Fig 2: 1 wk ABX gvg-treated APPSWE/PS1ΔE9 mice display altered peripheral and brain inflammatory profiles. (A) Representative density dot plots of T-bet and Foxp3 intracellular expression in TCRβ+ CD4+ T cell populations isolated from MLN, blood and brain tissue of vehicle and 1 wk ABX gvg-treated APPSWE/PS1ΔE9 mice analysed by flow cytometry. Quantified percentages of (B) Foxp3+ and (C) T-bet+ CD4+ T cells, representative of a T-reg and Th1 T cell phenotype respectively, are expressed relative to total live CD4+ T cell counts (n = 5–6, *p < 0.05, **p < 0.01, un-paired two-tailed Student’s t-test). (D) Immunoblot-based array of inflammatory mediators present in the serum of vehicle and 1 wk ABX gvg-treated APPSWE/PS1ΔE9 mice (n = 10 pooled sera). (E) Immunoblot-based array of inflammatory mediators present in the CSF of vehicle and 1 wk ABX gvg-treated APPSWE/PS1ΔE9 mice (n = 10 pooled CSF). (F) Heat map analysis of inflammatory mediator fold change expression in 1 wk ABX gvg-treated APPSWE/PS1ΔE9 mice relative to control. Data are displayed as loge(mean) or mean ± SEM. See Supp. Figs 2, 4, 5, 6, and statistical Table 2 for additional information.

Fig 3: LTßR expression by thymic epithelium controls the intrathymic iNKT cell pool.a Thymocytes from Foxn1Cre and LTßRTEC mice, analysed for expression of TCRß and reactivity with the PBS57-loaded CD1d tetramer to identify iNKT cells. TCRß+PBS57:CD1d+ cells were analysed for intracellular expression of PLZF, Tbet and ROR?t to identify NKT1, NKT2 and NKT17 subsets as indicated. b Absolute numbers of iNKT cells and iNKT subsets in Foxn1Cre controls (n = 11 biologically independent samples, closed symbols) and LTßRTEC mice (n = 12 biologically independent samples, open symbols), over three independent experiments. Percentages relative to Foxn1Cre controls are also shown. Significant P values using two-tailed unpaired t test as follows: no. of total iNKT P = 0.0002, no. of NKT1 P = 0.0003, no. of NKT2 P = 0.0001, no. of NKT17 P = 0.0021, % Total iNKT P = 0.0001, % NKT1 P = 0.0003, % NKT2 P = 0.0001, % NKT17 P = 0.0021. c Levels of expression of IL-17RB (IL-25R) on iNKT subsets; grey histograms represent isotype controls. Bar graphs show MFI of IL-17RB on indicated iNKT subsets, n = 5 biologically independent samples, over three independent experiments. Significant P values using two-tailed unpaired t test as follows: NKT1 vs. NKT2 P = 0.0001, NKT1 vs. NKT17 P = 0.0005. d Levels of CD122 expression on iNKT cell subsets; grey histograms represent isotype controls. Bar graphs show MFI of CD122 on indicated iNKT subsets, n = 5 biologically independent samples, over three independent experiments. Significant P values using two-tailed unpaired t test as follows: NKT1 vs. NKT2 P = 0.0001, NKT1 vs. NKT17 P = 0.0004. e qPCR analysis of Il25, Il15 and Il15ra mRNA in CD104+ (closed symbols) and CD104- (open symbols) mTEClow isolated from adult WT mice. f qPCR analysis of Il25 in CD104- mTEClow, and expression of Il15 and IL15ra in CD104+ mTEClow from control Foxn1Cre (closed symbols) and LTßRTEC mice (open symbols). All data are represented as mean ± SEM, **P < 0.01, ***P < 0.001 and ****P < 0.0001. qPCR was performed in replicate, and data shown are representative of three independent biological sorted samples. Source data are provided as a Source Data file.

Fig 4: TrIP KO T cells display a higher tendency for Th1 differentiation. (A–D) Naive T cells from WT and KO mice were cultured under neutral, Th0, Th1, Th17, and iTreg conditions for 3 d, restimulated, and analyzed for IFN-? and T-bet expression (A), IL-17a and ROR?t expression (B), and Foxp3 and CD25 expression (C and D). (E) Naive CD4+ T cells from the indicated mice were stimulated under the indicated conditions, and RNA was extracted and analyzed by quantitative PCR for PIk3ip1 message. Het, Pik3ip1 heterozygous. Data shown are normalized to naive WT T cells. P values were calculated using two-way ANOVA with Sidak’s multiple comparisons test. P values are represented with the following symbols: *, P = 0.01–0.05; **, P = 0.001–0.01; and ***, P < 0.001. Data in each panel are representative of at least three experiments. Data shown represent the mean ± SEM.

Fig 5: AIEC induces ER-stress-dependent Th17 cell ileocolitis in gnotobiotic Agr2−/− mice(A and B) SPF and GF Agr2+/− and Agr2−/− mice were monitored for 6 months. Flow cytometry of live, CD4+ T cells was used to evaluate RORγt and FOXP3 expression in ileal lamina propria (A). Percentage of RORγt+ and T-bet+ CD4+ T cells was evaluated (B). n = 4 from one of three experiments. Error bars represent SEM. *p < 0.05, **p < 0.01, ANOVA.(C–F) GF Agr2+/− and Agr2−/− mice were mono-colonized at weaning with non-pathogenic T75, AIEC MSL1, or AIEC MSL6 for 10 weeks. Representative flow cytometry of live, CD4+ T cells was used to evaluate IL-17A and IFNγ expression in the ileal lamina propria following a 4 h stimulation with PMA/ionomycin and brefeldin A (C). Percentage of ileal IL-17A+ (D) and RORγt+/T-bet+ (E) CD4+ T cells was evaluated. IL-23 production in ileal tissue explants was quantified by ELISA (F). n = 4 from one of four experiments. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.005, ANOVA.(G–I) GF Agr2+/− and Agr2−/− mice were mono-colonized at weaning with AIEC MSL1 for 3 weeks. Mice were treated with ER stress inhibitor 4-PBA or PBS control every 3 days. Expression of Il23p19 was measured in ileal LPMCs by quantitative PCR (G). Representative flow cytometry of live, CD4+ T cells was used to evaluate IL-17A and IFNγ expression in the ileal lamina propria (H). Percentage of ileal RORγt+ and IL-17A+ CD4+ T cells was evaluated following a 4 h stimulation with PMA/ionomycin and brefeldin A (I). n = 3–7 mice per group from one of two experiments. Error bars represent SEM. *p < 0.05, **p < 0.01, ANOVA.