title: Skewed primary Igκ 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 Igκ mRNAs from bone marrow, splenic, and lymph node B cells of C57BL/6 mice combined with amplicon pyrosequencing to assess the functional and nonfunctional Vκ 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 Vκ genes are used but at frequencies ranging from ~0.001 to ~10%. Usage of seven Vκ genes made up >40% of the repertoire. A small class of transcripts from apparently nonfunctional Vκ genes was found, as were occasional transcripts from several apparently functional genes that carry aberrant recombination signals. Of 404 potential V-J combinations (101 Vκs × 4 Jκs), 398 (98.5%) were found at least once in our sample. For most Vκ transcripts, all Jκs were used, but V-J association biases were common. Usage patterns were remarkably stable in different selective conditions. Overall, the primary κ repertoire is highly skewed by preferred rearrangements, limiting Ab diversity, but potentially facilitating receptor editing.
authors.affiliation: Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Miyo
authors.initials: M
authors.lastname: Aoki-Ota
authors.forename: Ali
authors.initials: A
authors.lastname: Torkamani
authors.forename: Takayuki
authors.initials: T
authors.lastname: Ota
authors.forename: Nicholas
authors.initials: N
authors.lastname: Schork
authors.forename: David
authors.initials: D
authors.lastname: Nemazee
mesh_terms: D000818:Animals; D000916:Antibody Diversity / Q000235:genetics; D016175:B-Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism; D001854:Bone Marrow Cells / Q000276:immunology / Q000378:metabolism; D005260:Female; D015328:Gene Rearrangement, B-Lymphocyte, Light Chain; D007133:Immunoglobulin Joining Region / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D007145:Immunoglobulin kappa-Chains / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D017393:RNA Editing / Q000235:genetics* / Q000276:immunology*; D011995:Recombination, Genetic / Q000276:immunology*; D017422:Sequence Analysis, DNA
chemical_list: D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains