Figure 3. PLZF^high cells are ILC progenitors.

a-c, CD45.2 Rag2^−/−Il2rg^−/− mice were injected with equivalent numbers of tdTomato⁺ PLZF^high cells and CD45.1 CLP (800-1200 of each) and the progeny of these populations analyzed 5-7 weeks later by FACS, as indicated. Summary bar graph of mean percentages ± s.e.m. in c, with significant differences between PLZF^high- (black bar) and CLP-derived (white bar) shown by *. LPL RORγt⁺ NCR⁺ cells were identified as CD3ε⁻CD19⁻RORγt⁺NKp46⁺CD4⁻; LPL RORγt⁺NCR⁻ cells as CD3ε⁻CD19⁻RORγt⁺NKp46⁻CD4⁻; and LPL RORγt⁺CD4⁺ cells as CD3ε⁻CD19⁻RORγt⁺CD4⁺. Remaining populations were gated as indicated in Fig. 1. Data representative of 6-9 chimeras analyzed in at least 2 independent experiments. d, FACS analysis of Lin⁻IL-7Rα⁺α4β7^higheGFP⁻CXCR6⁻ cells sorted from PLZF^GFPcre+/− BM and cultured on OP9 or OP9-DL1 for 48 h. Data representative of 3 independent experiments. e, FACS analysis of sorted PLZF^high BM cells cultured on OP9 for indicated periods. Data representative of at least 4 replicate cultures for each time point from 2 or more independent experiments. f, FACS analysis of PLZF^high or CLP cells from adult BM or fetal liver cultured on OP9 for 4 days. Data representative of at least 4 replicate cultures from 2 or more independent experiments. g, FACS analysis of representative colonies originating from single fetal liver PLZF^high cells that were sorted and cultured into 96-well plates containing irradiated OP9 stromal cells for 5-6 days. ILC1 were characterized as ICOS^lowα4β7⁻ NK1.1⁺ populations, ILC2 as ICOS^highα4β7⁻NK1.1⁻, and ILC3 as ICOS^int/highα4β7⁺NK1.1⁻. The table summarizes the analysis of thirteen 96-well plates analyzed in four independent experiments (three plates in experiments 1, 2, and 4; four plates in experiment 3), with the average colony size ± s.e.m. as indicated. In experiment 4, we mixed 1:1 CD45.1/5.2 and CD45.2 PLZF^GFPcre+/− fetal liver cells prior to single-cell sorting. All 122 colonies were either CD45.1/5.2 (n=59) or CD45.2 (n=63) ruling out doublet contamination as an explanation for the presence of mixed ILC colonies (p<0.01 Chi-Square). The cloning efficiency was 40% on average, with all but 17 colonies (not shown in the table) unambiguously assigned to defined ILC lineages.