Figure 5: KLRG1^high iILC2s are responsible for type-2 cytokine production in the lung 5 days after N. brasiliensis infection.

(A-C) RNA sequencing was performed on KLRG1^pos and KLRG1^neg Lin⁻CD90⁺ lung cells from mice treated with IL-33 as described in Figure 1. (A-B) Genes that are significantly different and increased at least two-fold in KLRG1^pos relative to KLRG1^neg cells were used in (A) Gene Ontology Molecular Function analysis and (B) KEGG Pathway analysis. Results are shown as -log10P values to denote pathways that are significantly associated with genes upregulated in KLRG1^high ILC2s. Activity groups or pathways that relate to cytokine signaling are highlighted in red. (C) Quantification of type-2 cytokine expression in KLRG1^pos and KLRG1^neg populations. (D-G) IL4^4get and Batf^−/−IL4^4get and (H-K) IL13^yetcre13 and Batf^−/−IL13^yetcre13 mice were infected with N. brasiliensis and pulmonary Lin⁻CD90⁺ ILC2s were assessed 5 days later by flow cytometry. (D, H) Contour plot showing IL-4 (GFP) (D) or IL-13 (YFP) (H) cytokine reporter expression of KLRG1^mid (blue) and KLRG1^high (red) ILC2s. Gates were set according to a reporter negative control. (E, I) Bar graphs comparing the percent of KLRG1^mid and KLRG1^high ILC2s that express either IL-4 (GFP) with n=8 mice combined from 3 separate experiments (E) or IL-13 (YFP) with n=9-10 mice combined from 4 experiments (I). (F, J) Representative contour plots of IL-4 (GFP) (F) or IL-13 (YFP) (J) and KLRG1 expression on Lin⁻CD90⁺ lung ILC2s. (G, K) Bar graphs comparing the percent of KLRG1⁺ ILCs expressing IL-4 (GFP) with n=6-8 mice combined from 3 separate experiments (G) or IL-13 (YFP) with n=6-7mice combined from 3 separate experiments (K). *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001 as determined by two-tailed unpaired t test.