The Restricted DH Gene Reading Frame Usage in the Expressed Human Antibody Repertoire Is Selected Based upon its Amino Acid Content

The Ab repertoire is not uniform. Some variable, diversity, and joining genes are used more frequently than others. Nonuniform usage can result from the rearrangement process, or from selection. To study how the Ab repertoire is selected, we analyzed one part of diversity generation that cannot be driven by the rearrangement mechanism: the reading frame usage of D-H genes. We have used two high-throughput sequencing methodologies, multiple subjects and advanced algorithms to measure the D-H reading frame usage in the human Ab repertoire. In most D-H genes, a single reading frame is used predominantly, and inverted reading frames are practically never observed. The choice of a single D-H reading frame is not limited to a single position of the D-H gene. Rather, each D-H gene participates in rearrangements of differing CDR3 lengths, restricted to multiples of three. In nonproductive rearrangements, there is practically no reading frame bias, but there is still a striking absence of inversions. Biases in D-H reading frame usage are more pronounced, but also exhibit greater interindividual variation, in IgG(+) and IgA(+) than in IgM(+) B cells. These results suggest that there are two developmental checkpoints of D-H reading frame selection. The first occurs during VDJ recombination, when inverted D-H genes are usually avoided. The second checkpoint occurs after rearrangement, once the BCR is expressed. The second checkpoint implies that D-H reading frames are subjected to differential selection. Following these checkpoints, clonal selection induces a host-specific D-H reading frame usage bias.