期刊
JOURNAL OF IMMUNOLOGY
卷 188, 期 5, 页码 2305-2315出版社
AMER ASSOC IMMUNOLOGISTS
DOI: 10.4049/jimmunol.1103484
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资金
- National Institutes of Health [RO1AI059714, R01DE017568]
- National Institutes of Health/National Center for Research Resources [UL1 RR025774]
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 kappa 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 kappa 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 similar to 90 Mbp, representing >250,000 individual transcripts from 59 mice, revealed that 101 distinct functional V kappa genes are used but at frequencies ranging from similar to 0.001 to similar to 10%. Usage of seven V kappa genes made up >40% of the repertoire. A small class of transcripts from apparently nonfunctional V kappa 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 kappa s x 4 J kappa s), 398 (98.5%) were found at least once in our sample. For most V kappa transcripts, all J kappa s were used, but V-J association biases were common. Usage patterns were remarkably stable in different selective conditions. Overall, the primary kappa repertoire is highly skewed by preferred rearrangements, limiting Ab diversity, but potentially facilitating receptor editing. The Journal of Immunology, 2012, 188: 2305-2315.
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