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. 2020 Mar 25;204(9):2600–2611. doi: 10.4049/jimmunol.2000025

FIGURE 6.

FIGURE 6.

Monitoring Gata3 expression in immature BM cell subsets. (A) Flow cytometric analysis of Gata3-vYFP reporter expression in Lin− BM cells. Left panels, Gating strategy and representative histograms of vYFP reporter expression. Scatter plot: percentage of Lin− BM cells with low to intermediate (low/int) and high reporter expression in homozygous GATIR mice. Classification in low/int and high cells is based on the gating shown in the histogram to the left. (B) Immunophenotypic characterization of vYFPhigh cells from homozygous GATIR mice for eight informative cell surface markers (dark histograms). As internal reference for staining intensities, open histogram overlays represent the corresponding staining pattern on Lin− cells (CD25, CD90, CD117, CD135, and Sca-1 panels), on all live cells (CD127 panel), on cells with low-to-intermediate reporter expression (CD2 panel), and on BM Lin− (CD11b−, CD11c−, CD19−, NK1.1−, GR1−, and TER119−) CD3+ T cells (CD44 panel), analyzed in the same sample. (C) All ILC2Ps identified as Lin−, CD90high, Sca-1high, CD127+, and CD25+ cells following a published gating scheme (10) are homogenously Gata3-vYFPhigh cells. (D) No difference in total number of ILC2Ps in BM of wild-type (WT) (WT/WT) and homozygous GATIR (knock-in [KI]/KI) mice. ILC2Ps were identified based on the gating scheme shown in (C) (i.e., independent of reporter expression). (E) GATIR mice allow identification of ILC2Ps within a previously defined CTP population based on high vYFP reporter expression. Dot plots show the gating scheme that has been described for the identification of CTPs and CIPs as discrete developmental stages of extrathymic T lymphopoiesis (53). Histograms show Gata3-vYFP reporter expression in gated cell subsets. (F) Comparison of Gata3-vYFP reporter expression intensities in CIP, CTP, and total Lin− BM cells. (G) Percentage of Gata3-vYFPint and Gata3-vYFPhigh (ILC2P) cells within the population gated as CTP. Data are from 10 homozygous GATIR mice. (H) Selective expression of the ILC2 marker T1/ST2 (IL-33Rα) on Gata3-vYFPhigh CTPs.