Germinal centres, the structures in which B cells evolve to produce antibodies with high affinity for various antigens, usually form transiently in lymphoid organs in response to infection or immunization. In lymphoid organs associated with the gut, however, germinal centres are chronically present. These gut-associated germinal centres can support targeted antibody responses to gut infections and immunization1. But whether B cell selection and antibody affinity maturation take place in the face of the chronic and diverse antigenic stimulation characteristic of these structures under steady state is less clear2,3,4,5,6,7,8. Here, by combining multicolour ‘Brainbow’ cell-fate mapping and sequencing of immunoglobulin genes from single cells, we find that 5–10% of gut-associated germinal centres from specific-pathogen-free (SPF) mice contain highly dominant ‘winner’ B cell clones at steady state, despite rapid turnover of germinal-centre B cells. Monoclonal antibodies derived from these clones show increased binding, compared with their unmutated precursors, to commensal bacteria, consistent with antigen-driven selection. The frequency of highly selected gut-associated germinal centres is markedly higher in germ-free than in SPF mice, and winner B cells in germ-free germinal centres are enriched in ‘public’ clonotypes found in multiple individuals, indicating strong selection of B cell antigen receptors even in the absence of microbiota. Colonization of germ-free mice with a defined microbial consortium (Oligo-MM12) does not eliminate germ-free-associated clonotypes, yet does induce a concomitant commensal-specific B cell response with the hallmarks of antigen-driven selection. Thus, positive selection of B cells can take place in steady-state gut-associated germinal centres, at a rate that is tunable over a wide range by the presence and composition of the microbiota.
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Understanding repertoire sequencing data through a multiscale computational model of the germinal center
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We thank all members of the Victora and Mucida laboratories, past and present, for assistance with experiments, fruitful discussions and critical reading of the manuscript. In particular we thank: A. Rogoz and G. Fayzikhodjaeva for maintaining gnotobiotic mice; S. Gonzalez for maintaining SPF mice; K. Gordon and K. Chhosphel for FACS; the Rockefeller University Genomics Center for RNA sequencing; and employees of the Rockefeller University for continuous assistance. We thank A. Vale (Universidade Federal do Rio de Janeiro, Brazil) for help with analysis of public clonotypes; K. McCoy (University of Calgary, Canada), S. Y. Wong and K. Cadwell (New York University, USA) for providing Oligo-MM12 strains; J. Faith (Mount Sinai School of Medicine, USA) for other bacterial strains; and J Däbritz and E. Wirthgen (University of Rostock, Germany) for contributing to the training and supervision of C.W. This work was supported by National Institutes of Health (NIH)/National Institute of Allergy and Infectious Diseases (NIAID) grants R01AI119006 and R01AI139117 (to G.D.V.), and NIH/NIAID/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grants R01DK093674, R01DK113375 and R21AI144827 (to D.M.), with additional support from NIH grant DP1AI144248 (Pioneer Award) and from the Robertson Foundation to G.D.V. and NIH grant R01DK116646 (Transformative Award) to D.M. C.R.N. is a Human Frontier of Science Program postdoctoral fellow. G.P.D. is a Robert Black Fellow of the Damon Runyon Cancer Research Foundation. A.S. is a Boehringer-Ingelheim Fonds PhD fellow. G.D.V. and D.M. are Burroughs-Wellcome Investigators in the Pathogenesis of Infectious Disease. G.D.V. is a Searle Scholar, a Pew-Stewart Scholar, and a MacArthur Fellow.
The authors declare no competing financial interests.
Peer review information Nature thanks Ramy Arnaout, Rachael Bashford-Rogers and Hai Qi for their contribution to the peer review of this work.
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Extended data figures and tables
a, Gating strategy for PA-GFP mice used in Fig. 1a–d. GC, germinal centre. b, Gating strategy and efficiency of labelling in germinal centres of AID-Confetti mice seven days after the administration of tamoxifen, as in Fig. 1e. The labelling efficiency is calculated as 100% minus the product of the percentage of unlabelled cells in the GFP/YFP, RFP and CFP channels. c, Gating strategy for the S1pr2CreERT2/tdTomato fate-mapping experiments shown in g and Fig. 1h. All flow plots are representative of multiple experiments. d, Multiphoton images of Peyer’s patches from SPF mice at different times after tamoxifen treatment (see Fig. 1f). Values in parentheses in images are NDS values. e, Quantification of multiple images as exemplified in d (see also Fig. 1g). Data are from three to five mice per time point at days 14–35, and one to two mice per time point for day 7 and later times. f, Size of germinal centres in Peyer’s patches (PPs) versus mLNs, calculated from samples obtained seven days after tamoxifen treatment as in d and Fig. 1f, plotted as the cross-sectional area of the largest available z-section. Each symbol represents one germinal centre. Lines show medians; P values are from two-tailed Mann–Whitney U tests. Data are pooled from multiple mLNs and Peyer’s patches of two mice from two independent experiments. g, Turnover of B cell clones in germinal centres of Peyer’s patches from S1pr2CreERT2 × Rosa26Stop-tdTomato mice (see Fig. 1h).
a, Gating strategy for isolating AID-Confetti single germinal centres shown in b, c, Figs. 2a, 3d, e and Extended Data Figs. 6a, b, 8a. CR, CFP and/or RFP; non CR, non-CFP, non-RFP, GY, GFP and/or YFP. b, c, Additional Igh sequence relationships among B cells from high-NDS germinal centres (b) and one low-NDS germinal centre (c) (see Fig. 2a). Scale bars, 50 μm. In c, each tree is for a separate clone (defined as a unique V(D)J rearrangement). Only clones with more than five cells are shown (grey slices in pie charts). d, Gating strategy for bacterial flow cytometry, performed in e, Figs. 2b, 3f, h, i and Extended Data Fig. 6d. e, Flow-cytometry analysis of the binding of recombinant monoclonal antibodies to faecal bacteria isolated from SPF mice. Plots gated as in d. All plots are representative of data obtained from at least two separate experiments. f, Summary of the reactivity of SPF monoclonal antibodies, assayed by ELISA against food protein extracts, autoantigens (anti-nuclear antibody, ANA), and a five-antigen polyreactivity panel comprising single-stranded DNA, double-stranded DNA, keyhole limpet haemocyanin (KLH), insulin and LPS. Shown are background-subtracted OD450 values. Data representative of assays repeated in at least three separate experiments.
a–c, qPCR of total (a) and strain-specific (b, c) 16S DNA from faecal samples of mice stably colonized with the Oligo-MM12 consortium. In a, ΔCt values were calculated in respect to a reference SPF sample, marked by the black filled symbols, with which all other values were compared. In c, Ct values were used to quantify the relative abundance of each species (see Methods). LOD, limit of detection. F1 refers to the first generation after the parental strain (P, colonized by gavage). Note that Bifidobacterium animalis (YL2) is usually undetectable in faeces19.
Extended Data Fig. 4 Frequency and isotype distribution of gaGCs in germ-free and Oligo-MM12-colonized mice.
a, Gating strategy for analysing the frequency of germinal centres and distribution of isotypes (results shown in b–d). b, Frequency of cells with the phenotype of germinal centres (CD38– FAShi) among total B220+ B cells in the indicated organs of mice raised under the indicated conditions. Each symbol represents one mouse. SPF, n = 25; germ-free (GF), n = 16; Oligo-MM12, n = 11. c, Frequency of germinal-centre B cells positive for the indicated surface BCR isotype in different organs of mice raised under the indicated conditions. Data are from at least three mice per group, as in d. Data are presented as means ± s.e.m. d, Statistical analysis of selected isotypes and anatomical locations, using data from c. Each symbol represents one mouse. Lines indicate medians; P values are obtained from two-tailed Kruskall–Wallis tests carried out on each trio, with Dunn’s multiple comparisons post-test. All P values below 0.05 are reported.
a, Gating strategy for germ-free AID-Confetti single germinal centres used in b–d. b–d, Sequencing of Igh genes from B cells obtained from individual mLN germinal centres. Germinal-centre B cells were single-cell-sorted from fragments of vibratome slices containing single germinal centres. To avoid biased selection of germinal centres based on NDS or loss of germinal centres with a low density of coloured cells, mLNs were harvested at five to seven days after treatment with tamoxifen, before extensive selection or clonal turnover; both fluorescent and non-fluorescent cells were included in the sample. This unbiased selection ensures that data are comparable to those obtained using in situ photoactivation (Fig. 1a–d), which we could not perform because the photoactivatable GFP-transgenic strain is not available under germ-free status. b, Clonal composition of individual germinal centres from five mice (GF1–GF5). C, caecal-colonic mLN; J, jejunal mLN. c, Quantification of data from b. Each symbol represents one germinal centre. d, Proportion of germinal centres in which the largest clone accounts for more than 50% of all B cells in mLNs of SPF mice (data from Fig. 1b) and germ-free mice (data from b). P values are from two-tailed Fisher’s exact tests. Centre bars represent the proportion in the sample; error bars show the exact binomial 95% confidence interval. e, Multiphoton images of Oligo-MM12 mLNs and Peyer’s patches at different times after treatment with tamoxifen. Blue represents collagen (second harmonics); white shows autofluorescence; other colours are from the Confetti allele. Scale bars, 200 μm (overviews), 50 μm (close-ups). N/D, NDS not determined owing to a low density of coloured cells. f, Quantification of images as in e for mLNs (top) and Peyer’s patches (bottom). Each symbol represents one germinal centre. Medians are indicated. Only germinal centres with a density of more than 0.4 fluorescent cells per 100 μm2 are included in the NDS calculations. g, Proportion of germinal centres with NDS values of more than 0.75 in mLNs (top) and more than 0.5 in Peyer’s patches (bottom) under SPF, germ-free and Oligo-MM12 conditions at 20–23 days after tamoxifen; SPF and germ-free data are as in Fig. 3c. For SPF, Oligo-MM12 and germ-free mLN gaGCs, n = 57, 16 and 27, respectively; for gaGCs from Peyer’s patches, n = 20, 10 and 9, respectively. P values obtained by two-tailed Fisher’s exact tests. Error bars represent exact binomial 95% confidence intervals. All data are from three to five mice per time point.
Extended Data Fig. 6 Characteristics of ‘winner’ gaGC clones from germ-free and Oligo-MM12-colonized mice.
a, b, Additional Igh sequence relationships among B cells from high-NDS germinal centres of germ-free (a) and Oligo-MM12-colonized (b) mice. Details are as in Fig. 2a. Scale bars, 50 μm. c, Reactivity summary of germ-free monoclonal antibodies assayed by ELISA against food protein extracts, autoantigens (anti-nuclear antibody, ANA), and a five-antigen polyreactivity panel. Shown are background subtracted OD450 values. d, Flow-cytometry analysis of the binding of monoclonal antibodies from germ-free mice to faecal bacteria from SPF mice. Details are as in Fig. 2b. e, ELISA analysis of the binding of monoclonal antibodies from germ-free mice to faecal bacterial fractions from SPF mice. MG053 was assayed at three dilutions only. Other monoclonal antibodies were assayed at dilutions indicated on the x-axis. Lines show the means of two assays. f, Western blot (WB) analysis of the binding of monoclonal antibodies from germ-free mice to a protein extract from mouse ileum tissue, run on a single-well 4–15% gel and blotted using a multiwell mask. Monoclonal antibody 3H9 is a DNA-specific negative control. Data in c–f are representative of two or more independent experiments.
a, Dendrograms showing the sequence relationships between VH1–47 and VH1–12 clones in different mice. All clones with up to two-amino-acid differences from the public-clonotype CDRH3 motifs are included. b, Heat maps showing the frequency of amino-acid replacements along the VH1–47 and VHH1–12 families in germ-free (blue) and Oligo-MM12 (green) mice, using the same data as in Fig. 4b. Only mice with more than two cells within the specified clone were included in the analysis. The number of cells analysed per mouse is indicated at the top of each column. Only those amino acids mutated in at least three (VH1–47) or two (VH1–12) mice are listed on the left, using Immunogenetics (IMGT; http://www.imgt.org) numbering; to the right, the most frequent amino-acid replacement in each mouse is given. Arrows indicate recurrent amino-acid mutations found in five of six mice (VH1–47) or three of three mice (VH1–12).
Extended Data Fig. 8 Stereotypical germ-free IgH clonotypes are present in Oligo-MM12 and germ-free/dietary-protein-free conditions.
a, Massive expansion of a public VH1–12 clonotype across different secondary lymphoid organs of mouse MM12 1 (from Fig. 4b), at 21 days after tamoxifen treatment. Multiphoton images show all three germinal centres sequenced from this mouse (yellow dotted boxes), magnified in the side panels. Scale bars, 200 μm (overviews) and 50 μm (close-ups). mLN close-ups are from different image acquisitions of the same germinal centre. A clonal tree of all cells from this clone is shown at the bottom right. Arrowheads indicate clonal bursts and the organ of origin of cells with that particular sequence. b, Frequency of cells with a germinal-centre phenotype (CD38dim FAShi) among total B220+ B cells in the indicated organs of mice raised on protein-free chow (PFC). Data for SPF and germ-free mice are reproduced from Extended Data Fig. 4b. Each symbol represents one mouse. For PFC, n = 8 mice. c, Clonal distribution of germinal-centre B cells sequenced from the indicated tissues of three separate mice (PFC1–3), with public clonotypes colour-coded. See also Fig. 4b. C, caecal colonic mLN; D, duodenal mLN; I, ileal mLN; PP, Peyer’s patch.
Extended Data Fig. 9 Multiwell incidence-based Igh sequencing reveals clonal overlap among individual mice and between microbial colonization conditions.
a, Overview of the incidence-based Igh sequencing method used for c–g and Fig. 4c, d. To identify expanded public clonotypes among gaGC samples from multiple mice with high confidence, we developed an incidence-based sequencing strategy based on repeated sampling of the same germinal-centre B cell population. We sorted multiple samples of 100 germinal-centre B cells (usually 32 for mLN and 16 for Peyer’s patches) from 6 germ-free, 6 SPF, and 7 Oligo-MM12-colonized mice, and sequenced all BCRs in each sample, for a total of roughly 80 thousand input B cells, plus 32 wells each of non-germinal-centre B cells from the mLN of 3 germ-free and 3 SPF mice as controls. To avoid counting as ‘public’ sequences that were spuriously present in different mice owing to barcode misassignment or DNA contamination, we included in our analysis only those clones that were represented by more than five reads in any single well and found in at least two wells from the same sample. Key bioinformatics steps are described in the figure; see Methods for a full description of the bioinformatic pipeline. b, Gating strategy used for data in c–g and Fig. 4c, d, described in a. c, Number of distinct clones per well, after collapsing sequences with matching VH, JH, and CDRH3 nucleotide sequences. Each symbol represents one well. Boxes represent medians and interquartile ranges. As expected, non-germinal-centre B cell samples had many more total clones per well than did germinal-centre B cells. d, Proportion of expanded clones (present in more than one well per sample) in germinal-centre and non-germinal-centre samples from mLNs and Peyer’s patches of mice held under the specified conditions. e, Histograms showing Levenshtein distances between the indicated consensus CDRH3 sequence and the CDRH3 sequence of all clones in the indicated category. For ARGSNYXXXXDY, distances are plotted for clones carrying the ‘correct’ VH1–47 gene or two ‘control’ VH regions with similar usage frequency in our sample. P values were obtained by Kruskall–Wallis test comparing all three conditions. Owing to the very low number of total VH1–12 clones outside of the germ-free condition, distances to the AREGFAY CDRH3 are compared between VH1–12 clones and all clones. P values obtained by two-tailed Mann–Whitney U test. f, Fraction of clone*wells containing public clonotypes in each condition, pooled from all mice. P values were obtained by Fisher’s exact test. g, Venn diagram showing the number of clones per condition (pooled from all mice) and overlap between conditions. The clone in the centre of the graph (SPF/Oligo-MM12/germ-free overlap) corresponds to the VH1–47 public clonotype. In f, g, data are as in Fig. 4d.
Supplementary Spreadsheet 1. Ig sequence information for single-cell sequences used throughout the manuscript, organized in labeled tabs according to colonization status/sequence use (for monoclonal antibody production, for clonal trees etc).
PDF containing further information under the following headings: Use of the AID-Confetti model to estimate the rate of GC selection, Supplementary discussion and Supplementary text references.
Supplementary Table S1. List of mABs produced and their characteristics.
Supplementary Table S2. Ingredients in protein-free chow.
Supplementary Table S3. Antibodies used for flow cytometry.
Supplementary Table S4. Bacterial strains/isolates used.
Supplementary Table S5. Primers used for QPCR amplification of individual Oligo-MM12 bacteria.
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Nowosad, C.R., Mesin, L., Castro, T.B.R. et al. Tunable dynamics of B cell selection in gut germinal centres. Nature 588, 321–326 (2020). https://doi.org/10.1038/s41586-020-2865-9
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