Fig 2: Characterization of macrophages and T cells.(A, B, C) Representative plots for the identification of the macrophage population (A) and of their two subtypes, M1-like (B) and M2-like (C) macrophages versus days after implantation. (D, E) Percentage of macrophages (D) and macrophage subtypes (E) versus days after implantation. (F, G) T cells (CD3) identification strategy (F) for the CD4+ and CD8+ subpopulations (G). (H, I) Percentage of T cells among viable cells (H) and T cells subpopulations (I) among all T cells, both versus days after implantation. (J, K, L) Representative plots for identifying T regulatory cells (CD4+CD127-, J, CD25+, K) among T cells, versus days after implantation. (L). Data shown as mean ± SEM. *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.

Fig 3: TNF-a Enhances ILC2 Survival and Activation via TNFR2(A) BALB/cByJ mice were challenged i.n. on days 1–3 with 0.5 µg rmIL-33. On day 4, aILC2s were FACS sorted as lineage- CD45+ ST2+ CD127+ and cultured ex vivo (50 × 104/mL) for 24 h with rmIL-2 (10 ng/mL) and rmIL-7 (10 ng/mL) with or without rmTNF-a (40 ng/mL).(B) Representative flow cytometry plots of AnnexinV and DAPI expression on cultured ILC2s.(C) Frequency of AnV+DAPI+ ILC2s ± SEM on cultured ILC2s.(D) Representative flow cytometry plots of intranuclear Ki67-expressing ILC2s among cultured ILC2s and corresponding quantitation, presented as mean frequency ± SEM.(E) Levels of IL-5 and IL-13 in cultured supernatants, measured by ELISA and normalized to the number of viable cells after culture.(F) BALB/cByJ mice were challenged i.n. on days 1–3 with 0.5 µg rmIL-33. On day 4, aILC2s were FACS sorted as lineage- CD45+ ST2+ CD127+ and cultured ex vivo (50 × 104/mL) for 4 h with rmIL-2 (10 ng/mL) and rmIL-7 (10 ng/mL) with or without rmTNF-a (40 ng/mL) and GolgiPlug.(G) Intracellular expression of IL-5 and IL-13 in cultured ILC2s and corresponding quantitation, presented as mean frequency ± SEM.(H) BALB/cByJ mice were challenged i.n. on days 1–3 with 0.5 µg rmIL-33. On day 4, aILC2s were FACS sorted as lineage- CD45+ ST2+ CD127+ and cultured in vitro for 24 h with rmIL-2 (10 ng/mL) and rmIL-7 (10 ng/mL) with or without rmTNF-a (40 ng/mL) and aTNFR2 (10 µg/mL). aTNFR2 was added 30 min prior to rmTNF-a.(I) Representative flow cytometry plots of AnnexinV/DAPI expression on cultured ILC2s.(J) Frequency of AnV+DAPI+ ILC2s ± SEM on cultured ILC2s.(K) Levels of IL-5 and IL-13 in culture supernatants, measured by ELISA.Data are representative of 4 individual experiments (n = 5). *p < 0.05, **p < 0.01. See also Figure S3.

Fig 4: aILC2s and TNF-a Promote Development of AHR(A) BALB/cByJ mice were challenged i.n. on days 1–3 with 0.5 µg rmIL-33. On day 4, aILC2s were FACS sorted as lineage- CD45+ ST2+ CD127+ and cultured ex vivo (50 × 104/mL) for 48 h with rmIL-2 (10 ng/mL) and rmIL-7 (10 ng/mL) with or without recombinant mouse (rm) TNF-a (40 ng/mL).(B) Levels of IL-5, IL-6, IL-9, IL-13, and GM-CSF in culture supernatants.(C) Rag2-/- mice received i.v. injections of 100 µg aTNF-a or an isotype control and 0.5 ag rmIL-33 or PBS i.n. on days 1–3. On day 4, lung function, BAL eosinophils, lung ILC2s, and histology were analyzed.(D and E) Lung resistance (D) and dynamic compliance (E) in response to increasing doses of methacholine.(F and H) Representative FACS plots of BAL eosinophils (F) and lung ILC2s (H). Eosinophils were gated as CD45+, SiglecF+ CD11c- and ILC2s as lineage- CD45+ ST2+ CD127+.(G and I) Total number of eosinophils in the BAL fluid (G) and of ILC2s in the lungs (I), presented as mean numbers ± SEM.(J) Lung histology. Scale bars, 50 µm.(K) Rag2-/- mice received i.v. injections of 100 µg aTNF-a or an isotype control and 100 µg A. alternata or PBS i.n. on days 1–4. On day 5, lung function, BAL eosinophils, and lung ILC2s were analyzed.(L) BAL fluid was collected, and supernatant TNF-a was measured by ELISA.(M) Lung resistance in response to increasing doses of methacholine.(N and P) Representative FACS plots of BAL eosinophils (N) and lung ILC2s (P).(O and Q) Total number of eosinophils in the BAL fluid (O) and of ILC2s in the lungs (Q), presented as mean numbers ± SEM.Data are representative of 3 individual experiments (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S2 and S4.

Fig 5: Derepression of the Il7r gene increases IL7R expression and promotes downstream signaling. (A) IL-7 receptor expression of FACS-sorted pro-B cells (c-KIT+CD25-) of miR-15a/bfl/fl Vav-Cre and control mice as analyzed by quantitative PCR. (B, C) Flow cytometric analysis of IL-7 receptor surface expression in pro- (c-KIT+CD25-), intermediate- (c-KIT-CD25-) and pre-B cells (c-KIT-CD25+) from control or miR-15a/bfl/fl Vav-Cre mice (B) as well as in vitro cultured progenitors thereof (C). For the in vivo data, the MFI values were normalized to the mean IL-7 receptor expression of control mice for each respective experimental day. (D) Western blot analysis of control and miR-15a/bfl/fl Vav-Cre progenitors depicting pAKTSer473, AKT and GAPDH protein levels. (E) Representative histograms of flow cytometric analysis of intracellular pAKTSer473 levels in control and miR-15a/bfl/fl Vav-Cre progenitors. The bar graph summarizes the pAKTSer473 MFI normalized to the respective Isotype. (F) Representative FACS plots depicting the “proliferating” progenitors defined as the FSChi population (dark grey) upon culturing the cells with 5 ng/ml, 1 ng/ml or 0.1 ng/ml IL-7 for 48 hours. Bar graphs summarize the quantification of the FSChi population among all living progenitors derived from miR-15a/bfl/fl Vav-Cre or control mice. (G) Summary of the percentage of FSChi cells among pro-, intermediate and pre-B cells upon culturing the progenitors with 0.1 ng/ml IL-7 for 48 hours. MiR-15a/bfl/fl Vav-Cre progenitors were statistically compared to miR-15a/b+/+ Vav-Cre progenitors by an unpaired two-tailed Student’s t-test. *P < 0.05; **P < 0.005; ***P < 0.0005.