Lactobacillus reuteri induces gut intraepithelial CD4+CD8αα+ T cells

Abstract

The small intestine contains CD4⁺CD8αα⁺ double-positive intraepithelial lymphocytes (DP IELs), which originate from intestinal CD4⁺ T cells through down-regulation of the transcription factor Thpok and have regulatory functions. DP IELs are absent in germ-free mice, which suggests that their differentiation depends on microbial factors. We found that DP IEL numbers in mice varied in different vivaria, correlating with the presence of Lactobacillus reuteri This species induced DP IELs in germ-free mice and conventionally-raised mice lacking these cells. L. reuteri did not shape the DP-IEL-TCR (TCR, T cell receptor) repertoire but generated indole derivatives of tryptophan that activated the aryl-hydrocarbon receptor in CD4⁺ T cells, allowing Thpok down-regulation and differentiation into DP IELs. Thus, L. reuteri, together with a tryptophan-rich diet, can reprogram intraepithelial CD4⁺ T cells into immunoregulatory T cells.

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.

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.

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.

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.