Lactobacillus reuteri induces gut intraepithelial CD4⁺CD8αα⁺ T cells

Abstract

The gut microbiota drives maturation and function of the immune system (1–3). Several bacterial taxa shape the differentiation of naïve T cells: segmented filamentous bacteria (SFB) bias CD4⁺ T cells towards a Th17 fate (4), Clostridia clusters IV and XIVa promote CD4⁺ T regulatory cells (Treg) differentiation (5, 6); and Bacteroides fragilis (7, 8) and Faecalibacterium prausnitzii (9) induce CD4⁺ T cells that secrete IL-10. The small intestinal epithelium contains a unique population of CD4⁺CD8αα⁺ TCR αβ T cells (referred to as double-positive intraepithelial lymphocytes, or DP IELs) (10). These cells have a regulatory function complementary to that of Tregs and promote tolerance to dietary antigens (11). DP IELs originate from lamina propria CD4⁺ T cells that include but are not limited to Tregs (11). Upon reaching the epithelium, these cells reactivate the CD8⁺ T cell lineage program through downregulation of Thpok and upregulation of Runx3 and T-bet (12–14). This process is facilitated in part by transforming growth factor-β (TGFβ), retinoic acid (RA), IFN-γ and IL-27 (12, 14). The lack of DP IELs in germ-free (GF) mice (11, 13) indicates that the intestinal microbiota is required for their differentiation, although the bacterial taxa and metabolites required are not known.

We noticed that mice born at one of our vivaria [specialized research facility (SRF)] harbored substantial numbers of DP IELs (DP⁺), whereas DP IELs were negligible or absent (DP⁻) in mice from another vivarium [Clinical Sciences Research Building (CSRB)] (Fig. 1A). Moreover, DP IELs in SRF mice expressed low levels of the transcription factor Thpok, Thpok^lo, whereas CSRB mice lacked CD4⁺ Thpok^lo IELs (13), consistent with the observed lack of DP IELs (fig. S1). Thus, the microbiota in SRF mice might contain taxa capable of inducing DP IELs. Consistent with this, DP IELs appeared in CSRB mice after gavage with fecal or ileal microbiota from SRF, but not CSRB animals (Fig. 1B).

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Fig. 1

Specific microbiota components induce DP IELs

(A) Representative plots and frequencies of DP IELs (gated on CD45⁺CD3⁺TCRγδ⁻CD8β⁻CD4⁺ IELs) from C57BL/6 mice born in clinical sciences research building (CSRB) and specialized research facility (SRF) facilities. (B) DP-IEL frequencies in CSRB mice four weeks after oral gavage with ileal or fecal microbiota harvested from the indicated mice. (C) DP-IEL frequencies in C57BL/6 mice from Jackson (JAX), Taconic and Charles River (CR) Laboratories. (D) DP-IEL frequencies in F0 and F1 generations of JAX and CR mice bred in CSRB. (E) DP-IEL frequencies in JAX or CR mice housed separately (JAX, CR), or cohoused (JAX co, CR co). (F) DP-IEL frequencies in JAX mice treated with ileal or fecal microbiota from the indicated mice. (G–H) Frequencies of DP IELs in CR mice that were either untreated (Ctrl), or treated with (G) vancomycin, neomycin, ampicillin , and metronidazole (VNAM), or (H) ampicillin plus vancomycin (Amp/Van), neomycin (Neo), or metronidazole (Met). Dots represent individual mice. Data are pooled from 2–3 independent experiments. Statistical analysis was performed using Mann−Whitney U test between groups or Kruskal−Wallis test for multiple comparison analysis. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Bars represent means.

Most SRF (DP⁺) mice are embryo-rederived using female recipient mice from Charles River (CR) Laboratories and thus acquire a CR microbiota. In contrast, most CSRB (DP⁻) mice originate from the Jackson Laboratories (JAX). Thus, taxa present in CR but not JAX mice might induce DP IELs. Indeed, C57BL/6 mice purchased from CR had a DP⁺ phenotype, whereas C57BL/6 mice purchased from JAX had a DP⁻ phenotype (Fig. 1C). DP⁺ and DP⁻ phenotypes were vertically transmissible (Fig. 1D). Moreover, the DP⁺ phenotype was laterally transferable from CR to JAX mice by co-housing animals (Fig. 1E) or by colonizing JAX mice with CR-ileal or fecal microbiota (Fig. 1F). Treatment of CR mice with vancomycin, ampicillin, neomycin and metronidazole (VNAM) abrogated DP IELs (Fig. 1G), as did ampicillin and vancomycin only, which target Gram-positive bacteria; neomycin, which targets Gram-negative bacteria, led to a slight increase in DP IELs (Fig. 1H); metronidazole did not affect DP IELs. Thus, DP IELs are induced by neomycin-resistant Gram-positive bacterial taxa.

To define bacterial taxa that induce DP IELs, we sequenced PCR amplicons from 16S ribosomal RNA genes present in the ileum of CR and JAX mice, as well as neomycin-treated and untreated CR animals. Six operational taxonomic units (OTUs) were selectively present in the CR microbiota and were also present or enriched after neomycin treatment (fig. S2A). These OTUs included Lactobacillus reuteri and five members of the Bacteroidales S24-7 lineage (Fig. 2A, fig. S2A, and Table S1). L. reuteri was present in the ileum of CR control mice and CR mice treated with neomycin or metronidazole, but absent in CR mice treated with ampicillin plus vancomycin (fig. S2B).

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Fig. 2

L. reuteri induces DP IELs

(A) Relative abundance of L. reuteri operational taxonomic unit ID 411486 as determined by sequencing of the V4 region of 16S rRNA genes present in the ileal microbiota of 8-week-old JAX mice and CR mice (n = 4); and 4 week-neomycin treated (Neo) or untreated (Ctrl) CR mice (n=5 mice per treatment group). Statistical analysis was performed using Mann−Whitney U test (*, p < 0.05). (B–D) DP-IEL frequencies in JAX mice colonized with L. reuteri WU, L. reuteri strain 100-23, L. johnsonii WU, L. murinus, or a mixture of Bacteroides vulgatus, B. uniformis, and B. acidifaciens. Untreated JAX mice were used as controls. (E) DP IEL-frequencies in specific pathogen free (SPF) and germ-free (GF) C57BL/6 mice. Duo = duodenum, je = jejunum, ile = ileum. (F) Total number of DP IELs in germ-free mice colonized with: JAX mice ileal microbiota; JAX mice ileal microbiota combined with L. reuteri W; L. reuteri WU alone; or CR ileal microbiota. Dots represent individual mice. Data are pooled from 1–3 independent experiments. Statistical analysis was performed using Mann–Whitney U test between groups or Kruskal−Wallis test for multiple comparison analysis. (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). Bars represent means; error bars represent the SEM.

Two L. reuteri strains (WU and 100-23) induced DP IELs after colonization of JAX mice (Fig. 2B), whereas L. johnsonii, L. murinus (Fig. 2C), and various Bacteroides species (Fig. 2D) did not. As expected, DP IELs were absent throughout the small intestine of GF animals (11, 13)(Fig. 2E). Concurrent inoculation of GF mice with L. reuteri (WU strain) and ileal microbiota from JAX mice induced DP IELs, whereas JAX ileal microbiota alone was ineffective (Fig. 2F). Mono-colonization with L. reuteri resulted in a slight increase of DP IELs. As expected, transfer of CR intestinal microbiota induced DP IELs. Thus, L. reuteri induces DP IELs, but the presence of other microbes enhances this effect perhaps by expanding the CD4⁺ T cells in the lamina propria and epithelium.

We next examined how L. reuteri induces DP IELs. If L. reuteri antigens shape the specificity of DP IELs, these cells would express quasi-clonal TCR αβ repertoires, as shown for SFB-specific Th17 cells (15). Analysis of TCR Vβ expression on DP IELs, CD4 IELs and mesenteric lymph node (MLN) naïve CD4⁺ T cells showed that TCR Vβ usage of DP IELs resembled that of CD4 IELs, but was different from that of naïve CD4⁺ T cells (Fig. 3A). In each mouse analyzed, DP IELs exhibited a preferential Vβ usage (> 2-fold compared to MLN), although no enrichment for a specific Vβ pattern was shared by all mice, suggesting mouse-to-mouse variability in DP IEL TCRs (Fig. 3A and B; and fig. S3A). Because the great diversity of polyclonal T cells complicates analysis at the individual TCR level, we analized the TCR α repertoires of DP IELs, CD4 IELs, CD8 IELs, and MLN naïve CD4⁺ T cells from mice that express a fixed transgenic TCR β chain (TCli⁺ mice) (16, 17). Tcli⁺ T cells were very similar in phenotype and frequency to WT T cells (fig. S3B). TCR Renyi diversity profiles showed both decreased diversity (lower entropy at each order) and evidence for increased clonal expansion (greater downward slope) in the repertoires of all three IEL subsets compared to the repertoire of naïve CD4⁺ T cells (fig. S3C). The TCR α repertoires of CD4 IELs, CD8 IELs, and DP IELs were distinct from those of naïve CD4⁺ T cells (Fig. 3C), consistent with the expansion of T cell clones in response to specific antigens. Moreover, the TCR α repertoires of DP IELs and CD4 IELs were similar, but distinct from those of CD8 IELs (Fig. 3D and E), corroborating that DP IELs differentiate from CD4 IELs. Although the TCR α repertoires of naïve T cells were similar in all mice analyzed, the TCR α repertoires of DP IELs and CD4 IELs were quite distinct in each mouse (Fig. 3F), suggesting that the specificity of CD4⁺ IELs and DP IELs is shaped by various environmental and/or self-antigens in each mouse, rather than by a single antigen.

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Fig. 3

DP IELs have diverse TCR repertoires in different mice

(A) Frequencies of indicated TCR Vβ in DP IELs, CD4 IELs and mesenteric lymph node (MLN) naïve CD4⁺ T cells from 10 individual DP⁺ C57BL/6 mice. (B) For each TCR Vβ, the ratio between its frequency in DP IELs and its frequency in MLN naïve CD4⁺ T cells was calculated for individual mice; values are displayed as a heat map. Each column represents a single mouse. (C–D) Morisita-Horn similarity analysis of the TCR α-chain repertoires of intraepithelial CD4 IELs, CD8 IELs, DP IELs, and MLN naïve CD4⁺ T cells. Each symbol represents a comparison between the indicated two subsets within a mouse. Index values of 0 indicate that the two samples are completely dissimilar whereas index values of 1 indicate that they are indistinguishable. (E) Venn diagram showing the number of unique and overlapping complementarity determining region 3 (CDR3) sequences found in TCR α-chains of CD4 IELs, CD8 IELs, and DP IELs. (F) Morisita-Horn similarity analysis of the TCR α-chain CDR3 repertoires of CD4 IELs, CD8 IELs, and DP IELs. Each symbol represents a comparison of the TCR-α sequences of the same T cell subset in different mice. Lines indicate mean values. Populations from 4 individual animals were analyzed.

Some bacterial taxa shape T cell differentiation via their physical components (e.g., polysaccharide A (7, 18)), or their metabolic products (e.g., short-chain fatty acids (19–21)). L. reuteri releases reuterin (22), and histamine (23), and also has the ability to metabolize tryptophan (L-Trp) to indole derivatives, some of which activate the aryl-hydrocarbon receptor (AhR) in group 3 innate lymphoid cells (ILC3s) (24, 25). Accordingly, culture supernatants of L. reuteri grown in L-Trp-containing medium activated an AhR reporter cell line, whereas supernatants from L. johnsonii or L. murinus did not (Fig. 4A). To determine whether L. reuteri induces DP IELs through the release of AhR ligands, we added L. reuteri culture supernatants to naïve ovalbumin-specific CD4⁺ T cells (OTII) stimulated with ovalbumin peptide-pulsed dendritic cells (DCs) with or without TGFβ (12). L. reuteri culture supernatant induced CD8αα⁺CD4⁺ T cell differentiation of OTII T cells, as did the AhR agonist 2,3,7,8-tetrachlorodibenzodioxin (TCDD) (Fig. 4B and fig. S4A) but only in the presence of TGFβ. The AhR antagonist CH223191 inhibited CD8αα⁺CD4⁺ T cell differentiation, confirming that the L. reuteri supernatant acts through AhR. AhR agonists plus TGFβ were as effective as TGFβ plus retinoic acid and IFN-γ (12, 14) (Fig. 4B). We also generated a strain of L. reuteri lacking a functional aromatic aminotransferase and hence the ability to convert amino acids into indolic AhR ligands (L. reuteri ΔArAT) (24, 26). Culture supernatants from L. reuteri ΔArAT failed to stimulate the AhR reporter cells (Fig. 4C). Moreover, L. reuteri ΔArAT did not induce DP IELs in JAX mice (Fig. 4D), although this strain colonized mice efficiently (fig. S4B).

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Fig. 4

L. reuteri induces DP IELs through AhR activation in T cells

(A) Representative plots and quantification of GFP⁺ cells in an AhR reporter cell line after stimulation with medium (minimum essential medium +10% bovine calf serum), peptone–tryptone water + L-Trp (PT-T), L.r., L. reuteri WU, L. reuteri 100-23; L.j., L. johnsonii, or L.m., L. murinus supernatants (grown in PT-T) or TCDD 2,3,7,8-tetrachlorodibenzodioxin. FSC= Forward Scatter (B) CD4⁺CD8β⁻CD8α⁺ T cell frequencies after culture of naïve OTII CD4⁺ T cells with spleen CD11c⁺ DCs, OVA_329–337 peptide, and the indicated stimuli. T = TGFβ; L.r. = L. reuteri WU supernatant; CH = CH223191; R = retinoic acid. (C) Quantification of GFP⁺ cells in an AhR reporter cell line after stimulation with medium, PT-T, L. reuteri 100-23 supernatant, L. reuteri ΔArAT supernatant, or TCDD. (D) DP-IEL frequencies in JAX C57BL/6 mice four weeks after colonization with either L. reuteri 100-23, L. reuteri ΔArAT, or not colonized (Ctrl). (E) DP-IEL frequencies in CR C57BL/6 mice fed for four weeks with low (0.11%), standard (0.24%) or high (0.48%) L-Trp diets. (F) CD4⁺CD8αα⁺ T cell frequencies after culture of naïve OTII CD4⁺ T cells with spleen CD11c⁺ DCs, OVA_329–337 peptide, and the indicated stimuli. IAId = indole-3-aldehyde; ILA = indole-3-lactic acid. (G) DP-IEL frequencies in cohoused WT, AhR^−/+ and AhR^−/− littermate mice. (H) CD4⁺CD8αα⁺ T cell frequencies after culture of WT or AhR^−/− CD4⁺ T cells with WT or AhR^−/− DCs, a combination of SEB, SEE, and TSST1 superantigens, and the indicated stimuli. (I) DP IEL frequencies in Rorc-Cre⁺ × Ahr^fl/fl and Rorc-Cre⁻ × Ahr^fl/fl littermates. (J) Representative flow cytometry plots showing DP IELs and CD4 IELs (left) and Thpok expression (right) in CD4 IELs (colored blue) and DP IELs (colored green) in AhR^−/+ and AhR^−/− littermates. Dots represent single animals; data have been pooled from 2–3 independent experiments. Statistical analysis was performed using Mann−Whitney U test (I), Kruskal−Wallis test (D, E and G), and One-way ANOVA with Tukey's post hoc test (A, B, C, F and H) *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Bars represent means, error bars represent SEM.

We next assessed the impact of dietary L-Trp levels in DP IELs development. More DP IELs were observed in CR mice fed a high L-Trp diet (0.48%) than in mice fed standard (0.24%) or low (0.11%) L-Trp diets (Fig. 4E). However, a high L-Trp diet failed to induce DP IELs in germ-free mice mono-colonized with either L.reuteri 100-23 or L. reuteri ΔArAT (fig. S4C). Thus, L. reuteri acts together with a complex microbiota and dietary L-Trp to induce DP IELs. Bioactivity-guided fractionation of L. reuteri 100-23 supernatant identified the presence of indole-3-lactic (ILA) acid, an indole derivative of L-Trp, that activated the AhR reporter cell line and induced CD8αα⁺CD4⁺ T cells (fig. S5). This compound was as effective as indole-3-aldehyde (IAId), which activates AhR in ILC3s (24) (Fig. 4F and fig. S4D). AhR^−/− mice lacked DP IELs, whereas these cells were present in Ahr^+/− littermates and WT controls supporting our observation that L. reuteri induces DP IELs through the release of AhR ligands (Fig. 4G). We next asked whether AhR drives DP IEL differentiation in a T cell intrinsic or extrinsic fashion. TGFβ with either L. reuteri culture supernatant or TCDD induced CD8αα⁺CD4⁺ T cells from naïve WT CD4⁺ T cells stimulated in vitro with AhR^−/− or WT splenic DCs pulsed with a combination of three superantigens. In contrast, AhR^−/− T cells did not differentiate into CD8αα⁺CD4⁺ T cells, whether or not DCs lacked AhR, implying that AhR is required in T cells (Fig. 4H). Moreover, stimulation of OTII T cells with Ahr^−/− or WT DCs in the presence of TGFβ and either L. reuteri supernatant or TCDD induced CD8αα⁺CD4⁺ T cells (fig. S4E). Finally, DP IELs were significantly reduced in Ahr^fl/fl × Rorc-Cre mice (which lack AhR in T cells and ILCs), as well as in Ahr^fl/fl × Cd4-Cre mice, (which lack AhR in T cells) in comparison to littermate controls (Fig. 4I, fig. S4 F and G), demonstrating that DP IEL-development requires AhR activation in T cells. To determine when AhR signaling intervenes in the DP IEL-developmental pathway, we analyzed the expression of Thpok in IELs from Ahr^+/− and AhR^−/− mice. Thpok^hi CD8αα⁻CD4⁺ IELs, ThpoK^lo CD8αα⁺CD4⁺ IELs (DP IELs) and a small intermediate population of ThpoK^lo CD8αα⁻CD4⁺ IELs were evident in Ahr^+/− mice, whereas a single Thpok^hi CD4⁺ IEL population predominated in AhR^−/− mice (Fig. 4J). Thus, lack of AhR signaling arrests DP IEL-development before Thpok downregulation occurs.

Our study reveals that L. reuteri provides indole-derivatives of dietary L-Trp, such as indole-3-lactic acid, that activate AhR and lead to downregulation of Thpok and reprogramming of CD4⁺ IELs into DP IELs. Other indole derivatives may have a similar effect. This Ahr-mediated mechanism is distinct from those by which AhR impacts other T cells with regulatory functions (Tr1, Tregs), intraepithelial γδ T cells and DCs (27–32). Yet undefined microbial species, dietary and/or self-antigens are necessary to expand the CD4⁺ IEL population with a diverse TCR repertoire that converts into DP IELs. The complete reliance of DP IELs on a single species, L. reuteri, and its tryptophan metabolites for their final maturation may provide a basis for the use of L. reuteri as a probiotic and tryptophan-rich food to treat disorders that may be modifiable by DP IELs, such as inflammatory bowel diseases (11).

Supplementary Material

Acknowledgments

Footnotes

References and Notes