Enhanced activation of B cells and myeloid cells in ABIN1[D485N] mice. (A) Naive purified splenic B cells from WT and ABIN1[D485N] mice were stimulated with 200 ng/ml LPS, 10 µg/ml LTA, 200 ng/ml of the TLR7 agonist R848, 10 µg/ml α-IgM, or 1 µg/ml α-CD40 for 72 h or left unstimulated (control) before pulsing for a further 16 h with [³H]thymidine (0.5 µCi/well). [³H]thymidine incorporation into DNA was measured by harvesting and washing the cells followed by measurement of radioactivity incorporated. (B) Flow cytometric analysis of surface expression of the activation marker CD86 after stimulation of purified B cells with the agonists indicated. The filled histograms show results for WT cells and the empty histograms show the ABIN1[D485N] cells. (C) IL-6 and IL-12p40 secreted into the culture medium of B cells 48 h after exposure to the agonists indicated. (D) IL-6 and TNF secreted into the culture medium of BMDC 24 h after exposure to 100 ng/ml LPS, 2 µg/ml LTA, 1 µg/ml Pam3CSK4, 1 µg/ml R848, or 1 µM of the TLR9 agonist ODN 1826. Data are representative of five (A) or three (B–D) independent experiments with three to four mice of each genotype analyzed together. Error bars represent mean ± SD. *, P ≤ 0.05; **, P ≥ 0.005; ***, P ≤ 0.005 (two-tailed Student’s t test).