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Antibody Repertoire Deep Sequencing Reveals Antigen-Independent Selection in Maturing B Cells

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Antibody Repertoire Deep Sequencing Reveals Antigen-Independent Selection in Maturing B Cells Antibody repertoire deep sequencing reveals antigen-independent selection in maturing B cells Joseph Kaplinskya,b, Anthony Lia,b, Amy Sunc, Maryaline Coffrec, Sergei B. Koralovc,1, and Ramy Arnaouta,b,d,1,2 aDepartment of Pathology and dDivision of Clinical Informatics, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, MA 02215; bDepartment of Systems Biology, Harvard Medical School, Boston, MA 02115; and cDepartment of Pathology, New York University Medical Center, New York, NY 10016 Edited* by Phillip A. Sharp, Massachusetts Institute of Technology, Cambridge, MA, and approved May 15, 2014 (received for review February 24, 2014) Antibody repertoires are known to be shaped by selection for “innate-like” (7) cells in the spleen that are theorized not to antigen binding. Unexpectedly, we now show that selection also undergo B-cell receptor (BCR)-based selection during matura- – acts on a non antigen-binding antibody region: the heavy-chain tion (8–11). These studies also found no differences in VH use, – “ ” variable (VH) encoded elbow between variable and constant although, again, the number of sequences studied was small. In domains. By sequencing 2.8 million recombined heavy-chain genes contrast, studies have found differences in D segment use between from immature and mature B-cell subsets in mice, we demonstrate antibodies with short and long CDR3s, including the appearance of a striking gradient in VH gene use as pre-B cells mature into follicular tandem D gene segments in very long CDR3s, suggesting a poten- and then into marginal zone B cells. Cells whose antibodies use VH tial causative relationship (4, 12). However, these studies did not genes that encode a more flexible elbow are more likely to mature. investigate whether these differences were a sign of selection during This effect is distinct from, and exceeds in magnitude, previously B-cell maturation. described maturation-associated changes in heavy-chain comple- mentarity determining region 3, a key antigen-binding region, The advent of high-throughput antibody repertoire sequencing makes it possible to investigate the forces that govern B-cell se- which arise from junctional diversity rather than differential VH gene use. Thus, deep sequencing reveals a previously unidentified lection with statistical rigor (13, 14). This is what we set out to do. mode of B-cell selection. Results immunomics | principal component analysis | development We sorted pre-B, FO, and MZ cells from the bone marrow and spleen of nine unimmunized 2.5-month-old C57BL/6J mice according to surface expression of CD19, B220, CD21, CD23, he mature antibody repertoire is shaped by selective forces Tthat influence B-cell survival (1). Comparison of immature and IgM (Fig. S1). We prepared heavy-chain libraries from ge- and mature B-cell subsets has shown that selection acts specifi- nomic DNA and sequenced V(D)J-recombined genes for 2.8 cally on complementarity determining region 3 (CDR3) of the million recombination events at 4.3-fold coverage. We annotated antibody heavy-chain molecule, an antigen-binding region that is reads for VH,D,andJH gene segment use; CDR3 length and a key determinant of antigen specificity (2). On average, mature charge; and VH-D, D-JH, and total junctional nucleotides; as well B-cell subsets express antibodies that have shorter and more as whether the reads were productive (full-length and free of negatively charged CDR3s, which is the result of selection against stop codons) or nonproductive, as previously described (14, 15). autoreactive and polyreactive B cells (3, 4). Each recombined antibody heavy-chain gene is composed of Significance a variable (VH), diversity (D), and joining (JH) gene segment. Because the CDR3 region spans the VH-D-JH joint, investigators Antibodies play essential roles in vaccination, infection, auto- have asked whether selection might favor B cells whose anti- immunity, aging, and cancer. A key question is how the anti- bodies use specific VH,D,orJH gene segments. Selection in body repertoire achieves its remarkable diversity. Part of the favor of specific gene segments during B-cell maturation might answer is that B cells, which express antibodies on their sur- help to explain the observed maturation-associated changes in face, are selected for survival based on the specific antigens CDR3 length and charge, and might suggest a preference for that their antibodies bind, with antigen specificity determined “hard-wired” antigen specificities. Evidence against selection by the protein sequence of antibodies’ antigen-binding re- would suggest that differences in CDR3 result exclusively from gions. Unexpectedly, we find that B cells are also selected the nontemplated addition and deletion of nucleotides at the based on whether their antibodies have a loose or tight “elbow ” VH-D and D-JH junctions, and therefore that the death of B cells joint, independent of the sequence of their antigen-binding with counterselected CDR3s during maturation is simply the evo- regions. This discovery, enabled by sequencing technology and lutionary cost (3) of maintaining this mechanism of generating mathematics, adds a surprising new dimension to our under- antibody diversity. standing of antibody repertoires, and might one day help us Nearly two decades ago, a low-throughput sequencing study shape them ourselves. in mice suggested that specific VH gene segments were used at different frequencies by pre-B cells (in which heavy-chain re- Author contributions: J.K., S.B.K., and R.A. designed research; J.K., A.L., A.S., M.C., S.B.K., and R.A. performed research; J.K., A.L., A.S., M.C., S.B.K., and R.A. contributed new re- combination has been completed) and mature B cells in the agents/analytic tools; J.K. and R.A. analyzed data; and J.K., S.B.K., and R.A. wrote spleen (5). This observation was interpreted as selection for the paper. hard-wired specificities. However, the statistical robustness of The authors declare no conflict of interest. this observation was limited by the small number of recombined *This Direct Submission article had a prearranged editor. genes that were sequenced, and although subsequent investigations Data deposition: The antibody heavy-chain sequences reported in this paper have have detected differences in VH use between pre-B and upstream been deposited in the Sequence Read Archive, www.ncbi.nlm.nih.gov/sra (submission pro-B cells, they have failed to confirm such differences between no. 432110; NCBI BioProject number PRJNA248676). pre-B and mature B cells (6). 1S.B.K. and R.A. contributed equally to this work. More recent sequencing studies have looked for differences in 2To whom correspondence should be addressed. E-mail: [email protected]. VH gene segment use between follicular (FO) B cells, which This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. circulate through the spleen, and marginal zone (MZ) B cells, 1073/pnas.1403278111/-/DCSupplemental. E2622–E2629 | PNAS | Published online June 9, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1403278111 −9 PNAS PLUS Similarity among VH and D gene segments and junctional rearrangements than for productive rearrangements (P = 5.1 × 10 × −11 diversity at the VH-D and D-JH junctions preclude unique an- to 6.2 10 for pairwise comparisons between nonproductive and notation of all reads (16); we obtained unique VH and JH gene productive rearrangements within each subset; compare Fig. 1A, segment annotations in 89% and 100% of reads, respectively, Right vs. Left), suggesting selection against extreme-length CDR3s and unique VJH and VDJ combinations in 89% and 63% of even in pre-B cells. reads, respectively (Table 1). The remaining reads were excluded from subsequent analysis of gene segment use. Comparison of Junctional Diversity and Differential D Gene Segment Use also 32 pairs of PCR replicates showed excellent reproducibility for Account for Most of the Trend Toward More Negatively Charged frequency of gene segment use and for CDR3 length and charge CDR3s During B-Cell Maturation. CDR3 charge was also approxi- mately normally distributed in all three subsets (Fig. 1C). Again, (Fig. S2). VH, D, and JH gene segment use among mature subsets also agreed well with our previous high-throughput study of distributions largely overlapped. The mean charge across animals − − mature subsets, which used a different primer set (14). ranged from 0.18 to 0.48 per CDR3 for productive rearrange- ments, compared with +0.92 to +0.96 predicted for nonproductive = × −24– × −17 Junctional Diversity and Differential D Gene Segment Use Account for rearrangements (P 4.6 10 5.7 10 ), consistent with Most of the Trend Toward Shorter CDR3s During B-Cell Maturation. previous reports (3, 14). Among productive rearrangements, CDR3s – We first measured differences in CDR3 length and charge in from FO and MZ cells were, on average, 0.13 0.15 unit more = × −8 × −7 pre-B, FO, and MZ subsets, treating productive rearrangements, negative than those from pre-B cells (P 3.2 10 and 8.4 10 , which contribute to functional BCRs and are thereby subject to respectively; Fig. 1C, Right). selection, separately from nonproductive rearrangements, which As with CDR3 length, the distribution of charges was slightly narrower among productive rearrangements than among non- are not subject to selection. = × −6– × −14 CDR3 lengths followed an approximately normal (Gaussian) productive rearrangements (P 6.8 10 3.0 10 ), con- distribution among both productive and nonproductive rear- sistent with selection against extremes of charge in addition to rangements in all three B-cell subsets (Fig. 1A). Distributions selection for more negative charge. Among productive rear- rangements, the distribution of charges in MZ and FO cells was largely overlapped. Among productive rearrangements, pre-B − − P = × 11 × 3 and FO subsets had the longest CDR3s, at 13.3 ± 3.1 aa and 13.4 ± narrower than in pre-B cells ( 2.4 10 and 3.5 10 , respectively; Fig.
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