Abstract
BackgroundThe naïve antibody repertoire is initially dependent upon the number of germline V(D)J genes and the ability of recombined heavy and light chains to pair. Individual VH and VL genes are not equally represented in naïve mature B cells, suggesting that positive and negative selection also shape the antibody repertoire. Among the three member murine Vκ10 L chain family, the Vκ10C gene is under-represented in the antibody repertoire. Although it is structurally functional and accessible to both transcriptional and recombination machinery, the Vκ10C promoter is inefficient in pre-B cell lines and productive Vκ10C rearrangements are lost as development progresses from pre-B cells through mature B cells. This study examined VH/Vκ10 pairing, promoter mutations, Vκ10 transcript levels and receptor editing as possible factors that are responsible for loss of productive Vκ10C rearrangements in developing B cells.ResultsWe demonstrate that the loss of Vκ10C expression is not due to an inability to pair with H chains, but is likely due to a combination of other factors. Levels of mRNA are low in sorted pre-B cells and undetectable in B cells. Mutation of a single base in the three prime region of the Vκ10C promoter increases Vκ10C promoter function in pre-B cell lines. Pre-B and B cells harbor disproportionate levels of receptor-edited productive Vκ10C rearrangements.ConclusionsOur findings suggest that the weak Vκ10C promoter initially limits the amount of available Vκ10C L chain for pairing with H chains, resulting in sub-threshold levels of cell surface B cell receptors, insufficient tonic signaling and subsequent receptor editing to limit the numbers of Vκ10C-expressing B cells emigrating from the bone marrow to the periphery.
Highlights
The naïve antibody repertoire is initially dependent upon the number of germline V(D)J genes and the ability of recombined heavy and light chains to pair
We examined the effect of mutations in the Vκ10C promoter to drive luciferase expression in transient transfection assays
The Vκ10A and Vκ10C promoters differ by four nucleotides over an approximately 130 bp region (Figure 2A)
Summary
The naïve antibody repertoire is initially dependent upon the number of germline V(D)J genes and the ability of recombined heavy and light chains to pair. This study examined VH/Vκ10 pairing, promoter mutations, Vκ10 transcript levels and receptor editing as possible factors that are responsible for loss of productive Vκ10C rearrangements in developing B cells. The potential diversity of the naïve antibody repertoire is dependent in part upon the number of functional VH and VL genes that are able to undergo successful V(D)J recombination and H-L chain pairing, followed by the selection events that occur during B cell development. In one study, skewing of the repertoire by preferred Vκ-Jκ rearrangements was such that seven Vκ genes represented greater than 40% of the repertoire in C57BL/6 mice [16] It is not known why other potentially functional Vκ genes would be underutilized, but it was hypothesized that the observed preferential Vκ gene usage could be the result of intrinsic rearrangement preferences and/or differences in expression levels
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