(A) Expression of GFP and IL-17 in total cells from lung and liver of Rorγt ^gfp/+ and C57BL/6 mice. Data are representative of 3 independent experiments. Lung and liver cells were isolated as described in Methods. All plots were gated on lymphocytes.
(B) Representation of different cell populations among the IL-17⁺ cell fraction in lungs and small intestines (SI LP) of C57BL/6 mice. Data are combined from three independent experiments. N = 5 mice for lung and N = 8 mice for the SI LP. Error bars represent standard deviation of the mean.
(C, D) Expression of GFP (C) and IL-17 (D) in total cells from lung of Rorγt ^gfp/+ (C) and C57BL/6 (D) mice. Data are representative of 3 independent experiments. Lung cells were isolated as described in Methods. All plots are gated on lymphocytes.

(A) Requirement for TGF-β, but not for IL-21R or IL-23, in SI LP Th17 cell differentiation.
(B) TLR signaling is dispensable for SI LP Th17 cell differentiation.
Lamina propria lymphocytes were isolated from the small intestines of mice with the indicated genotypes as described in the Methods. Immediately after isolation the cells were incubated for 4 hours with PMA/Ionomycin and GolgiPlug (BD). All plots represent individual mice and were gated on TCRβ⁺CD4⁺ lymphocytes. For analysis of IL-23 requirement, Il23a^-/- mice from Jackson Laboratory were co-housed for 17 days with Taconic B6 mice to equalize the intestinal microbiota. N = 5 (Tgfb1^C33S/C33S), 6 (Il21r^-/-), 3 (IL23a^-/-), 2 (Trif^-/-), 2 (Myd88^-/-,Tlr3^-/-).

Lamina propria lymphocytes were isolated from the small intestines of Rorγt ^gfp/+ mice at the indicated ages. For day 4 and day 16 studies, small intestines from 3-4 pups from the same litter were combined in order to obtain sufficient cell numbers for analysis. For day 25 and 33, plots represent individual mice. Top panels — surface staining and RORγt-GFP analysis; bottom panels — intracellular staining for IL-17 after 4 hour stimulation with PMA/Ionomycin in the presence of GolgiPlug (BD). Data are representative of two independent experiments with 2-3 samples/mice in each. Plots are gated on lymphocytes.

(A) SI LP Th17 cells in 4-month-old 129S6/SvEv, C57BL/6, and 6-week-old Swiss-Webster germ-free (GF) or conventionally raised (SPF) mice. Representative plots shown are samples from individual mice gated on TCRβ⁺CD4⁺ cells. Two independent experiments with identical results were performed for each strain and 5-9 mice from each group were analyzed.
(B) SI LP Th17 cells in 6-month-old C57BL/6 SPF, GF, and GF mice reconstituted with fecal slurries from SPF mice for the indicated times. Data are combined from two independent experiments with identical results. A total of 6 SPF, 7 GF, and 7 SPF reconstituted GF mice were analyzed.
(C) Proportion of Foxp3⁺ cells within the TCRβ⁺CD4⁺ SI LP lymphocytes from the mice in (B).
(D) Proportion of IL-17⁺ cells within the TCRγδ ⁺ SI LP lymphocytes from the mice in (B).
Error bars represent standard deviation of the mean. Statistics, unpaired Student t test. *p<0.05, ***p<0.005, ******p < 0.0001. Lines represent the two populations being compared for the statistical analysis.

(A, C) Th17 cells in SI LP of 16-week-old C57BL/6 mice treated with antibiotics in the drinking water for 4 weeks. In (A), all four antibiotics were included. In (C), N — no treatment (N=9), A — all four antibiotics (N=6), M/N — metronidazole plus neomycin sulfate (N=9), V — vancomycin only (N=6). 2-3 independent experiments for each condition were performed with similar results. Combined histograms from all experiments (C) as well as representative plots from individual mice gated on TCRβ⁺CD4⁺ lymphocytes (A) are shown.
(B, D, E) Lamina propria lymphocyte populations in mice treated with antibiotics in the drinking water from birth. Representative data from individual mice (gated on TCRβ⁺CD4⁺ lymphocytes) are shown from one of multiple experiments. Error bars represent standard deviation of the mean. N — no treatment, V — vancomycin only, Amp — Ampicillin only, M/N — metronidazole plus neomycin sulfate.
(F) Relative proportions of IgA⁺ cells. Data were combined from two experiments. Percentage of IgA⁺ cells in the lymphocyte gate was calculated for each condition and the data are presented as percentage of the untreated control (N) mean value.
(G, H) Mice were treated with vancomycin (Vanco) or all four antibiotics (A) from birth. At 6.5 weeks of age some mice were transferred into cages with regular water with (Vanco then SPF) or without (Vanco then H₂O) co-housing with SPF mice not treated with any antibiotics. Th17 cell numbers in the SI LP were analyzed 4.5 weeks later. Results shown are from one of two independent experiments with similar results. Individual mouse plots (G) were gated on TCRβ⁺CD4⁺ lymphocytes. Histogram (H) is from one of two independent experiments with similar results. N = 4 mice for the 6.5 week time point and N = 2 mice for the 11 week time point; error bars represent standard deviation of the mean. Statistics, unpaired Student t test. *p<0.05, ** p<0.01, *** p<0.005, ****p<0.001, *****p<0.0005. For the statistical analysis, in all cases the corresponding population was compared to the WT (untreated) control.

(A, C) LPL populations in 10-week-old C57BL/6 mice from Taconic Farms (black bars) or The Jackson Laboratory (white bars). Data are combined from two out of three experiments with similar results. Error bars represent standard deviation from the mean. In (C) γδ T refers to percentage of IL-17⁺ cells in the TCRγδ ⁺ lymphocyte gate; Foxp3 refers to percentage of Foxp3⁺ cells within the TCRβ⁺CD4⁺ lymphocyte gate; and IgA⁺ refers to percentage of IgA⁺ cells within the lymphocyte gate.
(B) Jackson B6 mice were co-housed with Taconic B6 mice for 2 weeks before analyzing SI LP Th17 cells. Data are representative of two experiments with identical results (total of 4-6 mice in each group). Individual plots represent individual mice (2 mice of each group are shown) and were gated on TCRβ⁺CD4⁺ lymphocytes. (D) 4 week-old Swiss-Webster germ-free (GF) mice were colonized with homogenized cecal content from Jackson B6 (GF+Jack) or Taconic B6 (GF+Tac) mice and SI LP Th17 cells were analyzed on days 10 and 12 after colonization (results combined from both days). All GF mice were littermates. SPF, age and sex-matched controls raised under conventional conditions. N = 4 (SPF, GF) and 5 (GF+Jack, GF+Tac). Statistics, unpaired Student t test. *p<0.05, ***p<0.005, ****p<0.001, *****p<0.0005, ****** p < 0.0001, ns, not statistically significant (p>0.05). For statistics on (D), p values refer to comparisons to the SPF group, except on the horizontal line, which compares GF+Jack to the GF group.

(A) Gram staining of undiluted cecal material from 8-week-old Taconic B6 mice, Jackson B6 mice, and Taconic B6 mice treated with vancomycin (Vanco) from birth.
(B) SYBR green staining of cecal bacteria after dilution and filtration of the solid matter in Taconic B6, Jackson B6, vancomycin-treated from birth, and vancomycin-treated mice exposed subsequently to SPF mice to recover the SI LP Th17 cells.
(C) FISH analysis of intestinal bacteria from ceca of Taconic B6 (Tac B6) mice, mice treated from birth with vancomycin (Tac+Vanco), vancomycin-treated mice subsequently co-housed with non-treated mice (Tac+Vanco+SPF), and Jackson B6 (Jackson B6) mice, performed as described in the Methods.
(D) FISH analysis of intestinal bacteria from terminal ileum of Taconic B6 (Tac B6), mice treated from birth with vancomycin (Tac+Vanco), and Jackson B6 (Jackson B6) mice, performed as described in the Methods. For (C) and (D) data are presented as number of bacteria per gram tissue. Error bars represent standard deviation of the mean. 3 samples for each group were analyzed. Statistics, unpaired Student t test. p-values were calculated in comparison to the control Taconic B6 mice value. *p<0.05, ** p<0.01, *** p<0.005, nd — not detected.