Mitochondria are key regulators of immune cell function, going beyond their traditional role in ATP and metabolite production to support anabolic processes and act as hubs for intracellular signaling. A key aspect of this signaling function is the production of mitochondrial reactive oxygen species (mtROS), which act as critical second messengers in both adaptive and innate immune regulation. Immune cells maintain an optimal concentration of mtROS to maintain physiological responses, and excessive or lack of mtROS production contributes to chronic inflammation, autoimmunity, and cancer. Here, we review the molecular mechanisms controlling mtROS production and detoxification, their role in shaping macrophage and T cell fate and function, and their implications for disease pathogenesis.

Tregs require Foxp3 expression and induction of a specific DNA hypomethylation signature during development, after which Tregs persist as a self-renewing population that regulates immune system activation. Whether maintenance DNA methylation is required for Treg lineage development and stability and how methylation patterns are maintained during lineage self-renewal remain unclear. Here, we demonstrate that the epigenetic regulator ubiquitin-like with plant homeodomain and RING finger domains 1 (Uhrf1) is essential for maintenance of methyl-DNA marks that stabilize Treg cellular identity by repressing effector T cell transcriptional programs. Constitutive and induced deficiency of Uhrf1 within Foxp3⁺ cells resulted in global yet nonuniform loss of DNA methylation, derepression of inflammatory transcriptional programs, destabilization of the Treg lineage, and spontaneous inflammation. These findings support a paradigm in which maintenance DNA methylation is required in distinct regions of the Treg genome for both lineage establishment and stability of identity and suppressive function.

The tumor microenvironment (TME) enhances regulatory T (T _reg ) cell stability and immunosuppressive functions through up-regulation of lineage transcription factor Foxp3, a phenomenon known as T _reg fitness or adaptation. Here, we characterize previously unknown TME-specific cellular and molecular mechanisms underlying T _reg fitness. We demonstrate that TME-specific stressors including transforming growth factor–β (TGF-β), hypoxia, and nutrient deprivation selectively induce two Foxp3-specific deubiquitinases, ubiquitin-specific peptidase 22 ( Usp22 ) and Usp21 , by regulating TGF-β, HIF, and mTOR signaling, respectively, to maintain T _reg fitness. Simultaneous deletion of both USPs in T _reg cells largely diminishes TME-induced Foxp3 up-regulation, alters T _reg metabolic signatures, impairs T _reg -suppressive function, and alleviates T _reg suppression on cytotoxic CD8 ⁺ T cells. Furthermore, we developed the first Usp22 -specific small-molecule inhibitor, which dramatically reduced intratumoral T _reg Foxp3 expression and consequently enhanced antitumor immunity. Our findings unveil previously unappreciated mechanisms underlying T _reg fitness and identify Usp22 as an antitumor therapeutic target that inhibits T _reg adaptability in the TME.

Mitochondrial electron transport chain (ETC) function is linked to the generation of ATP, signaling molecules including reactive oxygen species (ROS), pyrimidines and tricarboxylic acid cycle metabolites¹. Mitochondrial electron transport is required for T cell proliferation^2,3,4. However, which mitochondrial ETC functions are necessary for each dynamic state of CD8⁺ T cell responses is unknown. Here we report that impairing mitochondrial complex III function, which diminishes respiration, proton pumping linked to ATP production and superoxide production, decreases peripheral naive numbers, antigen-induced CD8⁺ T cell proliferation and memory formation. Acute stimulation of mitochondrial complex III-deficient CD8⁺ T cells induced an exhausted-like phenotype. Expression of Ciona intestinalis alternative oxidase (AOX) in mitochondrial complex III-deficient CD8⁺ T cells restores respiration without generating ROS or proton pumping, and rescues proliferation and the exhausted phenotype but not naive or memory formation. Thus, T cell development, proliferation and memory formation have distinct requirements for mitochondrial complex III ROS.

Significance: We show CD1d-restricted natural killer (NK)T cells have distinct metabolic profiles compared with CD4 ⁺ conventional T cells. Mature NKT cells have poor fatty acid oxidation and exhibit reduced mitochondrial respiratory reserve in the steady state. In addition, NKT cell development is more sensitive to alterations in mitochondrial electron transport chain function than conventional T cells. Using T cell-specific mitochondrial complex III ablation in mice, we further demonstrate that mitochondrial metabolism plays a crucial role in NKT cell development and function by modulating T cell receptor/interleukin-15 signaling and NFAT activity. Collectively, our data provide evidence for a critical role of mitochondrial metabolism in NKT cell development and activation, opening a new avenue for NKT cell-based immunotherapy by manipulating NKT cell metabolism.