Research ArticleMUCOSAL IMMUNOLOGY

Diet modulates colonic T cell responses by regulating the expression of a Bacteroides thetaiotaomicron antigen

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Science Immunology  08 Feb 2019:
Vol. 4, Issue 32, eaau9079
DOI: 10.1126/sciimmunol.aau9079
  • Fig. 1 Generation and characterization of the BθOM TCR transgenic mouse.

    (A and B) IL-2 levels in picogram per milliliter after generated T cell hybrid clones were cultured with BMDMs loaded with (A) B. theta (n = 2, one experiment) or (B) OMVs (n = 2, one experiment). (C) IL-2 levels in picogram per milliliter after the BθOM T cell hybrid was cultured with BMDMs loaded with B. theta grown in TYG or mTYG (n = 2; both replicates are shown). (D) Representative flow cytometry plot with Vβ12 staining on blood leukocytes of C57BL/6J mice (left) or BθOM transgenic mice (middle) (n = 3, three experiments). Representative TCRα1 PCR on DNA isolated from tails of C57BL/6J mice and BθOM transgenic mice (right) (x = 3, three experiments). (E) Representative histograms of CD69, CD25, and CD44 expression (left) and quantification of the percentage of CD69, CD25, and CD44 cells among all CD4 cells (right) isolated from the mLNs and spleen of C57BL/6J mice (red) or BθOM transgenic mice (blue) (x = 5, three experiments). (F) Representative flow cytometry plots of CD4 and CD8 staining of thymic cells isolated from C57BL/6J mice or BθOM transgenic mice (x = 5, three experiments) and quantification of the percentage of CD8 T cells among the thymic leukocyte population. (G) Percentage of Tregs in the thymus (n ≥ 6, three experiments), cdLN (n ≥ 10, six experiments), spleen (n ≥ 10, six experiments), and colon (n = 4, four experiments) of C57BL/6J mice (black) or BθOM transgenic mice (gray). Student’s t test: (E) *P < 0.1 and **P < 0.01; (F) ***P = 0.0004; (G) ****P < 0.0001 and ***P = 0.0001.

  • Fig. 2 B. theta activates BθOM T cells in a nutrient-dependent manner.

    (A and B) Percentage of CD69 expressing BθOM T cells after a 24-hour culture with BMDM loaded with (A) Bacteroidaceae family [human: B. thetaiotaomicron (n = 4, four experiments); mouse: B. fragilis, B. vulgaris, Parabacteroides goldsteinii, E. coli, B. sartorii (n = 3, three experiments)] or (B) human B. theta OMVs (75 μg/ml: n = 7, six experiments; 37.5 μg/ml: n = 6, six experiments; 18.75 μg/ml: n = 5, four experiments; 10 μg/ml: n = 8, six experiments; 1 μg/ml: n = 3, three experiments; 0.1 μg/ml: n = 4, four experiments; 0.01 μg/ml: n = 3, three experiments). Flow cytometry plots are gated on CD4+ CD45.1+ leukocytes. (C) Percentage of CD69 expressing BθOM hybridoma T cells after a 24-hour culture with BMDM loaded with human B. theta grown in TYG (n = 13, five experiments) or mTYG medium (n = 5, five experiments). One-way ANOVA analysis: (A) ***P < 0.001 and ****P < 0.0001. Means with asterisks are significantly different by Tukey’s multiple comparisons test. Student’s t test: (C) ****P < 0.0001, ***P = 0.0001, and **P < 0.01.

  • Fig. 3 BθOM T cells proliferate in the colon in B. theta–colonized mice.

    (A) Schematic of adoptive transfer of BθOM T cells into Rag1−/− mice gavaged with PBS or B. theta. (B) Representative flow cytometry plots of CD45.1+CD4+ BθOM T cells in the colon of B. theta–gavaged mice compared with PBS-gavaged mice. (C) Number of BθOM T cells among live leukocytes that are CD45.2CD45.1+CD4+ in PBS or B. theta–gavaged mice in the colon (n ≥ 6, ≥ five experiments), cdLN (n ≥ 5, ≥ three experiments), and spleen (n ≥ 6, ≥ four experiments). (D) Representative histograms of adoptively transferred carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled BθOM T cells in the colon (n ≥ 3, ≥ three experiments), cdLN (n ≥ 3, three experiments), and spleen (n ≥ 3, ≥ three experiments) of B. theta–gavaged mice compared with PBS-gavaged mice. (E) Quantification of the percentage of proliferated CFSE low CD45.2CD45.1+CD4+ T cells in the colon (n ≥ 3, ≥ three experiments), cdLN (n ≥ 3, three experiments), and spleen (n ≥ 3, ≥ three experiments). Mann-Whitney test for non-normally distributed data: (C) ****P < 0.0001 and ***P = 0.0006. Student’s t test: (E) ****P < 0.0001, ***P = 0.0005, and *P = 0.0160.

  • Fig. 4 BθOM T cells in the colon differentiate into Tregs.

    (A) Flow cytometry plots of CD45.1+CD4+ BθOM T cells in the colon, cdLN, and spleen of PBS or B. theta–gavaged Rag1−/− mice transferred with naive CD25 BθOM T cells. (B) The number of CD4+ CD45.1+FoxP3+ BθOM Tregs cells in the colon (n ≥ 6, ≥ five experiments), cdLN (n ≥ 5, ≥ three experiments), and spleen (n ≥ 6, ≥ four experiments) of PBS or B. theta–gavaged Rag1−/− mice after CD25 BθOM T cell transfer. (C) Percentage of FoxP3+ Tregs in the colon (n = 27, nine experiments), cdLNs (n = 25, seven experiments), and spleen (n = 20, seven experiments) of Rag1−/− mice that received naive CD25 BθOM T cells and were gavaged with B. theta. (D) Percentage of CD25high versus CD25low CD4+FoxP3+ Tregs in the colon (n = 27, nine experiments) and cdLNs (n = 25, seven experiments) of Rag1−/− mice gavaged with B. theta and injected with naive BθOM T cells. Mann-Whitney test for non-normally distributed data: (B) ****P < 0.0001 and **P = 0.004. Kruskal-Wallis with Dunn’s posttest for non-normally distributed data: (C) ****P < 0.0001. Two-way ANOVA analysis: (D) ****P < 0.0001 and *P = 0.0161.

  • Fig. 5 Depletion of BθOM Tregs drives BθOM CD4+ Teff to cause colitis.

    (A) Schematic of adoptive transfer of BθOM or BθOM-FoxP3-DTR T cells into Rag1−/− mice gavaged with PBS or B. theta and treated with DT (31) to deplete BθOM Tregs. (B) Percentage of BθOM Tregs after depletion in the mLN (n ≥ 12, five experiments) or spleen (n ≥ 14, five experiments). (C to E) Histology (C), quantification of the number of mitotic figures/10 crypts (D), and average crypt height (E) in cecal sections from Rag1−/− mice given BθOM T cells and DT (n = 6, three experiments) compared with those given BθOM-FoxP3-DTR T cells and DT (n = 10, three experiments). Scale bars, 120 μm. (F) Cytometric bead array used to quantify IFN-γ (n ≥ 10, three experiments), IL-17A (n ≥ 10, three experiments), and IL-6 (n ≥ 10, three experiments) after cells isolated from the mLN were stimulated with PMA for 5 hours. Student’s t test: (B) ***P = 0.0002 and **P = 0.0055; (D) **P = 0.0029; (E) ****P < 0.0001; (F) *P = 0.0205 and **P = 0.098.

  • Fig. 6 BθOM T cells specifically recognize the BT4295(541–554) epitope.

    (A) Two parallel methods, T cell Western with proteomics (left) and transposon mutagenesis (TM) screen (20) (right), used to identify the antigen that stimulates BθOM T cells. (B) Schematic of the PUL80 affected by BT4298 disruption by TM. The arrow represents the direction of transcription. (C to G) Percentage of CD69 expressing BθOM T cells after culture with BMDM loaded with (C) E. coli expressing the full-length BT4295 (n = 3, three experiments for each dilution) or three consecutive segments of BT4298 (BT4298A, BT4298B, and BT4298C) (n = 3, three experiments for each dilution), (D) B. theta (n = 4, four experiments) or Δ4295 (n = 4, four experiments), or (E) E. coli expressing two consecutive segments of BT4295 (BT4295A and BT4295B) (n = 3, three experiments for each dilution). (F) Synthetic 20-amino acid peptides overlapping by 12 amino acids. The asterisks represent the P5 position. (G) B. theta (n = 4, four experiments, same data as Fig. 2E) or Δ4295 (n = 3, three experiments). One-way ANOVA analysis: (C) **P < 0.01, ***P < 0.001, and ****P < 0.0001. Means with asterisks are significantly different by Tukey’s multiple comparisons test. Student’s t test: (D) ***P < 0.001 and ****P < 0.0001; (E) *P < 0.1, **P < 0.01, and ***P < 0.001; (G) ***P < 0.001 and ****P < 0.0001.

  • Fig. 7 Salt and glycan regulate BT4295 expression and alter BθOM T cell activation.

    (A) Percentage of CD69 expressing BθOM T cells after a 24-hour culture with BMDM loaded with B. theta grown in mTYG (n = 4, four experiments), TYG (n = 2, two experiments), and mTYG supplemented with TYG salts (n = 4, four experiments). (B) The concentration in microgram per milliliter of BT4295 protein expressed in B. theta grown in TYG, mTYG, and mTYG supplemented with TYG salts (n = 4, four experiments) as determined by a quantitative ELISA. (C) Percentage of CD69 expressing BθOM T cells after a 24-hour culture with BMDM loaded with B. theta grown in mTYG, TYG, mTYG supplemented with MOG and TYG supplemented with MOG (n = 2, two experiments). (D) The concentration in microgram per milliliter of BT4295 protein expressed in B. theta grown in mTYG, TYG, mTYG supplemented with MOG and TYG supplemented with MOG (n = 3, three experiments) as determined by a quantitative ELISA. One-way ANOVA analysis: (A) *P < 0.1, **P < 0.01, ***P < 0.001, and ****P < 0.0001; (B) **P = 0.0093; (D) ****P < 0.0001 and **P = 0.0065. Means with asterisks are significantly different by Tukey’s multiple comparisons test.

  • Fig. 8 Dietary glucose represses BT4295 expression, decreasing the activation of BθOM T cells in vivo.

    (A) Representative plot of the percentage of CD69 expressing BθOM T cells after culture with BMDM loaded with B. theta grown in TYG and mTYG media with or without glucose (n = 6, three experiments). (B) The concentration in microgram per milliliter of BT4295 protein expressed in B. theta grown in TYG and mTYG media with or without glucose (n = 6, three experiments). The percent difference in the number of (C) CD4+CD45.1+ BθOM T cells or (D) CD4+CD45.1+CD44+CD62L activated BθOM T cells in the colon (n = 26, x = 3 experiments) and cdLN (n = 16, two experiments) of B. theta–colonized mice given water or 30% glucose water and adoptively transferred with 200,000 CD4-enriched BθOM T cells. (C and D) The percent difference was calculated from the mean of each experiment. ANOVA multiple comparison analysis: (A) *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; (B) ****P < 0.0001 and *P = 0.0190. Means with asterisks are significantly different by Tukey’s multiple comparisons test. Mann-Whitney test for non-normally distributed data: (C) ***P = 0.0002 and **P = 0.0052; (D) ***P = 0.0002 and *P = 0.0115.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/32/eaau9079/DC1

    Fig. S1. Sorting strategy and B. theta colonization for in vivo BθOM T cell transfer experiments.

    Fig. S2. BθOM T cells do not cause weight loss in B. theta–colonized mice.

    Fig. S3. Cytokines not altered by BθOM Treg depletion.

    Fig. S4. BθOM T cells primarily differentiate into TH1 cells in vivo in the colon lamina propria and mLN.

    Fig. S5. Identification of the epitope recognized by BθOM T cells.

    Fig. S6. BθOM T cells recognize BT4295(541–554) and schematic of the BT4295 PUL.

    Fig. S7. The effect of various nutrients on BθOM T cell activation.

    Fig. S8. The addition of 30% glucose to the drinking water has no effect on B. theta colonization or Treg differentiation.

    Table S1. Composition of TYG medium versus mTYG medium.

    Table S2. BT4295 and BT4298 primers.

    Table S3. Raw data.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Sorting strategy and B. theta colonization for in vivo BθOM T cell transfer experiments.
    • Fig. S2. BθOM T cells do not cause weight loss in B. theta–colonized mice.
    • Fig. S3. Cytokines not altered by BθOM Treg depletion.
    • Fig. S4. BθOM T cells primarily differentiate into TH1 cells in vivo in the colon lamina propria and mLN.
    • Fig. S5. Identification of the epitope recognized by BθOM T cells.
    • Fig. S6. BθOM T cells recognize BT4295(541–554) and schematic of the BT4295 PUL.
    • Fig. S7. The effect of various nutrients on BθOM T cell activation.
    • Fig. S8. The addition of 30% glucose to the drinking water has no effect on B. theta colonization or Treg differentiation.
    • Table S1. Composition of TYG medium versus mTYG medium.
    • Table S2. BT4295 and BT4298 primers.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). Raw data.

    Files in this Data Supplement:

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