IL-25 simultaneously elicits phenotypically distinct populations of MPP^type2 cells and ILC2. (a–r) C57BL/6 WT mice (The Jackson Laboratory) were treated i.p. with PBS (control), 0.3 µg of recombinant IL-33, or 0.3 µg of recombinant IL-25 daily for 2, 4, or 7 d. Indicated tissues were harvested at day 7 (or as indicated), and the frequency and total cell numbers of ILC2 and MPP^type2 cells were assessed by flow cytometry. (a) Frequency of T1/ST2^pos IL-7Rα^pos ILC2 in the Lin^neg cell compartment from the MLNs of control or IL-33–treated mice. (b) CD90.2 (Thy1.2) and CD25 expression on ILC2 (black histograms) from IL-33–treated mice. (c) Total cell numbers of ILC2 from control or IL-33–treated mice. (d) Expression of c-kit on gated T1/ST2^neg IL-7Rα^neg cells from control or IL-33–treated mice. (e) CD90 and CD25 expression on c-kit^pos MPP^type2 cells (black histograms) from IL-33–treated mice. (f) Total cell numbers of MPP^type2 cells from control or IL-33–treated mice. (g) Frequency of ILC2 in the Lin^neg cell compartment from the MLN of control or IL-25–treated mice. (h) CD90 and CD25 expression on ILC2 (black histograms) from IL-25–treated mice. (i) Total cell numbers of ILC2 from control or IL-25–treated mice. (j) Expression of c-kit on gated T1/ST2^neg IL-7Rα^neg cells from control or IL-25–treated mice. (k) CD90 and CD25 expression on c-kit^pos MPP^type2 cells (black histograms) from IL-25–treated mice. (l) Total cell numbers of MPP^type2 cells from control or IL-25–treated mice. Plots are gated on live, Lin^neg (CD4, CD8, CD11b, CD11c, and CD19) cells or as indicated. Gray shaded histograms represent CD90 or CD25 expression on Lin^neg cells from control mice. (m and n) Frequencies of ILC2 (m) or MPP^type2 cells (n) as gated in a, g, d, and i from the MLNs of control (open bars), IL-33–treated, or IL-25–treated mice at days 2, 4, and 6. (o–r) Frequencies of ILC2 and MPP^type2 cells as gated in a and g and d and i from the blood (o), caudal LN (p), lung (q), and PEC (r) of control (open bars), IL-33–treated (black bars), or IL-25–treated (gray bars) mice at day 6. Data in a–n are representative of two or more independent experiments (control, n = 4; IL-33 treated, n = 8; IL-25 treated, n = 8). Data in o–r are representative of two independent experiments (control, n = 4; IL-25 treated, n = 8; IL-33 treated, n = 8). *, P < 0.05; **, P < 0.01; ***, P < 0.0001. Lung IL-33–elicited ILC2 versus IL-25–elicited ILC2, P = 0.07; PEC IL-33–elicited MPP^type2 cells versus IL-25–elicited MPP^type2 cells, P = 0.31. Error bars indicate SEM.