Figure 3. TNF-α 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-α (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-α (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-α (40 ng/mL) and αTNFR2 (10 μg/mL). αTNFR2 was added 30 min prior to rmTNF-α.

(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.