TF cytoplasmic domains signaling coupling to the NAPDH oxidase is required for aPL development. (A) Flow cytometric analysis of endosomal ROS production in MM1 cells stimulated with aPL HL5B and the effect of inhibitors. Cells were loaded with H2DCFDA before stimulation with HL5B without or with NAPc2, anti-TF 5G9 (aPL signaling noninhibitory) and 10H10 (aPL signaling inhibitory), or anti-PAR1 ATAP/WEDE; n = 5, means ± SD of mean fluorescence intensity; ∗P ≤ .001; 2-way ANOVA, Sidak multiple comparisons test. (B-C) Serum anticardiolipin (CL) (B) or anti-β2GPI (C) titers in mice of the indicated genotypes immunized with aPL HL5B; n = 5 mice, ∗P ≤ .03; 2-way ANOVA, Sidak multiple comparisons test. (D-E) The indicated mouse strains mice were infected with mCMV and serum anti-LBPA (D) or anti-β2GPI (E) titers were measured 5 and 12 weeks after infection; n = 6 mice, ∗P ≤ .0001; 2-way ANOVA, Sidak multiple comparisons test. (F) Representative example of flow cytometric detection of peripheral blood B cells reactive with fluorescently labeled PL vesicles or fluorescently labeled β2GPI. Seven weeks after CMV infection, circulating lipid-reactive as well as β2GPI-reactive B1 cells are found in C57BL/6J WT mice but not in TFΔCT mice.
