The standard-of-care therapeutics for the treatment of ocular neovascular diseases like wet age-related macular degeneration (AMD) are biologics targeting vascular endothelial growth factor signaling. There are currently no FDA approved small molecules for treating these blinding eye diseases. Therefore, therapeutic agents with novel mechanisms are critical to complement or combine with existing approaches. Here, we identified soluble epoxide hydrolase (sEH), a key enzyme for epoxy fatty acid metabolism, as a target of an antiangiogenic homoisoflavonoid, SH-11037. SH-11037 inhibits sEH in vitro and in vivo and docks to the substrate binding cleft in the sEH hydrolase domain. sEH levels and activity are up-regulated in the eyes of a choroidal neovascularization (CNV) mouse model. sEH is overexpressed in human wet AMD eyes, suggesting that sEH is relevant to neovascularization. Known sEH inhibitors delivered intraocularly suppressed CNV. Thus, by dissecting a bioactive compound's mechanism, we identified a new chemotype for sEH inhibition and characterized sEH as a target for blocking the CNV that underlies wet AMD.

Background: Bcl6 is required for the development of T follicular helper and regulatory (Tfh, Tfr) cells that regulate germinal center responses. Bcl6 also impacts the function of regulatory T (Treg) cells. Objective:The goal of this study is to define the functions of Bcl6 in Treg cells including Tfr cells in the context of allergic airway inflammation (AAI). Methods: We employed a model of house dust mite (HDM) sensitization to challenge wild type, Bcl6 fl/fl Foxp3-Cre and Prdm1(Blimp1) fl/fl Foxp3-Cre mice to study the reciprocal roles of Bcl6 and Blimp1 in AAI. Results: In the HDM model, Tfr cells repress the production of IgE and Bcl6+ Treg cells suppress the generation of type 2 cytokine producing cells in the lungs. In mice with Bcl6deficient Treg cells, twice as many ST2 (IL-33R) + Tregs develop as observed in wild type mice. ST2 + Tregs in the context of AAI are Blimp1-dependent, express type 2 cytokines, and share features of visceral adipose tissue Treg cells. Bcl6-deficient Tregs are more susceptible, and Blimp1-deficient Tregs are resistant, to acquiring the ST2 + Treg cell phenotype in vitro and in vivo in response to IL-33. Bcl6-deficient ST2+ Tregs but not Bcl6-deficient ST2+ T conventional cells strongly promote AAI when transferred into recipient mice. Lastly, ST2 is required for the exacerbated AAI in Bcl6 fl/fl Foxp3-Cre mice. Conclusions: During AAI, Bcl6 and Blimp1 play dual roles in regulating Tfr activity in the germinal center and in the development of ST2 + Tregs that promote type 2 cytokine responses. Koh page 4 4 Key Messages: • Tfr cells limit IgE production in mice challenged by airway allergen • Bcl6 and Blimp1 reciprocally regulate ST2 + Treg development • ST2 + Tregs promote allergic airway inflammation Capsule Summary: Bcl6 attenuates allergic disease by promoting Tfr cell development to repress the allergen-specific humoral response and by limiting expansion of ST2 + Tregs.

Asthma is a chronic inflammatory lung disease with intermittent flares predominately mediated through memory T cells. Yet, the identity of long-term memory cells that mediate allergic recall responses is not well defined. In this report, using a mouse model of chronic allergen exposure followed by an allergen-free rest period, we characterized a subpopulation of CD4 + T cells that secreted IL-9 as an obligate effector cytokine. IL-9–secreting cells had a resident memory T cell phenotype, and blocking IL-9 during a recall challenge or deleting IL-9 from T cells significantly diminished airway inflammation and airway hyperreactivity. T cells secreted IL-9 in an allergen recall–specific manner, and secretion was amplified by IL-33. Using scRNA-seq and scATAC-seq, we defined the cellular identity of a distinct population of T cells with a proallergic cytokine pattern. Thus, in a recall model of allergic airway inflammation, IL-9 secretion from a multicytokine-producing CD4 + T cell population was required for an allergen recall response.

T helper cell differentiation requires lineage-defining transcription factors and factors that have shared expression among multiple subsets. BATF is required for development of multiple Th subsets but functions in a lineage-specific manner. BATF is required for IL-9 production in Th9 cells but in contrast to its function as a pioneer factor in Th17 cells, BATF is neither sufficient nor required for accessibility at the Il9 locus. Here we show that STAT5 is the earliest factor binding and remodeling the Il9 locus to allow BATF binding in both mouse and human Th9 cultures. The ability of STAT5 to mediate accessibility for BATF is observed in other Th lineages and allows acquisition of the IL-9-secreting phenotype. STAT5 and BATF convert Th17 cells into cells that mediate IL-9-dependent effects in allergic airway inflammation and anti-tumor immunity. Thus, BATF requires the STAT5 signal to mediate plasticity at the Il9 locus.

The basic leucine zipper (bZIP) transcription factor BATF is expressed in multiple Th subsets and cooperates with other factors to regulate gene transcription. BATF activates lineage-specific cytokines in Th subsets, activating IL-9 in Th9 cells and IL-17 in Th17 cells, but not IL-9 or IL-17 in the reciprocal subset. The mechanism for this restricted activity is unclear. In this report, we define BATF binding partners that contribute to Th subset-specific functions. Although BATF and IRF4 are expressed in greater amounts in Th9 than Th17, increased expression of both factors is not sufficient to induce IL-9 in Th17 cells. BATF also requires heterodimer formation with Jun family members to bind DNA and induce gene expression. Using primary mouse T cell culture, we observed that JunB and c-Jun, but not JunD, promote IL-9 production in Th9 cells. Ectopic expression of BATF with either JunB or c-Jun generates modest, but significant, increases in IL-9 production in Th17 cells, suggesting that the low expression of Jun family members is one factor limiting the ability of BATF to induce IL-9 in Th17 cells. We further identified that Bach2 positively regulates IL-9 production by directly binding to the Il9 gene and by increasing transcription factor expression in Th9 cells. Strikingly, cotransduction of Bach2 and BATF significantly induces IL-9 production in both Th9 and Th17 cells. Taken together, our results reveal that JunB, c-Jun, and Bach2 cooperate with BATF to contribute to the specificity of BATF-dependent cytokine induction in Th subsets.