Fig. 6

TACI interacts with active TLR9 and MyD88 through mTORC1. a ELISA of total IgG and IgA from human splenic MZ B cells cultured with or without APRIL and/or CpG and in the presence of control vehicle (vehi) or rapamycin (rapa) for 5 days. Ctrl, medium alone. b Frequency of viable cells, proliferated CFSE^lo cells and CD19⁺CD27^hiCD38^hi plasmablasts after culturing human splenic MZ B cells as in a. Proliferation data derive from experiments identical to those in Fig. 1e, which includes comparisons between MZ and follicular B cells with no exposure to rapamycin. c qRT-PCRs of mRNAs for BLIMP-1 (PRDM1), XBP-1 (XBP1), and PAX5 (PAX5) from human splenic MZ B cells cultured for 72 h as in a. Results are normalized to β-actin mRNA and presented as relative expression (RE) compared to B cells incubated with medium alone (ctrl). d IB of mTOR, TLR9, MyD88, and TACI following IP with an anti-TACI antibody of protein lysates from splenic IgD⁺ B cells cultured for 60 min with or without APRIL and/or CpG in the presence of control vehicle or rapamycin. Ctrl, medium alone. Red numbers indicate band intensity relative to TACI bands. e Top schematics: structure of mCherry-tagged WT TACI or D1, D2, and D3 TACI deletion mutants (acceptors) used in FRET assays. Bottom graphs: FCM of FRET signal in co-transfected 293 cells expressing donor TLR9-eYFP together with mCherry-tagged WT or mutant acceptor TACI proteins as well as frequency of FRET⁺ 293 cells (f) IFA of 293 cells stained for transfected TACI (red) and TLR9 (green) as well as endogenous endosomal Rab7 (magenta). Bar, 10 μm. White arrows indicate endosomal TACI-TLR9 co-localization. Data summarize two experiments with at least two donors in each experimental group (a, c, e), show one representative experiment of at least three with similar results (d, f), or correspond to three biological replicates for each cell type (b). Error bars, s.e.m.; *p < 0.05, **p < 0.01, ***p < 0.001 (two-tailed Student’s t test)