Fig 2: ILC2s are altered in infected mice after DSS treatment. (A) Gating strategy for ILCs. ILCs are identified by gating on CD45+ live cells, and then looking for a population positive for CD127 and negative for lineage (Lin) markers CD11c dendritic cells (DCs), CD11b (myeloid cells), Ter-119 (granulocytes), B220 (B cells), Ly-6g, and CD3 (lymphocytes). From the CD127+ Lin- population, we gated on GATA3+ cells (ILC2), RORγT+ cells (ILC3), and cells negative for GATA3 and RORγT that were positive for T-bet (ILC1). Dot plots are representative of ILCs in the mesenteric lymph nodes. (B) T-bet+ ILC1s do not display significant differences between the 2 groups (unpaired t test, 99.55 ± 1.96 vs 88.90 ± 2.10; P = NS; N ≥ 4). (C) GATA3+ ILC2s (gated on CD127+ Lin-) of the infected mice show a significant increase compared with the uninfected group (unpaired t test, 10.28 ± 2.28 vs 2.08 ± 0.34; P < .05). (D) RORγT+ ILC3s show no significant differences between the 2 groups (unpaired t test, 0.85 ± 0.44 vs 2.35 ± 1.27; P = NS). (E) T-bet+ ILC1s do not display significant differences in the colonic mucosa between the 2 groups (unpaired t test, 43.33 ± 5.97 vs 36.45 ± 7.13; P = NS; N ≥ 4). (F) GATA3+ ILC2s do not display significant differences in the colonic mucosa between the 2 groups (unpaired t test, 43.82 ± 9.14 vs 33.90 ± 6.42; P = NS; N ≥ 4). (G) RORγT+ ILC3s show no significant difference in the colonic mucosa between the 2 groups (unpaired t test, 15.30 ± 7.70 vs 9.45 ± 2.65; P = NS). Data are presented as means ± SEM, and are representative of 3 independent experiments.

Fig 3: Age-dependent output of blood td-ILCs and their differentiation(A) Peripheral blood of indicated strains of mice were analyzed. The frequencies of Lin−Thy1+CD127+ and subsequent CD62L+icCD3ε+ subsets are quantified.(B) The average frequency of blood CD62L+icCD3ε+ cells of the Lin−Thy1+CD127+ compartment in WT mice of indicated ages. Data are represented as mean ± SD. One-way ANOVA was used for determining the statistical significance of data shown in (A) and (B). ∗p<0.05, ∗∗<0.01, ∗∗∗p<0.001, ∗∗∗∗p<0.0001, ns, not significant.(C) In vitro differentiation on OP9-DL stroma of the indicated cell populations (800 cells per well) in 30 ng/mL of each IL-2, IL-7, and stem cell factor. Cells were analyzed on day 9 of the culture and the frequencies of indicated subsets are shown in the graphs. Data shown are representative of three independent experiments.

Fig 4: icCD3ε+ cells mature into ILC2 and ILC3 in the lung(A) Lung Lin−CD127+ cells of 2-month-old WT mice were analyzed for the expression of indicated transcription factors in triplicates. Subsequent gates from ST2− and ST2+ cells are color coded in red and blue. Mean fluorescence intensities of icCD3ε and GATA3 in indicated subsets are plotted on the right.(B) Analyses of Lin−CD127+ cells of WT lung at indicated time points post subcutaneous infection with 500 stage3 Nb. Cell numbers or MFI of icCD3ε of indicated subsets are shown in the bar graphs.(C) Cytokine expression in the indicated subsets from experiments described in (B) was measured after stimulation with PMA and Ionomycin for 3.5 h. One-way ANOVA was used for determining the statistical significance. ∗p<0.05, ∗∗<0.01, ∗∗∗p<0.001, ∗∗∗∗p<0.0001. Data are represented as mean ± SD.

Fig 5: Blocking 6PGD induces an effector T cell phenotype(A and B) CD8+ T cells were isolated from C57BL/6 mice and were stimulated for 4 days with aCD3 + aCD28 mAbs and IL-2 in the presence of either 6-aminonicotinamide (6-AN) (10 µM), DHEA (20 µM), or vehicle control (DMSO) (A). IFN-? expression was assessed by intracellular staining and flow cytometry, and (B) viability was calculated as percentage of total. The plot shown is representative of three independent experiments with n = 4 per experiment.(C and D) Thymic T cells were collected from 6PGD-/- and 6PGDfl/fl mice, and fractions of CD4+ and CD8+ T cells as well as expression of CD44 and CD25 on gated double-negative (DN) T cells were assessed by flow cytometry. Results are representative of three independent experiments.(E and F) Percentage of CD4+ and CD8+ T cells in spleen (E) and lymph nodes (F) of 6PGD-/- and 6PGDfl/fl mice was examined by flow cytometry.(G and H) Absolute numbers of CD4+ (G) and CD8+ (H) T cells in thymus, lymph nodes, and spleen of 6PGD-/- and 6PGDfl/fl mice were calculated from three replicates.(I–K) Splenocytes from 6PGD-/- and 6PGDfl/fl mice were analyzed for expression of CD44 and CD62L (I), KLRG1 and CD127 (J), and CD69 (K) by flow cytometry. Results are representative of five independent experiments with n = 4 mice per group. Error bars represent ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.