Fig 2: Effects of TNF on the suppressive function of TNFR2+Tregs. Purified CD4+ T cells were cultured in medium alone, TNF (50 ng/mL), TNF combined with anti-TNFR2 mAbs (10 µg/mL), or isotype IgG, as indicated, for 72 hours. The representative FACS analysis of (A) CTLA-4+ cells and (B) PD-L1+ cells in TNFR2+ Tregs as indicated, gated on live CD4+Foxp3+ cells. Summary of the proportions of (C) CTLA-4+ cells (n=3) and (D) PD-L1+ cells (n=3) in Tregs within each condition. Data are expressed as means ± SEM. *, P<0.05, **, P<0.01, ****, P<0.0001 by one-way ANOVA test. NS represents no statistics difference. TNFR2, tumor necrosis factor receptor type II.

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: TNF-a Signaling via NIK Enhances ILC2-Dependent AHR(A–E) 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 for 24 h with rmIL-2 (10 ng/mL) and rmIL-7 (10 ng/mL) with or without rmTNF-a (40 ng/mL), aTNFR2 (10 µg/mL), or NIK inhibitor (10 µM). aTNFR2 and the NIK inhibitor were added 30 min prior to rmTNF-a.(A and B) NF-?B p52 representative flow cytometry plots in TNFR2 (A) and NIK (B) blocking experiments on ILC2s and corresponding quantitation, presented as MFI ± SEM.(C) Levels of IL-5 and IL-13 secretion in culture supernatants, measured by ELISA.(D) Frequency of AnnexinV+DAPI+ late apoptotic/necrotic ILC2s, presented as mean frequency ± SEM.(E) Representative flow cytometry plots of AnnexinV+DAPI+ late apoptotic/necrotic ILC2s.(F) C57BL/6 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 for 3 days with rmIL-2 (10 ng/mL), rmIL-7 (10 ng/mL), and rmIL-33 (50 µg/mL) prior to transfer of 50 × 103 aILC2s in 2 separate cohorts of Rag-/- Il2rg-/- mice, followed by i.n. administration of 50 ng rmTNF-a and i.p. injection of 250 µg NIK inhibitor or vehicle on days 1–3. On day 4, lung function, BAL, and lung ILC2s were analyzed.(G) Lung resistance in response to increasing doses of methacholine.(H) Total number of eosinophils in the BAL fluid, presented as mean numbers ± SEM.(I) Total number of ILC2s in the lungs, presented as mean numbers ± SEM.Data are representative of 3 individual experiments (n = 5). *p < 0.05, **p < 0.01. See also Figure S3.

Fig 5: The relationships between TNFR2+Treg and clinical parameters. (A) Correlations between the TNFR2+ cell proportion in Tregs and the tumor cell proportion in MPE (n=26). (B) Correlations between TNFR2+ cell proportion in Tregs and the maximum PEAD (n=30). (C) The graphs summarize the frequencies of patients with different ECOG PS scores within the total patients in the indicated group (n=15 for high proportions of TNFR2+ cells; and n=26 for low proportions of TNFR2+ cells). (D) Kaplan-Meier survival curves for patients with “high (n=15)” and “low (n=21)” frequency of TNFR2+ cells in Tregs. Data are expressed as means ± SEM (A and B). The proportions of Tregs and TNFR2+ cells in Tregs were determined by flow cytometry. The proportions of tumor cells in MPE were determined by immunohistochemical staining (n=26). The maximum PEAD was measured by ultrasonography. Correlations were determined by Spearman’s rank correlation coefficients. NS represents no statistics difference. Overall survival was estimated by the Kaplan-Meier method and compared by the log-rank test. TNFR2, tumor necrosis factor receptor type II; MPE, malignant pleural effusion.