Human group 2 innate lymphoid cells do not express the IL-5 receptor
Authors: Adam K.A. Wright, Cathryn Weston, Batika M.J. Rana, Christopher E. Brightling, David J. Cousins
Journal: Journal of Allergy and Clinical Immunology (2017)

Eosinophils, cardinal effector cells of type 2 inflammation, contribute to the clinical and immunopathologic manifestations of asthma1Russell R. Brightling C. Mepolizumab for the reduction of exacerbations in severe eosinophilic asthma.Expert Rev Respir Med. 2016; 10: 607-617Crossref PubMed Scopus (10) Google Scholar and chronic obstructive pulmonary disease2Brightling C.E. Bleecker E.R. Panettieri Jr., R.A. Bafadhel M. She D. Ward C.K. et al.Benralizumab for chronic obstructive pulmonary disease and sputum eosinophilia: a randomised, double-blind, placebo-controlled, phase 2a study.Lancet Respir Med. 2014; 2: 891-901Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar inflammatory endotypes. Eosinophil biology is governed by IL-5, a cytokine that binds with high affinity to a specific IL-5 receptor α subunit (IL-5Rα) before forming a heterodimeric receptor complex with the β subunit.3Tavernier J. Devos R. Cornelis S. Tuypens T. Van der Heyden J. Fiers W. et al.A human high affinity interleukin-5 receptor (IL5R) is composed of an IL5-specific alpha chain and a beta chain shared with the receptor for GM-CSF.Cell. 1991; 66: 1175-1184Abstract Full Text PDF PubMed Scopus (494) Google Scholar IL-5 signaling promotes differentiation, maturation, and survival of eosinophil-committed progenitors while acting on mature eosinophils to enhance their migratory potential and effector responses.4Varricchi G. Bagnasco D. Borriello F. Heffler E. Canonica G.W. Interleukin-5 pathway inhibition in the treatment of eosinophilic respiratory disorders: evidence and unmet needs.Curr Opin Allergy Clin Immunol. 2016; 16: 186-200Crossref PubMed Scopus (147) Google Scholar IL-5 signaling also promotes alternative splicing of the IL-5Rα gene to generate transmembrane forms of IL-5Rα.5Tavernier J. Tuypens T. Plaetinck G. Verhee A. Fiers W. Devos R. Molecular basis of the membrane-anchored and two soluble isoforms of the human interleukin 5 receptor alpha subunit.Proc Natl Acad Sci U S A. 1992; 89: 7041-7045Crossref PubMed Scopus (120) Google Scholar Because the IL-5-IL-5R axis appears to be restricted to eosinophils and basophils (and their progenitors),6Kolbeck R. Kozhich A. Koike M. Peng L. Andersson C.K. Damschroder M.M. et al.MEDI-563, a humanized anti-IL-5 receptor alpha mAb with enhanced antibody-dependent cell-mediated cytotoxicity function.J Allergy Clin Immunol. 2010; 125: 1344-1353.e2Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar therapeutic regulation of these cells through the neutralization of circulating IL-5 (eg, mepolizumab and reslizumab)1Russell R. Brightling C. Mepolizumab for the reduction of exacerbations in severe eosinophilic asthma.Expert Rev Respir Med. 2016; 10: 607-617Crossref PubMed Scopus (10) Google Scholar or IL-5Rα ligation (eg, benralizumab),1Russell R. Brightling C. Mepolizumab for the reduction of exacerbations in severe eosinophilic asthma.Expert Rev Respir Med. 2016; 10: 607-617Crossref PubMed Scopus (10) Google Scholar, 2Brightling C.E. Bleecker E.R. Panettieri Jr., R.A. Bafadhel M. She D. Ward C.K. et al.Benralizumab for chronic obstructive pulmonary disease and sputum eosinophilia: a randomised, double-blind, placebo-controlled, phase 2a study.Lancet Respir Med. 2014; 2: 891-901Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar have emerged as effective strategies to deplete blood, tissue, and airway eosinophils and consequently reduce exacerbation rates and improve lung function.1Russell R. Brightling C. Mepolizumab for the reduction of exacerbations in severe eosinophilic asthma.Expert Rev Respir Med. 2016; 10: 607-617Crossref PubMed Scopus (10) Google Scholar Basophils and group 2 innate lymphoid cells (ILC2s) are important innate sources of type 2 cytokines, including IL-5, in response to epithelial-derived cytokines such as IL-33.7Martinez-Gonzalez I. Steer C.A. Takei F. Lung ILC2s link innate and adaptive responses in allergic inflammation.Trends Immunol. 2015; 36: 189-195Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 8Smithgall M.D. Comeau M.R. Yoon B.R. Kaufman D. Armitage R. Smith D.E. IL-33 amplifies both Th1- and Th2-type responses through its activity on human basophils, allergen-reactive Th2 cells, iNKT and NK cells.Int Immunol. 2008; 20: 1019-1030Crossref PubMed Scopus (494) Google Scholar ILC2s, however, have emerged as central and critical innate coordinators of steady-state eosinophilopoiesis and epithelial cell–driven, type 2 immunopathology in asthma.8Smithgall M.D. Comeau M.R. Yoon B.R. Kaufman D. Armitage R. Smith D.E. IL-33 amplifies both Th1- and Th2-type responses through its activity on human basophils, allergen-reactive Th2 cells, iNKT and NK cells.Int Immunol. 2008; 20: 1019-1030Crossref PubMed Scopus (494) Google Scholar As an important upstream regulator of eosinophil function we asked whether ILC2s, like their basophil counterparts, express the IL-5Rα subunit because this may have important implications for our understanding of the role of the IL-5-IL-5R axis in disease and therapeutic targeting of these rare but important innate immune cells. Detailed methods are provided in this article's Online Repository at www.jacionline.org. ILC2s are rare innate lymphocytes that lack the T-cell receptor complex and all known lineage markers but, similar to other type 2 cytokine-producing cells such as TH2 cells, eosinophils, and basophils, express the type 2 prostaglandin D2 receptor, DP2/CRTH2 (CD294).9Jackson D.J. Makrinioti H. Rana B.M. Shamji B.W. Trujillo-Torralbo M.B. Footitt J. et al.IL-33-dependent type 2 inflammation during rhinovirus-induced asthma exacerbations in vivo.Am J Respir Crit Care Med. 2014; 190: 1373-1382Crossref PubMed Scopus (114) Google Scholar We defined blood ILC2s as cells with singlet, lymphocyte light scatter properties (Fig 1, A and B), lineage (CD2, 3, 14, 16, 19, 56, and 235a)− but CD294+ (Fig 1, C). A large proportion of cells within this gate were basophils, (CD123+ cells in Fig 1, D); however, CD294+, CD123− ILC2s were present (Fig 1, D, and light scatter in E). Basophils displayed a higher level of CD125-phycoerythrin (PE) staining than did the ILC2s (Fig 1, D) that could be blocked in the presence of rhIL-5 (Fig 1, F), confirming the specificity of the antibody and basophil IL-5Rα expression. In contrast to basophils, there was no change in CD125-PE signal intensity when ILC2s were incubated with rhIL-5, indicating that there was no IL-5Rα expression on these cells (Fig 1, F). To confirm the reproducibility of these findings, we recruited 6 control and 13 volunteers with asthma (see clinical details in Table E1 in this article's Online Repository at www.jacionline.org) and measured IL-5Rα expression. The total mean (SD) % of ILC2s within the lymphocyte gate for both control and asthma groups were 0.1% ± 0.1% and 0.03% ± 0.03%, respectively (P = .06, Mann-Whitney). In total, combining asthma and control data sets, the basophil CD125 geometric mean fluorescence intensity (GMFI) (mean ± SD) in the absence or presence of rhIL-5 was 1285 ± 614 and 401 ± 315 respectively, representing a significant reduction in GMFI (Fig 1, G and I). In contrast, the ILC2 CD125 GMFI (mean ± SD) in the absence or presence of rhIL-5 was 45 ± 45 and 42 ± 33, respectively (Fig 1, H and I). In a subset of samples (n = 3), where basophils and ILC2s could be detected in whole blood, data were qualitatively similar. There were no significant differences between the control and asthma subjects for any of the flow cytometric measurements obtained. We next sought to confirm whether the IL-5Rα data were reflected at the RNA level using real-time PCR probes spanning the exon boundaries present within IL-5Rα subunit variants (see Fig E1, A, in this article's Online Repository at www.jacionline.org). For this, ILC2s were isolated from 3 additional donors and cultured in the absence or presence of cytokines known to enhance survival (IL-2 and IL-7) and activate ILC2s (IL-25 and IL-33). RNA was isolated at 6 time points (day 0, 1, 2, 4, 7, and 14) and converted into cDNA. Eosinophil cDNA was included as a positive control. The IL-5Rα transcripts were not detected in ILC2s at any time point; however, an increase in IL-5 mRNA was observed, indicating that the culture conditions were sufficient to activate the cells (Fig E1, B and C). Finally, using a similar approach to the blood analyses (Fig 1) and using surgically removed lung tissue from 7 subjects (see Table E2 in this article's Online Repository at www.jacionline.org), we asked whether tissue-derived ILC2s and basophils expressed IL-5Rα. Tissue-derived basophils were defined as viable, CD45+Lin−CD294+CD123+CD127− cells and ILC2s as viable, CD45+Lin−CD294+CD123−CD127+ cells present within the singlet lymphocyte gate (Fig 2, A-E). Basophil CD125-PE GMFI (mean ± SD, 612 ± 327) could be reversed in the presence of rhIL-5 to a mean of 317 ± 91 (example in Fig 2, F, and cumulative data in Fig 2, H). Using the super enhanced Dmax (SED) algorithm to compare basophil populations stained in the absence or presence of IL-5 for each of the 7 donors revealed that 71.1% ± 4.2% basophils were CD125 positive (Fig 2, I). In contrast, ILC2 CD125 GMFI (mean ± SD) was significantly lower than that of basophils and showed little change in the presence of rhIL-5 (190 ± 42 and 145 ± 49, respectively) (example in Fig 2, G, and cumulative data in Fig 2, H). SED analysis of ILC2s stained in the absence or presence of rhIL-5 for each of the 7 donors (Fig 2, I) did not reveal a CD125-positive subset (mean ± SD, 3.3% ± 0.89%). Data from these 7 subjects consistently show that tissue-derived basophils express the IL-5Rα whereas tissue-derived ILC2s do not (Fig 2, H). The strength(s) of this study lies in the fact that we have measured protein IL-5Rα subunit expression in a rare critical regulator of type 2 inflammation, ILC2s, and in the context of rhIL-5–mediated receptor blockade/downregulation, rather than an isotype control, which is less robust. The robustness of our data using human peripheral blood (from donors with asthma), ex vivo activated cells, and lung tissue cells diminishes the likelihood that IL-5Rα–expressing ILC2s are present in asthmatic tissue. Although it would be desirable to extend our blood and tissue observations to investigate whether ILC2s in bronchial biopsies from patients with asthma express IL-5Rα, the paucity of tissue ILCs and the need for multiple immunological markers to positively identify them severely limits this approach. These results extend the list of cells6Kolbeck R. Kozhich A. Koike M. Peng L. Andersson C.K. Damschroder M.M. et al.MEDI-563, a humanized anti-IL-5 receptor alpha mAb with enhanced antibody-dependent cell-mediated cytotoxicity function.J Allergy Clin Immunol. 2010; 125: 1344-1353.e2Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar that are known not to express the IL-5Rα subunit, specific for the biological activities of IL-5. Moreover, we show that rare tissue-derived basophils, like their blood counterparts, express the IL-5Rα. Our data suggest that the success of therapeutic interventions targeting IL-5/R is unlikely to be mediated directly on ILC2s but may function via both eosinophils and basophils. We thank all research participants for taking part in this study and Tracy Thornton and Marcia Soares for their assistance with volunteer recruitment and data collection. We also thank Prof Andy Wardlaw, Paige Tongue, Malgorzata Rekas, Will Monteiro, Dr Amanda Sutcliffe, Beverley Hargadon, Sarah Parker, and Hilary Marshall for their assistance in the procurement and processing of lung tissue. Control subjects (without a history of respiratory disease) and subjects with an asthma diagnosis were recruited at University Hospitals of Leicester NHS Trust, Leicester, United Kingdom (UK), and provided informed written consent, under ethics (REC reference no. 08/H0406/189) approved by the Leicestershire, Northamptonshire, and Rutland ethics committee, to take part in this study. The recruited subjects with asthma were mostly diagnosed with late-onset asthma. Lung resection material was ethically obtained at University Hospitals of Leicester NHS Trust, under the auspices of the Midlands lung tissue consortium (REC reference no. 07/MRE08/42), from a separate cohort of patients undergoing lung surgery. Clinical characteristics can be found in Table E1 (blood) and Table E2 (lung tissue). Additional control subjects (without a history of respiratory disease) were also recruited under ethical approval from the research ethics committee of Guy's Hospital, London, UK (REC reference no. 09/H804/077) as described previouslyE1Jackson D.J. Makrinioti H. Rana B.M. Shamji B.W. Trujillo-Torralbo M.B. Footitt J. et al.IL-33-dependent type 2 inflammation during rhinovirus-induced asthma exacerbations in vivo.Am J Respir Crit Care Med. 2014; 190: 1373-1382Crossref PubMed Scopus (417) Google Scholar to isolate ILC2 cDNA for downstream experiments. Human eosinophils were purified from whole blood using CD16 microbead depletion of neutrophils over CS columns (both Miltenyi Biotec, Surrey, UK) to a purity of 97% as described previously.E2Wright A. Mahaut-Smith M. Symon F. Sylvius N. Ran S. Bafadhel M. et al.Impaired P2X1 receptor-mediated adhesion in eosinophils from asthmatic patients.J Immunol. 2016; 196: 4877-4884Crossref PubMed Scopus (9) Google Scholar Purified eosinophils were immediately placed into Qiazol (Qiagen, Manchester, UK) for downstream RNA isolation and used as a positive control for the detection of transmembrane and soluble mRNA variants of the IL-5Rα gene. ILC2s were isolated from peripheral blood by flow cytometric cell sorting and cultured in vitro as described in detail previously.E1Jackson D.J. Makrinioti H. Rana B.M. Shamji B.W. Trujillo-Torralbo M.B. Footitt J. et al.IL-33-dependent type 2 inflammation during rhinovirus-induced asthma exacerbations in vivo.Am J Respir Crit Care Med. 2014; 190: 1373-1382Crossref PubMed Scopus (417) Google Scholar Briefly, ILC2s were grown at 37°C, 5% CO2, in a humidified incubator at a density of 10,000 cells/mL in RPMI 1640 supplemented with 10% FBS, glutamine, penicillin, and streptomycin (all Thermofisher, Paisley, UK). Recombinant cytokines were added as described in the figure legend (rhIL-2 [50 U/mL; Eurocetus, Amsterdam, The Netherlands], rhIL-7 [50 ng/mL; R&D Systems, Abingdon, UK], rhIL-25 [125 ng/mL; R&D Systems], and rhIL-33 (50 ng/mL; R&D Systems]). Lung tissue cells were obtained from enzymatically digested lung obtained within 1 hour of resection (n = 7). Lung tissue bathed in media (Dulbecco modified Eagle medium, with 2% FBS) was cut into small pieces and enzymatically digested using hyaluronidase (0.75 mg/mL final, H3506, Sigma, Gillingham, Dorset, UK) and collagenase (0.75 mg/mL final, C2674, Sigma) for 75 minutes at 37°C. Digested tissue was passed through a syringe, filtered through a 100- and 50-μm gauze, then washed twice with media (centrifuging at 230g for 8 minutes, 4°C). Cell counts were performed using hemocytometer and Kimura stain, washed again, and the cell suspension was prepared for flow cytometry, as described below. PBMCs were isolated following centrifugation of whole blood over Lymphoprep (Stem Cell Technologies, Cambridge, UK). PBMCs were washed in PBS containing 2% FBS (Thermofisher) and 2 mM EDTA (Thermofisher) and the cell density was adjusted to 1 × 106/100 μL before cell surface staining. For lung tissue cells, at least 5 × 105 cells were resuspended in PBS and stained with Zombie Aqua fixable viability kit (as described by the manufacturer [Biolegend, London, UK]) for 20 minutes at room temperature. Following incubation, lung tissue cells were washed and resuspended in 50 μL brilliant violet stain buffer (Becton Dickinson, Oxford, UK) before cell surface staining. A total of 1 × 106 PBMCs or lung tissue cells were then incubated at room temperature for 15 minutes with Human Trustain (Biolegend, London, UK) to block Fc receptors and either a vehicle control (PBS/2%FBS) or rhIL-5 (50 or 100 ng/mL final for PBMCs and lung tissue cells, respectively [Miltenyi Biotec]), to block cell surface staining of IL-5Rα. Cells were then incubated at room temperature with the following anti-human mAbs in a fluorescence minus oneE3Herzenberg L.A. Tung J. Moore W.A. Herzenberg L.A. Parks D.R. Interpreting flow cytometry data: a guide for the perplexed.Nat Immunol. 2006; 7: 681-685Crossref PubMed Scopus (336) Google Scholar set-up: BV421-conjugated CD123 (clone 6H6, Biolegend), fluorescein isothiocyanate–conjugated lineage (α-CD2, α-CD3, α-CD14, α-CD16, α-CD19, α-CD56, α-CD235a) cocktail (Affymetrix, Cheshire, UK), phycoerythrin-conjugated α-CD125 (clone A14, Becton Dickinson), and AF647-conjugated α-CD294 (clone BM16, Biolegend). In some experiments, whole blood (100 μL) was stained as described above. For single-cell suspensions derived from lung tissue, the lineage cocktail, CD125, and CD294 antibodies (described above) were supplemented with BV421-conjugated CD127 (clone A019D5), BV605-conjugated CD123 (clone 6H6), and BV785-conjugated CD45 (clone HI30) (all Biolegend) to enable identification of tissue-derived ILC2s and basophils. PBMCs were washed with PBS/2%FBS before acquisition, whereas whole blood or lung tissue cells were fixed and lysed (1-step fix and lyse, Affymetrix) to remove erythrocytes according to the manufacturer's protocol. Cell acquisition was performed on an Attune flow cytometer operating NxT software v2.2 (Life Technologies). CD125-PE and rhIL-5 (for blocking purposes) were both titrated in pilot experiments. Compensation beads (Becton Dickinson, High Wycombe, UK) and unstained cells were used to determine compensation settings. Analysis was performed using FlowJo v10 (Tree star, Ashland, Ore). RNA was isolated from purified cell populations using the miRNeasy mini kit (Qiagen), followed by DNAse digestion (Thermofisher) and RNeasy minElute cleanup (Qiagen) according to the respective manufacturer's instructions. RNA integrity was assessed using a Bioanalyser (Agilent, Edinburgh, UK), according to the manufacturer's instructions. For eosinophil samples, cDNA synthesis was performed using superscript II reverse transcriptase (Thermofisher), random hexamers (Roche, West Sussex, UK), and dNTPs (Thermofisher) according to the manufacturer's instructions. Because of low cell numbers, ILC2 cDNA samples were amplified using the Ovation PicoSL WTA system V2 kit (NuGEN, Leek, Netherlands) as per the manufacturer's instructions. Real-time RT-PCR was performed as described previouslyE4Lam E.P. Kariyawasam H.H. Rana B.M. Durham S.R. McKenzie A.N. Powell N. et al.IL-25/IL-33-responsive TH2 cells characterize nasal polyps with a default TH17 signature in nasal mucosa.J Allergy Clin Immunol. 2016; 137: 1514-1524Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar using TaqMan Master mix II (Thermofisher) and TaqMan probe sets Hs00602482_m1 (transmembrane and soluble variants of the IL-5Rα), Hs00236871_m1 (transmembrane only variant of IL-5Rα), or Hs00174200_m1 (IL-5) (Thermofisher) and compared with an 18S rRNA endogenous control (Thermofisher), using FAM-MGB (IL-5Rα probe sets) and VIC-MGB (18S rRNA) dyes, respectively. Cycling conditions were as follows: 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds then 60°C for 1 minute for 50 cycles on ViiA7 real-time PCR system (Thermofisher). Because IL-5Rα transcripts were undetectable, to confirm that a sufficient amount of ILC2 cDNA from each condition was present in the reaction, the mean (±SEM) 18S CT values were as follows: D0 (18.4 ± 0.18), D1 (18.5 ± 0.23), D2 (18.0 ± 0.17), D4 (17.4 ± 0.01), D7 (17.4 ± 0.03), D14 (16.8 ± 0.08), eosinophils (16.2 ± 0.4) (representative results from 1 donor). Furthermore, to confirm that ILC2s were activated in the presence of rhIL-2/7/25 and 33, we have presented IL-5 transcript data alongside the IL-5Rα data in Fig E1. Descriptive and comparative (super enhanced Dmax SED) subtraction % positiveE5Roederer M. Treister A. Moore W. Herzenberg L.A. Probability binning comparison: a metric for quantitating univariate distribution differences.Cytometry. 2001; 45: 37-46Crossref PubMed Scopus (95) Google Scholar analyses of flow cytometric population data were performed using FlowJo version 10 (Tree Star). Descriptive and comparative analyses were performed using Graph Pad PRISM v6 (GraphPad software, Inc, La Jolla, Calif), and Shapiro-Wilk normality test was used to determine data distribution. Statistical comparisons were made using paired or unpaired tests where relevant, as indicated, with statistical significance determined if the P value was less than .05.Table E1Clinical characteristics of the study participants recruited for blood collectionCharacteristicControlAsthmaP value∗Unpaired t test.n613Age (y)†Mean ± SD.50 ± 1559 ± 13.18Sex: M:F4:26:7.63Smoking history (never, ex, current)‡All ex-smokers have quit smoking for 3+ years.5, 1, 09, 4, 01.0BMI (kg/m2)†Mean ± SD.27 ± 429 ± 6.47Post-BD FEV1 (L)†Mean ± SD.3.6 ± 0.82.5 ± 0.9.02Post-BD FEV1 (% predicted)†Mean ± SD.109 ± 1692 ± 24.17FEV1/FVC (ratio)†Mean ± SD.0.82 ± 0.060.71 ± 0.09.01WBC (×109/L)†Mean ± SD.6.4 ± 0.9§n = 5.7.1 ± 2.8‖n = 12..63Eosinophils (×109/L)†Mean ± SD.0.16 ± 0.04§n = 5.0.28 ± 0.16‖n = 12..12Eosinophil (%)2.5 ± 0.6§n = 5.4.3 ± 2.4‖n = 12..13Basophils (×109/L)†Mean ± SD.0.04 ± 0.02§n = 5.0.04 ± 0.02‖n = 12..97GINA 1, 2, 3, 4, 5NA1, 1, 0, 7, 4NABeclomethasone dipropionate equivalents (μg)¶Median (interquartile range).NA1000 (650-1600)NABD, Bronchodilator; BMI, body mass index; F, female; FVC, forced vital capacity; GINA, Global Initiative for Asthma; M, male; NA, not applicable; WBC, white blood cell.∗ Unpaired t test.† Mean ± SD.‡ All ex-smokers have quit smoking for 3+ years.§ n = 5.‖ n = 12.¶ Median (interquartile range). Open table in a new tab Table E2Clinical characteristics of the study participants recruited for lung tissue collectionCharacteristicControln7Age (y)∗Mean ± SD.66 ± 9Sex: M:F4:3Smoking history (never, ex, current)†All ex-smokers have quit smoking for 2+ years.0, 4, 3WBC (×109/L)∗Mean ± SD.9.5 ± 3.7Eosinophils (×109/L)∗Mean ± SD.0.24 ± 0.18Eosinophil (%)∗Mean ± SD.2.4 ± 1.1Basophils (×109/L)∗Mean ± SD.0.04 ± 0.03DiagnosisAdenocarcinoma (n = 4)Squamous cell carcinoma (n = 2)Lung volume reduction surgery (n = 1)F, Female; M, male; WBC, white blood cell.∗ Mean ± SD.† All ex-smokers have quit smoking for 2+ years. Open table in a new tab BD, Bronchodilator; BMI, body mass index; F, female; FVC, forced vital capacity; GINA, Global Initiative for Asthma; M, male; NA, not applicable; WBC, white blood cell. F, Female; M, male; WBC, white blood cell.