Abstract
Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response1,2,3,4,5. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with complex defined communities (roughly 100 bacterial strains) and profile T cell responses to each strain. The pattern of responses suggests that many T cells in the gut repertoire recognize several bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all the bacteria-specific TCRs show a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that each recognize 18 Firmicutes. By screening three pooled bacterial genomic libraries, we discover that these 13 clonotypes share a single target: a conserved substrate-binding protein from an ATP-binding cassette transport system. Peripheral regulatory T cells and T helper 17 cells specific for an epitope from this protein are abundant in community-colonized and specific pathogen-free mice. Our work reveals that T cell recognition of commensals is focused on widely conserved, highly expressed cell-surface antigens, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected.
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Data availability
The data have been deposited as follows: Cell Ranger out files, https://doi.org/10.5281/zenodo.8008419; raw sequence data: scTCR-seq of T cells from the small intestine of germ-free mice, https://doi.org/10.5281/zenodo.8007653; scTCR-seq of T cells from the small intestine of hCom1 colonized mice, https://doi.org/10.5281/zenodo.8007692; scTCR-seq of T cells from the small intestine of hCom2-colonized mice, https://doi.org/10.5281/zenodo.8007696; scTCR-seq of T cells from the large intestine of germ-free mice, https://doi.org/10.5281/zenodo.8007700; scTCR-seq of T cells from the large intestine of hCom1 colonized mice, https://doi.org/10.5281/zenodo.8007704; scTCR-seq of T cells from the large intestine of hCom2-colonized mice, https://doi.org/10.5281/zenodo.8007709; scRNA-seq of T cells from the small intestine of germ-free mice, https://doi.org/10.5281/zenodo.8007733; scRNA-seq of T cells from the small intestine of hCom1 colonized mice, https://doi.org/10.5281/zenodo.8007735; scRNa-seq of T cells from the small intestine of hCom2-colonized mice, https://doi.org/10.5281/zenodo.8007737; scRNA-seq of T cells from the large intestine of germ-free mice, https://doi.org/10.5281/zenodo.8007740; scRNA-seq of T cells from the large intestine of hCom1 colonized mice, https://doi.org/10.5281/zenodo.8007742; scRNA-seq of T cells from the large intestine of hCom2-colonized mice, https://doi.org/10.5281/zenodo.8007744. Source data are provided with this paper.
Code availability
The custom code for scRNA-seq and scTCR-seq analyses is deposited at Zenodo (https://doi.org/10.5281/zenodo.8011206). The following software packages were used: R v.3.6.2 (2019-12-12), platform x86_64-apple-darwin15.6.0 (64-bit); macOS v.10.16; the R packages Seurat v.3.1.5 and scRepertoire v.1.0.0; Microsoft Excel v.16.73; FlowJo (TreeStar) v.10.8.1; Prism 9 for macOS (graphpad) v.9.5.1; PyMOL (Schrödinger, Inc.) v.2.5.
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Acknowledgements
We are deeply indebted to D. Bousbaine and other members of the Fischbach Group for helpful suggestions and comments on the manuscript. We thank H. Takayanagi, S. Sawa, R. Muro, N. A. Bracey, M. M. Davis and members of their laboratories for useful discussions. We are grateful to the Stanford Shared FACS Facility for access to flow cytometry and the Stanford Genomics Facility for constructing and sequencing libraries for scRNA-seq and scTCR-seq experiments, and the Harvard Faculty of Arts & Sciences Research Computing Cluster for computational resources. This work was supported by the Stanford Microbiome Therapies Initiative, the Human Frontier Science Program grant no. LT000493/2018-L (K.N.), a Fellowship from the Astellas Foundation for Research on Metabolic Disorders (K.N.), a research grant from Kanae Fundation for the Promotion of Medical Science (K.N.), NIH grant no. K99AI173524 (K.N.), an HHMI-LSRF Award (J.E.B.), a fellowship from the Kwanjeong Educational Foundation (M.B.), the Howard Hughes Medical Institute (E.P.B.), the Alan T. Waterman Award from the National Science Foundation (E.P.B., grant no. CHE-20380529), an HHMI-Simons Faculty Scholar Award (M.A.F.), the Leona M. and Harry B. Helmsley Charitable Trust (M.A.F.), NIH grant no. DK110174 (M.A.F.), the Chan Zuckerberg Biohub (M.A.F.), Stand Up to Cancer (M.A.F.) and the MAC3 Impact Philanthropies (M.A.F.).
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K.N., M.B., E.P.B. and M.A.F. conceived and designed the experiments. K.N., A.Z., K.A., M.B., J.E.B., A.W., S.J., X.M., A.G.C., M.W., S.H., A.D., J.J.M. and P.M. performed the experiments. K.N., M.B. and M.A.F. analysed data and wrote the manuscript. M.B., E.S.S. and E.P.B. edited the manuscript. All authors discussed the results and commented on the manuscript.
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Stanford University and the Chan Zuckerberg Biohub have patents pending for microbiome technologies on which the authors are co-inventors. M.A.F. is a cofounder and director of Federation Bio and Kelonia, a cofounder of Revolution Medicines and a member of the scientific advisory boards of the Chan Zuckerberg Initiative and NGM Bio. The remaining authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 T cell profiling for hCom1d- and hCom2d-colonized mice.
a,b, hCom1d or hCom2d were used to colonize germ-free C57BL/6 mice by oral gavage. Mice were housed for two weeks before sacrifice. Intestinal immune cells were extracted, stimulated by PMA/ionomycin and analyzed by flow cytometry. Th cell subtypes, as a percentage of the total Th cell pool, were analyzed in the large intestine (a) or in the small intestine (b). See Supplementary Fig. 1b for the gating strategy. Statistical significance was assessed using a one-way ANOVA (NS > 0.05; **p < 0.01; ***p < 0.001). Data shown are mean ± standard deviations. n = 8, 8, 4 mice per group from 2 independent experiments (a). n = 4 mice per group from one experiment (b).
Extended Data Fig. 2 Introduction of a gut bacterial community to germ-free mice by horizontal transfer.
a, Metagenomic analysis of germ-free mice colonized with hCom2d by oral gavage or cohousing (horizontal transfer, HT). Faecal samples were collected 2 weeks after colonization and subjected to metagenomic sequencing; the resulting data were analyzed by NinjaMap to measure the composition of each community. The average of 4 mice per group is displayed above. Each dot is an individual strain; the collection of dots in a column represents the community at a single time point. Strains are coloured according to their rank-order abundance in the oral gavage sample. Relative abundances in the oral gavage and HT samples are highly correlated, indicating that coprophagy (horizontal transfer) results in a similar community architecture to that of oral gavage. b, Intestinal T cells from mice colonized with hCom2d by oral gavage and HT show similar phenotypes. Mice were sacrificed after two weeks of colonization. Immune cells were isolated from the large intestine, stimulated by PMA/ionomycin and analyzed by flow cytometry. See Supplementary Fig. 1b for the gating strategy. Statistical significance was assessed using a two-sided t-test (NS > 0.05). Data shown are mean ± standard deviations. n = 6 mice per group from one experiment.
Extended Data Fig. 3 The mixed lymphocyte assay detects TCR activation while preserving T cell phenotype.
a-c, We colonized germ-free mice with hCom1d, waited two weeks, and then sacrificed the mice. Immune cells from the colon were co-cultured for 4 h with three non-community bacterial strains (Bacillus cereus, Bacillus subtilis, or Staphylococcus epidermidis) or two strains from hCom1d (Tyzzerella nexilis and Clostridium bolteae). For antigen presentation, we used either MHCII+ wild-type DCs, MHCII-deficient DCs or a no DC control. pTreg (Helios- Foxp3+) cells and Th17 (RORgt+ Foxp3-) cells were analyzed by flow cytometry. a, Co-culture with strains and wild-type DCs doesn’t affect the number of pTreg cells and Th17 cells. b,c, Nur77 expression is upregulated when T cells were cocultured with strains in hCom1d and wild-type DCs in pTreg cells (b) and in Th17 cells (c). p-values were calculated by comparison to PBS treatment as a negative control using a one-way ANOVA (NS > 0.05; **p < 0.01; ***p < 0.001). Data shown are mean ± standard deviations. n = 4 mice per group from one experiment.
Extended Data Fig. 4 Analysis of scRNA-seq data by unbiased clustering.
a, Uniform manifold approximation and projection (UMAP) plot of all the cells. The data is generated by merging three groups: hCom1d-colonized, hCom2d-colonized, and germ-free mice. Cells were clustered into 25 groups using the FindClusters function of the Seurat R package. b–d, Gene expression profiling of cell clusters. To investigate the identity of each cell cluster, expression levels of cell subset markers were visualized by dot plots (b), feature plots (c) and violin plots (d) using Seurat.
Extended Data Fig. 5 scRNA-seq analysis of immune modulation by synthetic community colonization.
a, Left panel: Uniform manifold approximation and projection (UMAP) plot. These data represent three merged samples: hCom1d-colonized, hCom2d-colonized and germ-free mice. Right panel: Frequency of TCR clonotypes on the UMAP plot. Expanded TCRs (red) represent clonotypes observed in more than five cells, multiple (orange) are clonotypes found in 2–5 cells, and single (light blue) were seen in only one cell. Most of the expanded TCR clonotypes have an expression profile consistent with effector T cells, whereas naïve T cells are rich in unique (that is, non- expanded) TCR clonotypes. b, UMAP plot of intestinal immune cell clusters in each colonization condition. Immune cells were isolated from the large and small intestine from three groups of mice: hCom1d-colonized, hCom2d-colonized, and germ-free. c, Analysis of the frequency of T cell subsets in each group. The percentage of each T cell subset on the UMAP plot was calculated by the Seurat R package; fold changes compared to GF mice are shown. Colonization of germ-free mice with hCom1d and hCom2d increased pTreg, Th17, Fr4 Th and other effector T cells in the large intestine, and Th17 and other effector T cells in the small intestine. d, Analysis of expanded TCR clonotypes in each sample. Each dot represents one TCR clonotype found in multiple T cells (red, shared between effector T cells and pTreg cells; grey, effector T cell; black, pTreg). e, Differentially expressed genes in T cell subsets upon colonization with hCom1d and hCom2d. The FindMarkers function of Seurat was used to find differentially expressed genes. The two-sided non-parametric Wilcoxon rank sum test was used to calculate the adjusted p-value. White bars show mean values. Each dot represents one cell. ***p < 0.001. f, Criteria used to select TCR clonotypes for making hybridoma cells. Red genes: Upregulated by hCom1d and hCom2d colonization. Black genes: T cell subset markers.
Extended Data Fig. 6 Reactivity of TCR hybridomas against reported commensal epitopes.
a, The list of previously reported commensal epitopes tested in this experiment. Epitopes were pooled into sets of 6-7 for testing the ability to stimulate TCR hybridomas. b, Previously reported commensal epitopes were co-cultured with TCR hybridomas and dendritic cells. The SFB-specific 7B8 TCR transgenic T cells, a positive control, showed a response to Pool1, which includes the SFB antigen SFB3340. The 92 TCR hybridomas generated in this work were not responsive to any of the previously reported commensal epitopes.
Extended Data Fig. 7 Metagenomic analysis for hCom1d and hCom2d strains in the colonization of the mouse intestine.
a,b, Each dot is an individual strain; the collection of dots in a column represents the community at a single time point in mice colonized by hCom1d (a) or hCom2d (b). Strains are colored according to their rank-order relative abundance. Strain names colored red harbor the conserved substrate-binding protein (SBP). Germ-free mice were colonized with synthetic communities, and fecal pellets were collected two weeks after community colonization. The results are an average of 5 mice.
Extended Data Fig. 8 Identification of the minimal antigen epitopes of SBP using a truncated peptide library.
a,b, Prediction of signal sequences and subcellular localization of SBP. SignalP-5.0 (a) and PRED-LIPO (b) predict that the SBP has a lipoprotein signal peptide. c, BLAST search for the antigenic epitopes of SBP in strains from hCom1d and hCom2d. 19 strains were found to harbor homologs of the SBP. d, To search for the minimal antigenic epitope in SBP, a library of truncated peptides was synthesized. 13 Firmicutes-reactive TCR hybridomas were mixed and co-cultured with truncated peptides and dendritic cells. The degree of TCR stimulation was estimated by assaying the concentration of IL-2 in the culture supernatant by ELISA. Truncated peptides containing the 9-mer YDAFAINMV stimulated the mixed TCR hybridomas.
Extended Data Fig. 9 Discovery of an N-terminal epitope from SBP.
a, Identification of the N-terminal epitope SBP76-84. N-terminal SBP peptides from strains from hCom1d and hCom2d were synthesized and co-cultured with the T cell hybridomas. Truncated peptides were also tested to identify the minimal epitope. 7 of the 13 TCRs were responsive to the synthetic peptides. The remaining 6 were stimulated by C-terminal peptide SBP405-413 as shown in Fig. 4f. There is a strong correlation between the reactivity of TCRs and the sequences of TCR CD3 regions. b, The T cell response to SBP is conserved in different murine settings. We colonized: (1) germ-free C57BL/6 mice with hCom2d by co-housing (horizontal transfer recipient), (2) germ-free C57BL/6 mice with a human fecal community (humanized), (3) germ-free C57BL/6 mice with oral gavage of hCom2d under a condition of a high-fat diet (HFD), and (4) germ-free Swiss Webster mice (SW) with oral gavage of hCom2d. After two weeks, intestinal immune cells were isolated and cocultured with a mix of SBP76-84 and SBP405-413, or PBS as a negative control, using dendritic cells for antigen presentation. Nur77 expression in pTreg or Th17 cells was analyzed by FACS to monitor TCR stimulation (see Supplementary Fig. 1a, c for the gating strategy). SBP-specific T cells were detected in pTreg and Th17 from HT recipient, humanized, and HFD groups, while T cells from SW mice failed to respond to SBP76-84 and SBP405-413. c, We hypothesized that T cells from SW mice recognize a distinct epitope in SBP because of the difference in MHC haplotype between C57BL/6 and SW mice. To test this hypothesis, we co-cultured immune cells from hCom2d-colonized SW mice with a mixture of peptides that tile the whole SBP. We detected SBP-specific pTreg and Th17 cells in hCom2-colonized SW mice. This suggests that SBP-specific T cell induction is preserved across different genetic backgrounds of mice, but—as expected—the epitope recognized is distinct because of the difference in MHC haplotype. p-values were calculated using a two-sided t-test by comparison to PBS treatment as a negative control. *p = 0.05. **p = 0.01. ***p = 0.005. NS > 0.05. Data shown are mean ± standard deviations. n = 8, 8, 14, 8 mice per group from 2 independent experiments (b). n = 15 mice per group from one experiment (c).
Extended Data Fig. 10 Identification of TPRL as an antigen from Bacteroides species.
a, To search for the antigenic epitope in TPRL from Bacteroides eggerthii, a library of truncated peptides was synthesized. 4 Bacteroides-reactive TCR hybridomas were mixed and co-cultured with truncated peptides and dendritic cells. The degree of TCR stimulation was estimated by assaying the concentration of IL-2 in the culture supernatant by ELISA. TPRL29-53 (Peptide 3, SDYFTVTPQVLEAVGGKVPATINGK) stimulated the mixed TCR hybridomas. b, We found that TCR H2-11, H2-30, and H1-14 are reactive to Peptide 3 from TPRL by coculturing each Bacteroides-reactive TCR hybridoma with Peptides 2 and 3. c, Results of a BLAST search using the antigenic epitope TPRL29-53 as a query; the results shown are from strains in hCom1d and hCom2d. d, Predicted crystal structure of the TPRL from AlphaFold2. The TPRL29-53 epitope, shown in red, lies within a beta-sheet in the N-terminal domain. e, Induction of TPRL-specific T cells in vivo. Germ-free C57BL/6 mice were colonized with hCom2d. After two weeks, intestinal T cells were isolated and cocultured with TPRL29-53 and dendritic cells. Nur77 expression in T cell subsets was analyzed by FACS to monitor TCR stimulation (see Extended Data Fig. 1a, c for the gating strategy). In hCom1d and hCom2d-colonized mice, Th17 and pTreg cells showed an antigen-specific response to SBP. p-values were calculated using a two-sided t-test by comparison to PBS treatment as a negative control. *p < 0.05. Data shown are mean ± standard deviations. n = 5, 8 mice per group from one experiment.
Extended Data Fig. 11 T cell targets are highly expressed in vitro and in vivo.
a, The SBP is highly expressed in in vitro transcriptomic data from two species in our community: Clostridium bolteae and Clostridium hathewayi. In C. bolteae, it is the 10th most highly expressed gene out of 5,982, and in C. hathewayi, it is the 4th most highly expressed gene out of 6,712. The expression level was normalized to transcripts per million (TPM). b, The SBP and TPRL are highly expressed in vivo. Reads were recruited from 378 metatranscriptomic samples38 and mapped to the whole genomes of select SBP and TPRL-encoding strains in our community (Tyzzerella nexilis DSM 1787 and Clostridium bolteae DSM 15670 (SBP); Bacteroides stercoris ATCC 43183, Bacteroides eggerthii DSM 20697, and Bacteroides cellulosilyticus DSM 14838 (TPRL)). To account for gene copy number variation, we measured expression as the ratio between RNA and DNA level from paired metagenomic and metatranscriptomic samples. In all cases, we found that the protein is very highly expressed in vivo.
Extended Data Fig. 12 SBP and TPRL are present in human stool samples.
We tested whether SBP and TPRL exist at a detectable level in human stool samples. We cocultured SBP- or TPRL-reactive TCR hybridomas with: (1) PBS as a negative control, (2) a mix of SBP and TPRL epitopes as a positive control, (3) heat-treated hCom1d, (4) heat-treated SPF mouse fecal pellets, 5) heat-treated mouse fecal pellets from Jackson, (5) 6 human fecal communities. For antigen presentation, we used MHCII+ wild-type DCs, or MHCII-deficient DCs as a negative control. IL-2 concentration was measured as a readout for TCR stimulation. We found that 6/6 human fecal communities restimulate the SBP405-413 and SBP76-84-specific hybridomas and 5/6 restimulate the TPRL29-53-specific hybridomas. These data suggest that the SBP and TPRL are expressed by human gut isolates under native conditions.
Supplementary information
Supplementary Figures
This file contains Supplementary Figs. 1–3. Supplementary Fig. 1 Gating strategy for T cell in flow cytometry analysis. Supplementary Fig. 2 Establishment of the mixed lymphocyte assay. Supplementary Fig. 3 Representative flow cytometry images in Fig. 2. See PDF for full legends.
Supplementary Table 1
List of strains in hCom1d and hCom2d. Strains in hCom1d and hCom2d and their respective growth media are shown.
Supplementary Table 2
TCR clonotypes chosen for hybridoma cell lines. Information on TCR clonotypes picked up for hybridoma cells is described.
Supplementary Table 3
Plasmid and gBlock sequences. Sequences of plasmids and gBlocks used in this study are listed.
Supplementary Table 4
P values. The P values from this study are listed.
Supplementary Data
Source data for Supplementary Figs.
Source data
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Nagashima, K., Zhao, A., Atabakhsh, K. et al. Mapping the T cell repertoire to a complex gut bacterial community. Nature 621, 162–170 (2023). https://doi.org/10.1038/s41586-023-06431-8
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DOI: https://doi.org/10.1038/s41586-023-06431-8
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