Extended Data Fig. 5: Systemic effects of 11 strain colonization.

a, Representative flow cytometry histograms showing the expression of CD103, PD-1, CD44, ICOS and KLRG1 by inguinal lymph node (iLN) or colonic IFNγ⁺ CD8 T cells from the indicated mice from two independent experiments. b, Frequencies of TCR Vβ gene usage among IFNγ⁺ CD8 T cells (left) and IFNγ⁻ CD8 T cells (right) from the inguinal lymph nodes of germ-free (grey) or GF+11-mix (red) mice, and those from the colons of GF+11-mix mice (blue). c, Principal component analysis plots of the water-soluble caecal metabolome from gnotobiotic mice colonized with the indicated bacterial mixtures. d–f, Heat maps depicting differentially elevated metabolites in the caecum (d, e) or serum (f) of gnotobiotic mice colonized with the indicated bacterial mixtures. Raw metabolomic data was screened for metabolites specifically increased in GF+11-mix mice only (d, e) or in GF+11-mix and GF+4-mix mice only (d) with at least fourfold enrichment as compared to GF controls and at least twofold enrichment compared to the other gnotobiotic groups, with a cut-off P value of 0.05. Overlapping metabolites between the two datasets were selected (highlighted in green), and their levels in the serum were queried (f). The most promising candidate effector molecules for the local and systemic effects observed in GF+11-mix mice were identified by considering the metabolomic data in light of the phenotypic immunomodulatory data, and are highlighted in red font (d–f). For a more detailed discussion of how these metabolites were chosen, see Supplementary Discussion. The area under each individual metabolite peak was normalized by dividing by the area under the peak of the corresponding internal standard. Heat map colours represent the z-score (red and blue indicate high and low abundance, respectively). Caecal contents were isolated 4 weeks after gavage unless otherwise noted (1w, 1 week; 4w, 4 weeks). 2PG, 2-phospho-d-glycerate; 3PG, 3-phospho-d-glycerate; GlcNAc 6P, N-acetyl-d-glucosamine 6-phosphate; GlcNAc 1P, N-acetyl-d-glucosamine 1-phosphate; Rib5P, ribose-5-phosphate; Ru5P, ribulose-5-phosphate; Xu5P, xylulose-5-phosphate. Each circle represents an individual mouse (b, iLN, c), a pool of mice (b, colon). The number of mice in each group is shown. Data are mean and s.d. *P < 0.05; two-tailed unpaired t-test. See Source Data for exact P values.

Source Data