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

Previous estimates of the diversity of the mouse antibody repertoire have been based on fragmentary data owing to many technical limitations, in particular the many samples necessary to provide adequate coverage. Here, 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 non-functional 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. In order to focus on preimmune B cells, our cell sorting strategy excluded memory B cells and plasma cells. Analysis of approximately 90 Mbp, representing >250,000 individual transcripts from 59 mice, revealed that 101 distinct functional V? genes are used, but at frequencies ranging from ~.001% to ~10%. Usage of seven V? genes made up over 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 X 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 antibody diversity, but potentially facilitating receptor editing.