Mitochondrial metabolism is essential for invariant natural killer T cell development and function

Conventional T cell fate and function are determined by coordination between cellular signaling and mitochondrial metabolism. Invariant natural killer T (iNKT) cells are an important subset of “innate-like” T cells that exist in a preactivated effector state, and their dependence on mitochondrial metabolism has not been previously defined genetically or in vivo. Here, we show that mature iNKT cells have reduced mitochondrial respiratory reserve and iNKT cell development was highly sensitive to perturbation of mitochondrial function. Mice with T cell-specific ablation of Rieske iron-sulfur protein (RISP; T-Uqcrfs1^−/−), an essential subunit of mitochondrial complex III, had a dramatic reduction of iNKT cells in the thymus and periphery, but no significant perturbation on the development of conventional T cells. The impaired development observed in T-Uqcrfs1^−/− mice stems from a cell-autonomous defect in iNKT cells, resulting in a differentiation block at the early stages of iNKT cell development. Residual iNKT cells in T-Uqcrfs1^−/− mice displayed increased apoptosis but retained the ability to proliferate in vivo, suggesting that their bioenergetic and biosynthetic demands were not compromised. However, they exhibited reduced expression of activation markers, decreased T cell receptor (TCR) signaling and impaired responses to TCR and interleukin-15 stimulation. Furthermore, knocking down RISP in mature iNKT cells diminished their cytokine production, correlating with reduced NFATc2 activity. Collectively, our data provide evidence for a critical role of mitochondrial metabolism in iNKT cell development and activation outside of its traditional role in supporting cellular bioenergetic demands.

Cellular metabolic pathways are interwoven with traditional signaling pathways to regulate the function and differentiation of T cells (1–3). Upon activation, effector T cells display a marked increase in glycolytic metabolism even in the presence of ample oxygen, termed aerobic glycolysis (4). We have previously shown that despite increased aerobic glycolysis, T cell activation depends on mitochondrial metabolism for generation of reactive oxygen species (ROS) for signaling (5). As activated T cells progress to a memory or regulatory phenotype, they preferentially oxidize fatty acids to support mitochondrial metabolism, and enhanced fatty acid oxidation (FAO) and spare respiratory capacity (SRC) are essential to maintenance of their phenotype (6, 7).CD1d-restricted invariant natural killer T (iNKT) cells are a unique subset of lymphocytes that exhibit a preactivated phenotype with rapid effector responses (8, 9). iNKT cells are capable of producing large amount of proinflammatory and antiinflammatory cytokines thus have broad immunomodulatory roles (8–10). Given that these cells are poised for rapid proliferation and cytokine production, we hypothesized that coordination of cellular signaling with cellular metabolism will be especially critical for optimal iNKT function. In support of this hypothesis, several studies suggest that modulation of cellular metabolism affects iNKT cell development and function. iNKT cell development is diminished upon deletion of the miR-181 a1b1 cluster, which regulates phosphoinositide 3-kinase signaling and decreases aerobic glycolysis (11, 12). In addition, T cell-specific deletion of Raptor (a component of mTORC1), a metabolic regulator, leads to defects in iNKT cell development and function (13, 14). Loss of folliculin-interacting protein 1 (Fnip1), an adaptor protein that physically interacts with AMP-activated protein kinase, also results in defective NKT cell development, and interestingly conventional T cells develop normally (15). Furthermore, a number of studies targeting bioenergetics processes or related molecules, like alteration of glucose metabolism, mitochondrial-targeted antioxidant treatment, and receptor-interacting protein kinase 3-dependent activation of mitochondrial phosphatase, showed significant effects on iNKT cell ratio and function (16–19). A recent study showed that iNKT cells are less efficient in glucose uptake than CD4⁺ T cells. Furthermore, activated iNKT cells preferentially metabolize glucose by the pentose phosphate pathway and mitochondria, instead of converting into lactate, since high lactate environment is detrimental to their homeostasis and effector function (20).In conventional lymphocytes, mitochondria clearly play a role in coordination of cell signaling and cell fate decisions outside of production of energy (5, 21–23). During T cell activation mitochondria localize at immune synapses that T cells form with antigen-presenting cells (22). T cell receptor (TCR) stimulation triggers mitochondrial ROS (mROS) production as well as mitochondrial ATP production that are released at the immune synapses and are critical for Ca²⁺ homeostasis and modulation of TCR-induced downstream signaling pathways (22). We previously showed that mice with T-cell–specific deletion of Rieske iron sulfur protein (RISP), a component of mitochondrial complex III of the mitochondrial electron transport chain (ETC), are defective in antigen-specific T cell activation due to deficiency of mROS required for cellular signaling (5). Several recent studies showed that ROS or factors that affect ROS production are also important in iNKT cell development and effector functions (24–27). In addition, inhibition of mitochondrial oxidative phosphorylation (OXPHOS) by oligomycin has been shown to decreased survival and cytokine production by splenic iNKT cells (20). However, the requirement of mitochondrial metabolism for iNKT cell development and function has not been previously defined genetically or in vivo.Here we showed that iNKT cells have comparable basal mitochondrial oxygen consumption to conventional T cells but displayed lower SRC and FAO, which are thought to impart cells with mitochondrial reserve under stress. Using Uqcrfs1^fl/fl;CD4-Cre⁺ (hereafter referred as T-Uqcrfs1^−/−) mice, we showed that abrogation of mitochondrial metabolism resulted in a cell-autonomous defect in iNKT cell development in thymus and periphery. The iNKT cells were able to proliferate but exhibited impaired activation, suggesting that they were not lacking bioenergetically but rather had aberrant TCR signaling in vivo, leading to altered expression of downstream factors required for their terminal maturation. Accordingly, T-Uqcrfs1^−/− iNKT cells displayed lower T-bet and CD122 levels and did not respond to interleukin (IL)-15 stimulation. Knockdown of RISP in mature iNKT cells also limited NFATc2 translocation to the nucleus. Collectively, our data highlighted an important role of mitochondrial metabolism in modulating TCR signaling in vivo and regulating iNKT cell development and function.