Abstract
The antigen-binding variable regions of the B cell receptor (BCR) and of antibodies are encoded by exons that are assembled in developing B cells by V(D)J recombination1. The BCR repertoires of primary B cells are vast owing to mechanisms that create diversity at the junctions of V(D)J gene segments that contribute to complementarity-determining region 3 (CDR3), the region that binds antigen1. Primary B cells undergo antigen-driven BCR affinity maturation through somatic hypermutation and cellular selection in germinal centres (GCs)2,3. Although most GCs are transient3, those in intestinal Peyer’s patches (PPs)—which depend on the gut microbiota—are chronic4, and little is known about their BCR repertoires or patterns of somatic hypermutation. Here, using a high-throughput assay that analyses both V(D)J segment usage and somatic hypermutation profiles, we elucidate physiological BCR repertoires in mouse PP GCs. PP GCs from different mice expand public BCR clonotypes (clonotypes that are shared between many mice) that often have canonical CDR3s in the immunoglobulin heavy chain that, owing to junctional biases during V(D)J recombination, appear much more frequently than predicted in naive B cell repertoires. Some public clonotypes are dependent on the gut microbiota and encode antibodies that are reactive to bacterial glycans, whereas others are independent of gut bacteria. Transfer of faeces from specific-pathogen-free mice to germ-free mice restored germ-dependent clonotypes, directly implicating BCR selection. We identified somatic hypermutations that were recurrently selected in such public clonotypes, indicating that affinity maturation occurs in mouse PP GCs under homeostatic conditions. Thus, persistent gut antigens select recurrent BCR clonotypes to seed chronic PP GC responses.
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Data availability
The next-generation sequencing data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database under the accession number GSE140795. All figures have associated raw data deposited. Source data for Figs. 1–3 and Extended Data Figs. 2, 3, 6, 8, 9 are provided with the paper.
Code availability
The computational pipeline of Rep-SHM-Seq and the code for statistical analysis tools used in this study are available at https://github.com/Yyx2626/HTGTSrep.
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Acknowledgements
We thank M. Tian for providing reagents and other members of the Alt laboratory for discussions and comments; the Protein-Glycan Interaction Resource of the CFG (supporting grant R24 GM098791) and the National Center for Functional Glycomics (NCFG) at Beth Israel Deaconess Medical Center, Harvard Medical School (supporting grant P41 GM103694); the Department of Hematology/Oncology flow cytometry research facility at Boston Children’s Hospital for assistance with cell sorting; and R. Chen and Y. Zhang for assistance with scRNA-seq library preparation. This work was supported by NIH grant R01AI077595 to F.W.A., NIH grant R01DK103358 to D.R.L. and a grant from the NYU Colton Center for Autoimmunity to D.R.L. F.W.A. and D.R.L. are Howard Hughes Medical Institute Investigators. H.C. is an NRSA Fellow (T32 AI07386) and was supported by a Leukemia and Lymphoma Society Fellow Award; Y.Z. is supported by a Damon Runyon Fellowship Award; M.X., C.S.-L. and Z.B. were supported by Cancer Research Institute Fellow Awards; J.K.H. was supported by an NIH MD/PhD grant F30AI114179-01A1; and D.N. was supported by the Dana-Farber/Harvard Cancer Center Support Grant 5P30 CA006516.
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H.C. and F.W.A. designed the study; H.C., Y.Z., M.X., C.-S.L. and J.K.H. performed experiments; Z.B. constructed the mouse Cer/Sis-deleted v-Abl pro-B cell line; Z.D., H.C. and N.K. designed the Rep-SHM-Seq bioinformatics pipeline; A.Y.Y. and H.C. designed and performed statistical analyses; H.C., A.Y.Y., Z.D., Y.Z., C.-S.L., N.K. and F.W.A. analysed and interpreted data; H.C., Y.Z., A.Y.Y. and F.W.A. drafted the manuscript; D.R.L. and M.X. discussed experiments, provided reagents and helped to improve the manuscript; D.N. discussed data analyses and helped to improve the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Overview of Rep-SHM-Seq.
a, Schematic showing the experimental procedure of the Rep-SHM-Seq method. MID, multiple identifier; NP, non-productive; P, productive. b, Left, diagram of the Igh locus in the hVh1-2 mouse model. Positions of bait primers are indicated. Right, the proportions of each Jh segment in hVh1-2DJ junctions are compared between libraries made from splenic B cells with hVh1-2 bait and mixed Jh1-4 bait primers. c, Left, diagram of the Igl locus of the v-Abl kinase-transformed pro-B cell line. Positions of bait primers are indicated. Top right, the Jκ proportions in Vκ3-2,7,10,12Jκ junctions are compared between libraries made from Vκ3d bait and mixed Jκ1,2,4,5 bait primers. Bottom right, the Jλ proportions in Vλ1,2Jλ junctions are compared between libraries made from Vλd1,2 bait and mixed Jλ1,2,3 bait primers. Three biological repeats were performed for each set of primers. r, Spearman’s correlation coefficient. P values were determined by two-sided Spearman’s correlation test. d, Schematic showing the bioinformatic pipeline of Rep-SHM-Seq. PE, paired-end; QC, quality control.
Extended Data Fig. 2 Rep-SHM-Seq detects NP-antigen-specific selection in GCs.
a, b, Vh (a) and Vl (b) repertoire of productive V(D)J junctions in splenic GC B cells versus naive B cells in five mice that were immunized intraperitoneally with NP-CGG. Data are mean +s.e.m. Vh and Vl segments are each ordered linearly based on their chromosome coordinates, with respect to relative proximity to the D or Jl segments. Vλ segments are plotted on the left of Vκ segments. The distribution of productive clonotypes of each indicated Vh or Vl segment in the GCs is shown as a long-tail-distribution plot, with the y axis representing the fraction of each productive clonotype among all productive clonotypes and the x axis representing the top clonotypes (rank-ordered). The number of mice that contained the clonotype is indicated for dominant recurrent clonotypes. P values for Vh or Vl usage were calculated by two-sided Mann–Whitney U test with FDR correction (**P < 0.05). See Supplementary Table 4 for exact P values. Enrichment of Vh14-3, the key Vh segment in the response of BALB/c mice to NP antigen59,60, is detected here by Rep-SHM-Seq with two recurrent clonotypes (Supplementary Table 2). c, Representative junctional structure of indicated common IgH clonotypes aligned with germline Vh (red), D (blue) and Jh (orange) sequences, with resected sequences shown in grey. The underline indicates microhomology. All possible segment assignments by IGoR with probabilities are shown in Supplementary Table 10. Canonical CDR3 sequences are shown in black, amino acid sequences in purple and the clonotype consensus (CDR3 nucleotide sequences with the same V and J segments, same CDR3 length and more than 90% similarity) in sequence-logo pictures. The number of mice containing the canonical CDR3 sequence or the clonotype consensus is indicated in parentheses after each sequence. d, SHM profile of Vh segments of the indicated PP GC clonotypes and intrinsic mutation pattern, plotted as the mutation rate at each nucleotide. Sequences were stratified by the overall Vh mutation rate19 (see Methods). Data are mean + s.e.m. from five mice. Significance was determined by hierarchical Bayesian modelling with PEP0.1 (see Methods; **PEP0.1 < 0.01, ***PEP0.1 < 0.005). PEP0.1 values and amino acid changes are denoted for significantly enriched SHMs.
Extended Data Fig. 3 Stable composition of variable-region exon-segment usage in naive B cells across tissues and mice.
a–d, Vh (a), D and Jh (b), Vl (c) and Jl (d) repertoires of productive V(D)J junctions in splenic (SP) versus PP naive B cells. Data are mean + s.e.m. from the five NP-CGG-immunized mice. r, Spearman’s correlation coefficient. P values were determined by two-sided Spearman’s correlation test.
Extended Data Fig. 4 Representative junctional structure of indicated recurrent PP GC IgH clonotypes in SPF mice.
Junctional structures are aligned with germline Vh (red), D (blue) and Jh (orange) sequences, with resected sequences shown in grey. The underline indicates microhomology. All possible segment assignments by IGoR with probabilities are shown in Supplementary Table 10. For certain clonotypes, the D segment could not be accurately annotated because of the short, aligned D sequence. Canonical CDR3 sequences are shown in black, amino acid sequences in purple and the clonotype consensus in sequence-logo pictures. The number of mice containing the canonical CDR3 sequence or the clonotype consensus is indicated in parentheses after each sequence.
Extended Data Fig. 5 FACS results of PP GC B cells and recurrent PP GC IgH CDR3s in different gnotobiotic mouse categories.
a, FACS plots showing the representative proportion of GC (GL7+CD38−) cells among B220+ cells in PPs. The FACS analysis was performed for 9 SPF mice, 10 pools of 3 germ-free mice, 5 germ-free mice with faecal transfer from SFB-mono mice (GF + SFB mice), 10 germ-free mice with faecal transfer from SPF mice (GF + SPF mice) and 15 Aid−/− SPF mice. Representative results are shown. b, Plot comparing percentage of GC B cells in PPs from different mouse categories. c, Plot comparing the number of sorted PP GC B cells per mouse from different mouse categories. Data are mean ± s.e.m. from 9 SPF mice, 10 × 3 germ-free mice, 5 GF + SFB mice, 10 GF + SPF mice and 15 Aid−/− SPF mice. P values were calculated by two-sided Mann–Whitney U test. d, f, h, Representative junctional structure of common IgH clonotypes aligned with germline Vh, D and Jh sequences for Vh1-47RC and Vh1-12RC in germ-free, GF + SFB and GF + SPF mice (d), Vh1-64RC in GF + SFB mice (f) and Vh6-3RC in GF + SPF mice (h). All possible segment assignments by IGoR with probabilities are shown in Supplementary Table 10. For certain clonotypes, the D segment could not be accurately annotated because of the short, aligned D sequence. Canonical CDR3 sequences are shown in black, amino acid sequences in purple and the clonotype consensus in sequence-logo pictures. The number of mice containing the canonical CDR3 sequence or the clonotype consensus is indicated in parentheses after each sequence. e, g, The distribution of productive clonotypes of each indicated Vh segment enriched in PP GC B cells versus naive B cells in germ-free mice (e) and GF + SPF mice (g) is shown as a long-tail-distribution plot, with the y axis representing the fraction of each productive clonotype among all productive clonotypes and the x axis representing the top clonotypes (rank-ordered). The number of mice that contained the clonotype is indicated for dominant recurrent clonotypes. Note that the Vh5-17 recurrent clonotype in germ-free mice is different from the one in GF + SPF mice.
Extended Data Fig. 6 SHM selection in recurrent PP GC clonotypes.
a, b, SHM profile of Vh segments of the indicated PP GC clonotypes and intrinsic mutation pattern, plotted as the mutation rate at each nucleotide. Sequences were stratified by the overall Vh mutation rate19 (see Methods). Significance was determined by hierarchical Bayesian modelling with PEP0.1 (see Methods; *PEP0.1 < 0.05, **PEP0.1 < 0.01, ***PEP0.1 < 0.005). PEP0.1 values and amino acid changes are denoted for significantly enriched SHMs. Data are mean + s.e.m. from mice containing the indicated clonotype: n = 11 SPF mice, 9 pools of 3 germ-free mice, 4 GF + SFB mice and 8 GF + SPF mice (a) and 3 GF + SFB mice and 4 GF + SPF mice (b). c, Microbial glycan microarray reactivities of mutated Vh1-12RC monoclonal antibody. For full antibody sequences, see Supplementary Table 5. Data are shown as the average fold enrichment over a negative control monoclonal antibody (anti-human PD-1) of four technical repeats.
Extended Data Fig. 7 Some recurrent PP GC clonotypes do not show selected SHMs.
a–c, SHM profile of Vh segments of the indicated PP GC clonotypes and intrinsic mutation pattern, plotted as the mutation rate at each nucleotide. Sequences were stratified by the overall Vh mutation rate19 (see Methods). Significance was determined by hierarchical Bayesian modelling with PEP0.1 (see Methods; NS, PEP0.1 ≥ 0.05). Data are mean + s.e.m. from mice containing the indicated clonotype: n = 7 SPF mice, 10 pools of 3 germ-free mice, 4 GF + SFB mice and 6 GF + SPF mice (a), 4 SPF mice (b) and 9 pools of 3 germ-free mice (c).
Extended Data Fig. 8 Recurrent PP GC IgH clonotypes are often associated with recurrent IgL clonotypes.
a, Vl repertoire of productive VJ junctions in PP GC B cell versus naive B cells. Data are plotted as mean + s.e.m. from 9 SPF mice (related to Fig. 1). The distribution of productive clonotypes of each indicated Vl segment in the GCs is shown as a long-tail-distribution plot, with the y axis representing the fraction of each productive clonotype among all productive clonotypes and the x axis representing the top clonotypes (rank-ordered). The number of mice that contained the clonotype is indicated for dominant recurrent clonotypes. P values for Vl usage were calculated by two-sided Mann–Whitney U test with FDR correction (*P < 0.1, **P < 0.05, ***P < 0.01, NS, not significant). See Supplementary Table 4 for exact P values. b, Table showing the paired IgL clonotype of recurrent PP GC IgH clonotypes, as inferred by Rep-SHM-Seq (see Methods) from nine SPF mice and/or detected by scRNA-seq from four SPF mice.
Extended Data Fig. 9 Recurrent clonotypes in chronic splenic and PP GCs in Aid−/− mice.
a, Vh repertoire of productive V(D)J junctions in splenic GC B cells versus naive B cells. Data are mean + s.e.m. from 9 Aid−/− SPF mice. b, Vh repertoire of productive V(D)J junctions in PP GC B cells versus naive B cells. Data are mean + s.e.m. from 15 Aid−/− SPF mice. The distribution of productive clonotypes of each indicated Vh segment in the GCs is shown as a long-tail-distribution plot, with the y axis representing the fraction of each productive clonotype among all productive clonotypes and the x axis representing the top clonotypes (rank-ordered). The number of mice that contained the clonotype is indicated for dominant recurrent clonotypes. The brown colour indicates a clonotype that was common to the splenic and PP GCs of Aid−/− mice. The green colour indicates a clonotype found in wild-type mice of different gnotobiotic categories (related to Fig. 1). P values for Vh usage were calculated by two-sided Mann–Whitney U test with FDR correction (*P < 0.1, ***P < 0.01, NS, not significant). See Supplementary Table 4 for exact P values. c, Schematic summarizing the main findings of the paper. Junctional biases during V(D)J recombination generate a diverse CDR3 repertoire for naive B cells in PPs, with a set of CDR3s occurring at higher frequency. Gut microbial or non-microbial antigens select recurrent IgH clonotypes from this set of CDR3s in multiple mice, most of which have a canonical CDR3 sequence and recurrent pairing of IgL. These recurrently selected antibodies contain selected SHMs, suggesting affinity maturation. The asterisk on Vh6-3RC–Vκ2-109 indicates that this pairing was picked up from scRNA-seq data (using which we expressed the antibody in vitro), rather than being confirmed as a recurrent pairing by Rep-SHM-Seq. The frequency of the BCRs and SHMs represented in this schematic does not correspond to their actual frequency in the naive or GC B cell repertoire of PPs.
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Chen, H., Zhang, Y., Ye, A.Y. et al. BCR selection and affinity maturation in Peyer’s patch germinal centres. Nature 582, 421–425 (2020). https://doi.org/10.1038/s41586-020-2262-4
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DOI: https://doi.org/10.1038/s41586-020-2262-4
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