Research ArticleINTESTINAL BOWEL DISEASE

Helicobacter species are potent drivers of colonic T cell responses in homeostasis and inflammation

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Science Immunology  21 Jul 2017:
Vol. 2, Issue 13, eaal5068
DOI: 10.1126/sciimmunol.aal5068
  • Fig. 1 Colonic Treg TCRs (CT2 and CT6) drive Teff cell development in inflammation.

    (A) Experimental model. Four- to 5-week-old CD45.1 specific pathogen–free (SPF) mice were administered αIL10R (1 mg per mouse) on day 0 and kept on 1% DSS water for 7 days to initiate colitis. Control mice were given isotype IgG (1 mg per mouse) on day 0. Four days after initiation of colitis, congenically marked naïve CT2/CT6 Tg cells (105) were intravenously transferred and analyzed 7 days later. (B to D) Effector cell induction and expansion with colitis. Transferred TCR Tg cells from the colon lamina propria (cLP) were analyzed by flow cytometry for (B) the development of Teff (CD44hi CD62Llo Foxp3) and Treg (Foxp3+) markers (P = 0.000003, P = 0.0007, P = 0.0024, P = 0.0157, Student’s t test; n = 10), (C) up-regulation of cytokines using IL-17AGFP and IFNγYFP (P = 0.000001, P = 0.0007, P = 0.0029, Student’s t test; n = 10 for CT2 and 7 for CT6), (D) proliferation indicated by CTV dilution (P = 0.000002, P = 0.00002, Student’s t test; n = 10 for CT2 and 7 and 8 for CT6), and (E) expansion indicated by the in vivo frequency among the host CD4+ T cells (P = 0.00005, P = 0.0275, Student’s t test; n = 10 for CT2 and 10 and 11 for CT6). Bars indicate mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns, not significant.

  • Fig. 2 Treg and Teff cell subsets show increased TCR overlap during colitis.

    (A to C) Analysis of TCRα repertoires from Tnaïve (CD44lo CD62hi), Treg (Foxp3+), and Teff (CD44hi CD62Llo) cells in the cLP of TCli TCRβ Foxp3IRES-Thy1.1 Tcra+/− mice 2 weeks after initiation of IgG or DSS + αIL10R. n = 5 and 6. (A) Morisita-Horn similarity comparison between two different T cell subsets within each mouse (left) or between different mice within each T cell subset (right). An index value of 1 indicates that the two samples are completely similar, and an index value of 0 means that they are completely dissimilar (left: P = 0.009, Student’s t test; n = 5 and 6; right: P = 0.033, P = 0.014, Student’s t test; n = 10 and 15). (B) Heat map showing the top 25 Teff TCRs in one mouse per row and their corresponding percentage in the Treg subset. Each column does not represent one TCR across all mice. (C) Percentage of T7-1 TCR in repertoire of Tnaïve, Treg, and Teff cell subsets. Each dot represents data from an individual mouse. (D) In vivo analysis of T7-1 TCR. T7-1 was retrovirally transduced into in vitro activated naïve TCliβ Rag1−/− Tg cells, and 2 × 105 cells were transferred into IgG or DSS + αIL10R hosts as indicated in Fig. 1A. Seven days after transfer, cells from the cLP were analyzed by flow cytometry for Teff and Treg cells, and the in vivo frequency among the host CD4+ T cells (P = 0.029, Student’s t test; n = 4). Bars indicate mean ± SEM. *P < 0.05, **P < 0.01.

  • Fig. 3 Colonic Treg TCRs react to MA Helicobacter species.

    (A) Treg TCRs preferentially react to MA antigens (Ags). Hybridoma cells expressing different TCRs were cultured with CD11c+ DCs, and the indicated antigen was obtained 2 weeks after the initiation of colitis. NFAT (nuclear factor of activated T cells)–GFP up-regulation was assessed by flow cytometry 1.5 days later. (B) Selective elimination of MA antigen using individual antibiotics. TCRs from (A) that react to MA antigen were stimulated with colonic MA antigens isolated from antibiotic-treated or untreated mice as per (A). (C) Changes in H. typhlonius and H. apodemus OTUs correlate with in vitro reactivity to MA antigen in (B). Data are the percentage of 16S OTUs from the MA preparations of individual antibiotic-treated or untreated mice. (D) In vitro recognition of H. typhlonius or H. apodemus. Cultured isolates were tested for TCR reactivity in vitro as per (A). Two to three independent experiments. Bars indicate mean.

  • Fig. 4 Helicobacter species induce pTreg cell differentiation during homeostasis.

    (A) In vivo validation of TCR reactivity to Helicobacter species. Three-week-old SPF mice were treated with ampicillin (amp) for 2 weeks via drinking water. Two days after the last treatment, H. typhlonius or H. apodemus were gavaged for a total of three times every other day. With the last gavage, congenically marked naïve CT2/CT6 Tg cells or retrovirally expressed CT9/T7-1 cells were transferred. Seven days after transfer, cLP cells were analyzed by flow cytometry for (top) the development of Treg (Foxp3+) cells (P = 0.000003, P = 0.001, P = 0.0002, P = 0.0046, Student’s t test; n = 4 and 5 for CT2, 6 and 7 for CT9, 5 for T7-1, and 3 and 5 for CT6), (middle) frequency of transferred TCR-expressing cells among the host CD4+ T cells (P = 0.0442, P = 0.021, P = 0.0046, P = 0.0705, Student’s t test; n = 4 and 5 for CT2, 6 and 7 for CT9, 5 for T7-1, and 3 and 5 for CT6), or (bottom) CTV dilution (P = 0.00000002, P = 0.0000007, Student’s t test; n = 4 and 5 for CT2 and 3 and 5 for CT6). (B) T cell response to Helicobacter in vivo is species-specific. Three-week-old SPF mice obtained from Charles River Laboratories were gavaged with H. typhlonius or H. apodemus (for a total of three times every other day). With the last gavage, congenically marked naïve CT2 and CT6 Tg cells (105 each) were cotransferred. One week after transfer, cells from the distal mesenteric lymph node (dMLN) were analyzed by flow cytometry for the frequency of transferred TCR-expressing cells among the host CD4+ T cells (left), CTV dilution (middle), or development of Treg (Foxp3+) cells (right) (n = 4, 6, and 6). Bars indicate mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 5 DSS + αIL10R colitis is associated with differential changes of bacterial composition in the lumen and mucosa.

    (A to D) 16S rRNA sequencing of colonic lumen contents or MA preparations 2 weeks after initiation of IgG or DSS + αIL10R. n = 8. Data are mean bacterial changes at the phyla (A) or family (B) level, and principal coordinates analysis on unweighted UniFrac distances (C). (D) Bacteria enriched during colitis. OTUs enriched in DSS + αIL10R mice with average percentage of >1% and Benjamini-Hochberg–adjusted P value of <0.05 (Mann-Whitney U) from lumen contents or MA preparations. (E) Increase of H. typhlonius in the mucosa with colitis. Percentages of H. typhlonius and H. apodemus OTUs are shown (Benjamini-Hochberg–adjusted: P = 0.0009, P = 0.0023, P = 0.0277, Mann-Whitney U test; n = 8). (F) Marked expansion of Bacteroides species in the lumen with colitis. Percentages of B. vulgatus, B. acidifaciens, and B. uniformis OTUs are shown (Benjamini-Hochberg–adjusted: P = 0.0009, P = 0.0086, P = 0.0009, P = 0.0086, P = 0.0266, Mann-Whitney U test; n = 8). Bars indicate mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 6 Expansion of Bacteroides species during colitis does not enhance TCR-specific T cell responses.

    (A) Antigenic reactivity of Bacteroides-reactive TCRs was used. In vitro stimulation by the Bacteroides isolates are shown as per Fig. 3A. Two to three independent experiments. (B and C) In vivo expansion and effector cell development of Bacteroides-reactive T cells. Congenically marked naïve DP1 Tg cells (105) were transferred into CD45.1 hosts as indicated in Fig. 1A and analyzed 7 days after transfer by flow cytometry for (B) CTV dilution in the dMLN (n = 7), (C) (left) frequency among the host CD4+ T cells (P = 0.0005, P = 0.047, Student’s t test; n = 7 for DP1 and NT2 and 9 for CT7), and (right) development of Teff cells (CD44hi CD62Llo Foxp3) (n = 7 for DP1 and NT2 and 11 for CT7; Student’s t test). NT2 and CT7 (C) were retrovirally transduced into in vitro activated naïve Vα2 TCliβ Tg cells from Rag1+/− (black) or Rag1−/− (blue) mice before transfer of 2 × 105 cells. (D) In vitro reactivity to in vivo antigen preparations is consistent with Bacteroides expansion by 16S rRNA analysis. Colonic lumen contents or MA preparations from IgG or DSS + αIL10R mice were tested as per Fig. 3A. Two independent experiments. Bars indicate mean ± SEM. *P < 0.05, **P < 0.01.

  • Fig. 7 Pathogenic potential of naïve Helicobacter-reactive CT6 cells in lymphopenic mice.

    Six-week-old Rag1−/− mice were given H. apodemus with or without naïve CT6 T cell transfer. H. apodemus was gavaged for a total of three times every other day. Naïve CT6 cells (105) were transferred at the time of last gavage. (A) Flow cytometry analysis for the frequency of Tg cells among the host CD45+ cells [P = 0.0002 CT6 versus CT6 + H. apodemus, one-way analysis of variance (ANOVA) with Tukey’s post hoc test; n = 3, 5, 5, and 5] and development of Treg (Foxp3+) and Teff (CD44hi CD62Llo Foxp3) cell markers (n = 5) in dMLN at 6 weeks. (B) Colon weight/length ratio at 6 weeks [P = 0.0003 (no treatment versus CT6 + H. apodemus), P = 0.00002 (CT6 versus CT6 + H. apodemus), P = 0.0005 (H. apodemus versus CT6 + H. apodemus), one-way ANOVA with Tukey’s post hoc test; n = 3, 5, 5, and 5]. (C) Representative hematoxylin and eosin (H&E)–stained section of the ascending colon at 6 weeks (original magnification, ×10) and quantification of crypt number [P = 0.00007 (no treatment versus CT6 + H. apodemus), P = 0.00005 (CT6 versus CT6 + H. apodemus), P = 0.0003 (H. apodemus versus CT6 + H. apodemus), one-way ANOVA with Tukey’s post hoc test; n = 3]. The number of crypts observed per 100 μm of ascending colon was averaged from five fields. Decreased crypt number reflects crypt dropout due to inflammation. Bars indicate mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/13/eaal5068/DC1

    Materials and Methods

    Fig. S1. DSS + αIL10R treatment induces colitis and Teff cell responses.

    Fig. S2. Gating strategy for TCR Tg transfer experiments in Figs. 1, 4A (CT2 and CT6), and 6 (B and C) (DP1).

    Fig. S3. Effects of DSS + αIL10R–mediated colitis on the TCR repertoire.

    Fig. S4. Gating strategy for T cell transfer experiments in Figs. 2D, 4A (CT9 and T7-1), and 6C (NT2 and CT7).

    Fig. S5. CBir1 TCR Tg cells become Teff, and not Treg, cells during DSS + αIL10R colitis.

    Fig. S6. Gating strategy for T cell hybridoma experiments in Figs. 3 (A, B, and D) and 6 (A and D) and figs. S7 (A and C), S8, and S11.

    Fig. S7. Colonic Treg TCRs react to MA Helicobacter species.

    Fig. S8. CT2/CT6/CT9/T7-1 TCRs do not react to other bacterial isolates tested.

    Fig. S9. Gating strategy and confirmation of Helicobacter spp. inoculation in Fig. 4B.

    Fig. S10. Quantification of 16S rRNA gene abundance in the lumen or MA preparation.

    Fig. S11. Specificity of DP1/NT2/CT7 TCRs for Bacteroides spp.

    Fig. S12. Gating strategy for Fig. 7A and fig. S13A.

    Fig. S13. Bacteria-specific induction of colitis in lymphopenic mice.

    Fig. S14. Helicobacter species are detected in fecal and colonoscopy samples from IBD patients.

    Fig. S15. Isotype controls for antibodies used in this study.

    Table S1. TRAV multiplex primer sequences.

    Table S2. TRAV designation, CDR3 amino acid sequences, and in vitro bacterial reactivity of indicated TCR clones.

    Table S3. Excel file containing source data for all the figure panels.

    References (65, 66)

  • Supplementary Materials

    Supplementary Material for:

    Helicobacter species are potent drivers of colonic T cell responses in homeostasis and inflammation

    Jiani N. Chai, Yangqing Peng, Sunaina Rengarajan, Benjamin D. Solomon, Teresa L. Ai, Zeli Shen, Justin S. A. Perry, Kathryn A. Knoop, Takeshi Tanoue, Seiko Narushima, Kenya Honda, Charles O. Elson, Rodney D. Newberry, Thaddeus S. Stappenbeck, Andrew L. Kau, Daniel A. Peterson, James G. Fox, Chyi-Song Hsieh*

    *Corresponding authors. Email: chsieh{at}wustl.edu

    Published 21 July 2017, Sci. Immunol. 2, eaal5068 (2017)
    DOI: 10.1126/sciimmunol.aal5068

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. DSS + αIL10R treatment induces colitis and Teff cell responses.
    • Fig. S2. Gating strategy for TCR Tg transfer experiments in Figs. 1, 4A (CT2 and CT6), and 6 (B and C) (DP1).
    • Fig. S3. Effects of DSS + αIL10R–mediated colitis on the TCR repertoire.
    • Fig. S4. Gating strategy for T cell transfer experiments in Figs. 2D, 4A (CT9 and T7-1), and 6C (NT2 and CT7).
    • Fig. S5. CBir1 TCR Tg cells become Teff, and not Treg, cells during DSS + αIL10R colitis.
    • Fig. S6. Gating strategy for T cell hybridoma experiments in Figs. 3 (A, B, and D) and 6 (A and D) and figs. S7 (A and C), S8, and S11.
    • Fig. S7. Colonic Treg TCRs react to MA Helicobacter species.
    • Fig. S8. CT2/CT6/CT9/T7-1 TCRs do not react to other bacterial isolates tested.
    • Fig. S9. Gating strategy and confirmation of Helicobacter spp. inoculation in Fig. 4B.
    • Fig. S10. Quantification of 16S rRNA gene abundance in the lumen or MA preparation.
    • Fig. S11. Specificity of DP1/NT2/CT7 TCRs for Bacteroides spp.
    • Fig. S12. Gating strategy for Fig. 7A and fig. S13A.
    • Fig. S13. Bacteria-specific induction of colitis in lymphopenic mice.
    • Fig. S14. Helicobacter species are detected in fecal and colonoscopy samples from IBD patients.
    • Fig. S15. Isotype controls for antibodies used in this study.
    • Table S1. TRAV multiplex primer sequences.
    • Table S2. TRAV designation, CDR3 amino acid sequences, and in vitro bacterial reactivity of indicated TCR clones.
    • References (65, 66)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S3 Excel file containing source data for all the figure panels.

    Files in this Data Supplement:

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