Figure 6.

Inducible ablation of E proteins promotes ILC2 differentiation in vivo. (a) ROSA26-CreERT2; E2A^f/f; HEB^f/f and control (E2A^f/f; HEB^f/f or ROSA26-CreERT2) mice were treated to induce the deletion of E proteins as diagrammed on the top. The definitions of the BM populations are ILCP (Lin⁻Thy1.2⁺IL-7⁺ST2⁻Sca-1⁻cKit⁺α4β7⁺PD-1⁺), ILC2P (Lin⁻Thy1.2⁺IL-7⁺ST2⁺Sca-1⁺CD25⁺α4β7⁺), B cells (B220⁺CD19⁺), and myeloid cells (Mac-1⁺). ILC2s in the thymus and lung are Lin⁻Thy1.2⁺ST2⁺. Total cell numbers of each indicated subset were quantified. Data shown are pooled from three experiments (n = 5–8). (b) Lineage cocktails for BM ILC1, BM ILC3, and BM NK include antibodies against FcεRI, B220, CD19, Mac-1, Gr-1, Ter-119, CD3ε, CD5, and CD8α. BM ILC1 were defined as Lin⁻Thy1.2⁺IL-7R⁺CD62L⁻CD27⁺ST2⁻; BM ILC3 as Lin⁻ Thy1.2⁺IL-7R⁺CD62L⁻CD27⁻ST2⁻; and BM NK as Lin⁻Thy1.2⁺IL-7R⁻CD62L⁺NK1.1⁺DX5⁺. Lineage markers for BM pre-cDC and BM pDCs are CD3, CD8α, CD5, TCRβ, γδTCR, NK1.1, Ter119, and Ly6G. BM pre-cDCs were gated as Lin⁻B220⁻MHCII⁻CD11c⁺Flt3⁺Sirpα⁻ and BM pDCs as Lin⁻B220⁺CD19⁻SiglecH⁺. Thy NKs were considered as Lin (CD3, CD8α, CD4, TCRβ, ΥδTCR)⁻CD122⁺NK1.1⁺DX5⁺. Data shown are pooled from three independent experiments (n = 8–10). Student’s t test was used to determine statistical significance. Error bars are SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. (c) Thymocytes from mice used in panel a were cultured in the presence of PMA and ionomycin plus monensin for 4 h before intracellular staining and analyzed with indicated antibodies. Data shown are representatives from two independent experiments.