Skewed primary Ig? repertoire and V-J joining in C57BL/6 mice: implications for recombination accessibility and receptor editing.

ABSTRACT: Previous estimates of the diversity of the mouse Ab repertoire have been based on fragmentary data as a result of many technical limitations, in particular, the many samples necessary to provide adequate coverage. In this study, we used 5'-coding end amplification of Ig? mRNAs from bone marrow, splenic, and lymph node B cells of C57BL/6 mice combined with amplicon pyrosequencing to assess the functional and nonfunctional V? repertoire. To evaluate the potential effects of receptor editing, we also compared V/J associations and usage in bone marrows of mouse mutants under constitutive negative selection or an altered ability to undergo secondary recombination. To focus on preimmune B cells, our cell sorting strategy excluded memory B cells and plasma cells. Analysis of ~90 Mbp, representing >250,000 individual transcripts from 59 mice, revealed that 101 distinct functional V? genes are used but at frequencies ranging from ~0.001 to ~10%. Usage of seven V? genes made up >40% of the repertoire. A small class of transcripts from apparently nonfunctional V? genes was found, as were occasional transcripts from several apparently functional genes that carry aberrant recombination signals. Of 404 potential V-J combinations (101 V?s × 4 J?s), 398 (98.5%) were found at least once in our sample. For most V? transcripts, all J?s were used, but V-J association biases were common. Usage patterns were remarkably stable in different selective conditions. Overall, the primary ? repertoire is highly skewed by preferred rearrangements, limiting Ab diversity, but potentially facilitating receptor editing.

SUBMITTER: Aoki-Ota M 

PROVIDER: S-EPMC3288532 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature