Generation of ST2-GFP reporter mice and characterization of ILC1 cells following infection

Transcription

1 Supplementary Figure 1 Generation of ST2-GFP reporter mice and characterization of ILC1 cells following infection with influenza virus. (a) ST2-GFP reporter mice were generated as described in Methods. (b) Representative flow cytometric plots demonstrating Lineage vs. ST2-GFP staining in lung of littermate control or ST2-GFP reporter mice; and expression of CD90, IL-7R, CD44 and CD25 on Lin - ST2-GFP + cells. Representative flow cytometric plots comparing ILC populations in naive lung, infected lung and draining lymph nodes at Day 7 post-infection (c). Representative flow cytometry plots comparing ILC

2 populations in the bronchoalveolar lavage fluid from naive and infected mice (d). Live/dead fixable blue (e) or Annexin V (f) staining on ILCs in naïve and infected lungs at Day 7 post-infection. (g) Expression of GATA-3, T-bet, CD44, CD25, IL-7r, ckit and ICOS on lung-resident ST2 + (red) or IL- 18r + (blue) ILCs at Day 7 post-infection. (h) Percentage of lung ILCs positive for Ki67. (i) IL-5, IL-13 and IFN- levels in culture supernatants from lung ILCs isolated from the lung at day 7 post-infection and stimulated as indicated for 24 hours. Data are expressed as Mean±S.E.M., b-h are representative of 2 independent experiments with 3-5 mice per group; 1l is representative of 2 separate experiments with 9 mice/group. *P<.01 according to the Mann-whitney Wilcoxon test.

3 Supplementary Figure 2 Bacterial infection and/or exposure cigarette smoke trigger(s) phenotypic changes in lungresident ILC2 cells. (a-c) Quantification of MFI for IL-18R, Tbet and IL-12R 2 on lung-resident ILCs in naïve mice and mice infected with Staphylococcus aureus (S.aureus) at day 5 post-challenge. (d) Representative flow cytometric plots of GATA-3 expression in naive mice or mice infected with nontypeable Haemophilus influenzae (NTHi). at day 2 post-infection. (e-f) Representative flow cytometric plots and MFI of IL-12R 2 expression on lung-resident ILCs in naive mice and mice infected with NTHi at day 5 post-infection (g). Percentage of cells expressing GATA-3 in naïve mice, mice infected with influenza,

4 or mice exposed to cigarette smoke prior to influenza infection. (h) Representative flow cytometric plots of ST2 and IL-18R expression on lung-resident ILCs in naive mice, influenza-infected mice or mice exposed to cigarette smoke prior to influenza infection. Correlation (i) and percentage (j) of ILCs expressing ST2 or IL-18R in naive mice, influenza-infected mice or mice exposed to cigarette smoke prior to influenza infection. (k-l) GATA-3 and T-bet expression in lung-resident ILCs from naive mice, influenza-infected mice or mice exposed to cigarette smoke prior to influenza infection. *p<.01, **p<.001. Data in a-f are representative of 2-3 independent experiments with 5 mice per group. Data in g are pooled from three independent experiments with 3-15 mice/group. Data in h-l are representative of 3-4 independent experiments with 5 mice per group. *p<.01, **p<.001.

5 Supplementary Figure 3 IL-12 and IL-18 induce IFN- production by ILC2 cells in vitro and in vivo. (a) Representative flow cytometric plots of ILCs following double depletion of lineage positive cells. ILCs were defined as CD45 +, viable, CD3 -, CD49b -, Lin - CD44 + CD90 + IL-7r +. Levels of IL-5 (b) and IL-13 (c) as measured in the culture supernatants after 4 days of culture with IL-12 and IL-18. MFI of GATA-3 (d), ST2 (e), T-bet (f) and IL-18R (g) on lung-resident ILCs from mice treated intranasally as indicated. (h-j) Representative flow cytometric plots showing IL-5 and IFN- on ILCs were purified from lungs of mice treated as indicated. Data in a-c are representative of three independent

6 experiments, where ILC2 cells were pooled from n 8 mice. Data in d-g are representative of two independent experiments. For h-j, ILCs were pooled from n 5 mice per treatment group. *p<.01, **p<.001.

7 Supplementary Figure 4 GFP + ILC2 cells adopt an ILC1 phenotype following infection with influenza virus. (a) ST2 + IL-18R - ILC2 were sorted and transferred in RAG/ c -/- mice, which were subsequently infected with influenza A. Representative flow cytometric plots showing GATA-3 (b), IL-18R (c) and IL-12R 2 (d) expression on adoptively transferred ILC2 cells 10 days after virus infection. Representative flow cytometric plots showing IFN (d) or IL-13 (e) expression on adoptively transferred ILC2 cells purified from the lung and stimulated overnight with IL-12 and IL-18 (d) or IL-33 (e). MFI of GATA-3 (f), IL-18R (g) and IL-12R 2 (h) on adoptively transferred ILCs. (i) IFN- levels

8 in the supernatants of ILCs cultured overnight in indicated conditions. Data are expressed as Mean and are representative of 2 independent experiments with 5-9 mice per group (g-i) or from 2 independent experiments where each group was pooled (j). ***p< All data was analyzed at day 10 post-infection.

9 Supplementary Figure 5 Rate of influenza-virus infectivity and imaging of transferred GFP + ILC2 cells in tissue. (a) Quantification of influenza A staining in RAG/ c -/- deficient mice at day 10 post-infection. Data are expressed as Mean and are representative of 2 independent experiments with 8-11 mice/group, ***p< (b) Positive control of GFP immunostaining with brown DAB chromogen in an infected RAG/ c -/- mouse that received GFP + ILC2 cells. (c) Negative control with the same staining protocol in a virus-infected mouse that had not received any GFP cells. (d) Double in situ hybridization (ISH) with simultaneous visualization of IL-12 and IL18 mrna by green and red chromogen, respectively.

10 The image is from 6 d post infection and influenza virus particles are immunostained with brown DAB chromogen. (e) Positive ISH control with probe for the eukaryote house-keeping gene PPIB. (f) Negative control with the non-eukaryote bacterial gene DapB. (g,h) Positive staining control for IL-12 and IL-18 alone, respectively. Scale bars: b-h= 20 m.

11 Supplementary Figure 6 T-bet promotes IFN- production by ILC2 cells and effects of CD90 depletion on lung-resident cells. Representative flow cytometric plots (a) and quantification (b) of GATA-3 expression in lung-resident ILCs in C57BL/6 and Tbx21 -/- mice after intranasal treatment with PBS or IL-12 and IL-18. Representative flow cytometric plots (c) of ST2 and IL-18R expression, quantified in (d), in lungresident ILCs in C57BL/6 and Tbx21 -/- mice treated intranasally with PBS or IL-12 and IL-18. Representative flow cytometric plots (e) and quantification (f) of IL-12R 2 expression in lung-resident

12 ILCs in C57BL/6 and Tbx21 -/- mice treated intranasally with PBS or IL-12 and IL-18. (g) Intracellular IFN- expression in purified ILCs from PBS- or IL-12/IL-18-treated C57BL/6 and Tbx21 -/- mice that were stimulated overnight with IL-12 and IL-18. (h) IFN- levels in culture supernatants from purified ILCs stimulated overnight as indicated. Representative flow cytometric plots (i) showing Lin - CD90 + cells (gated on CD45 +, viable, CD3 - CD49b - cells) in mice treated with isotype control or anti-cd90.2 antibody. (j) Quantification of the percentage of ILCs in naive and infected mice treated with isotype control or anti-cd90.2 antibody. Representative flow cytometric plots (k) showing NK cell populations (defined as viable, CD45 +, CD3 - CD49b + cells) in naive and infected SCID mice treated with isotype control or anti CD90.2 antibody. (l) Quantification of NK cells in the lungs of naïve and infected SCID mice treated with isotype control or anti CD90.2 antibody. Representative flow cytometric plots (m) showing mesenchymal stem cell populations (msc, defined as viable, CD45 -, CD90 + cells) in naive and infected mice treated with isotype control or anti CD90.2 antibody. (n) Quantification of msc cells in the lungs of naïve and infected mice treated with isotype control or anti CD90.2 antibody. (o-p) CD166 and Sca-1 expression on CD90 + CD45 - cells in the lung. (q) ILC2 and ILC1 cells were purified from mice treated with IL-33 or IL-33 +IL-12+IL-18, respectively, and transferred into RAG/ c deficient mice. Data are shown as Mean and are representative of 2 independent experiments with 4-9 mice/group (a-g) or 7-10 mice/group (j-n) or groups were pooled from 2 independent experiments (h). *p<.01, **p<.001, ***p<.0001.

13 Supplementary Figure 7 Flow cytometry gating strategy for ILC2 cells from healthy human donors. Human PBMCs were pooled from 3-4 healthy donors and enriched for NK cells/ilcs using an NK cell enrichment kit. Purity of ILCs in post-nk-enrichment demonstrated by representative flow cytometric plots showing pre-sort (a) and post-sort (b) samples for in vitro culture of human ILC2 cells, defined here as CD45 + Viable CD3 - CD19 - Lin - IL-7R + CRTH2 + CD (c) Representative flow cytometric plots demonstrating post-culture purity human ILC2 cells cultured with IL-2 and IL-33.

14 Supplementary Figure 8 Flow cytometry gating strategy for the identification and quantification of ILC subsets in patients with COPD. (a) Representative flow cytometric plots demonstrating ILC gating strategy for identifying ILCs in the peripheral blood of non-smokers (aged matched healthy controls), healthy smokers or COPD patients. Samples were depleted of T and B cells and ILCs were defined as CD45 + Viable CD3 - CD19 - Lin - IL-7R + CD56 -. (b) Correlative analysis between FEV1/FVC and % ILC1 cells. (c) Correlative analysis between FEV1 predicted and % ILC2 cells. (d) Correlative analysis between

15 FEV1/FVC and % ILC2 cells in the peripheral blood of non-smokers, healthy smokers and COPD patients. (e) Quantification of the sum of % ILC1 and ILC2 cells in non-smokers, healthy smokers and COPD patients, dotted line indicates the mean ± SD across all groups; 42.3%±13.1%. Group sizes: b-d GOLD I=4; GOLD II=11; GOLD III=12; GOLD I V = 1 4 ; e Non-smokers=11; Smokers=18; total COPD=42.

16 Demographic Table 1 Non-Smoker Control (N=10 a ) Smoker Control (N=18 a ) COPD (N=42 a ) p-value Age ± ± ± Gender(%Male) 4 (40.00) 5 (27.78) 21 (50.00) BMI ± ± ± Smoker(%Current) 0 (0.00) 4 (22.22) 5 (11.90) FEV1 (%) ± ± ± <.0001 FEV1/FVC 0.81 ± ± ± 0.13 <.0001 Ave Exacerbations/Year, Median [Min,Max] NA 0 [0, 3.6] 0.3 [0, 4.4] Medication, n (%) bagonist 0 (0.00) 1 (9.09) 24 (60.00) bagonistlongact 0 (0.00) 0 (0.00) 5 (12.50) CombcCSBagon 0 (0.00) 0 (0.00) 22 (55.00) combivent 0 (0.00) 1 (9.09) 12 (30.00) cortsterinhal 0 (0.00) 0 (0.00) 7 (17.50) CortsterOral 0 (0.00) 0 (0.00) 0 (0.00) ipratrop 0 (0.00) 0 (0.00) 4 (10.00) MacAntibiotic 0 (0.00) 0 (0.00) 1 (2.50) nebulizer 0 (0.00) 1 (9.09) 19 (47.50) OsteoporosisMed 2 (22.22) 0 (0.00) 5 (12.20) PhosphoInhibitor 0 (0.00) 0 (0.00) 2 (5.00) tiotrop 0 (0.00) 1 (9.09) 22 (55.00) * Subjects may have missing information in some demographic variables.

17 Demographic Table 2 COPD (GOLD I) (N=4 a ) COPD (GOLD II) (N=11 a ) COPD (GOLD III) (N=12 a ) COPD (GOLD IV) (N=14 a ) p-value Age ± ± ± ± Gender(%Male) 2 (50.00) 7 (63.64) 5 (41.67) 6 (42.86) BMI ± ± ± ± Smoker(%Current) 2 (50.00) 2 (18.18) 1 (8.33) 0 (0.00) FEV1 (%) ± ± ± ± 4.62 <.0001 FEV1/FVC 0.60 ± ± ± ± 0.04 <.0001 Ave Exacerbations/Year, Median [Min,Max] 0 [0, 0.2] 0 [0, 2.8] 0.7 [0, 4.4] 1.05 [0, 3.5] Medication, n (%) bagonist 0 (0.00) 5 (50.00) 9 (75.00) 9 (64.29) bagonistlongact 0 (0.00) 1 (10.00) 2 (16.67) 1 (7.14) CombcCSBagon 1 (33.33) 2 (20.00) 8 (66.67) 10 (71.43) combivent 0 (0.00) 3 (30.00) 4 (33.33) 4 (28.57) cortsterinhal 0 (0.00) 1 (10.00) 2 (16.67) 3 (21.43) CortsterOral 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00) ipratrop 0 (0.00) 1 (10.00) 1 (8.33) 1 (7.14) MacAntibiotic 0 (0.00) 0 (0.00) 1 (8.33) 0 (0.00) nebulizer 0 (0.00) 2 (20.00) 4 (33.33) 12 (85.71) OsteoporosisMed 1 (25.00) 1 (9.09) 1 (8.33) 2 (15.38) PhosphoInhibitor 0 (0.00) 0 (0.00) 1 (8.33) 1 (7.14) tiotrop 1 (33.33) 4 (40.00) 6 (50.00) 11 (78.57) * Subjects may have missing information in some demographic variables.