in male WT and Stim1/2^Foxp3 mice; means ± SEM of four mice. e Detection of IgG autoantibodies in the sera of male WT and Stim1/2^Foxp3 mice using a multiplexed auto-antigen array. Sera of 9–11 individual mice were tested. f Analysis of anti-red blood cell (RBC) IgG and IgM autoantibodies using ELISA; means ± SEM of six mice. g Detection of relative IgG binding to RBCs of male WT and Stim1/2^Foxp3 mice by flow cytometry; means of eight mice. h Representative blood smears of WT and Stim1/2^Foxp3 mice; scale bar: 10 µm. i Analyses of RBC counts and hemoglobin levels in the blood of young (<30 days) and older (>40 days) male WT and Stim1/2^Foxp3 mice; means of 6–13 mice. j–l Depletion of B cells in Stim1/2^Foxp3 mice inhibits RBC destruction but not the premature death of mice. j Analysis of blood B cells of male WT and Stim1/2^Foxp3 mice 4 days after injection of 1 mg isotype or 1 mg anti-CD20 (5D2) antibody; means of 4–6 mice. k RBC counts and hemoglobin levels in the blood of WT and Stim1/2^Foxp3 mice 2–4 weeks after antibody injection; means of 4–6 mice. l Cumulative survival of WT and Stim1/2^Foxp3 mice after antibody injection; 8–10 mice per cohort. Mean survival times (MST): Stim1/2^Foxp3 mice isotype control: 42.0 days; Stim1/2^Foxp3 mice anti-CD20: 47.5 days; WT mice: >80 days. Each dot in g, j and l represents one mouse. Statistical analysis in b, d, g, i–k by unpaired Student’s t-test; in (l) using the Mantel–Cox test. *p < 0.05; **p < 0.01; ***p < 0.001