Autoantibodies encoding the V-region, IGHV4-34 cause cold agglutinin disease by binding a carbohydrate self-antigen, poly-N-acetyllactosamine (pNAL), on the surface of erythrocytes and B-cells. Yet, 5-10% of naïve B-cells express IGHV4-34 in healthy people. Previously, we demonstrated IGHV4-34 can contribute to a productive immune response during vaccination by clonal redemption – somatic hypermutation of V-regions to reduce binding to self and increase reactivity to immunogen. Notably mutations within a hydrophobic patch in IGHV4-34 that reduced self-reactivity were selected, despite also reducing binding to the immunogen. However, antibody sequencing data reveals <50% of IGHV4-34 memory B-cells contain mutations in the HP. This suggests regions outside the HP may contribute to IGHV4-34 self-reactivity or additional mechanisms are involved in tolerating IGHV4-34. The aim of this study was to track the development and mutational trajectories of IGHV4-34 in vivo.
Using a CRISPR-Cas9 knock in system, we generated mice expressing a human IGHV4-34 autoantibody that was elicited during immunisation with vaccinia virus. Flow cytometry with 9G4 antibody against the IGHV4-34 V-region bound 40-50% of B-cells in the spleen and bone marrow. 9G4+ (IGHV4-34) B-cells were present at all stages of development except the marginal zone. Similar to IGHV4-34 B-cells in healthy humans, 9G4+ B-cells in mice have reduced surface IgM expression. Mouse IGHV4-34 B cells (CD45.2+) were adoptively transferred into CD45.1 mice inoculated with vaccinia virus. IGHV4-34 antibody sequencing 10, 16 and 24-days post-vaccination revealed a similar mutational trajectory to the human antibody with the exception of HP mutations, which were rare in mice. Four nonsynonymous mutations outside the HP were selected, which decreased binding to self-antigen and increased binding to immunogen. These findings demonstrate the utility of human antibody knock in mice to track autoantibodies in vivo.