Functional characterization PF-associated CR3/4 variants. (A-B) WT and variant complement receptors were cloned and expressed in HEK-293T cells, and surface expression levels of CR3 (A) and CR4 (B) were evaluated by flow cytometry for integrin β₂ (ITGB2/CD18). WT CR3 and WT CR4 are depicted in orange. Receptors containing a variant integrin β₂ subunit are depicted in blue, those with a variant integrin α_M (ITGAM) subunit are depicted in red, and those with a variant integrin α_X (ITGAX) subunit are depicted in green. (C-D) The ability of complement receptor-expressing cells to bind immobilized iC3b was assessed using a solid-phase functional assay. iC3b binding activity of WT and variant CR3 (C) and WT and variant CR4 (D) are shown. ∗P < .05 compared to WT/WT control. (E-F) TNFα-mediated activation of an NF-ĸB dual-luciferase reporter in cells expressing the indicated PF-associated CR3 (E) or CR4 variants (F) in the presence or absence of iC3b. Control HEK-293T cells not expressing CR3 or CR4 (Untransfected) and HEK-293T cells expressing WT CR3 or WT CR4 were used as negative and positive controls, respectively. Data are shown as the mean fluorescence intensity ratio of the iC3b-plus condition to the iC3b-minus condition. Error bars represent standard error of the mean. (G) PF-associated variants in ITGAM (M∗) reduce the ability of variant CR3 (CR3∗) to suppress inflammatory signaling. (H) PF-associated variants in ITGAX (X∗) enhance the ability of variant CR4 (CR4∗) to activate inflammatory signaling. (I) PF-associated variants in the shared subunit ITGB2 (B2∗) simultaneously decrease suppression of inflammatory signaling by CR3∗ and increase activation of inflammatory signaling by CR4∗. ITGAM H687R, ITGAM T1001N, ITGAX V1019M, and ITGB2 P7Q were predicted in silico to be neutral. Shown in panels A-F are representative results of at least 3 independent experiments.