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Cutting Edge: Glycolytic Metabolism and Mitochondrial Metabolism Are Uncoupled in Ag-Activated CD8+ Recent Thymic Emigrants This information is current as of September 25, 2021. Cody A. Cunningham, Suzanne Hoppins and Pamela J. Fink J Immunol published online 1 August 2018 http://www.jimmunol.org/content/early/2018/07/31/jimmun ol.1800705 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2018/07/31/jimmunol.180070 Material 5.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 1, 2018, doi:10.4049/jimmunol.1800705 Cutting Edge: Glycolytic Metabolism and Mitochondrial Metabolism Are Uncoupled in Ag-Activated CD8+ Recent Thymic Emigrants Cody A. Cunningham,*,1 Suzanne Hoppins,† and Pamela J. Fink* Recent thymic emigrants (RTEs) are peripheral T cells imparts lasting functional differences that distinguish that have most recently completed selection and thymic RTE-derived memory cells from mature T cell–derived memory egress and constitute a population that is phenotypi- cells (8). Importantly, the unique physiology of RTEs is not just cally and functionally distinct from its more mature a feature of the mouse but also of humans (9–12). counterpart. Ag-activated RTEs are less potent effec- RTE maturation is independent of many of the signals required for mature but naive (MN) T cell homeostasis tors than are activated mature T cells, due in part to Downloaded from reduced aerobic glycolysis (correctable by exogenous including IL-7, self-MHC molecules, and CD80/CD86 IL-2), which in turn impacts IFN-g production. (13, 14). However, thymic egress and access to secondary Mitochondria serve as nodal regulators of cell function, lymphoid organs are strictly required for RTE maturation but their contribution to the unique biology of RTEs is (15). Recently, the lymphocyte migration factor sphingosine unknown. In this study, we show that activated mouse 1-phosphate, which is present at high concentrations in the RTEs have impaired oxidative phosphorylation, even blood, has been shown to regulate MN T cell homeostasis http://www.jimmunol.org/ in the presence of exogenous IL-2. This altered respi- by maintaining mitochondrial content (16). These data ratory phenotype is the result of decreased CD28 sig- underscore the possibility that RTE maturation has a metabolic naling, reduced glutaminase induction, and diminished component. mitochondrial mass in RTEs relative to mature T cells. MN T cells undergo profound metabolic reprogramming These results suggest an uncoupling whereby IL-2 following activation (17) that is associated with an increase in the rate of aerobic glycolysis (18). However, even in the tunes the rate of RTE glycolytic metabolism, whereas presence of this large glycolytic flux, mitochondrial meta- the unique profile of RTE mitochondrial metabolism bolism remains critically important both for T cell activation is “hard wired.” The Journal of Immunology, 2018, by guest on September 25, 2021 and effector function (19, 20). The requirement for ongoing 201: 000–000. respiration in activated T cells extends beyond the requisite ATP generation (reviewed in Ref. 21). In CD4+ T cells, cells were once thought to be fully mature following production of mitochondrial reactive oxygen species is re- thymic selection and egress (1). This notion has quired for NFAT-mediated IL-2 production (20). Addi- T been progressively revised as improved models have tionally, mitochondria-derived acetyl-CoA is critical for allowed for the study of intrinsic peripheral T cell age histone acetylation at the ifng locus in the context of on- (reviewed in Ref. 2). The Rag2p-GFP transgenic (Tg) mouse going aerobic glycolysis (22). Mitochondrial metabolism also (3) has been used to isolate live, untouched recent thymic regulates memory T cell formation (23–25). Specifically, + emigrants (RTEs), facilitating previously unfeasible experi- mitochondrial morphology distinguishes CD8 effector from ments (4). Using this model, it has become clear that for the memory T cells. The former have round mitochondria with first 3 wk in the lymphoid periphery, T cells occupy a distinct loose cristae and the latter have elongated mitochondria phenotypic and functional niche. Relative to mature T cells, with tight cristae, in which key membrane components are CD8+ RTEs are poor proliferators and producers of effector brought into close spatial proximity, thereby enhancing the cytokines (IFN-g, TNF-a, and IL-2) following activation in efficacy of electron transport (26–28). Thus, mitochondria the absence of inflammation (4–7). Critically, Ag exposure represent a central hub through which T cell fate decisions are during the period when T cells occupy the RTE compartment orchestrated. *Department of Immunology, University of Washington, Seattle, WA 98109; and The online version of this article contains supplemental material. †Department of Biochemistry, University of Washington, Seattle, WA 98195 Abbreviations used in this article: Cpt1a, carnitine palmitoyltransferase 1a; GLS, glutaminase; 1Current address: Mayo Clinic School of Medicine–Arizona, Scottsdale, AZ. MN, mature but naive; OCR, oxygen consumption rate; OxPhos, oxidative phosphorylation; PGC-1a, peroxisome proliferator–activator receptor g coactivator-1a; RTE, recent thymic ORCIDs: 0000-0002-6507-3804 (C.A.C.); 0000-0002-7559-5948 (P.J.F.). emigrant; SRC, spare respiratory capacity; Tg, transgenic; VDAC1, voltage-dependent anion Received for publication May 21, 2018. Accepted for publication July 13, 2018. channel1;WT,wildtype. This work was supported by National Institutes of Health Grants R01 AI 064318 and R21 AI Ó 132960 (to P.J.F.), R01 GM 118509 (to S.H.), and T32 DK 007247 (to C.A.C.). Copyright 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 Address correspondence and reprint requests to Dr. Pamela J. Fink, Department of Immunology, University of Washington, South Lake Union 3.1, 750 Republican Street, E471, Box 358059, Seattle, WA 98109. E-mail address: pfi[email protected] www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800705 2 CUTTING EDGE: ALTERED MITOCHONDRIAL METABOLISM IN CD8+ RTEs In addition to signals triggered through the TCR, costim- Intracellular staining ulatory signals delivered through CD28 play a well-established Surface-stained cells were fixed and permeabilized using the Cytofix/Cytoperm role in regulating the induction of glycolytic metabolism in Kit (BD Biosciences) and stained with Abs against GLS (polyclonal), activated T cells (29) and an emerging role in modulating ATPB (3D3), Cpt1a [EPR12740(B)], and voltage-dependent anion channel 1 (VDAC1; 20B12AF2), all from Abcam, and PGC-1a (D-5; Santa Cruz mitochondrial function (30). CD28 signals are required for Biotechnology), p-S6 S240/S244 (D68F8), p–NF-kB (931H1) and p-p38 the establishment of tight cristae and carnitine palmitoyl- (D3F9), all from Cell Signaling Technology. For IL-2 staining, cells were transferase 1a (Cpt1a)–mediated spare respiratory capacity plated and activated as above for 6 h; GolgiPlug (BD Biosciences) was added (SRC). Additionally, 4-1BB signaling has been implicated after the first hour. Cells were stained with a labeled Ab against IL-2 in the regulation of mitochondrial metabolism (31), being (JES6-5H4; eBioscience). critical for optimal oxidative phosphorylation (OxPhos) via Metabolic bioassays g a peroxisome proliferator–activator receptor coactivator-1 Oxygen consumption rate (OCR) measurements (in picomoles per minute) (PGC-1a)–mediated accumulation of mitochondrial mass. were generated using an XF24 bioanalyzer (Seahorse Bioscience). T cells were We recently found that activated CD8+ RTEs have a re- plated (1 3 106 per well) into a XF24 microplate coated with Cell-Tak duced rate of glycolysis relative to mature T cells, a defect that (Sigma-Aldrich). Cells were allowed to equilibrate in nonbuffered medium (Seahorse Bioscience) containing 10 mM glucose for 30 min in a non-CO2 underlies their diminished IFN-g production (32). Activation incubator. Four baseline OCR readings were acquired before sequential in- in the presence of exogenous IL-2 corrects this deficit. As jection of 2.5 mM oligomycin A, 500 nM carbonyl cyanide-4-(trifluoromethoxy) mitochondria are central drivers of effector function, we now phenylhydrazone, and 2 mMrotenone+2mM antimycin A, all from Sigma-Aldrich. To determine the contribution of glutamine to OCR, basal OCR was measured in assess mitochondrial metabolism in RTEs to better under- Downloaded from cells plated in nonbuffered medium containing 10 mM glucose and glutamine at stand their unique biology. In this study, we show that either 0.2 mM (2Gln) or 2 mM (+Gln). activated