Allergen-induced Increases in Sputum Levels of Group 2 Innate Lymphoid Cells. Guangzhou Medical University, Guangzhou, China.

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1 Online Supplemental Data Title: Allergen-induced Increases in Sputum Levels of Group 2 Innate Lymphoid Cells in Asthmatic Subjects Authors: Ruchong Chen 1,2, Steven G Smith 1, Brittany Salter 1, Amani El-Gammal 1, John Paul Oliveria 1, Caitlin Obminski 1, Rick Watson 1, Paul M O Byrne 1, Gail M Gauvreau 1 and Roma Sehmi 1. Address: 1 Asthma Research Group, Department of Medicine, McMaster University 1, Hamilton, Ontario, Canada. 2 State Key Laboratory of Respiratory Disease, Guangzhou Institute of Respiratory Diseases, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.

2 METHODS Subjects Ten non-smoking subjects with mild stable asthma were recruited for this study. Subjects (aged years) were not on any regular asthma treatment, infrequently used inhaled ß 2 - agonists as reliever therapy and were asymptomatic for 4 weeks prior to the study. All subjects were studied when not naturally exposed to the seasonal allergen to which they were sensitized. All experimental procedures were reviewed and approved by the Hamilton Integrated Research Ethics Board and all subjects provided informed consent. Study Design Subjects completed a randomized, diluent-controlled, allergen-challenge crossover study (Figure 1). Subjects underwent skin prick testing, methacholine PC20 and a medical history during a screening period. Eligible subjects participated in two series of experiments. In series 1, baseline measurements of, blood and a bone marrow aspirates were collected pre-challenge. On the following day subjects were randomized to inhale diluent or allergen. Spirometry was performed hourly to 7hr post- challenge, blood was drawn 6hr and 24hr post-challenge, and a bone marrow aspirate was collected 24h post-challenge. After a washout period of 4 weeks subjects returned for the corresponding challenge (allergen or diluent), and matching measurements of blood, bone marrow and spirometry were repeated. Series 2 was similar to series 1 however bone marrow and blood sampling was replaced by methacholine PC20 tests, and sputum induction was conducted at baseline pre-challenge and 24h and 48h post-challenge. E2

3 Allergen Inhalation Challenge Asthma was required to be stable (FEV 1 within 10% of baseline) to proceed with the inhalation challenges. Allergen-inhalation was performed as described by O Byrne et al. E1, E2. Specifically, subjects inhaled allergen to which they were sensitized, and allergen was selected using results from a skin prick test to a panel of allergens and the starting concentration was determined as described by Cockcroft et al. E3. Using a Wright s nebulizer, doublingconcentrations of allergen were inhaled until 20% fall in FEV 1 was reached. Spirometry was performed at regular intervals until 7 hours after allergen inhalation. The maximum percent fall in FEV 1 was recorded for the early (0-2h post-allergen) and late (3-7h post-allergen) phase responses. Where mentioned physiologic saline was used for diluent control challenges. Sputum Induction Sputum was induced by inhalation of 3, 4 and 5% saline and mucous plugs were selected and processed as previously described E4,E5. Briefly, sputum was treated with 4 times volume of Dulbecco's phosphate buffered saline (DPBS) and 4 times volume of dithiothreitol (DTT) and filtered through a nylon mesh, the cells were pelleted by centrifugation. Two cytospins were prepared and stained with Diff Quik for cell differentials and the remaining cells were stored at 4 C flow cytometry staining. The fluid phase was frozen for measurements of cytokines. Cell Isolation from Peripheral Blood and Bone Marrow Peripheral blood from venipuncture drawn into heparin (1000 units/ml) and bone marrow samples (5-10 ml) aspirated from the prosterior iliac crest were subject to density gradient E3

4 sedimentation on Accuprep (Accurate Chemical & Scientific Corp., New York, USA) as previously described in detail E6 (Dorman 2004). Immunofluorescence Staining and Flow Cytometric Gating Strategy Sputum-extracted cells and mononuclear cells from blood and bone marrow were stained for ILC2 and CD4-enumerations with antibodies to surface markers Lin-eFluor450, FcεR1- efluor450, CD45-BV786, ST2-PE, CRTH2- BV421, IL7Rα (CD127)-PE-cy7, and CD4-AF700 or relevant isotype controls (BD Bioscience, Mississauga, ON, Canada; ebiosciences, San Diego, CA; R&D Systems, MN, USA). Lin-cocktail antibodies to (CD2, CD3, CD14, CD16, CD19, CD56, CD235a) plus FcεR1 excluded T and B lymphocytes, B-cells, monocytes, eosinophils, NK cells and basophils. Cells were then incubated in Perm/Fix Buffer and optimal concentrations of intracellular stains for anti-il-5-fitc and anti-il-13 PerCP-Cy5.5 (BD Biosciences) were added. After incubation, cells were washed and resuspended in fixed in PBS with 1% paraformaldehyde and analyzed by a FACS LSRII flow cytometer within 24 h. Gating in the lympho-mononuclear region (low side scatter/low forward scatter) and following acquisition of 300,000 events, data were analyzed using FlowJo program to enumerate intracellular IL-5 and IL-13 levels in ILC2s (lin-cd45+cd127+st2+/crth2+) and CD3+CD4+ T lymphocytes. Detailed gating strategies are described in online supplement Figure E1 and E2 respectively. Absolute cell numbers were calculated from the percent positive population obtained from FACS analysis and total white cell count from sputum, or the total lympho/mononuclear cell count in blood and bone marrow. E4

5 FACS Sorting of ILC2 and In vitro Cell culture Blood derived mononuclear cells were stained with BV786-CD45, efluor450-lineage cocktail, AF647-CD127, and PE-ST2 or appropriate isotype-matched controls and sorted using a FACS Aria (BD Biosciences) into CD45+Lin+ and CD45+Lin-. The latter was further sorted into Lin-CD127+ST2+ (ILC2) and Lin-CD127+ST2- populations. Sorted cell populations were cultured at cells/ml, in 96-well tissue culture plates for 6 days (37 o C, 5% CO2) in the presence of medium alone or IL-33 (10 ng/ml) plus IL-2 (20 U/mL). Supernatants from each culture condition were collected and assayed for type 2 cytokines by multiplex ELISA as described below. Multiplex Analysis of Cytokines Cytokines in blood plasma, bone marrow plasma, sputum supernatants and cell culture supernatants were detected the multiplex cytokine assay performed at Eve Technologies (Eve Technologies Corp, Calgary, AB, Canada) by using the Bio-Plex 200 system (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and a Milliplex human cytokine array kits (Millipore, St. Charles, MO, USA). The Multiplex tested for IL-5, IL-13, IL-25, TSLP and IL33 and the lower limit of detection was IL-5:0.6 pg/ml; IL-13: 0.7 pg/ml; IL-25: 0.07 pg/ml; TSLP: 1.05 pg/ml and IL-33: 0.58 pg/ml). No correction factor was applied to the cytokine levels to adjust for sputum processing. E5

6 REFERENCES E1. O'Byrne, P. M., Dolovich, J., & Hargreave, F. E. (1987). Late asthmatic responses. Am.Rev.Respir.Dis., 136, E2. O'Byrne, P. M. (1988). Allergen-induced airway hyperresponsiveness. J.Allergy Clin.Immunol., 81, E3. Cockcroft, D. W., Davis, B. E., Boulet, L. P., Deschesnes, F., Gauvreau, G. M., O'Byrne, P. M. et al. (2005). The links between allergen skin test sensitivity, airway responsiveness and airway response to allergen. Allergy, 60, E4. Pizzichini E, Pizzichini MM, Efthimiadis A, Evans S, Morris MM, Squillace D, et al. Indices of airway inflammation in induced sputum: reproducibility and validity of cell and fluid-phase measurements. Am J Respir Crit Care Med 1996 Aug;154(2 Pt 1): E5.Sohani ZN, Strinich TX, Watson B, Smith SG, Torek A, Howie KJ, et al. Reproducibility of Sputum Differential Cell Counts Is not Affected by Squamous Epithelial Cells. J Asthma 2011;48: E6. Dorman SC, Sehmi R, Gauvreau GM, Watson RM, Foley R, Jones GL, Denburg JA, Inman MD, O'Byrne PM. Kinetics of bone marrow eosinophilopoiesis and associated cytokines after allergen inhalation. Am J Respir Crit Care Med Mar 1;169(5): E6

7 FIGURE LEGENDS Supplemental Figure E1: Representative gating strategy for absolute and ILC2 with isotype controls in sputum. (A) Representative plots from a patient show sequential gating of sputum cells to identify (A) Lineage-negative and CD45+ region, (B) Isotype controls, and (C) CD127+ST2+ stained cells. Intracellular staining for IL-5 and IL-13 compared to relevant isotype controls were assessed for lin-cd45+cd127+st2+ cells using a 98% confidence limit. Similar strategies were used for blood and bone marrow samples. Supplemental Figure E2: Representative gating strategy for CD4+ cells with isotype controls in sputum. Representative plots show sequential gating of sputum cells identified as (a) SSC(low) CD45+(region R1), (B) CD3+ cells (region R2), and (C) CD4+cells region R3). (D-I) Intracellular staining of cells in region R3 for IL-5, IL-13 or both compared to relevant isotype controls were assessed using a 98% confidence limit. Similar strategies were used for blood and bone marrow samples. Supplemental Figure E3: Spirometry was measured hourly post-allergen and diluentchallenge for the first 7 hours in series one and series 2 of the study. All subjects had a dual response following inhaled allergen, with a mean maximal fall in FEV 1 from baseline of 41.02±3.70% during the early response (p<0.05) and 23.67±4.57% during the late response (p<0.05). In contrast, there was no significant change in FEV 1 from baseline during the diluent challenge for both series 1 and series 2 challenges. E7

8 Supplemental Figure E4: Expression of CRTH2 and TSLPR on ILC2 (lin- CD45+CD127+ST2+ cells) and CD4+ lymphocytes (CD3+4+ cells) in sputum following diluent (open symbols) or allergen (closed symbols) challenge. There was a significant increase in the absolute number of ILC2 expressing (A) CRTH2 and (B) TSLPR at 24h post-ag which reduced to baseline by 48h post-ag. Although no change in CRTH2 expression on CD4+ T cells (C), a trend for an increase in TSLPR expression on CD4+ lymphocytes was observed at 48hr post-ag compared to baseline levels (p=0.08) (D). Horizontal lines represent median values of each data set. (n=10; *P<0.05; **P<0.01). Supplemental Figure E5: Expression of type 2 cytokines, TSLPR and ST2 on sputum ILC2 gated as lin-cd45+cd127+crth2+ cells following diluent (open symbols) or allergen (closed symbols) There was a significant increase in the absolute number of (A) ILC2, (B) IL- 5+, (C ) IL-13+, and (D) IL-5+IL13+ ILC2s 24h post-ag only. There was a significant increase in (E ) ST2, but not (F) TSLPR expression on sputum ILC2, 24 post-ag which returned to pre- Ag levels at 48h post-ag. Horizontal lines represent median values of each data set. (n=10; *P<0.05; **P<0.01). Supplemental Figure E6: In correlational assessments absolute numbers of (A) IL5+ILC2s and (B) CD4+ T Lymphocytes correlated significantly with absolute numbers of sputum eosinophil numbers enumerated at all time points (pre, 24 and 48h post-allergen for all ten subjects in this study). Data are presented as correlations with Spearman rank r-values and P<0.05 was considered significant. E8

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