Abstract The long-term goal of this project is to develop better therapies for respiratory inflammation and allergic asthma. Group 2 innate lymphoid cells (ILC2) are a recently identified cell population producing type 2 cytokines in response to a growing number of environmental signals and epithelial cell-derived cytokines. Studies show increased ILC2 activity in asthma and many widespread diseases, as ILC2s are sufficient to induce airway inflammation independent of adaptive immunity in mice. The proposed research plan is motivated by recent new observations from our laboratory and others that the TNF/TNFR2 axis controls ILC2- dependent airway inflammation (Cell Report 2019, J Allergy Clin Immunol. 2020). High levels of TNF are found in the lungs of asthmatic patients, however anti-TNF therapy is generally associated with systemic toxicity due to the existence of two distinct functionally different receptors for TNF: TNFR1 and TNFR2. Our results suggest that TNF enhanced the secretion of ILC2 effector cytokines IL-5 and IL-13 and increased survival via TNFR2 signaling, leading to airway inflammation. However, how the TNF/TNFR2 axis mechanistically affects ILC2s and subsequent development of airway inflammation remains to be explored. Based on our data, we hypothesize that blocking TNFR2 on ILC2s induces an immunoregulatory phenotype fueling on a distinct metabolic source, together favoring the reduction of AHR. In Specific Aim 1 (SA1), we have designed several approaches to characterize the effects of TNF on ILC2 effector functions and lung inflammation. Our preliminary data suggest that TNFR2 is heterogeneously expressed on activated ILC2s. We will therefore characterize the transcription factors driving the effects of TNF in ILC2s using a combination of single cell genomic and transcriptomic analysis. Furthermore, data from our laboratory and others suggest that metabolic processes in ILC2s are dependent on the generation of energy from fatty acid oxidation (FAO) and oxidative phosphorylation. Interestingly our results clearly show a metabolic shift towards glycolysis in TNFR2-/- ILC2s. Based on these results we intend to assess glycolysis and FAO mechanisms in WT and TNFR2-/- ILC2s in SA2. Finally, we previously showed that human ILC2s express TNFR2 and humanized ILC2 mice developed TNFR2-dependent AHR in response to TNF. Therefore, we intend to assess in the SA3 the relevance of our findings in asthmatic patients. We will collect lung and blood ILC2s from carefully selected cohorts of mild/moderate, severe asthmatics and healthy donors and correlate the levels of TNF in the BAL to the numbers of ILC2s/expression of TNFR2, as well as monitor other cells that express TNFR2. These studies, based on strong preliminary data, will focus on developing novel therapy for allergic asthma. In order to achieve these results we have assembled a team including leading experts in lung biology and the chief of clinical pulmonology to complement our extensi...