Differential expression of functional chemokine receptors on human blood and lung group 2 innate lymphoid cells Cathryn A. Weston, PhD, Batika M.J. Rana,

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Differential expression of functional chemokine receptors on human blood and lung group 2 innate lymphoid cells Cathryn A. Weston, PhD, Batika M.J. Rana, PhD, David J. Cousins, PhD Journal of Allergy and Clinical Immunology Volume 143, Issue 1, Pages 410-413.e9 (January 2019) DOI: 10.1016/j.jaci.2018.08.030 Copyright © 2018 The Authors Terms and Conditions

Fig 1 ILC2s and T cells isolated from human lung display different chemokine receptors to those derived from blood. A, Percentage of CD3+ T cells from blood (white) and lung tissue (gray) expressing the indicated chemokine receptors. B, Percentage of ILC2s from blood (white) and lung (red) expressing the indicated chemokine receptors. Data are mean ± SEM of 11 (PBMCs) and 5 (lung) independent donors. Journal of Allergy and Clinical Immunology 2019 143, 410-413.e9DOI: (10.1016/j.jaci.2018.08.030) Copyright © 2018 The Authors Terms and Conditions

Fig 2 ILC2s respond to various chemokine receptor ligands in a dose-dependent manner. Activation of (A) CCR2, (B) CCR4, (C) CCR5, and (D) CCR6 receptors on ILC2s and T cells was determined following 10-second stimulation with a range of concentrations of the indicated ligands. E, Normalized ILC2 response for each receptor/ligand combination. All data are mean ± SEM from 5 independent experiments. MFI, Mean fluorescence intensity. Journal of Allergy and Clinical Immunology 2019 143, 410-413.e9DOI: (10.1016/j.jaci.2018.08.030) Copyright © 2018 The Authors Terms and Conditions

Fig E1 Gating strategy to identify ILC2s and T cells from human peripheral blood (A-E) and lung (F-K). A, Total PBMCs displayed with singlet gate overlaid. B, Lymphocytes (gated cells) selected from single-cell population based on light scattering properties. C, T cells identified as the CD3+ population of single-cell lymphocytes. D, Lineage−, CD294+ cells (boxed region) containing the CD123− ILC2s were also selected from the single-cell lymphocyte population (E). F, Total cells displayed with singlet gate overlaid. G, Lymphocytes (gated cells) selected from single-cell population based on light scattering properties. H, Live (zombie aqua−), CD45+ cells shown in gated region were selected and T cells were identified as the CD3+ population (I). J, Lineage−, CD294+ cells (boxed region) containing the CD123− ILC2s were also selected from the live, CD45+, single-cell lymphocyte population (K). FSC-A, Forward scatter-area; FSC-H, forward scatter-height; SSC-A, side scatter-area. Journal of Allergy and Clinical Immunology 2019 143, 410-413.e9DOI: (10.1016/j.jaci.2018.08.030) Copyright © 2018 The Authors Terms and Conditions

Fig E2 Chemokine receptor expression on T cells and ILC2s in human blood. Representative flow cytometry data showing the expression of the indicated chemokine receptors on CD3+ T cells (left) and ILC2s (right). Receptor+ gate determined using bimodal distribution observed on T-cell populations; numbers within gates indicate percentage cells. Journal of Allergy and Clinical Immunology 2019 143, 410-413.e9DOI: (10.1016/j.jaci.2018.08.030) Copyright © 2018 The Authors Terms and Conditions

Fig E3 Expression of chemokine receptors on ILC2s and T cells isolated from lung tissue. Representative flow cytometry data showing the expression of the indicated chemokine receptors on CD3+ T cells (left) and ILC2s (right). Receptor+ gate determined using bimodal distribution observed on T-cell populations; numbers within gates indicate percentage cells. Journal of Allergy and Clinical Immunology 2019 143, 410-413.e9DOI: (10.1016/j.jaci.2018.08.030) Copyright © 2018 The Authors Terms and Conditions

Fig E4 A-J, Quantification of chemokine receptor RNA expression in T cells and ILC2s. Relative quantities of the indicated genes were calculated relative to 18S mRNA. Plots show mean values (±SEM) for T cells (black boxes) and ILC2s (gray boxes) sorted from PBMCS obtained from 5 independent donors. Journal of Allergy and Clinical Immunology 2019 143, 410-413.e9DOI: (10.1016/j.jaci.2018.08.030) Copyright © 2018 The Authors Terms and Conditions

Fig E5 Chemokines can be used to stimulate human blood cells in a time-dependent manner. (A) CD3+ T cells and (B) Lineage−, CD123−, CD294+ ILC2s were identified from within a single-cell, lymphocyte gate. C, CCR4 expression on T cells (gray) and ILC2s (red) with gates used to select positive and negative populations overlaid. Phalloidin staining was analyzed for (D) CCR4− T cells, (E) CCR4+ T cells, and (F) CCR4+ ILC2s at the indicated time points. Receptor response was determined for (G) chemokine receptor–positive T cells and (H) chemokine receptor–positive ILC2s at each time point by normalizing the phalloidin MFI to that observed on CCR4− cells. Data shown are mean ± SEM of 5 independent donors. MFI, Mean fluorescence intensity; SSC-A, side scatter-area. Journal of Allergy and Clinical Immunology 2019 143, 410-413.e9DOI: (10.1016/j.jaci.2018.08.030) Copyright © 2018 The Authors Terms and Conditions