Convergent antibody mutation trajectories convert functional self-tolerance to genetic self-tolerance in IGHV4-34 B cells.

Preventing autoantibody secretion by rendering self-reactive B cells functionally silent through clonal anergy has long posed the question of why fill the circulating B cell repertoire with cells that cannot secrete antibody? Here we address this question from the perspective of B cells that comprise 5-10% of the human circulating repertoire, expressing self-reactive surface immunoglobulins employing the IGHV4-34 heavy chain variable element. Using gene targeting to construct mice expressing a representative human IGHV4-34 antibody on the surface of many B cells, we show these cells are prevented from autoantibody secretion by B cell clonal anergy marked by downregulation of surface IgM, induction of tolerance-response mRNAs, and exclusion from the marginal zone and B1 cell subsets. This functionally tolerant state is overridden when the IGHV4-34 B cells cross-react with a virus, which stimulates the self-reactive B cells to hypermutate in germinal centres. Within 16 days of infection, 99% of daughter cells have acquired one of five heavy chain mutations that diminish binding to self but preserve virus binding, and 33% had combined 2 or 3 of these mutations to become genetically self-tolerant and virus specific. These results demonstrate, from the perspective of a pathologically important human autoantibody class and the world's most successful virus vaccine, how human antibody specificity is sculpted in the progeny of anergic B cells to yield antibodies that bind a virus but not self. Overall design: We generated knock-in transgenic mice expressing a representative human IGHV4-34 antibody on a large percentage of the nascent B cell repertoire. We focussed on the unmutated ancestor of antibody 589, a highly mutated IGHV4-34 antibody against A14L, a nonglycosylated major protein of the vaccinia virus inner membrane. Antibody 589 was isolated from a healthy adult who had received vaccinia virus during the global vaccination effort that led to smallpox eradication in the 1970's. Three decades later the donor was boosted with vaccinia virus and after 14 days individual plasma cells were sorted from the blood and antibody heavy and light chain mRNAs sequenced and expressed.