Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Mucosal or systemic microbiota exposures shape the B cell repertoire


Colonization by the microbiota causes a marked stimulation of B cells and induction of immunoglobulin, but mammals colonized with many taxa have highly complex and individualized immunoglobulin repertoires1,2. Here we use a simplified model of defined transient exposures to different microbial taxa in germ-free mice3 to deconstruct how the microbiota shapes the B cell pool and its functional responsiveness. We followed the development of the immunoglobulin repertoire in B cell populations, as well as single cells by deep sequencing. Microbial exposures at the intestinal mucosa generated oligoclonal responses that differed from those of germ-free mice, and from the diverse repertoire that was generated after intravenous systemic exposure to microbiota. The IgA repertoire—predominantly to cell-surface antigens—did not expand after dose escalation, whereas increased systemic exposure broadened the IgG repertoire to both microbial cytoplasmic and cell-surface antigens. These microbial exposures induced characteristic immunoglobulin heavy-chain repertoires in B cells, mainly at memory and plasma cell stages. Whereas sequential systemic exposure to different microbial taxa diversified the IgG repertoire and facilitated alternative specific responses, sequential mucosal exposure produced limited overlapping repertoires and the attrition of initial IgA binding specificities. This shows a contrast between a flexible response to systemic exposure with the need to avoid fatal sepsis, and a restricted response to mucosal exposure that reflects the generic nature of host–microbial mutualism in the mucosa.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Antibody repertoires in mucosal and systemic tissues after transitory oral or systemic exposure to a commensal microorganism.
Fig. 2: Differences between B cell repertoires after systemic or mucosal exposure.
Fig. 3: Antimicrobial antibody responses after combined mucosal and systemic exposure or exposure to two different microbial taxa.

Data availability

The raw files for the datasets generated during this study are available on the Sequence Read Archive, Bioproject accession PRJNA625440. The pre-processed files (clonotype list and properties) are available on the GitHub repository

Code availability

The associated codes for the analysis using R packages can be found at


  1. Lindner, C. et al. Age, microbiota, and T cells shape diverse individual IgA repertoires in the intestine. J. Exp. Med. 209, 365–377 (2012).

    Article  CAS  Google Scholar 

  2. Lindner, C. et al. Diversification of memory B cells drives the continuous adaptation of secretory antibodies to gut microbiota. Nat. Immunol. 16, 880–888 (2015).

    Article  CAS  Google Scholar 

  3. Hapfelmeier, S. et al. Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses. Science 328, 1705–1709 (2010).

    Article  ADS  CAS  Google Scholar 

  4. Berg, R. D. Bacterial translocation from the gastrointestinal tract. Adv. Exp. Med. Biol. 473, 11–30 (1999).

    Article  CAS  Google Scholar 

  5. Lockhart, P. B. et al. Bacteremia associated with toothbrushing and dental extraction. Circulation 117, 3118–3125 (2008).

    Article  CAS  Google Scholar 

  6. Xu, J. L. & Davis, M. M. Diversity in the CDR3 region of VH is sufficient for most antibody specificities. Immunity 13, 37–45 (2000).

    Article  CAS  Google Scholar 

  7. Soto, C. et al. High frequency of shared clonotypes in human B cell receptor repertoires. Nature 566, 398–402 (2019).

    Article  ADS  CAS  Google Scholar 

  8. Koch, M. A. et al. Maternal IgG and IgA antibodies dampen mucosal T helper cell responses in early life. Cell 165, 827–841 (2016).

    Article  CAS  Google Scholar 

  9. Gomez de Agüero, M. et al. The maternal microbiota drives early postnatal innate immune development. Science 351, 1296–1302 (2016).

    Article  ADS  Google Scholar 

  10. Zeng, M. Y. et al. Gut microbiota-induced immunoglobulin G controls systemic infection by symbiotic bacteria and pathogens. Immunity 44, 647–658 (2016).

    Article  CAS  Google Scholar 

  11. Chen, Y. et al. Microbial symbionts regulate the primary Ig repertoire. J. Exp. Med. 215, 1397–1415 (2018).

    Article  CAS  Google Scholar 

  12. Wilmore, J. R. et al. Commensal microbes induce serum IgA responses that protect against polymicrobial sepsis. Cell Host Microbe 23, 302–311 (2018).

    Article  CAS  Google Scholar 

  13. Pepys, M. B. Role of complement in induction of antibody production in vivo: effect of cobra factor and other C3-reactive agents on thymus-dependent and thymus-independent antibody responses. J. Exp. Med. 140, 126–145 (1974).

    Article  CAS  Google Scholar 

  14. Sörman, A., Zhang, L., Ding, Z. & Heyman, B. How antibodies use complement to regulate antibody responses. Mol. Immunol. 61, 79–88 (2014).

    Article  Google Scholar 

  15. Stoel, M. et al. Restricted IgA repertoire in both B-1 and B-2 cell-derived gut plasmablasts. J. Immunol. 174, 1046–1054 (2005).

    Article  CAS  Google Scholar 

  16. Bunker, J. J. et al. Natural polyreactive IgA antibodies coat the intestinal microbiota. Science 358, eaan6619 (2017).

    Article  Google Scholar 

  17. Mowat, A. M., Faria, A. M. & Weiner, H. L. in Mucosal Immunology Vol. 1 (eds J. Mestecky et al.) 487–537 (Elsevier, 2005).

  18. Pfister, S. P. et al. Uncoupling of invasive bacterial mucosal immunogenicity from pathogenicity. Nat. Commun. 11, 1978 (2020).

    Article  ADS  CAS  Google Scholar 

  19. Boursier, L., Dunn-Walters, D. K. & Spencer, J. Characteristics of IgVH genes used by human intestinal plasma cells from childhood. Immunology 97, 558–564 (1999).

    Article  CAS  Google Scholar 

  20. Casola, S. et al. B cell receptor signal strength determines B cell fate. Nat. Immunol. 5, 317–327 (2004).

    Article  CAS  Google Scholar 

  21. Dunn-Walters, D. K., Boursier, L. & Spencer, J. Hypermutation, diversity and dissemination of human intestinal lamina propria plasma cells. Eur. J. Immunol. 27, 2959–2964 (1997).

    Article  CAS  Google Scholar 

  22. Bergqvist, P. et al. Re-utilization of germinal centers in multiple Peyer’s patches results in highly synchronized, oligoclonal, and affinity-matured gut IgA responses. Mucosal Immunol. 6, 122–135 (2013).

    Article  CAS  Google Scholar 

  23. Levine, M. M. Immunogenicity and efficacy of oral vaccines in developing countries: lessons from a live cholera vaccine. BMC Biol. 8, 129 (2010).

    Article  Google Scholar 

  24. Valdez, Y., Brown, E. M. & Finlay, B. B. Influence of the microbiota on vaccine effectiveness. Trends Immunol. 35, 526–537 (2014).

    Article  CAS  Google Scholar 

  25. Rivera, M. C., Maguire, B. & Lake, J. A. Isolation of ribosomes and polysomes. Cold Spring Harb. Protoc. 2015, pdb.prot081331 (2015).

    Article  Google Scholar 

  26. Slack, E. et al. Innate and adaptive immunity cooperate flexibly to maintain host-microbiota mutualism. Science 325, 617–620 (2009).

    Article  ADS  CAS  Google Scholar 

  27. Macpherson, A. J. et al. A primitive T cell-independent mechanism of intestinal mucosal IgA responses to commensal bacteria. Science 288, 2222–2226 (2000).

    Article  ADS  CAS  Google Scholar 

  28. Tomayko, M. M., Steinel, N. C., Anderson, S. M. & Shlomchik, M. J. Cutting edge: hierarchy of maturity of murine memory B cell subsets. J. Immunol. 185, 7146–7150 (2010).

    Article  CAS  Google Scholar 

  29. Greiff, V. et al. Quantitative assessment of the robustness of next-generation sequencing of antibody variable gene repertoires from immunized mice. BMC Immunol. 15, 40 (2014).

    Article  Google Scholar 

  30. Krebber, A. et al. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J. Immunol. Methods 201, 35–55 (1997).

    Article  CAS  Google Scholar 

  31. Menzel, U. et al. Comprehensive evaluation and optimization of amplicon library preparation methods for high-throughput antibody sequencing. PLoS ONE 9, e96727 (2014).

    Article  ADS  Google Scholar 

  32. Bolotin, D. A. et al. MiXCR: software for comprehensive adaptive immunity profiling. Nat. Methods 12, 380–381 (2015).

    Article  CAS  Google Scholar 

  33. Greiff, V. et al. Systems analysis reveals high genetic and antigen-driven predetermination of antibody repertoires throughout B cell development. Cell Rep. 19, 1467–1478 (2017).

    Article  CAS  Google Scholar 

  34. Lefranc, M. P. et al. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. 27, 209–212 (1999).

    Article  CAS  Google Scholar 

  35. Shugay, M. et al. VDJtools: unifying post-analysis of T cell receptor repertoires. PLoS Comput. Biol. 11, e1004503 (2015).

    Article  Google Scholar 

  36. Stern, J. N. et al. B cells populating the multiple sclerosis brain mature in the draining cervical lymph nodes. Sci. Transl. Med. 6, 248ra107 (2014).

    Article  Google Scholar 

  37. Khan, T. A. et al. Accurate and predictive antibody repertoire profiling by molecular amplification fingerprinting. Sci. Adv. 2, e1501371 (2016).

    Article  ADS  Google Scholar 

  38. Vander Heiden, J. A. et al. pRESTO: a toolkit for processing high-throughput sequencing raw reads of lymphocyte receptor repertoires. Bioinformatics 30, 1930–1932 (2014).

    Article  Google Scholar 

  39. Hsieh, T., Ma, K. & Chao, A. iNEXT: an R package for rarefaction and extrapolation of species diversity (Hill numbers). Methods Ecol. Evol. 7, 1451–1456 (2016).

    Google Scholar 

  40. van der Loo, M. The stringdist package for approximate string matching. R Journal 6, 111–122 (2014).

    Article  Google Scholar 

  41. Miho, E., Roškar, R., Greiff, V. & Reddy, S. T. Large-scale network analysis reveals the sequence space architecture of antibody repertoires. Nat. Commun. 10, 1321 (2019).

    Article  ADS  Google Scholar 

  42. Csardi, G. & Nepusz, T. The igraph software package for complex network research. Interjournal Complex Syst., 2006, 1695 (2006).

Download references


We thank M. Gomez de Agüero, T. Leeb, P. Nicholson, M. Geuking and D. Candinas. The Clean Mouse Facility is supported by the Genaxen Foundation, Inselspital and the University of Bern. This work was funded by the Swiss National Science Foundation (SNSF CRSII5_177164, SNSF 310030_179479) and the European Research Council (H2020 ERC-2016-ADG HHMM_Neonates, Grant Agreement: 742195) to A.J.M. S.C.G.-V. was funded by a Marie Curie Intra-European Fellowship (FP7-PEOPLE-2013-IEF project no. 627206), a long-term fellowship from the European Molecular Biology Organization and the Peter Hans Hofschneider Endowed Professorship by Stiftung Experimentelle Biomedizin. V.G. acknowledges the support of UiO:LifeSciences Convergence Environment Immunolingo and EU H2020 iReceptorplus (no. 825821). S.H. was funded by SNSF grant 169791 and ERC StG 281904. K.D.M. was funded by the ERC StG 281785. M.H. was funded by an ERC CoG (HepatoMetaboPath). J.P.L. was supported by a SystemsX Transition Postdoc Fellowship (TPdF2013/139).

Author information

Authors and Affiliations



H.L., J.P.L., S.C.G.-V. and A.J.M. conceived the study, interpreted data and wrote the manuscript. H.L. and S.C.G.-V. performed most experiments. J.P.L. and V.G. carried out computational analysis. B.Y., M.A.E.L., S.R., M.H. and K.D.M. helped with repertoire experiments. O.S. and S.H. contributed bacterial strains and culture preparation. C.U., M.Z. and I.D.Y. helped with ex-vivo analyses of bacterial fractions and cellular responses.

Corresponding authors

Correspondence to Stephanie C. Ganal-Vonarburg or Andrew J. Macpherson.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Rodney Newberry, Duane R. Wesemann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Mucosal and systemic exposure differentially shape the repertoires of the various immunoglobulin isotypes.

ah, Germ-free mice were either orally or systemically primed three times every other day by intragastric (1010 CFU) or intravenous (108 CFU) delivery of E. coli HA107 (ag) or Clostridium orbicindens (h) and compared with germ-free control mice. Immunoglobulin heavy-chain-repertoire clonotypes at day 21 are defined by V and J segment use in combination with CDR3 nucleotide sequences, except for a, in which clonotypes are based on CDR3 amino acid sequences as in Fig. 1. a, MDS plot of IgA, IgG2b and IgM showing distinct isotype repertoires (in each case all isotypes were sequenced from each individual sample from at least 3 mice). bd, MDS plot from ileum, MLN, bone marrow (BM) or spleen (SPL) of immunoglobulin repertoire sequencing for IgA (b), IgG2b (c) and IgM (d). eh, Heat maps showing the 100 most abundant non-unique clonotypes for IgA (e, h), IgG2b (f) and IgM (g). Clonotype specifics for each panel are shown in the Supplementary Information. Samples of ileum, BM, MLN and SPL from each mouse are included. Individual mice are colour-coded on the x-axis of heat maps. i, j, Immunoglobulins from intestinal wash of intestinally exposed mice (i) or from serum of systemically exposed mice (j) on day 21 were assessed in bacterial flow cytometry for specific IgM, IgG1, IgG2b, IgG2c, IgG3 or IgA binding to E. coli HA107. k, l, E. coli HA107 was incubated with day 21 serum or intestinal wash immunoglobulin of the three groups of mice followed by detection of bound mouse IgG2b (k) or IgA (l) by flow cytometry. All data points are from organs of individual mice.

Extended Data Fig. 2 Comparison of computational correction method with UMIs for PCR artefacts and sequencing reproducibility on different occasions.

Germ-free mice were either orally or systemically primed three times every other day by intragastric (1010 CFU) or intravenous (108 CFU) exposure to E. coli HA107 and compared with germ-free control mice. Immunoglobulin repertoire sequencing at 21 days for IgA in MLN and IgG2b in spleen was performed in parallel from the same RNA samples with two different primers containing different UMIs or primers without UMIs. a, b, MDS plot of MLN IgA (a) or spleen IgG2b (b) repertoires showing Euclidean distance between points representing the similarity between results whether computational correction (filled symbols) or UMI correction (open and cross symbols for the different UMIs) was used. c, Heat map based on CDR3 sequence identity reflecting similarity between UMI-corrected and computational-corrected IgG2b repertoires. d, Identity matrix based on CDR3 sequences of technical replicates of the same biological sample that had been used on two separate occasions for the entire pipeline of cDNA preparation, IgA amplicon PCR, library preparation and MiSeq sequencing. The distinctions between replicate mice and technical repeats are shown in the diagram.

Extended Data Fig. 3 Threshold differences for shaping systemic or mucosal B cell repertoires and induction of antibody responses.

Reversible E. coli HA107 was given to germ-free mice at the indicated doses orally (a, b, e, f) or systemically (c, d, g, h), as in the legend to Fig. 1. a, c, Immunoglobulin repertoire analysis at 21 days showing heat maps of the top 100 non-unique clonotypes for IgA and IgG2b in MLN and spleen based on V, J segment use combined with CDR3 nucleotide sequences. b, d, Flow cytometric analysis of E.-coli-specific immunoglobulin binding from intestinal or serum samples from the corresponding mice in a and c, respectively. eh, Hierarchical clustering of the indicated immunoglobulin repertoires. Dendrogram of samples, branch length shows the distance between repertoires based on CDR3 amino acid sequence similarity of entire B cell repertoires. The dotted lines indicate the principal separations in unsupervised analyses as an independent assessment of thresholds of exposure required for repertoire shaping. Each column within the heat maps or each dilution series from antibody-binding bacterial flow cytometry are from individual mice; in every case, three mice were used for every experimental condition studied. The Supplementary Information contains a table specifying the top 100 clonotypes in each case for a, c. Data are representative of two independent experiments.

Extended Data Fig. 4 Differences in processing of microbial antigens, and presentation in the mucosal and systemic compartments.

a, Reversible E. coli HA107 were given to germ-free mice at the indicated doses orally or systemically, as in the legend to Fig. 1. Binding of systemic or intestinal antibodies to E. coli non-ribosomal versus ribosomal proteins of the cytoplasmic fraction evaluated using ELISA (\(\bar{x}\) ± s.d., n = 6, two-sided unpaired t-test). b, c, Enterobacter cloacae proteins were either separated by FPLC or subject to ribosomal protein purification, before 12% SDS-polyacrylamide gel electrophoresis. Silver stain for total protein (b). Western blot for immunoreactivity against serum IgG raised from specific pathogen-free C57BL/6 mice injected with 108 CFU E. cloacae 20 days previously (c). Control experiments verified previously published data27 that un-manipulated control mice showed no immunoreactivity against this dominant intestinal aerobe. Proteomic analysis of extracted bacterial ribosomal proteins confirmed identities as follows: 50SL24, 50SL11, 50SL4, 50SL3, 50SL1, 30SS4 and 30SS1. d, Germ-free mice were orally (n = 6) or systemically (n = 5) exposed once by intragastric (1010 CFU) or intravenous (108 CFU) delivery of E. coli HA107. Mesenteric lymph nodes and spleens were analysed 18 h later for LPS levels and 16S rRNA (\(\bar{x}\) ± s.d., two-sided unpaired t-test). e, f, Indicated doses of intact or ultrasound-lysed E. coli HA107 were administered orally (e) or systemically (f) three times to germ-free mice every other day and compared to germ-free control mice. Immunoglobulin heavy-chain repertoire analysis at 21 days for IgA in mesenteric lymph nodes (e) or IgG2b in the spleen (f). g, In vitro culture of leukocytes from the mesenteric lymph nodes or spleen of germ-free mice stimulated with either cytoplasmic or membrane fractions of E. coli HA107. Activated B cells were sorted on day 5 after stimulation and IgM heavy chain sequencing was carried out. eg, In all cases, Euclidean distance in MDS plots reflects the distance between indicated repertoires based on CDR3 amino acid sequences of the entire B cell repertoire. ad, Data are representative of two independent experiments. eg, Data are from two single experiments. Each data point is from the organ of an individual mouse.

Extended Data Fig. 5 Network formation of different isotypes depending on transitory microbial treatment, and comparison with strong cholera toxin immunogen.

Germ-free mice were orally or systemically primed three times every other day by a range of intragastric (102–1010 CFU) or intravenous (102–108 CFU) doses of E. coli HA107, compared with priming by cholera toxin or to germ-free control mice. a, Rarefaction plots of immunoglobulin repertoire sequencing at 21 days for IgM or IgG2b (inset shows same data as in Fig. 2b, but different y-axis scale) in MLN and spleen. Colour coding indicates the route of exposure. b, c, Median number of mutations per clonotype for IgA in the MLN (b) or IgG2b in splenic B cells (c). Tukey plots in each case are shown with whiskers at 1.5× interquartile range (n = 3 for each condition). d, e, Germ-free mice were orally or systemically primed three times every other day by intragastric (1010 CFU) or intravenous (108 CFU) delivery of E. coli HA107, or by intragastric (15 μg) or intravenous (15 μg) delivery of cholera toxin B (CTB) and compared with germ-free control mice. Immunoglobulin repertoire sequencing was carried out for IgA, IgM and IgG2b at 21 days. d, Tukey plots with whiskers at 1.5× interquartile range indicate proportions of expanded clonotypes (excluding singletons) within the entire CDR3 amino acid IgA repertoire in ileum, MLN and spleen, or within the IgG2b repertoire in MLN and spleen after the indicated exposures (n = 3 mice in each bacterial or cholera toxin priming group, n = 12 germ-free control mice, two-sided Wilcoxon rank-sum test). Adjusted P values as shown. e, Radial plot showing median values for mutational levels in MLN, ileum and spleen in individual mice. Peripheral displays of representative network structures of clonotypes showing relatedness with edges representing Levenshtein distance 1 (blue). Singletons are shown in orange. d, e, All conditions were repeated over 10 times, except for cholera toxin intravenous priming, which was carried out once, and cholera toxin intestinal priming, which was carried out twice. All data points are from individual mice.

Extended Data Fig. 6 Characteristics of naive B cell repertoires following systemic or mucosal exposure and sites of B cell activation.

a, b, Characteristics of naive single-cell B cell repertoires corresponding to class-switched single-cell repertoires shown in Fig. 2e, f, respectively. Germ-free mice were orally or systemically primed three times every other day by intragastric (1010 CFU) or intravenous (108 CFU) delivery of E. coli HA107 and compared with germ-free control mice. Single-cell VDJ sequencing analysis on day 21. Network built on combined heavy- and light-chain CDR3 amino acid sequences each from a single splenic (a) or MLN (b) naive IgD-expressing B cell, excluding singlets (n = 4,272 or 5,672 for a and b, respectively). Edges show Levenshtein distance 1 or 2. Networks show the immunoglobulin sequence relationships within and between different mice in the same experiment, colour-coded according to mouse and treatment. Left pie charts indicate the percentage of edge connections that are based on a Levenshtein distance of 1 on the light chain compared to the heavy chain; right pie charts indicate the distribution of edge connections between individual B cells within or between exposure conditions. c, d, Germ-free mice were systemically (i.v.) primed with two doses of 108 CFU E. coli HA107, 7 days apart, and compared with germ-free control mice. Lymphocytes from spleen were isolated 18 h after the intravenous injection, stained with fluorescent-labelled antibodies and analysed by flow cytometry. c, Overall quantifications (\(\bar{x}\) ± s.d., n = 11 germ-free and 9 systemic exposure) of live GL7+CD19+ lymphocytes as a proportion of all CD19+ lymphocytes from two independent experiments. d, Lymphocytes from spleen were isolated 3 d after the last injection for culture. IgA and IgG2b antibody levels were determined in culture supernatants at 5 d by ELISA. Plots show pooled data from two experiments (geometric mean, n = 7 germ-free and 8 systemic exposure for both isotypes). e, f, Germ-free mice were reversibly exposed with 3 gavage doses of 1010 CFU E. coli HA107 and compared with germ-free control mice. e, Overall quantifications (\(\bar{x}\) ± s.d., n = 4 for each condition) of live GL7+BCL6+CD19+ lymphocytes as a proportion of all CD19+ lymphocytes from MLN 24 h after the last gavage (\(\bar{x}\) ± s.d.). f, Lymphocytes from MLN were isolated 3 d after the last injection for culture. IgA and IgG2b antibody levels were determined at 5 d in culture supernatants by ELISA. Geometric means, n = 4 for each condition, except IgG2b intestinal exposure = 3. cf, P values with unpaired t-test are indicated as shown in the figure. All data points are from individual mice.

Extended Data Fig. 7 CD4 T cells are required for systemic immune memory following intestinal exposure to reversible E. coli HA107.

a, b, Schematic experimental designs to Figs. 3a–c. a, Germ-free mice were intestinally exposed to 1010 CFU reversible E. coli HA107 on alternate days or remained germ-free throughout. On day 21, all mice were reversibly intravenously exposed to 103–107 CFU HA107, as shown. Mice were analysed on day 42. b, As in a, except that the initial reversible exposure was given systemically with subsequent reversible intestinal exposures at different doses. c, Germ-free mice were mucosally exposed to 3 doses of 1010 CFU E. coli HA107 on days 0, 2 and 4, or left germ-free. In both groups, half the mice were treated with anti-CD4 depleting antibody intraperitoneally on day −3; the other half received a control isotype. CD4+ T cells were absent (<0.1% of blood leukocytes) from day 0 until at least day 10, but were shown to have recovered by day 21. On day 21, mice were intravenously primed with 107 CFU E. coli HA107. Control groups included intestinally only exposed mice and untreated germ-free mice. d, Representative dot plots of flow cytometric analysis of the blood on days 0 and 21 in both the isotype and anti-CD4 antibody treated groups. e, Bacterial flow cytometry at day 42 analysing specific bacterial surface binding against E. coli HA107 from serum IgG2b of the indicated groups. f, Immunoglobulin repertoire sequencing for IgG2b in the spleen on day 42. Euclidean distance in the MDS plot reflects the distance based on CDR3 amino acid sequences of the entire repertoires. Data in cf are representative of two independent experiments. Each dilution series (e) or data points (f) are from individual mice.

Extended Data Fig. 8 Experimental schemes.

This figure relates to Fig. 3. a, Schematic experimental design, related to Fig. 3d–g. Germ-free mice were mucosally primed with three doses of 1010 CFU E. coli HA107 or reversible S. typhimurium HA218 on alternate days or remained germ-free. At day 21, half of each group received a second schedule of priming, but with the opposite taxon. Recovery of germ-free status was verified after each stage. b, Schematic experimental design, related to Fig. 3h–k. As in a, except that both reversible taxa were given systemically at doses of 108 CFU, as shown.

Extended Data Table 1 Comparison of B cell receptor CDR3 sequences assessed in this study with previously reported data

Supplementary information

Supplementary Information

This file contains a) a summary table of sequenced data in the paper; b) tables with top 100 clonotypes in Extended Data Figure 1e-h; and c) tables with top 100 clonotypes in Extended Data Figure 3 a and c; d) SI Figure 1: gating strategy; e) tables with all primers used for bulk B cell repertoire sequencing.

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, H., Limenitakis, J.P., Greiff, V. et al. Mucosal or systemic microbiota exposures shape the B cell repertoire. Nature 584, 274–278 (2020).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing