Figure 3. Heterogeneity of Refined ChILP Cells Separated by the Differential Expression of GATA3 and Id2.

(A) A refined ChILP (rChILP) population in the BM was defined as the live Lin⁻CD127⁺Flt-3⁻T1/ST2⁻α₄β₇⁺c-Kit⁺ cells. The expression of GATA3 and Id2, and the proportions of three subsets with different GATA3 and Id2 expression among the rChILP cells were analyzed.

(B) Differential Id2-YFP expression in CLPs (live Lin⁻CD127⁺Flt-3⁺α4β7⁻Sca1⁺c-Kit⁺), rChILPs (live Lin⁻CD127⁺Flt-3⁻T1/ST2⁻α₄β₇⁺c-Kit⁺) and ILC2Ps (live Lin⁻CD127⁺Flt-3⁻α₄β₇⁺T1/ST2⁺) in the bone marrow.

(C) The GATA3 expression difference among the Lin⁻CD127⁺Flt-3⁻T1/ST2⁻α₄β₇⁺ including the rChILPs (red gate and red line) was further confirmed with a GATA3-GFP reporter mouse. Within the live Lin⁻CD127⁺Flt-3⁻ BM cells, GATA3 level in the T1/ST2⁺ BM ILC2s (green gate and line) and the α₄β₇⁻ cells (blue gate and line) were used as references for the GATA3 high- and negative/low- expression. The pink and blue shadows indicated the high and low amounts of GATA3-GFP.

(D) Id2^high and Id2^low/− rChILP cells were further analyzed for their expression association between GATA3 and PLZF.

(E) The previously defined Lin⁻PLZF⁺ ILC progenitors (ILCPs) (red gate and line) were compared with the rChILP cells (blue gate and line) in the same sample for Id2 and GATA3 expression.

(F) The GATA3^high and GATA3^low rChILP subsets were isolated from the GATA3-GFP reporter mice bearing a congenic marker (CD45.1XCD45.2) by cell sorting (see also Figure S3H), and were subsequently transferred into sublethally irradiated Rag2^−/−Il2rg^−/− recipients (CD45.2). After 4–6 weeks, the repopulated progenies derived from the CD45.1⁺CD45.2⁺ donor cells were analyzed in the siLPL of recipients. GATA3, RORγt, NKp46, T-bet, Eomes and CCR6 were used to distinguish distinct innate lymphoid populations, among which the CCR6⁺ cells (red gate) represent the LTi/LTi-like cells.

Data are representative of three (A-E) and two (F) independent experiments.

See also Figure S3.