Distribution of human ILCs during chronic lung disease.

Transcription

1 Supplementary Figure 1 Distribution of human ILCs during chronic lung disease. Quantification of flow cytometric analysis of lung tissue from patients with COPD or IPF identifying (a) frequencies of total Lin - CD127 + ILCs (Lin gating includes CD3, CD5, CD56, CD19, Fc R1, CD14, CD16). Cells are pregated on live CD45 + cells. (b) Frequency of ILC subsets defined by IL-33R (ST2L) and CRTH2 expression, pregated on Lin - CD127 + cells. (c) Mean fluorescence intensity (MFI) of intracellular Arg1 expression in each cell subset. Data shown are from one experiment. Data are the mean ± SEM, n = 8 COPD and n = 8 IPF tissue samples.

2 Supplementary Figure 2 Fate-mapping analysis of IL-7R expression in immune cell subsets during lung inflammation. Il7r cre/+ Rosa26 floxstop-eyfp and C57BL/6J WT mice were treated with 30 g papain or PBS intranasally (i.n.) for 5 days and analyzed on day 6. Representative flow cytometric histograms of eyfp expression from lung immune cell subsets, Il7r cre/+ Rosa26 floxstop-eyfp red line; WT, gray shaded. Numbers indicate the percentage of total cells that are marked by a history of IL-7R expression for ILC2s (Lin - CD45 + CD90 + CD25 + IL-33R + ), CD4 + T cells (CD3 + CD4 + ), B cells (CD19 + CD3 - ), NK cells (NK1.1 + CD3 - ) and eosinophils (CD11b + Siglec F + CD11c - ). All data are representative of two independent experiments with similar results. N = 3-4 mice per group.

3 Supplementary Figure 3 Analysis of Arg1 expression in immune cell subsets during lung inflammation. Arg1 YFP and C57BL/6J WT mice were treated with 30 g papain or PBS i.n. for 5 days and analyzed on day 6. Representative flow cytometric histograms of Arg1-YFP expression from lung immune cell subsets, Arg1 YFP, blue line; WT, gray shaded. Numbers indicate the percentage of total cells that express Arg1 for ILC2s (Lin - CD45 + CD90 + CD25 + IL-33R + ), CD4 + T cells (CD3 + CD4 + ), B cells (CD19 + CD3 - ), NK cells (NK1.1 + CD3 - ) and eosinophils (CD11b + Siglec F + CD11c - ). All data are representative of two independent experiments with similar results. N = 3-4 mice per group.

4 Supplementary Figure 4 Loss of ILC-intrinsic Arg1 impairs ILC2 responses during chronic lung inflammation. (a-d) Arg1 fl/fl and Arg1 ILC mice were infected with 500 L3 Nippostrongylus brasiliensis (Nb) larvae subcutaneously and assessed one month post infection. (a) Representative flow cytometric plots and (b) total frequencies of ILC2s (Lin - CD45 + CD90 + CD25 + ) in the lungs of naïve and Nb-infected mice. (c) mrna expression of (c) Arg1 in lung tissue, determined by RT-PCR and expressed relative to levels in naive Arg1 fl/fl mice. (d-g) Arg1 fl/fl and Arg1 ILC mice were instilled with 3 units of elastase or PBS intratracheally and assessed

5 one month post treatment. (d) Representative flow cytometric plots and (e) total frequencies of ILC2s (Lin - CD45 + CD90 + CD25 + ) in the lungs of PBS and elastase-treated mice. (f) mrna expression of Arg1 in lung tissue, determined by RT-PCR and expressed relative to levels in PBS-treated Arg1 fl/fl mice. (g) Representative histological sections of whole left lung lobes stained with H&E. Scale bar, 100 m. Data are representative of two independent experiments with similar results. N = 2 mice (PBS and naïve) and n = 4 mice (Nb and elastase). Data shown are the mean ± SEM. *p < 0.05, **p < 0.01, *** p < 0.001, as determined by unpaired Student s t test.

6 Supplementary Figure 5 ILC3 development and anti-bacterial immunity are independent of ILC-intrinsic Arg1. (a) Representative flow cytometric plots and (b) frequencies of total ILCs (Lin - CD45 + CD90 + CD25 + ) in the gutassociated mesenteric lymph nodes (mln) of naïve Arg1 fl/fl and Arg1 ILC mice. (c) Representative flow cytometric plots and (d) total frequencies of GATA3 hi ILC2 versus ROR t + ILC3 populations, parent gate is total ILCs as in (a,b). (e) RORc( t) gfp/gfp, Rag2 -/- Il2rg -/-, Rag1 -/-, Arg1 fl/fl and Arg1 ILC mice were infected with CFU of Citrobacter rodentium (C. rodentium) in 200 µl via oral gavage. Mice were monitored for morbidity and mortality over the course of 28 days. Data are representative of two independent experiments with similar results. N = 2-5 mice per group. Data shown are the mean ± SEM. NS, not significant.

7 Supplementary Figure 6 ILC-intrinsic Arg1 does not influence cell survival. (a-c) Arg1 fl/fl, Arg1 +/+ Il7r Cre/+ and Arg1 ILC were treated with 30 g papain or PBS i.n. for 5 days and assessed on day 6 for survival of lung ILC2s by Annexin V and 7AAD staining. (a) Representative flow cytometric plot showing validation of gating strategy to identify dead cells by 7AAD and Annexin V co-staining. (b) Representative flow cytometric plots and (c) quantification of Annexin V + 7AAD + ILC2 frequencies. ILC2s gated as Lin - CD45 + CD90 + CD25 +. (d) C57BL/6J WT mice were treated with 100 ng of rmil-33 intranasally every 3 days for 2 weeks. Sort-purified lung ILC2s (Lin - CD45 + CD90 + CD127 + CD25 + IL-33R + ) were labeled with cell trace violet and cultured for 48 h with rmil-2, rmil-7, rmil-33 and DMSO or N ω -hydroxy-nor-arginine (nor-noha). Cells were assessed for dilution of the cell trace dye using flow cytometry. Data are representative of two experiments (a-c) or three experiments (d) with similar results. N = 3-4 mice per group.

8 Supplementary Figure 7 ILC-intrinsic arginase metabolism. (a) Pathway diagram of arginine metabolism in activated ILC2s. Green indicates positive metabolism, black indicates no significant metabolism, grey indicates metabolite not detected or not examined. Blue indicates enzymes arginase (Arg1), nitric oxide synthase (NOS) and L-Arginine:glycine amidinotransferase (AGAT). (b) Experimental schematic for liquid chromatography mass spectrometry analysis. C57BL/6J WT mice were treated with 100 ng rmil-33 i.n. every 3 days for 2-3 weeks and sort-purified lung ILC2s (Lin CD45 + CD90 + CD127 + CD25 + IL-33R + ) were cultured with rmil-2, rmil-7 and rmil-33 in DMSO or 500 M nor- NOHA for 24 h. Methanol-fixed cells were subjected to liquid chromatography mass spectrometry analysis.