Research ArticleMICROBIOTA

B cell superantigens in the human intestinal microbiota

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Science Translational Medicine  28 Aug 2019:
Vol. 11, Issue 507, eaau9356
DOI: 10.1126/scitranslmed.aau9356
  • Fig. 1 A subset of human infants harbor microbiota with superantigen-type binding to mAbs expressing murine VH5/6/7 and human VH3 variable regions.

    (A) Bacterial flow cytometry analysis indicating the percent of fecal bacteria from 10 human donors grafted into GF mice that were coated with endogenous polyclonal IgA (open squares), or various negative control B2 mAbs (open circles), or microbiota-reactive SI IgA mAbs (closed circles) expressed with a human IgG1 backbone and detected with anti-hIgG reagents. hIgG, human immunoglobulin G; FSC, forward scatter. Each circle represents a distinct mAb. Red boxes highlight the four individuals with superantigen-type reactivity. Asterisks denote microbiota from cow’s milk–allergic infants, and the remaining samples were from healthy infants. Data compiled from four independent experiments. (B) Bacterial flow cytometry analysis of the four human infants’ microbiota from (A) that showed superantigen-type reactivity with a larger panel of 53 murine SI IgA–derived (closed circles) or 47 naïve B2–derived mAbs (open circles), grouped by heavy chain variable gene usage as indicated. (C) Representative flow cytometry plots (top) and summary of bacterial flow cytometry analysis (bottom) of indicated strains cultured in vitro and stained with 53 SI IgA– or 47 B2-derived mAbs, grouped by heavy chain variable gene usage as indicated. Data compiled from three independent experiments. (D) Representative flow cytometry plots (top) and summary bacterial flow cytometry analysis (bottom) of indicated strains cultured in vitro and stained with 26 fully human anti-influenza mAbs, grouped by heavy chain variable gene usage as indicated. Data compiled from three independent experiments. P values in (B) to (D) were calculated by unpaired t test.

  • Fig. 2 Identification of the superantigens.

    (A) Western blot analysis of total protein lysates from indicated strains probed with nonsuperantigen-reactive VH1 mAb 277C1 or superantigen-reactive VH5 mAb 338E6. Representative of >3 independent experiments. (B) Diagram of the two superantigens. aa, amino acid. (C) ELISA analysis of purified R. gnavus proteins expressed in E. coli with an N-terminal His tag. Proteins were coated on plates and probed for reactivity against the indicated dose titration of 11 VH5/6/7 or 11 non-VH5/6/7 mAbs from naïve B2 cells, as indicated (left panels) or (D) against seven human VH3 and five non-VH3 antibodies. Right panels summarize the area under the curve for each antibody shown in the left panels. OD450, optical density at 450 nm. Representative of two independent experiments. P values calculated by unpaired t test.

  • Fig. 3 IbpA and IbpB activate murine and human B cells in vitro.

    (A) Representative flow cytometry analyses and summary graphs depicting purified murine B cells from wild-type or Myd88−/− mice 6 hours after in vitro incubation with indicated stimuli and analyzed for surface expression of CD19 and IgM or (B) CD69. Data pooled from two independent experiments with a total of four wild-type and three Myd88−/− mice. (C) Representative flow cytometry analyses and summary plots depicting purified human B cells 6 hours after in vitro incubation with indicated stimuli and analyzed for surface expression of CD19 and IgM or (D) CD83 and CD69. Each data point represents one human sample. Samples were obtained from three individuals, and cells from one individual were isolated and analyzed in two independent experiments. Data pooled from two independent experiments. P values were calculated by one-way ANOVA.

  • Fig. 4 Superantigen-expressing strains stimulate and attract IgA in vivo.

    (A) Representative flow cytometry plots gated on FSChi lineage cells or (B) absolute number summaries of SI LP IgA plasma cells from GF or gnotobiotic mice 4 weeks after monocolonization with indicated bacterial strains. Data compiled from three independent experiments with 12 total mice, distributed as indicated. (C) Absolute number of SI IgA plasma cells expressing indicated variable gene families. Calculated from the total cell number shown in (B) multiplied by the percent of the repertoire expressing indicated variable region genes as determined by sequencing of plasmids containing cDNA clones. One-way ANOVA P values were 0.0048 for VH5/6/7, 0.5156 for VH1-47, and 0.6958 for VH3-5. P values in (B) and (C) were calculated by unpaired t test.

  • Fig. 5 Distribution of R. gnavus and its superantigens across human metagenomes.

    Dendrogram alignment of the R. gnavus ATCC 29149 genome to 424 human metagenomes (data files S3 and S4). Each spoke represents one gene in the R. gnavus genome, and each layer represents an individual human metagenome. The two superantigen genes are labeled. Intensity represents coverage of the open reading frame in the metagenome.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/507/eaau9356/DC1

    Fig. S1. mAb reactivity to mouse microbiota and cladogram of individual bacterial strains analyzed.

    Fig. S2. Immunoprecipitation, mass spectrometry, Coomassie, and Western blot analysis of purified proteins.

    Fig. S3. Additional analyses of GF or gnotobiotic monocolonized mice.

    Table S1. Distribution of superantigens across human metagenomes.

    Data file S1. List of mAbs used in this study.

    Data file S2. Primary data.

    Data file S3. List of metagenomes analyzed.

    Data file S4. Metagenomic recruitment analysis of R. gnavus genes including its superantigens.

  • The PDF file includes:

    • Fig. S1. mAb reactivity to mouse microbiota and cladogram of individual bacterial strains analyzed.
    • Fig. S2. Immunoprecipitation, mass spectrometry, Coomassie, and Western blot analysis of purified proteins.
    • Fig. S3. Additional analyses of GF or gnotobiotic monocolonized mice.
    • Table S1. Distribution of superantigens across human metagenomes.
    • Legends for data files S1 to S4

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). List of mAbs used in this study.
    • Data file S2 (Microsoft Excel format). Primary data.
    • Data file S3 (Microsoft Excel format). List of metagenomes analyzed.
    • Data file S4 (Microsoft Excel format). Metagenomic recruitment analysis of R. gnavus genes including its superantigens.

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