Fig. 7: Tfam deletion disrupts B cell mobility.
a, Representative Airyscan IHC images of spleen sections depicting GCs. Quantification of the dark zone area normalized to the GC area in SRBC-immunized Aicda-WT (n = 41 GCs) and Aicda-Tfam (n = 17 GCs) mice. Image analyses were performed including all GCs identified on two sections from n = 2 mice of each genotype. Scale bar, 100 μm. Representative of four independent experiments. b, Representative flow cytometry plots of the dark zone, gray zone and light zone subpopulations of tdTomato+CD38−GL-7+ GC B cells. Quantification of the dark zone to light zone ratio from NP-CGG-immunized Aicda-WT (n = 8) and Aicda-Tfam (n = 9) mice is shown. Representative of four independent experiments. c, 3D Airyscan confocal images of magnetic bead-sorted and F-actin phalloidin + 4,6-diamidino-2-phenylindole (DAPI)-stained GC B cells from Aicda-WT and Aicda-Tfam mice. Scale bar, 6 μm. Representative of two independent experiments. d, Representative flow cytometry histogram of F-actin phalloidin fluorescence of tdTomato+GL-7+ GC B cells and tdTomato−IgD+ naive B cells from immunized Aicda-WT (n = 6) and Aicda-Tfam (n = 8) mice. Quantification of F-actin phalloidin gMFI in GC B cells normalized to naive B cells from the same host is shown. Data were pooled from three independent experiments. e, Quantification of chemotaxis to CXCL12 and CXCL13 in GC B cells from Aicda-WT (n = 3) and Aicda-Tfam (n = 5) mice. Data are representative of four independent experiments. f, Representative Fluo-4 AM geometric mean signal intensity kinetics (moving average) of B-WT (n = 4) and B-Tfam (n = 4 mice) B220+ B cells stimulated with CXCL12 for 120 s. Quantification of the area under the curve (AUC) between 60 and 90 s (corresponding to the peak response) is shown. g, Representative Fluo-4 AM gMFI signal kinetics (moving average) of B-WT (n = 4) and B-Tfam (n = 4 mice) B220+ B cells stimulated with anti-IgM for 300 s. Quantification of the AUC between 60 and 300 s is shown. In f and g, experiments were run as technical duplicates in four independent replicate experiments consisting of one B-Tfam and one WT mouse in each. The data points from the B-Tfam mice were normalized to the WT data run in the same batch. h, Representative flow cytometry histogram of MCU fluorescence in B220+ IgD+ B cells from unimmunized B-Tfam and B-WT mice. Quantification of MCU gMFI in IgD+ B cells from B-Tfam and B-WT mice (n = 3). Data are representative of three independent experiments. i, Representative flow cytometry histogram of mtROS-Deep Red fluorescence in IgD+ B cells from B-WT (n = 4) and B-Tfam (n = 5) mice. Data are representative of two independent experiments. j, Quantification of chemotaxis to CXCL12 in B cells from unimmunized B-Tfam (n = 3) and B-WT (n = 5) mice in the presence of mitoTEMPO or Ru265. Data are representative of two independent experiments and were pooled after batch correction. Statistical significance was calculated using an unpaired two-tailed t-test (b,d,h,i), a two-tailed Mann–Whitney U-test (a,f,g) or a two-way ANOVA with Šidák’s correction (e), with batch as a covariate (j). Data are presented as the mean ± s.e.m.
