Somatic hypermutation of IGHV4-34 autoantibody Fog-1 removes self-reactivity while increasing binding to RhD alloantigen. (A and B) Variable domain amino acid sequence of the IMGT-predicted pFog-1 H chain (A) and L chain (B) and sequence substitutions acquired in the immune Fog-1 antibody (shown below in red). (C) Gating strategy for measuring self-reactivity of IgG by binding to mature naive B cells (CD19⁺, IgD⁺ CD27⁻, CD14⁻, and CD3⁻), flow cytometric histograms representative of four independent experiments, and dose-dependent binding measured as relative MFI. Data points are the mean and standard deviation of four separate experiments using PBMCs from four individual donors. Statistical significance was assessed using two-way ANOVA (*, P ≤ 0.05 for Fog-1 H26Y vs. Fog-1 and pFog-1 Y26H; ****, P ≤ 0.0001 for pFog-1 vs. Fog-1, pFog-1 Y26H, and Fog-1 H26Y). The key demonstrates the number of amino acid substitutions (#aa subs) in each antibody. Numbering follows IMGT. SHM, somatic hypermutation. (D) Binding of Fog-1 and pFog-1 IgG to RhD⁺ and RhD⁻ erythrocytes. Data in D are representative of three separate experiments using erythrocytes from three individual donors. Statistical significance was determined by two-way ANOVA (****, P ≤ 0.0001 for Fog-1 and Fog-1 H26Y vs. pFog-1 and pFog Y26H). (E) Binding of pFog-1 to mature naive B cells at 4°C (continuous line) and 37°C (dashed line) by flow cytometry. Data are representative of two independent experiments, and statistical significance was determined by Student’s paired t test (P = 0.10). (F) Structural models depicting the hydrophobic patch region of pFog-1 (left) and Fog-1 (right). Preimmune residues are depicted in blue with immune substitutions in dark red and oxygen in light red.