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

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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 CD8+ RTEs have reduced OxPhos and SRC rela- tive to their more mature counterparts. RTEs fail to increase Widefield microscopy glutaminase (GLS) expression and mitochondrial mass fol- CD8+ RTEs and MN T cells were stimulated as above, and mitochondria lowing activation, defects that persist even in the presence and nuclear DNA were visualized using MitoTracker Red CMXRos (Thermo Fisher Scientific) and NucBlue Live ReadyProbes Reagent (Molecular of exogenous IL-2. These alterations are driven by reduced http://www.jimmunol.org/ Probes), respectively. Cells were allowed to settle onto poly-D-lysine CD28 signaling in RTEs. Collectively, these results suggest (Sigma-Aldrich)–coated glass-bottom petri dishes (MatTek) for 30 min be- that although glycolytic metabolism in activated CD8+ RTEs fore imaging. A z-series with a 0.3-mm step size was collected at 37˚C and 3 is subject to modulation by IL-2, mitochondrial metabolism 7% CO2 using a Ti-E widefield microscope with a 100 numerical aperture 1.45 oil objective (Nikon), a solid-state light source (SPECTRA X; Lumencor), appears to be “hard wired” differently in RTEs than in mature and a scientific complementary metal oxide semiconductor camera (Zyla 5.5 T cells. These results have profound implications for T cell megapixel). Mitochondrial morphology was scored in a blinded fashion as responses that are dominated by RTEs, such as those in fragmented: spherical mitochondria ,1.5 mm in diameter; intermediate: , neonates and in adults recovering from therapeutic or accidental oblong and 2.5 mm in length; and reticular: elongated and highly connected.

lymphodepletion. Electron microscopy by guest on September 25, 2021 RTEs and mature T cells were activated as above, and 70-nm sections were Materials and Methods imaged using a JEM-1230 transmission electron microscope (JEOL USA) in Mice the University of Washington Vision Science Center. Image analysis was 2 2 performed blinded on 35–40 measurements of maximum crista width per C57BL/6J, B6.129P2-Tcrbtm1MomTcrdtm1Mom/J (TCRbd / ), B6.129S4- 2 2 condition across 20 image fields. Cd80tm1ShrCD86tm2Shr/J (CD80/86 / ), and OT-I [B6-Tg (TcraTcrb) 1100Mjb/J] mice expressing a transgene-encoded TCR that recognizes the Statistics chicken OVA-derived peptide SIINFEKL in the context of H-2Kb were 2 2 purchased from The Jackson Laboratory and maintained in-house. CD80/86 / The p values were calculated in Prism (GraphPad) using a paired two-tailed mice were cohoused with wild type (WT) controls for .14 d prior to ex- Student t test except where indicated. The p values ,0.05 were considered perimentation. Rag2p-GFP Tg mice (3) were backcrossed onto the C57BL/6J significant. background in our laboratory for at least 12 generations. Mice of both sexes were used at 8–10 wk of age. All mice were housed under specific pathogen- Results and Discussion free conditions and used in accordance with the University of Washington Defective glutamine-fueled OxPhos in activated RTEs Institutional Animal Care and Use Committee guidelines. Our previous work indicated that activated RTEs display Cell isolation and T cell stimulations glycolytic defects that can be corrected by exogenous IL-2 (32). However, mitochondrial metabolism and glycolytic RTE and MN OT-I Tg T cells were isolated from Rag2p-GFP Tg mice (7) and stimulated (32) as previously described. In brief, sort-purified CD45.1 RTEs metabolism play nonredundant roles in T cell activation and and MN T cells were plated at 1 3 105 per well + 5 3 105 irradiated CD45.2 effector function (19). Indeed, exogenous IL-2 failed to re- 2 2 2 2 TCRbd / or T cell–depleted CD80/86 / splenocytes as APCs in com- store defective IL-2 production by activated RTEs (Fig. 1A). plete RPMI 1640 (Life Technologies) containing 10% heat-inactivated FBS. Cells were stimulated with 10 nM SIINFEKL (Genmed Synthesis) 6 20 U/ml As IL-2 production requires mitochondrial metabolism (20), rIL-2 (Cetus) and maintained for 3 d at 37˚C in 7% CO2. we assessed the rate of OxPhos, which generates ATP and is coupled to NAD+/NADH homeostasis and the generation Abs, cell surface staining, and flow cytometry of mitochondrial reactive oxygen species in activated T cells (21). We found that RTEs and MN T cells measured directly Fc receptors were blocked using anti-16/32 (2.4G2; BD Biosciences), and cells were stained with fluorescent Abs to detect cell surface expression of 4-1BB ex vivo displayed equivalent OCRs. However, the robust, (17B5), CD4 (RM4-5), CD11b (M1/70), CD25 (PC61.5), CD44 (IM7), activation-dependent induction of OCR seen in mature CD45.1 (A20), B220 (RA3-6B2), ICOS (7E.17G9) NK1.1 (PK136), and T cells was significantly attenuated in RTEs (Fig. 1B, left). Ter119 (Ly-76), all from eBioscience, in addition to CD8a (53-6.7), CD28 Additionally, activated RTEs displayed less SRC (a measure of (37.51), and CD62L (MEL-14) from BD Biosciences. Data were acquired using a FACSCanto II (BD Biosciences) and analyzed with FlowJo software mitochondrial reserve). Importantly, exogenous IL-2 had no (Tree Star). effect on RTE basal OCR or SRC (Fig. 1B) despite restoring The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ FIGURE 1. Activated RTEs display defective OxPhos and GLS induction in the presence of exogenous IL-2. For each experiment, sort-purified CD8+ RTEs and MN T cells (Mat) were left unstimulated (non-stim) or stimulated in vitro for 3 d with peptide Ag and APCs 6 IL-2. (A) The percent of IL-2+ cells after restimulation was determined. (B) Basal (left) and maximal (right) OCR were determined on cells freshly isolated (non-stim) or stimulated with Ag 6 IL-2; arrows denote injection of the indicated compounds. A single timepoint of these data was previously published as supplemental figure 1C in Ref. 32. Intracellular expression of GLS (C)was determined on cells freshly isolated (non-stim) or stimulated with Ag 6 IL-2; representative histograms are shown on the left. The bar graphs in the right panels depict the average mean fluorescence intensity (MFI) 6 SD from .3 independent experiments. (D) Basal OCR was determined on activated RTEs and mature T cells plated in the presence of either 0.2 mM (2Gln) or 2 mM (+Gln) glutamine. *p , 0.05, **p , 0.01, *** p , 0.001, ****p , 0.0001 by unpaired (B and D)orpaired(A and C) Student t test. FCCP, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; Oligo, oligomycin A; Rot/AA, rotenone/antimycin A. by guest on September 25, 2021 maximum glycolytic rate (32). The TCA cycle is critical for may regulate a subtly different set of genes in RTEs and + generating NADH and FADH2. In activated CD8 T cells, mature T cells. glutaminolysis and fatty acid oxidation are critical for replenishing TCA-cycle intermediates depleted by cataplerotic Altered mitochondrial mass and morphology in activated RTEs processes (33). As RTEs display reduced OCR and SRC, we Respiratory capacity in activated CD8+ Tcellsisregulatedby next assessed the expression of rate-determining enzymes mitochondrial content (23) and morphology (26, 30). To de- for glutaminolysis (GLS) and fatty acid oxidation (Cpt1a). To termine mitochondrial mass, RTEs and mature T cells were left this end, we assessed enzyme expression in RTEs and mature resting or activated 6 exogenous IL-2 and the level of VDAC1 T cells left resting or activated 6 exogenous IL-2. Although was determined. Resting CD8+ RTEs and mature T cells had resting RTEs and mature T cells express similar levels of equivalent VDAC1 levels, similar to recent observations in GLS (Fig. 1C) and Cpt1a (Supplemental Fig. 1A), enzyme resting CD4+ RTEs (36). However, upon activation, RTEs induction is significantly diminished in activated RTEs. expressed significantly less VDAC1 than their mature T cell Critically, IL-2 fails to restore GLS expression in RTEs counterparts (Fig. 2A). This defect was also reflected in the ex- (Fig. 1C). To confirm that altered glutamine flux drives the pression of the ATPB subunit of Complex V (Fig. 2B), Tom20, differences in OCR seen in RTEs and mature T cells, OCR and staining with the mitochondrial membrane potential- was measured in cells activated in medium containing gluta- sensitive dye MitoTracker Red (data not shown). Critically, ac- mine and then plated in the presence of low (0.2 mM; 2Gln) tivation of RTEs in the presence of IL-2 failed to correct this or high (2 mM; +Gln) glutamine. Activated mature T cells defectinmitochondrialmass(Fig.2A,2B).ByMitoTracker had a significantly reduced OCR in the presence of low staining and live-cell microscopy, the majority of activated ma- glutamine (Fig. 1D). By contrast, glutamine access did not ture T cells contained mitochondria that were fragmented, with impact the OCR of activated RTEs. These results suggest that few cells having mitochondria of intermediate length or reticular the defective OCR observed in RTEs primed in the presence appearance (Fig. 2C). In contrast, fewer activated RTEs had of IL-2 is associated with a reduced rate of glutaminolysis. fragmented mitochondria, and a large proportion contained Interestingly, GLS is a Myc target gene (17) and IL-2 pro- mitochondria of intermediate length or reticular appearance. motes Myc expression (34) and restores Myc protein levels Crista structure is generally coupled to mitochondrial in RTEs (32). This discrepancy suggests that the GLS locus morphology (26), and both resting RTEs and mature may be in an altered epigenetic state in RTEs, as has been T cells had tight cristae (Fig. 2D). Upon activation, mature observed for certain cytokine loci (35). Alternatively, Myc T cells displayed greater crista width than RTEs, and this 4 CUTTING EDGE: ALTERED MITOCHONDRIAL METABOLISM IN CD8+ RTEs

difference was unaffected by activation in the presence of IL-2. Thus, activated RTEs have more elongated mito- chondria with tighter crista structure (parameters associated with greater respiratory capacity); however, this apparent respi- ratory advantage is offset by a profound reduction in mitochon- drial mass. Reduced mitochondrial mass even in the presence of IL-2 implies a defect in mitochondrial biogenesis following activation, a process that is regulated by PGC-1a (37–39). Resting RTEs and mature T cells had equivalent levels of PGC-1a; however, activated RTEs expressed signifi- cantly less PGC-1a than their more mature counterparts (Fig. 2E). Importantly, the addition of IL-2 failed to re- store PGC-1a levels in RTEs, suggesting that the reduced mitochondrial mass in RTEs is the result of lower PGC-1a expression.

Reduced CD28 expression and defective 4-1BB induction in Downloaded from activated RTEs Seminal work has shown that the costimulatory receptor CD28 regulates glycolytic metabolism in CD8+ T cells (29), a role that has been expanded to include respira- tory metabolism (30, 31). To determine if costimulatory

receptors contribute to the distinct mitochondrial metabolism http://www.jimmunol.org/ in RTEs, we assessed expression levels of 4-1BB, ICOS, and CD28. Compared with mature T cells, resting RTEs expressed equivalent levels of 4-1BB (Fig. 3A) and ICOS (Fig. 3B) but had a subtle, reproducible reduction in CD28 expression (Fig. 3C) (4). Upon activation, RTEs expressed significantly less 4-1BB, ICOS, and CD28 than their more mature T cell counterparts. However, in the presence of IL-2 (which fails to

restore OCR and SRC), RTEs expressed less 4-1BB but by guest on September 25, 2021 equivalent ICOS and CD28; hence, reduced CD28 expression on naive cells and reduced 4-1BB expression on activated cells correlates with the diminished respiratory capacity in activated RTEs.

CD28 signals drive alterations in mitochondrial mass Compared with mature T cells, resting RTEs express less CD28 and have more elongated mitochondria with tighter crista structure, alterations seen in CD8+ T cells primed FIGURE 2. RTE mitochondria have reduced mass and altered mor- without costimulation (30). To determine if altered phology. Sort-purified CD8+ RTEs and MN T (Mat) cells were freshly CD28 signaling accounts for the diminished mitochon- 6 isolated (nonstimulated [non-stim]) or stimulated with Ag IL-2 in vitro drial mass in RTEs, cells were left resting or activated with A B 2 2 for 3 d, and the intracellular level of VDAC1 ( )andATPB( ) was de- Ag presented by WT or CD80/86 / APCs. As expected, termined; representative histograms are shown on the left. The bar graphs in the right panels depict the average mean fluorescence intensity (MFI) 6 SD RTEs have lower mitochondrial mass than mature T cells from four independent experiments. CD8+ RTEs and MN T cells were when activated with WT APCs. By contrast, RTEs and stimulated with Ag for 3 d and imaged by widefield microscopy (C). Mi- mature T cells had equivalent mitochondrial mass when 2 2 tochondria (red) and nuclear DNA (blue) are shown in representative activated with CD80/86 / APCs (Fig. 4A). Mitochon- maximum intensity projections (scale bar, 1 mm). Bar graphs represent the drial morphology in both activated RTEs and mature fraction of cells containing mitochondria of the indicated morphology. T cells was unaffected by the loss of CD28 signaling at RTEs and mature T cells (.100 per experiment) were imaged across two independent experiments. (D) Cells were left resting or activated as in (A), and this timepoint (Figs. 2C, 4B). By contrast, crista width mitochondrial morphology was determined by electron microscopy. Original remained tight in both RTEs and mature T cells activated 2/2 magnification 350,000. The bar graph on the right depicts the average maxi- with CD80/86 APCs(Fig.4C).Insupportofthese mum crista width. Expression level of PGC-1a (E) was determined on cells left findings, RTEs and mature T cells expressed equivalent 2 2 resting or activated as in (A); representative histograms are shown on the left. PGC-1a levelswhenactivatedwithCD80/86 / APCs The bar graph depicts the average MFI 6 SD from three independent experi- , , , A B E (Fig. 4D). These data suggest that alterations in CD28 ments. *p 0.05, **p 0.01, ****p 0.0001 by paired ( , ,and )or signaling account for the diminished mitochondrial mass unpaired (D) Student t test. observed in activated RTEs. To determine whether the reduced CD28 expression by RTEs translates to quanti- tatively different signaling, we assessed the level of p-S6 as The Journal of Immunology 5 Downloaded from

FIGURE 3. RTEs display reduced basal CD28 expression and defec- tive 4-1BB induction in the presence of exogenous IL-2. Cells were left resting or activated as in Fig. 2A, and the surface expression levels of 4-1BB (A), http://www.jimmunol.org/ ICOS (B), and CD28 (C) were determined; representative histograms are shown on the left. The bar graphs on the right depict the average mean fluorescence intensity (MFI) 6 SD from four independent experiments. *p , 0.05, **p , 0.01 by paired Student t test. a readout of CD28 signaling intensity. As observed pre- viously (32), RTEs activated with WT APCs have re- duced p-S6 levels compared with their mature T cell by guest on September 25, 2021 counterparts (Fig. 4E). However, RTEs and mature T cells had similarly reduced levels of p-S6 when activated 2 2 with CD80/86 / APCs. These data argue that the re- duced surface expression of CD28 on RTEs results in a quantitatively reduced downstream signal. Additionally, 4-1BB can support mitochondrial biogenesis in acti- vated T cells (31). However, 4-1BB transduces a similar FIGURE 4. Altered CD28 signals account for the reduced mitochondrial downstream signal in RTEs and mature T cells as mea- mass in RTEs. Sort-purified CD8+ RTEs and MN T cells were freshly isolated 2 2 sured by p-p38 (Supplemental Fig. 1B) and p–NF-kB (nonstimulated [non-stim]) or stimulated with WT or CD80/86 / APCs + Ag (SupplementalFig.1C).Blocking4-1BBL/4-1BBinterac- in vitro for 3 d, and the intracellular expression levels of VDAC1 (A) tions did not affect VDAC1 levels in either RTEs or were determined; representative histograms are shown on the left. The bar mature T cells (Supplemental Fig. 1D), further suggesting that graphs on the right depict the average mean fluorescence intensity (MFI) 6 SD from three independent experiments. (B) RTEs and MN T cells diminished signaling via CD28, but not 4-1BB, accounts for 2 2 stimulated with WT or CD80/86 / APCs were imaged as in Fig. 2C. Bar the reduced mitochondrial mass observed in RTEs. graphs represent the fraction of cells containing mitochondria of the indi- In sum, our data show that activated RTEs have de- cated morphology. RTEs and mature T cells (.100 per experiment) were fective OxPhos even in the presence of exogenous IL-2. imaged across two independent experiments. (C) Cells were activated with 2 2 This reduced respiratory rate is due to defective GLS WT or CD80/86 / APC, and mitochondrial morphology was determined induction and reduced accumulation of mitochondrial by electron microscopy. Original magnification 350,000. The bar graph on mass following activation. Altered CD28 signals account the right depicts the average maximum crista width. Cells were stimulated as A D E for the reduced PGC-1a expression and diminished ac- in ( ), and PGC-1a ( ) and p-S6 ( ) levels were determined; representa- tive histograms are shown on the left. The bar graphs on the right depict cumulation of mitochondrial mass in RTEs. As shown the average MFI 6 SD (D) or fold induction 6 SD (E) from three inde- schematically in Supplemental Fig. 2, our study high- pendent experiments. *p , 0.05, ****p , 0.0001 by paired (A, D, and E)or lights that although defective glycolytic metabolism in unpaired (C) Student t test. RTEs is correctable by the addition of IL-2, the altered mitochondrial metabolism in these transitional T cells appears to be hard wired. The reduced mitochondrial important implications in the context of antitumor im- content in activated RTEs may also confer RTEs with a mune responses in which mitochondrial metabolism in survival advantage, as mitochondria serve as central hubs CD8+ T cells appears to be the central driver of disease in the regulation of apoptosis (40). Last, this work has outcome (31). 6 CUTTING EDGE: ALTERED MITOCHONDRIAL METABOLISM IN CD8+ RTEs

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A No cytokine + IL-2 20 * 2 15

10 % of Max 5 Cpt1a (MFI x 10 ) 0 Cpt1a Mat RTE Mat RTE Mat RTE Non-stim Activated Activated + IL-2 B No cytokine + IL-2 15 2

10

% of Max 5 p-p38 (MFI x 10 ) 0 p-p38 Mat RTE Mat RTE Mat RTE Non-stim Activated Activated C No cytokine + IL-2 + IL-2 8 2 * * 6

4 % of Max 2

p-NF-κB (MFI x 10 ) 0 p-NF-κB Mat RTE Mat RTE Mat RTE Non-stim Activated Activated + IL-2 D + Isotype + α-4-1BBL 15 3

10 * **

% of Max 5 VDAC1 (MFI x 10 ) 0 VDAC1 Mat RTE Mat RTE Mat RTE Mat (Non-stim) Mat (Activated) Non-stim Isotype α-4-1BBL RTE (Non-stim) RTE (Activated)

Supplemental Figure 1. Cpt1a and 4-1BB signaling are dispensable for the mitochondrial phenotype of RTEs (A) Sort-purified CD8+ RTEs and MN T cells were freshly isolated (non- stim) or stimulated with Ag ± IL-2 in vitro for 3 days and the expression level of intracellular Cpt1a (A), p-p38 (B), and p-NF-κB (C) determined; representative histograms are shown on the left. The bar graphs on the right depict the average MFI ± SD from 3 independent experiments. (D) RTEs and MN T cells left unstimulated or activated in the presence of α-4-1BBL or an isotype control (both at 10 μg/mL) and VDAC1 levels determined; representative histograms are shown on the left. The bar graphs on the right depict the average MFI ± SD from 3 independent experiments. *p < 0.05 ** p < 0.01 by paired Student t test. Supplemental Figure 2

Supplemental Figure 2. Altered metabolism in antigen-activated RTEs. Resting RTEs and mature T cells have similar metabolic profiles (top panels). However, upon activation in the absence of exogenous IL-2 (middle panels), RTEs display defective aerobic glycolysis and glutamine-fueled OxPhos relative to mature T cells. When RTEs are activated in the presence of exogenous IL-2 (bottom panels), aerobic glycolysis (and thus IFN-γ production) is restored. How- ever, IL-2 fails to restore GLS expression and mitochondrial mass accumulation in RTEs.