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. 2017 Jun 5;214(6):1663–1678. doi: 10.1084/jem.20161653

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© 2017 Taylor et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

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Figure 7. — PD-1 regulation of KLRG1⁺ILC-2 is conserved in human PBMCs. Human PBMCs were obtained from normal healthy volunteers and characterized for the expression of PD-1 in the various ILC subsets. Gating strategy included Lin⁻CD45⁺CD127⁺GATA3⁺KLRG1⁺ (A). PD-1 expression was analyzed in n = 5 donors (A and B). ILC-2 cells were gated as either GATA3⁺PD-1⁻ or GATA3⁺PD-1⁺, and then characterized for cytokine expression (C and D). PBMCs were stimulated with rhIL-2 (1,000 IU) + Vehicle (DMSO) + Isotype control for 15 min, and then p-STAT5 expression was measured by flow cytometry. Certain cohorts were treated with rhIL-2 + Vehicle + αPD-1 or rhIL-2 + Tofacitinib (0.3 µM) + αPD-1. The representative flowplot and mean fluorescence intensity of pSTAT5 in various cohorts were measured by flow cytometry (E and F; n = 4 donors). PBMCs were labeled with Cell Trace Violet. and then ILC-2s were stimulated with rhIL-2 + rhIL-7 (40 ng/ml) + Vehicle (DMSO) + Isotype control or with rhIL-2 + rhIL-7 + Vehicle + αPD-1 or with rhIL-2 + rhIL-7 + Tofacitinib + αPD-1. Dilution of Cell Trace Violet as a measure of proliferation was performed at day 5 after stimulation (G; n = 4 donors). Experiments were performed in multiple donors as stated, and data are represented as mean + SEM. Human PBMCs (4 million) were adoptively transferred into NSG murine recipients and treated with rhIL-2, rhIL-7, and rhIL-33 for 3 d, along with Vehicle (DMSO) and Isotype control antibody. This cohort is termed as control; certain cohorts received either αPD-1 or αPD-1 and Tofacitinib, in addition to rhIL-2, rhIL-7, and rhIL-33. At day 3 after adoptive transfer, lungs were harvested and stimulated with PMA/ionomycin, and then human ILC-2 subsets were characterized. Human CD45⁺ cells were gated followed by CD45⁺Lin⁻ CD127⁺GATA3⁺KLRG1⁺ gating. Cytokine expression of KLRG1⁺ ILC-2s were evaluated in the various cohorts (H). For in vivo experiments, each cohort had n = 4 mice; data are represented as mean ± SEM. A Student’s t test was performed to determine statistical significance in C and D, and a one-way ANOVA analysis followed by a multiple comparison test [Tukey] was performed to determine statistical significance between the various cohorts in F–H. P ≤ 0.05 was considered significant. Significant p-values are denoted in the figures.

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