published the paper; L.S. but save was possible by inhibiting mitochondrial fragmentation, implicating mitochondrial polarization like a central regulator of NK cell function. NKDim cells are heterogeneous, and mitochondrial polarization was associated with enhanced survival and function in adult NKDim cells, including memory-like > .05 was considered not significant (ns). *< .05, **< .01, ***< .001, ****< .0001 using 2-way analysis of variance with Tukeys correction. MFI, mean fluorescence intensity. Glucose and glutamine are key substrates of the mitochondrial bioenergetic pathways and have been reported to be essential nutrients for NK cell activation.12,13,28 We therefore characterized GLUT-1 (levels in NKDim cells, whereas expression was higher in NKBr cells (supplemental Number 1B). These subset-specific variations were confirmed in the protein level, with a higher percentage of NKDim cells expressing GLUT-1 and NKBr cells expressing ASCT2 (Number 1B; supplemental Number 1C). IL-15Cprimed NKDim cells did not significantly upregulate either of these receptors, whereas the rate of recurrence 21-Deacetoxy Deflazacort of ASCT2+ NKBr cells improved after IL-15 exposure. NK cell activation (IL-12/18) enhanced the average manifestation levels of both receptors on both subsets. Although we did not detect any switch in the already high percentage of ASCT2+ NKBr cells, we found improved frequencies of GLUT-1+ NKDim and NKBr cells as well as ASCT2+ NKDim cells after activation with IL-12 and IL-18 (Number 1B). We further analyzed nutrient receptors CD98 (a component of the L-amino acid transporter), CD71 (transferrin receptor), and CD36 (FA translocase). CD71 was indicated at higher levels in NKBr compared with NKDim cells at constant state; however, manifestation improved in response to cytokine activation for both subsets. CD98 and CD36 were upregulated in both subsets only upon activation (supplemental Number 1C). Analysis of [3H]L-glutamine and 2-DG uptake showed improved uptake in both subsets upon priming and activation, with NKDim cells showing significantly greater glucose uptake than NKBr cells (supplemental Number 1D). FAs are another important nutrient source that can gas mitochondrial pathways. Because multiple FA transporters exist, we measured FA uptake using BODIPY-FLC16.32 We found that upon activation, NKBr cells took up more long-chain FAs than NKDim cells (Number 1C). To clarify which of these numerous nutrients are required for NKBr and NOX1 NKDim cells to exert their function, we analyzed the effects of inhibitors focusing on glycolysis (2-DG), FA -oxidation (ETO), and glutamine conversion into glutamate (BPTES). We found that IFN- and TNF- were produced by NK cells only upon activation, and NKBr cells produced higher levels of both cytokines compared with NKDim cells15,27,29 (Number 1D; supplemental Number 1E). Treatment with 2-DG inhibited IFN- and TNF- production by both NK cell 21-Deacetoxy Deflazacort subsets, consistent with a requirement for increased glucose uptake in this process. Although BPTES treatment did not alter the production of effector 21-Deacetoxy Deflazacort cytokines in either subset, both IFN- and TNF- were significantly decreased in NKBr cells after treatment with ETO, suggesting that FA oxidation is definitely important for NKBr cell activation and function (Number 1D; supplemental Number 1E). None of these inhibitors had an impact on NK cell viability (supplemental Number 1F) or cytotoxic function (supplemental Number 1G). Distinct bioenergetics in NKBr and NKDim cells upon cytokine priming vs activation We used the Seahorse bioanalyzer33 to quantitate OCRs (indication of OXPHOS) and ECARs (reflecting lactate production and glycolysis) in purified NK cell subsets. NKDim cells exhibited improved maximal ECARs after FCCP compared with NKBr cells (Number 2A), indicating elevated maximal glycolysis capacity. NKDim cells also 21-Deacetoxy Deflazacort showed improved basal OCRs, higher maximal OCRs, and improved ATP-linked respiration when compared with NKBr cells (Number 2B; supplemental Number 2A), indicating that NKDim cells rely primarily on OXPHOS to produce ATP at constant state. SRC was higher in NKDim compared with NKBr cells (Number 2C), consistent with latent mitochondrial capacity. RNA sequencing analysis confirmed that NKDim cells indicated higher levels of.