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Lactobacillus reuteri induces gut intraepithelial CD4+CD8αα+ T cells

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Science  25 Aug 2017:
Vol. 357, Issue 6353, pp. 806-810
DOI: 10.1126/science.aah5825
  • Fig. 1 Specific microbiota components induce double-positive intraepithelial lymphocytes (DP IELs).

    (A) Representative plots and frequencies of DP IELs (gated on CD45+CD3+TCRγδCD8βCD4+ IELs) from C57BL/6 mice born in CSRB and SRF facilities. (B) DP IEL frequencies in CSRB mice 4 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 and 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). Symbols represent individual mice. Data are pooled from two or three independent experiments. Statistical analysis was performed using a Mann-Whitney U test between groups or a 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 mice) and 4-week-neomycin-treated (Neo) or untreated (Ctrl) CR mice (n = 5 mice per treatment group). Statistical analysis was performed using a Mann-Whitney U test. *P < 0.05. (B to 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 B. 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 GF mice colonized with JAX ileal microbiota, JAX ileal microbiota combined with L. reuteri WU, L. reuteri WU alone, or CR ileal microbiota. Symbols represent individual mice. Data are pooled from one to three independent experiments. Statistical analysis was performed using a Mann-Whitney U test between groups or a Kruskal-Wallis test for multiple comparison analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. NS, not significant. Bars represent means; error bars indicate 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 and 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 distinct 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 four individual animals were analyzed.

  • Fig. 4 L. reuteri induces DP IELs through AhR activation in T cells.

    (A) Representative plots and quantification of green fluorescent protein–positive (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. reuteri WU, L. reuteri (L.r.) 100-23, L. johnsonii (L.j.), or L. murinus (L.m.) supernatants (grown in PT-T) or TCDD (2,3,7,8-tetrachlorodibenzodioxin). FSC, forward scatter. (B) CD4+CD8βCD8α+ T cell frequencies after culture of naïve OTII CD4+ T cells with spleen CD11c+ dendritic cells (DCs), OVA329-337 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 4 weeks after colonization with either L. reuteri 100-23 or L. reuteri ΔArAT, or in mice that were not colonized (ctrl). (E) DP IEL frequencies in CR C57BL/6 mice fed for 4 weeks with diets low (0.11%), standard (0.24%), or high (0.48%) in l-Trp. (F) CD4+CD8αα+ T cell frequencies after culture of naïve OTII CD4+ T cells with spleen CD11c+ DCs, OVA329-337 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+ × Ahrfl/fl and Rorc-Cre × Ahrfl/fl littermates. (J) Representative flow cytometry plots showing DP IELs and CD4+ IELs (left) and Thpok expression (right) in CD4+ IELs (blue) and DP IELs (green) in Ahr+/– and Ahr–/– littermates. Symbols represent single animals; data have been pooled from two or three independent experiments. Statistical analysis was performed using a Mann-Whitney U test (I), a Kruskal-Wallis test [(D), (E), and (G)], and one-way analysis of variance (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 indicate SEM.

Supplementary Materials

  • Lactobacillus reuteri induces gut intraepithelial CD4+CD8αα+ T cells

    Luisa Cervantes-Barragan, Jiani N. Chai, Ma. Diarey Tianero, Blanda Di Luccia, Philip P. Ahern, Joseph Merriman, Victor S. Cortez, Michael G. Caparon, Mohamed S. Donia, Susan Gilfillan, Marina Cella, Jeffrey I. Gordon, Chyi-Song Hsieh, Marco Colonna

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods 
    • Figs. S1 to S7 
    • Tables S1, S2, S4, and S5 
    • Captions for Tables S3 and S6
    • References 
    Table S3
    TRAV sequencing database
    Table S6
    16S ribosomal RNA sequencing database

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