Cutting Edge: Hypoxia Sensing by the Histone Demethylase UTX (KDM6A) Limits Colitogenic CD4+ T Cells in Mucosal Inflammation.

Hypoxia is a hallmark of inflammatory conditions (e.g., inflammatory bowel disease [IBD]), and adaptive responses have consequently evolved to protect against hypoxia-associated tissue injury. Because augmenting hypoxia-induced protective responses is a promising therapeutic approach for IBD, a more complete understanding of these pathways is needed. Recent work has demonstrated that the histone demethylase UTX is oxygen-sensitive, but its role in IBD is unclear. In this study, we show that hypoxia-induced deactivation of UTX downregulates T cell responses in mucosal inflammation. Hypoxia results in decreased T cell proinflammatory cytokine production and increased immunosuppressive regulatory T cells, and these findings are recapitulated by UTX deficiency. Hypoxia leads to T cell accumulation of H3K27me3 histone modifications, suggesting that hypoxia impairs UTX's histone demethylase activity to dampen T cell colitogenic activity. Finally, T cell-specific UTX deletion ameliorates colonic inflammation in an IBD mouse model, implicating UTX's oxygen-sensitive demethylase activity in counteracting hypoxic inflammation.

Hypoxia-sensing by the Histone Demethylase UTX ( KDM6A ) Controls Colitogenic CD4 + T cell Fate and Mucosal Inflammation.

Hypoxia is a feature of inflammatory conditions [e.g., inflammatory bowel disease (IBD)] and can exacerbate tissue damage in these diseases. To counteract hypoxia's deleterious effects, adaptive responses have evolved which protect against hypoxia-associated tissue injury. To date, much attention has focused on hypoxia-activated HIF (hypoxia-inducible factor) transcription factors in these responses. However, recent work has identified epigenetic regulators that are also oxygen-sensitive, but their role in adaptation to hypoxic inflammation is currently unclear. Here, we show that the oxygen-sensing epigenetic regulator UTX is a critical modulator of colitis severity. Unlike HIF transcription factors that act on gut epithelial cells, UTX functions in colitis through its effects on immune cells. Hypoxia results in decreased CD4 + T cell IFN-γ production and increased CD4 + regulatory T cells, and these findings are recapitulated by T cell-specific UTX deficiency. Hypoxia impairs the histone demethylase activity of UTX, and loss of UTX function leads to accumulation of repressive H3K27me3 epigenetic marks at IL12/STAT4 pathway genes ( Il12rb2, Tbx21, and Ifng ). In a colitis mouse model, T cell-specific UTX deletion ameliorates colonic inflammation, protects against weight loss, and increases survival. Together these findings implicate UTX's oxygen-sensitive histone demethylase activity in mediating protective, hypoxia-induced pathways in colitis.

FOXP3 protects conventional human T cells from premature restimulation-induced cell death.

The adaptive immune response relies on specific apoptotic programs to maintain homeostasis. Conventional effector T cell (Tcon) expansion is constrained by both forkhead box P3 (FOXP3)+-regulatory T cells (Tregs) and restimulation-induced cell death (RICD), a propriocidal apoptosis pathway triggered by repeated stimulation through the T-cell receptor (TCR). Constitutive FOXP3 expression protects Tregs from RICD by suppressing SLAM-associated protein (SAP), a key adaptor protein that amplifies TCR signaling strength. The role of transient FOXP3 induction in activated human CD4 and CD8 Tcons remains unresolved, but its expression is inversely correlated with acquired RICD sensitivity. Here, we describe a novel role for FOXP3 in protecting human Tcons from premature RICD during expansion. Unlike FOXP3-mediated protection from RICD in Tregs, FOXP3 protects Tcons through a distinct mechanism requiring de novo transcription that does not require SAP suppression. Transcriptome profiling and functional analyses of expanding Tcons revealed that FOXP3 enhances expression of the SLAM family receptor CD48, which in turn sustains basal autophagy and suppresses pro-apoptotic p53 signaling. Both CD48 and FOXP3 expression reduced p53 accumulation upon TCR restimulation. Furthermore, silencing FOXP3 expression or blocking CD48 decreased the mitochondrial membrane potential in expanding Tcons with a concomitant reduction in basal autophagy. Our findings suggest that FOXP3 governs a distinct transcriptional program in early-stage effector Tcons that maintains RICD resistance via CD48-dependent protective autophagy and p53 suppression.

Multiplexed Functional Assessment of Genetic Variants in CARD11.

Genetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a "cloning-free" saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.

ORMDL3 and Asthma: Linking Sphingolipid Regulation to Altered T Cell Function.

Orosomucoid like 3 (ORMDL3) encodes an ER-resident transmembrane protein that regulates the activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme for sphingolipid biosynthesis in cells. A decade ago, several genome wide association studies revealed single nucleotide polymorphisms associated with increased ORMDL3 protein expression and susceptibility to allergic asthma. Since that time, numerous studies have investigated how altered ORMDL3 expression might predispose to asthma and other autoimmune/inflammatory diseases. In this brief review, we focus on growing evidence suggesting that heightened ORMDL3 expression specifically in CD4+ T lymphocytes, the central orchestrators of adaptive immunity, constitutes a major underlying mechanism of asthma pathogenesis by skewing their differentiation and function. Furthermore, we explore how sphingolipid modulation in T cells might be responsible for these effects, and how further studies may interrogate this intriguing hypothesis.

Fatty Acid Synthase Contributes to Restimulation-Induced Cell Death of Human CD4 T Cells.

Restimulation-induced cell death (RICD) is an apoptotic pathway triggered in activated effector T cells after T cell receptor (TCR) re-engagement. RICD operates at the peak of the immune response to ensure T cell expansion remains in check to maintain immune homeostasis. Understanding the biochemical regulation of RICD sensitivity may provide strategies for tuning the magnitude of an effector T cell response. Metabolic reprogramming in activated T cells is not only critical for T cell differentiation and effector functions, but also influences apoptosis sensitivity. We previously demonstrated that aerobic glycolysis correlates with optimum RICD sensitivity in human effector CD8 T cells. However, metabolic programming in CD4 T cells has not been investigated in this context. We employed a pharmacological approach to explore the effects of fatty acid and glycolytic metabolism on RICD sensitivity in primary human CD4 T cells. Blockade of fatty acid synthase (FASN) with the compound C75 significantly protected CD4 effector T cells from RICD, suggesting that fatty acid biosynthesis contributes to RICD sensitivity. Interestingly, sphingolipid synthesis and fatty acid oxidation (FAO) were dispensable for RICD. Disruption of glycolysis did not protect CD4 T cells from RICD unless glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymatic activity was targeted specifically, highlighting important differences in the metabolic control of RICD in effector CD4 vs. CD8 T cell populations. Moreover, C75 treatment protected effector CD4 T cells derived from naïve, effector memory, and central memory T cell subsets. Decreased RICD in C75-treated CD4 T cells correlated with markedly reduced FAS ligand (FASL) induction and a Th2-skewed phenotype, consistent with RICD-resistant CD4 T cells. These findings highlight FASN as a critical metabolic potentiator of RICD in human effector CD4 T cells.

Enhanced extracellular vesicle production and ethanol-mediated vascularization bioactivity via a 3D-printed scaffold-perfusion bioreactor system.

Extracellular vesicles (EVs) have garnered significant interest in the biotechnology field due to their intrinsic therapeutic properties as well as their ability to serve as vehicles for bioactive cargo. However, the lack of an established biomanufacturing platform and limited potency of EVs in vivo remain critical bottlenecks for clinical translation. In this study, we utilized a 3D-printed scaffold-perfusion bioreactor system to assess the response of dynamic culture on extracellular vesicle production from endothelial cells (ECs). We also investigated whether ethanol conditioning, which was previously shown to enhance vascularization bioactivity of EC-derived EVs produced in standard 2D culture conditions, could be employed successfully for the same purpose in a 3D production system. Our results indicate that dynamic culture in a perfusion bioreactor significantly enhances EV production from human ECs. Moreover, the use of ethanol conditioning in conjunction with dynamic culture induces pro-vascularization bioactivity of EC-derived EVs that is correlated with increased EV levels of pro-angiogenic lncRNAs HOTAIR and MALAT1. Thus, this study represents one of the first reports of rationally-designed EV potency enhancement that is conserved between static 2D and dynamic 3D EV production systems, increasing the potential for scalable biomanufacturing of therapeutic EC-derived EVs for a variety of applications. STATEMENT OF SIGNIFICANCE: Extracellular vesicles (EVs) have substantial therapeutic potential in a variety of applications. However, translation of EV-based therapies may be hindered by biomanufacturing challenges. EV production to date has predominantly involved the use of tissue culture flasks. Here, we report, for the first time, the use of a tubular perfusion bioreactor system with an integrated 3D-printed biomaterial scaffold for EV production from human endothelial cells. This system increases EV yield by over 100-fold compared to conventional tissue culture systems. Further, we show that an ethanol-conditioning approach that our group previously developed in 2D culture for enhancing EV potency is compatible with this new system. Thus, potency enhancement of EVs for vascularization applications is possible even with significantly increased production rate.