Human group 2 innate lymphoid cells do not express the IL-5 receptor

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Human group 2 innate lymphoid cells do not express the IL-5 receptor Adam K.A. Wright, PhD, Cathryn Weston, PhD, Batika M.J. Rana, PhD, Christopher E. Brightling, PhD, FRCP, David J. Cousins, PhD Journal of Allergy and Clinical Immunology Volume 140, Issue 5, Pages 1430-1433.e4 (November 2017) DOI: 10.1016/j.jaci.2017.04.025 Copyright © 2017 The Authors Terms and Conditions

Fig 1 CD125 protein is absent on blood ILC2s. A, Total PBMCs, singlets within boxed region. B, Singlet light scatter with lymphocyte gate overlaid. C, Lineage−, CD294+ cells were identified (boxed region) encompassing (D) CD123+ basophils and CD123− ILC2s, in the upper and lower left quadrant, respectively, with CD125 GMFI values. E, ILC2 light scatter properties. F, Overlay of basophil and ILC2 CD125-stained cells in the absence (red) or presence (blue) of rhIL-5. (G) Basophil and (H) ILC2 CD125 GMFI paired data (mean ± SD) from 6 healthy volunteers (open) and 13 (filled) volunteers with asthma, also shown in (I) unpaired on the same axes. Statistical comparisons were made using a Wilcoxon matched-pairs signed rank test. APC, Allophycocyanin; FITC, fluorescein isothiocyanate; FSC-A, forward scatter-area; FSC-H, forward scatter-height; SSC-A, side scatter-area. Journal of Allergy and Clinical Immunology 2017 140, 1430-1433.e4DOI: (10.1016/j.jaci.2017.04.025) Copyright © 2017 The Authors Terms and Conditions

Fig 2 CD125 protein is absent on lung-derived ILC2s. A, Total lung tissue cells, singlets within boxed region. B, Singlet light scatter properties with lymphocyte gate overlaid. C, Viable CD45+ cells (boxed region). D, Lineage− CD294+ cells (boxed region). E, ILC2s (CD123−CD127+) and basophils (CD123+CD127−) were identified. (F) Basophil CD125 fluorescence and (G) ILC2 CD125 fluorescence in the absence (red) or presence of rhIL-5 (blue). H, Collated basophil and ILC2 CD125 GMFI (mean ± SD) data (n = 7). I, Proportion of CD125+ basophils and ILC2s (calculated using SED); exemplar values given in Fig 2, F and G. Statistical comparisons were made using a Wilcoxon matched-pairs signed rank test. FITC, Fluorescein isothiocyanate; FSC-A, forward scatter-area; FSC-H, forward scatter-height; SSC-A, side scatter-area. Journal of Allergy and Clinical Immunology 2017 140, 1430-1433.e4DOI: (10.1016/j.jaci.2017.04.025) Copyright © 2017 The Authors Terms and Conditions

Fig E1 ILC2s do not express IL-5Rα transcripts. A, Schematic representation of IL-5Rα mRNA showing the splice variants and probe sets Hs00602482_m1 (detecting soluble and transmembrane variants) and Hs00236871_m1 (transmembrane only) used in this study. ΔCT values were calculated relative to 18S mRNA for (B) all IL-5Rα transcript variants using the Hs00602482_m1 probe and (C) IL-5 transcripts using Hs00174200_m1. In Fig E1, B and C, data are derived from isolated ILC2s (n = 3) that were grown for up to 14 days in the presence of rhIL-2, rhIL-7, rhIL-25, and rhIL-33. In Fig E1, B, cDNA generated from freshly isolated eosinophils was also included as a positive control in each IL-5Rα assay. In Fig E1, C, IL-5 transcript data are shown to confirm activation in the presence of cytokines; low ΔCT values reflect high abundance of the IL-5 transcript. n.d., Not detected. Journal of Allergy and Clinical Immunology 2017 140, 1430-1433.e4DOI: (10.1016/j.jaci.2017.04.025) Copyright © 2017 The Authors Terms and Conditions