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

Abstract

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

Multi Step Selection in Ig H Chains is Initially Focused on CDR3 and Then on Other CDR Regions

“…We built lineage trees from the sequences [see (17) for details of sequences, and production of lineage trees]. We measured the LONR distribution in all naïve and memory, IgM as well as IgA and IgG sequences trees (over 50,000 lineage trees) and compared the LONR distribution in NS and S mutations.…”

“…Over 500,000 BCRs were sampled from each donor in 12 donors (17), using 454 sequencing, and a RACE protocol. The details of the sequencing and the validity checks are beyond the scope of this manuscript.…”

The Shape of the Lymphocyte Receptor Repertoire: Lessons from the B Cell Receptor

“…IGHD segments can be utilized in all three reading frames, and each IGHD gene is therefore able to encode three distinct amino acid sequences. Analysis of IGH rearrangements in which the IGHJ is out-of-frame, and which are therefore non-productive, shows each IGHD gene rearranges at equal frequency in each of the three RFs, however among productive rearrangements there is a strong skewing of the utilization of each gene toward a dominant RF (48). This dominance is constant between individuals, and the preferred RF is gene family dependent.…”

“…This dominance is constant between individuals, and the preferred RF is gene family dependent. Analysis of in-frame and out-of-frame IGH rearrangements sequenced using the Illumina platform suggests that the underlying rearrangement processes have no reading frame bias, but that bias emerges from stronger negative selection of sequences in certain reading frames (48). Such negative selection particularly focuses on non-productive sequences that result from the presence of stop codons within the junction region.…”

“…Although bias in the reading frame of the IGHD gene is the result of selection, other biases appear to be intrinsic to the recombination process, for when analysis is confined to non-productive rearrangements which carry an out-of-frame J-REGION, preferential gene usage is still seen (48). Such sequences are not subject to positive or negative selection.…”