Fig 2: Homeostatic expansion and activation of RAG-deficient ILC2s is cell intrinsicA) Experimental schematic of AD-like disease in splenocyte chimera experiment. WT B6 or Rag1−/− mice received WT splenocytes and developed AD-like inflammation after subsequent topical treatment with 2 nmol MC903 in 10 μL ethanol vehicle to each ear daily for 10 days. B) Ear thickness measured daily in AD-like inflammation. Data representative of 2 independent experiments, 4–5 mice per group. **** P <0.0001 by 2-way ANOVA with Sidak’s multiple comparisons test, day 10. C) Proportion of CD90+, Lin− cells (Lin− defined as CD3−, CD5−, CD11b−, CD11c−, CD19−, NK1.1−, and FcεR1−) determined to be ILC2s (IL-33R+). Percent ILC2 from sdLN in splenocyte chimera mice with AD-like disease after PMA/iono stimulation positive for D) IL-5 or E) IL-13 staining. F) Schematic of bone marrow chimera experiment. Equal quantities of bone marrow cells from Rag1−/− (CD45.2, CD90.2 - orange) and WT (CD45.2, CD90.1 - blue) C57Bl/6J donor mice were used to reconstitute the immune systems of irradiated recipient WT (CD45.1 - black) C57Bl/6J mice. G) Proportion of donor (CD45.2+) ILC2 defined as in (C) in sdLN by donor source (CD90.1+ - WT, CD90.2− - Rag1−/−). Proportion of Lin− ILCs by donor source positive for H) IL-5 and I) IL-13 following PMA/iono stimulation and cytokine staining. C-E) Data representative of at least 2 independent experiments, 4–5 mice per group. ** P < 0.01, **** P < 0.0001 by two-tailed Welch’s t test. G-H) Data representative of at least 2 independent experiments, 4–5 mice per group. ** P < 0.01 by two-tailed ratio means paired t test. All data represented as mean with standard deviation.

Fig 3: A history of RAG expression marks a population of ILC2s in the sdLN.A) Schematic of RAG fate mapping in the lymphoid cell compartment using reporter mice expressing Cre-inducible tandem dimer red fluorescent protein (tdRFP) from the Rosa26 locus crossed to mice expressing Cre recombinase from the Rag1 locus. B-F) Histograms of tdRFP signal in CD45+ sdLN cells by cell type for B) CD4+ T cells (B220−, CD3+, CD4+), C) CD8+ T cells (B220−, CD3+, CD8+), D) B cells (MHCII+, B220+), E) NK cells (B220−, CD3−, CD4−, CD8−, CD49b+, NK1.1+), F) ILC2s (B220−, CD3−, CD4−, CD8−, CD49b−, NK1.1−, CD11b−, CD11c−, SiglecF−, CD90+, KLRG1+ or ICOS+ or IL-33R+), F) quantification of tdRFP+ proportion of each cell type.

Fig 4: A history of recombinase activating gene (RAG) expression marks a population of group 2 innate lymphoid cells (ILC2s) in the skin-draining lymph node (sdLN).(A) Schematic of RAG fate mapping in the lymphoid cell compartment using reporter mice expressing Cre-inducible tandem dimer red fluorescent protein (tdRFP) from the Rosa26 locus crossed to mice expressing Cre recombinase from the Rag1 locus. (B–F) Histograms of tdRFP signal in CD45+ sdLN cells by cell type for (B) CD4+ T cells (B220-, CD3+, CD4+), (C) CD8+ T cells (B220-, CD3+, CD8+), (D) B cells (MHCII+, B220+), (E) NK cells (B220-, CD3-, CD4-, CD8-, CD49b+, NK1.1+), (F) ILC2s (B220-, CD3-, CD4-, CD8-, CD49b-, NK1.1-, CD11b-, CD11c-, SiglecF-, CD90+, KLRG1+ or ICOS+ or IL-33R+), (G) quantification of tdRFP+ proportion of each cell type. Data representative of at least two independent experiments, 3-4 mice per group. Data in (G) represented as mean with scale bars representing standard deviation. Statistical analyses were not performed.

Fig 5: Homeostatic expansion and activation of recombinase activating gene (RAG)-deficient group 2 innate lymphoid cells (ILC2s) is cell intrinsic.(A) Experimental schematic of atopic dermatitis (AD)-like disease in splenocyte chimera experiment. Wild-type (WT) B6 or Rag1-/- mice received WT splenocytes and developed AD-like inflammation after subsequent topical treatment with 2 nmol MC903 in 10 μL ethanol vehicle to each ear daily for 10 days. (B) Ear thickness measured daily in AD-like inflammation. Data representative of two independent experiments, 4–5 mice per group. ****p<0.0001 by two-way ANOVA with Sidak’s multiple comparisons test, day 10. (C) Total number ILC2s normalized to 105 live cells and (D) proportion of CD90+, Lin- cells (Lin- defined as CD3-, CD5-, CD11b-, CD11c-, CD19-, NK1.1-, and FcεR1-) determined to be ILC2s (IL-33R+). Percent ILC2 from skin-draining lymph node (sdLN) in splenocyte chimera mice with AD-like disease after Phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation positive for (E) IL-5 or (F) IL-13 staining. (G) Schematic of bone marrow chimera experiment. Equal quantities of bone marrow cells from Rag1-/- (CD45.2, CD90.2 - orange) and WT (CD45.2, CD90.1 - blue) C57BL/6J donor mice were used to reconstitute the immune systems of irradiated recipient WT (CD45.1 - black) C57BL/6J mice. (H) Proportion of donor (CD45.2+) ILC2 defined as in (C and D) in sdLN by donor source (CD90.1+ - WT, CD90.2- - Rag1-/-). Proportion of Lin- ILCs by donor source positive for (I) IL-5 and (J) IL-13 following PMA/ionomycin stimulation and cytokine staining. (C–F) Data representative of at least two independent experiments, 4–5 mice per group. **p<0.01, ****p<0.0001 by two-tailed Welch’s t test. (H–J) Data representative of at least two independent experiments with 4–5 mice per group. **p<0.01 by two-tailed ratio means paired t test. All data represented as mean with scale bars representing standard deviation.