The restricted D_H 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.