Endoplasmic Reticulum Stress Contributes to Mitochondrial ... · Endoplasmic Reticulum Stress Contributes to Mitochondrial Exhaustion of CD8þ T Cells Katie E. Hurst1, Kiley A. Lawrence1,

Research Article

Endoplasmic Reticulum Stress Contributes toMitochondrial Exhaustion of CD8þ T CellsKatie E. Hurst1, Kiley A. Lawrence1, Matthew T. Essman1,2, Zeke J.Walton1,3,Lee R. Leddy1,3, and Jessica E. Thaxton1,3,4

Abstract

Tumor antigen–specific T cells rapidly lose energy andeffector function in tumors. The cellular mechanisms bywhich energy loss and inhibition of effector function occurin tumor-infiltrating lymphocytes (TILs) are ill-defined, andmethods to identify tumor antigen–specific TILs that expe-rience such stress are unknown. Processes upstream of themitochondria guide cell-intrinsic energy depletion. Wehypothesized that a mechanism of T-cell–intrinsic energyconsumption was the process of oxidative protein foldingand disulfide bond formation that takes place in the endo-plasmic reticulum (ER) guided by protein kinase R-likeendoplasmic reticulum kinase (PERK) and downstreamPERK axis target ER oxidoreductase 1 (ERO1a). To test thishypothesis, we created TCR transgenic mice with a T-cell–specific PERK gene deletion (OT1þLckcreþPERKf/f, PERK

KO). We found that PERK KO and T cells that were phar-macologically inhibited by PERK or ERO1a maintainedreserve energy and exhibited a protein profile consistentwith reduced oxidative stress. These T-cell groups displayedsuperior tumor control compared with T effectors. Weidentified a biomarker of ER-induced mitochondrialexhaustion in T cells as mitochondrial reactive oxygenspecies (mtROS), and found that PD-1þ tumor antigen–specific CD8þ TILs express mtROS. In vivo treatment with aPERK inhibitor abrogated mtROS in PD-1þ CD8þ TILs andbolstered CD8þ TIL viability. Combination therapy enabled100% survival and 71% tumor clearance in a sarcomamouse model. Our data identify the ER as a regulator ofT-cell energetics and indicate that ER elements are effectivetargets to improve cancer immunotherapy.

IntroductionIn response to antigen, T cells undergo rapid expansion, engag-

ing in up to 15 to 20 cell divisions, and proliferation is charac-terized by differentiation to an effector phenotype from a na€�vestate (1). To support the bioenergetic demands of growth andproliferation, na€�ve T cells that primarily engage oxidative phos-phorylation (OXPHOS) shift to generate adenosine triphosphate(ATP) via increased OXPHOS and aerobic glycolysis as theybecome functional effector cells (2, 3). Highly differentiatedeffector T cells rely primarily on glycolysis and are characterizedby a loss of mitochondrial integrity (4) that likely accounts fortheir inability to effectively continue OXPHOS. The cell-intrinsicmechanisms that consume metabolic energy and impart mito-chondrial exhaustion in T cells are unknown.

Programmed cell death protein 1 (PD-1) is expressed on highlydifferentiated effector T cells that have become exhausted due tochronic antigen exposure (5). In cancers, programmed death-ligand 1 (PD-L1) is expressed by tumor cells to further impairantitumor effector function of PD-1þ T cells (6). Checkpointblockade therapy is a means to reinvigorate effector function ofexhausted T cells by inhibition of the PD-1–PD-L1 interaction (7).Unfortunately, anti–PD-1 therapy remains ineffective for themajority of cancer patients due to the initial lack of response orloss of durable responses (8–10). In early-stage exhaustion,PD-1þ T cells show diminished OXPHOS and glycolysis, andlong-term chronically exhausted PD-1high T cells exhibit a depen-dence on glycolysis due to dysfunctional mitochondria (11).T-cell–intrinsic factors that drive PD-1þ CD8þ tumor-infiltratinglymphocyte (TIL) metabolic exhaustion in response to tumorantigen are unknown, and simple methods to characterize themetabolic state of PD-1þ TILs in patient tumors are ill-defined.

T-cell expansion initiated by antigen recognition requires Teffector cells to greatly increase new protein synthesis and initiateposttranslational modifications (12). Imbalances of unfoldedand misfolded proteins are detected by endoplasmic reticulum(ER) stress sensors inositol-requiring enzyme-1 (IRE1a), proteinkinase R-like endoplasmic reticulum kinase (PERK), and activat-ing transcription factor 6 (ATF6; ref. 13). The acute arm of thestress response aims to alleviate the burden of unfolded ormisfolded proteins in the ER to restore proteostasis. In the faceof insurmountable stress, the unfolded protein response (UPR)initiates cell death. PERK mediates the terminal UPR throughregulation of transcription factors activating transcription factor 4(ATF4) and C/EBPa homologous protein (CHOP), which inducedownstream target ER oxidoreductase 1 (ERO1a; refs. 14–17). Asa catalyst of disulfide bond formation in the ER, ERO1a facilitates

1Department of Orthopedics, College of Medicine, Medical University of SouthCarolina Medical School, Charleston, South Carolina. 2Medical University ofSouth Carolina Medical School, Charleston, South Carolina. 3Hollings CancerCenter, Medical University of South Carolina Medical School, Charleston, SouthCarolina. 4Department of Microbiology and Immunology, Medical University ofSouth Carolina Medical School, Charleston, South Carolina.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

K.E. Hurst and K.A. Lawrence contributed equally to this article.

CorrespondingAuthor: Jessica E. Thaxton,Medical University of SouthCarolina,68 President Street, Charleston, SC 29425. Phone: 843-876-2819; Fax: 843-876-2267; Email: [email protected]

doi: 10.1158/2326-6066.CIR-18-0182

�2019 American Association for Cancer Research.

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oxidation-reduction reactions (Redox). Excess ERO1a producedin response to chronic protein folding induces accumulation ofreactive oxygen species (ROS) and impairs survival (15, 16). ThePERK-specific cell stress response has not beenmeasured in T cellsor applied to the rubric of antitumor immunity.

Here, we report that the PERK axis contributes to ERO1aactivation in T effector cells. We find that this axis consumesmetabolic energy in T cells and drives a protein profile synony-mous with oxidative stress. We show that accumulation of mito-chondrial ROS (mtROS) is a hallmark of ER-induced mitochon-drial exhaustion, connecting ER biology to metabolic function inT cells. Memory T cells show reduced expression of mtROS andERO1a compared with T effectors. PERK KO, and PERK, orERO1a inhibitor–treated T cells exhibited superior tumor controlcompared with T effectors. Tumor antigen–specific PD-1þ CD8þ

TILs acquiremtROS in tumors. In sarcoma patients and a sarcomamousemodel, we find that high proportions of PD-1þCD8þ TILsexpress mtROS. In vivo treatment with a PERK inhibitor reducedmtROS in PD-1þ CD8þ TILs, increased tumor clearance, andextended animal survival in combination with anti–PD-1therapy.

Materials and MethodsMice

T-cell–specific deletion of PERKonaC57BL/6J backgroundwasaccomplished by cross of PERKloxP (Eif2ak3tm1.2Drc/J) mice withOT-1 (C57BL/6-Tg(TcraTcrb)1100Mjb/J) or Lck-Cre (B6.Cg-Tg(Lck-icre)3779Nik/J) mice to obtain OT-1xPERKf/f and LckCrex-PERKf/w mice, respectively. These lines were crossed to obtainOT1þLckcreþPERKf/f mice and OT1þLckcre�PERKf/f littermatecontrols. Lck-Cre, PERKloxP, OT-1, C57BL/6J, Ly5.2 (B6.SJL-Ptprca

Pepcb/BoyJ), and P-mel (B6.CgThy1a/CyTg(TcraTcrb)8Rest/J)mice were obtained from The Jackson Laboratory. All animalexperiments were approved by the Medical University of SouthCarolina (MUSC) Institutional Animal Care and Use Committee,and the Division of Laboratory Animal Resources at MUSCmaintained all mice.

Human samplesThis work was determined byMUSC Institutional Review Board

to be exempt under protocols Pro00055941 and Pro00055960.Normal donor patients undergoing routine non–cancer-associatedsurgery (Pro00055941) or patients undergoing surgical removalof high-grade deep pleomorphic undifferentiated sarcomas(Pro00055960) granted consent under MUSC Biorepositorysurgical consent forms. Tissue samples were deidentified.Studies were conducted in accordance with the Declaration ofHelsinki, International Ethical Guidelines for BiomedicalResearch Involving Human Subjects (CIOMS), Belmont Report,or U.S. Common Rule. Blood (8 mL) was collected in EDTAcoated tubes and peripheral blood mononuclear cells (PBMCs)were isolated via Histopaque-1077 centrifugation (Sigma).Sarcoma tissue was collected on ice and immediately cut into2 mm3 pieces and dissociated to a single-cell suspension usingHuman Tumor Dissociation Kit and gentleMACS dissociator(Miltenyi Biotec) according to the manufacturer's protocol.

RT-PCR and immunoblot analysisRNA was isolated with the RNeasy Mini Kit (QIAGEN) and

single-strand cDNA was made with High Capacity RNA-to-cDNA

Kit (Applied Biosystems, Thermo Fisher Scientific). TaqMan gene-expression assays (Applied Biosystems, Thermo Fisher) were usedto perform real-time PCR using the StepOnePlus Real-Time PCRsystem (Applied Biosystems, Thermo Fisher). Gene expression forPERK (Eif2ak3), ATF4 (Atf4), CHOP (Ddit3), and ERO1a (Ero1)were normalized to Gapdh. For immunoblots, cell lysates wereprepared in RIPA buffer (Sigma-Aldrich) and probed with PERK(Cell Signaling Technology), ERO1a (Santa Cruz Biotechnology)antibodies, or b-actin (Cell Signaling Technology) loadingcontrol.

Cell sorting, FACS staining, and analysisFluorochrome-conjugated monoclonal antibodies and respec-

tive isotype controls listed in Supplementary Table S1 werepurchased from Thermo Fisher (eBioscience). Extracellular stainswere performed in PBS supplemented with 2% FBS. IFNg wasprobed after 4 to 6 hours of cell restimulation with Cell Stimu-lation Cocktail (eBioscience) and GolgiPlug Protein TransportInhibitor (eBioscience). Foxp3/Transcription Factor IntracellularStaining Buffer Set was used according to the manufacturer'sprotocol (eBioscience). mtROS were measured with MitoSOXRed Mitochondrial Superoxide Indicator (3 mmol/L) loaded at37�C for 30 minutes in PBS. Extracellular stains were added postdye incubation. For Annexin staining, MitoSOX dye loaded cellswere washed and stained for using Annexin V–FITC ApoptosisDetection Kit (eBioscience) according to the manufacturer'sprotocol. Samples were run directly on a BD Accuri C6 flowcytometer. For FACS sorting of mtROSþ and mtROS� cell frac-tions, samples were stained with MitoSOX Red MitochondrialSuperoxide Indicator and CD8 and sorted on a FACSAria IIu (BDBiosciences). Further analysis was performed post-sort for con-firmation of population purity. For RNA ex vivo gene analysis,lymphocytes were isolated via Histopaque gradient (Sigma) andCD8þ T-cell Isolation Kit (Miltenyi) was used to obtain >90%purity from spleens and tumors.

Cell linesMCA-205-OVA (Gift of William Redmond) and MC38

(Kerafast Inc.) cell lines were maintained in DMEM 10% FBS.B16F1-OVA and B16F10 (Gifts of Mark Rubinstein) were main-tained in RPMI complete T-cell media. Cell lines were last deter-mined to be Mycoplasma free in March 2017 (MCA-205-OVA),January 2018 (MC38), and 2012 (B16F1-OVA, B16F10). Celllines were not authenticated in the past year. All growth mediawere supplemented with Plasmocin mycoplasma prophylactic(Invivogen). All cell lineswere passaged three times prior to in vivotumor inoculation, and a fresh vial of cells was prepared forindividual experiments.

T-cell activation and treatmentSpleens from OT-1 mice were dissociated, and RBC lysis was

performed using ACK Lysing (Thermo Fisher Scientific) to achievea single-cell suspension. T cells were activated and expanded forindicated time points from total splenocytes incubated with1mg/mLOVA257–264 (Invivogen)or hgp10025–33 (GenScript)peptide in complete T-cell media (200U rhIL2, NCI). Cells werewashed, and media were changed after 3 days of initial activationand expansion. For inhibitor treatments, PERK (100 nmol/L,GSK2606414, Tocris) or ERO1a inhibitors (10 mmol/L, EN460,Cayman Chemical) were incubated with splenocytes for 10 min-utes prior to introduction of peptide and reintroduced at the day 3

ER Stress Contributes to Oxidative Stress in T Cells

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cell split. For central memory T-cell development, IL2 wasreplaced in T-cell media with rhIL15 (50 ng/mL, Shenandoah)at the day 3 cell split.

Metabolic and proteomic analysisOxygen consumption rate (OCR) was measured in nonbuffered

RS media supplemented with HEPES under basal conditionsand in response to 1 mmol/L oligomycin, 1.5 mmol/L FCCP, and2 mmol/L rotenone þ 1 mmol/L Antimycin A using the XFe96Extracellular Flux Analyzer (Seahorse Bioscience). Cell-Tak(Corning) was used for T-cell adherence.

Liquid chromatography–tandem mass spectrometry. Proteomicswere performed by Bioproximity, LLC. Samples were preparedfor digestion using the suspension-trapping (S-trap, Protifi)meth-od. Digested peptides were collected by centrifugation. Peptideswere elutedwith 80%acetonitrile, 5%ammoniumhydroxide andlyophilized in a SpeedVac (Thermo Savant) to remove volatilecomponents. Digestion mixtures were analyzed by UHPLC-MS/MS. LC was performed on an Easy-nLC 1000 UHPLC system(Thermo) interfaced to a quadrupole-Orbitrapmass spectrometer(Q-Exactive HF-X, Thermo Fisher) via nano-electrospray ioniza-tion using a source with an integrated column heater (ThermoEasy Spray source).

Data processing and library searching. Tandem mass spectra weresearched using X!Tandem and Open Mass Spectrometry SearchAlgorithm (OMSSA), requiring expectation value scores of 0.01 orbetter to be considered a match. Protein intensity values werecalculated using OpenMS to measure the area under the curve ofidentified peptides. Searches were performed on Amazon WebServices–based cluster compute instances using the ProteomeCluster interface, which builds species- and genus-specific proteinsequence librariesmonthly fromcurrentUniProtKBdistributions.Gene annotations were obtained from Ensembl Release 93 data-base, Mouse genes (v93GRCm38.p6) data set through the Bio-Mart website. Official Gene Ontology categories to annotatemouse proteins based on corresponding gene symbols were used.

Tumor mouse models and in vivo treatmentsFor transfer of 7-day expanded OT-1þ or PERK KO T cells,

C57BL/6 mice were implanted subcutaneously (s.c.) with 2.5 �105 B16F1-OVA tumor cells, and 5 � 105 T cells were infused viatail vein to 5 Gy irradiated mice after 7 days of tumor growth. Fortransfer of 7-day expanded Pmel or Pmel inhibitor–treated T cells,C57BL/6 mice were implanted s.c. with 2.5 � 105 B16F10 tumorcells, and 2 � 106 T cells were infused via tail vein to 5 Gyirradiated mice after 7 days of tumor growth. Tumor growth wasmeasured every other day for 3 weeks. For tracking acquisition ofmtROS/PD-1þ status in tumor antigen–specific CD8þ TILs, 2.5�105 MCA-205-OVA were implanted s.c. to Ly5.2 mice. Na€�ve OT-1þ T cells were obtained via the Mouse CD8þ T-Cell Isolation Kit(Miltenyi Biotec), and 1 � 106 CD8þ T cells were transferred viatail vein on day 7 of tumor growth. After 7 days of in vivoexpansion, tumor-draining lymph nodes (TDLN) and tumorswere harvested. Tumors were processed to single-cell suspensionusing theMouse TumorDissociation Kit (Miltenyi Biotec) accord-ing to the manufacturer's protocol. For combination therapyexperiments, 2.5 � 105 MCA-205-OVA were injected s.c. toC57BL/6 mice, and tumors were established for 7 days. PERKinhibitor (50 mg/kg; GSK2606414, GlaxoSmithKline) or vehicle

was administered twice daily via oral gavage as a suspension of0.5% hydroxypropylmethyl celluloseþ 0.1% Tween-80 in water.Two hundred micrograms aAnti–PD-1 (RMP1-14) or RatIgG2aisotype control (2A3; Bio X Cell) was administered every 4 daysafter 12 days of initial tumor growth. For CD8a cell depletion,100 mg Anti-CD8a (53-6.7, Bio X Cell) was administered threetimes per week after 5 days of tumor growth.

ResultsPERK contributes to activation of chronic ER stress in T effectorcells

Previously, we found that ER activation contributed to mito-chondrial stimulation in CD4þ T cells through inositol 1,4,5-triphosphate receptor (IP(3)R)–mediated Ca2þ signaling. Inhibi-tion of IP(3)R shifted T-cell fate and promoted cell persistence intumors (18). Continuousdisruptions inCa2þhomeostasis impairproper protein folding and a high burden of misfolded proteinsinduces chronic stress on the ER. In the face of irresolvable stress,PERK integrates signals from Ca2þ binding protein BiP/grp78 toinitiate the terminal UPR through activation of transcriptionfactors ATF4 and CHOP. A key downstream target of ATF4/CHOPis ERO1a. As a facilitator of Redox reactions, excessive ERO1aactivity can induce oxidative stress and impair cell survival as cellslose capability to scavenge ROS through antioxidant systems (17,19, 20).We asked how the ER stress sensor PERK and the terminalUPR were regulated in differentiating T effector cells. We foundthat gene expression of PERK (Eif2ak3), ATF4 (Atf4), CHOP(Ddit3), and ERO1a (Eor1l) were significantly increased over thecourse of T-cell activation and differentiation (Fig. 1A–D). PERKprotein increased in T cells and peaked after 5 days of expansion.ERO1a protein increased abundance as T cells differentiated(Fig. 1E). PERK gene expression peaked 7 days after activation,but protein expressionwas reduced after the5-day timepoint.Ourdata point out a discrepancy between gene and protein regulationof PERK in T cells and indicate that further study of how PERK ispost-transcriptionally regulated is needed.

To measure the role of PERK in induction of the terminal UPRin T cells, we created T-cell receptor (TCR) transgenic T-cell–specific conditional knockout mice (OT1þLckcreþPERKf/f, PERKKO). We measured genes associated with the chronic PERKresponse; Atf4, Ddit3, and Ero1l in na€�ve and 7-day expanded WTand PERK KO T cells. As expected, Eif2ak3, Atf4, Ddit3, and Ero1lwere significantly reduced in PERK KO T cells (Fig. 1F–I). In linewith these data, PERK and ERO1a proteins were decreased inPERK KO T cells (Fig. 1J). A longer exposure for PERK wasperformed to convey absence of PERK protein. These data reveala role for chronic ER stress as a component of differentiatingT effector cells.

PERK axis consumes energy in CD8þ T effector cellsIncreased spare respiratory capacity (SRC) is a property asso-

ciated with superior antitumor function of T cells (21). Themolecular processes that consume T-cell metabolic energy arenot well understood. Redox reactions for protein folding anddisulfide bond formation in the ER are energy-consumptiveprocesses that integrate ER activation with mitochondrial bioen-ergetics (17, 19, 22). We measured OCR in WT and PERK KOT cells and found that PERK KO T cells maintained superiorSRC ompared with WT cells. To assess a potential contributionof ERO1a to this phenomenon, we treated T cells with a

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target-specific PERK (PERK I; ref. 23) or ERO1a inhibitor(ERO1a I; ref. 24) across the course of differentiation andexpansion. We found that PERK I or ERO1a I–treated T cellspreserved metabolic energy compared with WT controls(Fig. 2A–C). Loss of SRC is associated with a reduction inT-cell effector function (25). We measured capacity to secreteIFNg in the abovementioned T-cell groups, and found thatPERK KO, PERK I, or ERO1a I T cells had increased IFNgsecretion compared with WT controls (Fig. 2D). Inhibitortreatments did not significantly reduce cell numbers and exper-imental T-cell groups exhibited increased viability comparedwith respective WT controls (Supplementary Fig. S1A and S1B).

Activation of chronic PERK UPR mediated by ATF4/CHOP-ERO1a signaling has previously been implicated to drive pro-tein synthesis, consume cellular energy, and contribute to deaththrough oxidative cell stress (16, 17, 19). To gain insight intohow PERK and downstream target ERO1a shape T effector cells,we assayed the proteomes of T effector, PERK KO, and ERO1aI–treated T cells. Shotgun liquid chromatography–tandemmass spectrometry (LC-MS/MS) was used to assess the top100 proteins with greatest intensity expression in T effectorscompared with PERK KO or ERO1a I–treated T cells (Supple-mentary Table S2). User-defined categories were used to qualifyproteins into biologically functional groups. Proteomicsrevealed protein groups associated with energy and metabo-lism, redox reactions, and oxidative stress/DNA damage withincreased expression in T effectors compared with PERK KOand ERO1a I–treated T-cell groups (Fig. 2E and F). Data arerepresented as fold increased expression values in T effectorscompared with PERK KO or ERO1a I–treated T-cell groups. Ofuser-defined categories, the greatest overlap in PERK KO andERO1a-treated T cells was in the "ER transport/cell stress"group (n ¼ 6 proteins). Three of seven of redox-associatedproteins overlapped as highly expressed in T effectors compared

with both PERK KO and ERO1a I–treated T cells, highlightinglactate dehydrogenases, thioredoxins, and glutathione-S-transferases as protein families of potential relevance. Furtheranalysis to validate these protein pathways to elucidate the roleof Redox and oxidative stress in T cells is warranted.

Mitochondrial ROS are a hallmark of mitochondrialexhaustion in T cells

Persistent ERO1a activation is bioenergetically costly to cells,and OXPHOS may be stimulated in efforts to replenish deplet-ed ATP stores (19). We wished to establish a biomarker ofenergy loss and aging in T cells that may be associated withchronic stress on the ER consistent with pathogenic ERO1aactivation. We reasoned that persistent mitochondrial activa-tion through continued OXPHOS would result in accumulationof mtROS in T cells. The live-cell dye MitoSOX Red detects thesuperoxide anion, a precursor to the majority of cellular ROS, inmitochondrial membranes and is detectable by flow cytometry(26). Under normal physiologic conditions, detoxification ofsuperoxide occurs through superoxide dismutase-mediatedconversion to hydrogen peroxide (27). However, excess ROSis associated with disease and aging (28). We measured mtROSin OT-1þ T cells over the course of T effector cell differentiation.As na€�ve T cells lost CD62L expression, a measure of stemness,mtROS accumulated in T-cell mitochondria (Fig. 3A). HumanCD8þ T cells underwent the same pattern over the course of 3weeks of in vitro expansion (Fig. 3B).

To determine whether mtROS accumulation was a hallmark ofenergy loss in T cells, we FACS sorted day 3 and 5 mtROS� andmtROSþ CD8þ T-cell subsets. The lowest 25% mtROS� andhighest 25% mtROSþ cells were obtained (Fig. 3C), and SRCamong groups was measured. Accumulation of mtROS indicatedenergy loss in T cells (Fig. 3D). We next measured the capacity forIFNg secretion among day 5 and 7 expanded mtROS� and

Figure 1.

PERK contributes to chronic ER stress in C8þ T effector cells. Na€�veWT OT-1þ CD8þ T cells were activated and expanded with cognate peptide and harvested atindicated time points. A–D, PERK (Eif2ak3), ATF4 (Atf4), CHOP (Ddit3), and ERO1a (Ero1l) gene expression were measured by qPCR and (E) PERK and ERO1aproteins measured by immunoblot (5 mg, 2 minutes). Data from 4 biological replicates are quantified and represented as SEM; results from Student t testperformed for na€�ve versus day 7 T cells are displayed. Experiments were repeated with four different WT animals and immunoblot is representative. Na€�vePERK KO (OT-1xLckCreþxPERKf/f) and littermate controls (OT-1xLckCre�xPERKf/f) or 7-day expanded T cells were harvested. F–I, PERK (Eif2ak3), ATF4 (Atf4),CHOP (Ddit3), and ERO1a (Ero1l) gene expression measured by qPCR and (J) immunoblot (5 mg, 1 minute) for PERK and ERO1a proteins. PERK 8-minuteexposure is shown to convey lack of protein expression. Data from threeWT and littermate pairs are quantified and represented as SEM, Student t test.Experiments repeated twice and immunoblot data are representative of four independent experiments. � , P < 0.05, �� , P < 0.01; �� , P < 0.001.






mtROSþ T-cell groups.We found that IFNg secretion underwent asimilar pattern to SRC among sorted T-cell populations (Fig. 3E).In day 7 cells, we asked whether mtROSþmay indicate early-stageapoptosis. We found that �8% of CD8þ mtROShigh T cellsharvested on day 7 were in early-stage apoptosis (Fig. 3F). Thiswas detectable only in day 7 samples, not in cells harvested atearlier time points. Our data show that mtROS can be used as ahallmark of metabolic energy loss and aging in T cells.

PERKaxis contributes tomitochondrial exhaustionof T effectorcells

Through ERO1a activation, oxidative protein folding in thestressed ER has been linked to generation of cellular ROS andinhibition of cell survival (19). However, the specific effects of thePERK axis on mitochondrial activation have not been assessed.Given the maintenance of SRC and diminished oxidative stress–associated proteomic signature in PERK KO and ERO1a I T cells(Fig. 2), we reasoned that these T-cell groups may show reducedmitochondrial activation and low mtROS accumulation. Wefound that mtROS was significantly diminished in PERK I, ERO1I, and PERKKOT cells comparedwith T effector controls (Fig. 4A).Indeed, in the abovementioned T-cell groups, maintained expres-sion of CD62L was a mark of reduced cell aging (Fig. 4B).

Reacquisition (29) or continued expression of CD62L hasbeen associated with memory T-cell formation (25, 30). Wedevelopedmemory T cells in vitrowith IL15 cytokine conditioningand found that memory T cells expressed reduced mtROS com-pared with T effector controls (Fig. 4C). These data are consis-tent with maintenance SRC of memory cells (25). We asked ifmemory T cells experience reduced activation of the chronicPERK axis. We found that Atf4, Ddit3, and ERO1l genes werereduced inmemory T cells compared with T effectors (Fig. 4D). Asexpected, ERO1a protein expression was increased in T effectors,and ERO1a protein was not highly expressed in memory T cells.Gene expression of Eif2ak3was not significantly different betweenT effector and T memory cells, but protein expression of PERKwas reduced (Fig. 4D and E). These data, similar to data obtainedin Fig. 1, may suggest posttranscriptional regulation of PERK inT cells. Together, the data indicate that ER programs may affectT-cell lineage fate.

PERK and ERO1a hinder T-cell–mediated tumor controlMemory T cells promote durable antitumor responses (30).We

used adoptive T-cell therapymousemodels tomeasure howPERKand ERO1a affect T-cell–specific tumor control. Mice bearingB16F1-OVA-expressing tumors were left untreated or treated with

Figure 2.

PERK axis affects CD8þ T effector profiles. Representative OCR trace and quantification of SRC from day 7 (A) PERK KO or littermate controls or (B) WT andPERK I or (C) WT and ERO1 I–treated OT-1þ CD8þ T cells measured via Seahorse Bioanalysis. SRC calculated as the difference between initial OCR rate and themaximal OCR rates achieved after FCCP uncoupling. Data are quantified and represented as SEM, Student t test performed for each condition versus controlT cells. Experiments were repeated at least 3 times. D, IFNg production from day 7WT, PERK I, or ERO1 I–treated OT-1þ T cells andWT (littermate) and PERK KOCD8þ T cells. Data from four biological replicates are quantified and represented as SEM, Student t test performed for each condition versus WT cells. Individualexperiments were repeated 3 times. � , P < 0.05; ��, P < 0.01; �� , P < 0.0001. Gene symbol and expression intensity of proteins identified by LC-MS/MS-basedshotgun proteomics extracted from the top 100 proteins with greatest enrichment in WT OT-1þ (T eff) compared with (E) PERK KO or (F) ERO1a I-treated T cells.Biologically functional groups of energy andmetabolism, ER transport/cell stress, oxidative stress/DNA damage, and Redox are shown. Heat maps representfold increased intensity of proteins from average value of three replicates in each T-cell group. Acly (þ212.98) in Teff versus PERK KO T cells is representednumerically.

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OT-1 (T eff) or PERK KO T cells, and tumor control wasmeasuredevery other day for 3 weeks. OT-1-PERK KO T cells exhibitedsignificantly greater tumor control compared with T effectors(Fig. 5A). Survival was significantly extended in tumor-bearingmice treatedwithPERKKOTcells (Fig. 5B). AlthoughPERKcanbeacutely protective, the PERK axis can also impair T-cell–mediatedantitumor immunity. To further test how the PERK axis contri-butes to T-cell–specific tumor control, P-mel T cells conditionedwith PERK I or ERO1a I were transferred to mice bearing path-ogenic B16F10melanomas, and tumor growthwasmonitored for3 weeks. Both inhibitor treatments of T cells yielded significantincreases in tumor control compared with T effectors (Fig. 5Cand D).

Tumor antigen–specific PD-1þ CD8þ TILs exhibitmitochondrial exhaustion

We next asked how the terminal PERKUPR is expressed amongendogenous CD8þ TILs. CD8þ cells were sorted from spleens andtumors of mice bearing immunogenic MCA-OVA sarcomas, andgene expression wasmeasured between splenocytes and TILs. Thechronic PERK signaling axis was significantly upregulated inCD8þ TILs compared with splenic-matched controls (Fig. 6A).We next asked whether mtROS accumulated in CD8þTILs inmice bearing immunogenic tumors. In multiple tumor models,CD8þ TILs accrued mtROS compared with T cells in TDLNs. Toexpand this finding, we asked whether mtROS was a property ofexhausted T cells in tumors. Wemeasured coexpression ofmtROS

Figure 3.

Mitochondrial ROS signify mitochondrial exhaustion. Representative FACS plots of (A) na€�ve WT OT-1þ CD8þ T cells activated and expanded withcognate peptide or (B) human PBMCs expanded in high-dose IL2 (3,000 U/mL) and CD8þ T cells FACS stained at indicated time points. FACSgates are set from fluorescence minus one controls. Data points represent quantification of five individual mice or human samples and arerepresented as SEM, Student t test performed for each time point versus T0 control. Experiments repeated twice. C, Representative FACS plot withgating from FACS sorts of mtROS/ CD8þ T cells. Lowest 25% � and highest 25% þ mtROS populations were collected and (D) representative OCRtrace and quantification of SRC from sorted populations at indicated time points were measured via Seahorse Bioanalysis. SRC calculated as thedifference between initial OCR rate and the maximal OCR rates achieved after FCCP uncoupling. Data are quantified and represented as SEM,Student t test performed for each condition versus control T cells. E, IFNg production from sorted subsets at indicated time points. Post-sort puritywas >97%. F, Representative FACS plot and quantification of mtROS-Annexin costaining on day 7 WT OT-1þ T cells. FACS gates are set fromfluorescence minus one controls. Data are quantified and represented as SEM, Student t test. Individual experiments repeated three times.� , P < 0.05; �� , P < 0.0001.






Figure 4.

PERK axis drives mitochondrial exhaustion and is impaired in memory T cells. Representative FACS plots and quantification of day 7WT, PERK I,ERO1a I–treated, andWT (littermate) and PERK KOOT-1þ CD8þ T cells probed for (A) mtROS or (B) CD62L expression. Bar graphs from 4 biological replicatesare quantified and represented as SEM, Student t test performed for each condition versus control. FACS gates are set from fluorescence minus one controls.Experiments were repeated at least three times. T effector (Teff) or memory (Tmem) cells were developed and harvested. C, Representative FACS plot andquantification of mtROS expression (D) quantification of PERK (Eif2ak3), ATF4 (Atf4), CHOP (Ddit3), and ERO1a (Ero1l) gene and (E) immunoblot for PERK andERO1a proteins. FACS quantification from four biological replicates. The experiment was repeated 3 times. Gene expression bar graphs represent average of3 separate experiments and are shown as SEM; Teff values are expressed relative to respective Tmem values set to 1. Immunoblot is representative datafrom 3 experiments. �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001.

Figure 5.

Inhibition of the PERK axis augmentsT-cell–specific tumor control. A,Individual graphs of mice bearing 7-dayB16F1-OVA tumors left untreated (n¼ 5)or treated intravenously with 5� 105

7-day expanded OT-1þ (Teff; n¼ 8) orPERK KO (n¼ 7) T cells. Tumor size wasrecorded every other day for 3 weeks.Lines represent individual mice. B,Survival to 45 days or tumor size of 400mm2was recorded, log-rank test; �� , P <0.01 survival proportions of mice treatedwith Teff (12%) versus PERK KO T cells(86%). C,Mice bearing 7-dayB16F10 melanomas were treatedintravenously with 2� 106 7-dayexpanded Pmel (Teff) or PmelT cells developed in the presence of (B)PERK inhibitor (PERK I T) or (D) ERO1ainhibitor (ERO1a I T). Tumor size wasrecorded every other day for 3 weeks.Lines represent individual mice. Linearregression of Teff versus PERK KO orinhibitor-treated T-cell groups, n¼ 5–6mice per group, �� , P < 0.0001.Experiments were repeated twice.

Hurst et al.





andPD-1 inCD8þT cells fromTDLNs and tumors ofmice bearingMCA-205-OVA sarcomas or MC-38 colon carcinomas. In bothmouse models mtROSþ cells were localized to the PD-1þ com-partment and mtROS expression was significantly increased inTILs compared with TDLNs (Fig. 6B and C). In agreement withgene-expression data, splenocytes from MCA-205-OVA tumor-bearing mice also expressed reduced mtROS/PD-1þ CD8þ T cellscompared with TILs (Supplementary Fig. S2A).

PD-1þ TILs comprise a pool of T cells enriched for tumorantigen specificity (31) and tumor-specific T cells home to andproliferate in tumors within 7 days of adoptive transfer (32).We reasoned that metabolically exhausted TILs would be in thetumor antigen–specific T-cell compartment. To address thishypothesis, we transferred na€�ve OT-1þ T cells to mice bearing7 day-established MCA-205-OVA sarcomas and measuredmtROS/PD-1þ coexpression among endogenous (CD45.1) andtransferred (CD45.2) T cells recovered from tumors after 7 daysof expansion (Fig. 6D). Approximately 70% of transferredCD45.2 CD8þ TILs coexpressed mtROS/PD-1 compared with�20% to 30% in the endogenous polyclonal pool of TILs, andthis expression could be visualized in TDLNs (Fig. 6E). Our dataagree with reports that PD-1þ T cells experience mitochondrialdepolarization (33) and shed light on the molecular eventswithin T cells that contribute to mitochondrial exhaustion.

PERK inhibition improves anti–PD-1 therapyWe wished to determine whether mtROSþ CD8þ TILs could

be identified in patients. Bone and soft-tissue sarcomas areimmunogenic human tumor types, but have shown poorresponsiveness to anti–PD-1 and combination checkpointblockade therapies (34). We obtained tumor tissue frompatients undergoing routine surgical removal of high-gradedeep pleomorphic undifferentiated (HGD PU) sarcomas.

These patients had not received radiation and had not under-gone recent chemotherapy. Dissociation and analysis of CD8þ

TILs from fresh HGD PU sarcomas showed a CD8þ T-cellinfiltrate in all tumors and FACS analysis of PBMCs versusautologous TILs showed a significant increase in mtROS/PD-1þ CD8þ T cells in tumors (Fig. 7A). Our data show that PERKinhibition overcomes mitochondrial exhaustion in T cellsin vitro (Fig. 4). MCA-205-OVA tumor–bearing mice weretreated with PERK I for 7 days during the time period whenT cells become functionally exhausted. Day 14 harvest of TILsshowed that PERK I–treated PD-1þ CD8þ TILs expressed sig-nificantly reduced mtROS compared with vehicle-treated con-trols (Fig. 7B). In line with these data, PERK I–treated miceexhibited increased absolute numbers of CD8þ T cells intumors (Fig. 7C). mtROSþ TILs comprise the pool of highlyactivated TILs measured by CD44 (Supplementary Fig. S2B). Areduction in mtROSþ TILs was concordant with reducedCD44þ expression among PERK I–treated TILs in bothMCA-205-OVA and MC-38 tumor models (Supplementary Fig.S2C). These data suggest that PERK I effects on CD8þ TILs werenot due to increased activation associated with reduced for-mation of immunosuppressive TIL populations or reducedtumor growth. In vivo PERK I treatment augmented anti–PD-1therapy as 5 of 7 combination therapy-treated mice achievedcomplete response compared with 2 of 7 mice treated withanti–PD-1 therapy alone. Depletions of CD8þ cells over thecourse of therapy demonstrated that CD8þ cells were necessaryfor combination therapy effect (Fig. 7D and E). Combinationtherapy–treated mice exhibited 100% survival compared with�28% survival in the anti–PD-1 therapy condition (Fig. 7F).Together, our data indicate that targeting ER stress in combi-nation with traditional immunotherapy may improveresponses in patients.

Figure 6.

Tumor antigen–specific PD-1þ CD8þ TILs experience mitochondrial exhaustion. CD8þ cells were sorted from spleens and tumors of mice bearing 14-dayMCA-205-OVA tumors and qPCR was performed to quantify (A) PERK (Eif2ak3), ATF4 (Atf4), CHOP (Ddit3), and ERO1a (Ero1l) gene expression. Bar graphs representaverages of 4 mice per group and are shown as SEM; the experiment was repeated twice. Representative FACS plots and quantification of mtROS/PD-1þ CD8þ

populations in TDLNs and tumors (TILs) harvested frommice bearing 14-day (B) MCA-205-OVA sarcomas or (C) MC38 colon carcinomas. Populations representgating from CD8þ/CD45þ lymphocytes and quadrants are set from PD-1 isotype control expression. Bar graphs represent 4–5 mice per group and are shown asSEM. Individual experiments were repeated 3 times. D, 1� 106 na€�ve CD45.2 OT-1þ T cells were transferred via tail vein to CD45.1 C57BL/6mice bearing 7 dayestablished s.c. MCA-205-OVA sarcomas. Tumors were harvested 7 days after transfer. E, Representative FACS plot overlay and quantification of mtROS/PD-1costaining from CD45.1 (gray) or CD45.2 (black) CD8þ cells in TDLNs and tumors. Gates are set from isotype control data. Bar graphs represent 4 mice per groupand are shown as SEM. Individual experiments repeated twice. Student t test; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001.






DiscussionWorks by several groups have brought the ER to the forefront of

programming pro- and antitumor immunity (35). ROS in thetumor microenvironment drive MDSC cell death through an ERstress–mediated mechanism. The shortened lifespan of MDSCspromotes their continued expansion in bone marrow (36). Inmultiple human tumor types,MDSCs in peripheral blood directlycorrelate with lowoverall survival rates in patients (37). Similarly,the tumormicroenvironment appears to impair tumor-associateddendritic cell (tDC) antigen presentation, and antigen presenta-tion is enhanced by impairing the IRE1a stress pathway in tDCs(38). Lastly, the role of ER stress, and in particular PERK, inaugmenting tumor cell development is well defined (39). Togeth-er, these data indicate that targeting ER stress pathways in cancerpatients may be a formidable strategy to promote antitumorimmune function and impair immune suppression and tumorcell growth.

Few reports have focused on the role of ER stress in control ofT-cell antitumor immunity, and little attention has been paid tothe role of the ER in defining T-cell biology. Our previous workfound that inhibition of ER activation improved T-cell mitochon-drial function and aided antitumor immunity (18). Our proteo-

mic screen yielded increased expression of multiple proteinsassociated with ER–mitochondrial cross-talk enriched in T effec-tors compared with PERK KO T cells. A second report found thatinhibition of IRE1a stress signaling led to memory T-cell devel-opment in response to acute infection (40). These data agree withour data presented here that uncover a role for ER stress signaling,a response that affects T-cell differentiation and effector cellmaturation.

Modulation of ER stress elements alone was able to affectmitochondrial bioenergetics and enhance T-cell–specific tumorcontrol. Mechanistically, acute PERK-specific ER stress is protec-tive to cells through momentary attenuation of protein synthesismediated by phosphorylation of eukaryotic translation initiationfactor 2A (EIF2a; ref. 13). The chronic PERK axis drives proteinsynthesis, energy consumption, oxidative cell stress, and deaththrough activation of transcription factors ATF4 and CHOP (16,17). Though PERK gene and protein expression were reduced inour PERK KO T cells, we do not discount the possibility ofremnant PERK expression that may have conferred the protectiveeffect of the acute PERK response combined with reduced lethaleffects from the terminal UPR. Based on our findings, T cellspresent a paradigm in which to study the effect of biologicallyrelevant chronic ER stress on cell development and death. Our

Figure 7.

PERK inhibition reduces CD8þ TIL mtROS and augments anti–PD-1 therapy. A, Representative FACS plot and quantification of mtROS/PD-1þ CD8þ T cells fromPBMCs and tumor of three patients with pleomorphic undifferentiated high-grade deep (PU HGD) sarcoma. Gates are set from isotype controls. PERK inhibitor(PERK I) or vehicle control was administered for 1 week (days 7–14) to mice bearing MCA-205-OVA sarcomas. B, Representative FACS plots and quantification ofmtROS/PD-1 TILs gated from CD45þ/CD8þ populations. Gates are set from isotype control data. C,Absolute number of CD45þ/PI�/CD8þ TILs calculated pergram of tumor weight. Bar graphs represent 4–5mice per group and are shown as SEM. Individual experiments repeated twice. Student t test; � , P < 0.05; �� , P <0.001. PERK I or vehicle control was administered beginning after 7 days of tumor growth to mice bearing MCA-205-OVA sarcomas, and anti–PD-1 or isotypeantibody was administered on day 12 and every 4 days thereafter. Anti-CD8 was administered every 2–3 days beginning 5 days after tumor inoculation.D,Composite and (E) individual graphs of tumor growth were measured every other day for 40 dayswith complete response (CR) listed per group; composite datarepresented as SEM. Linear regression of combination measured against anti–PD-1 therapy; �� , P < 0.0001. F, Survival to 41 days or tumor size of 200mm2 wasrecorded, log-rank test; �� , P < 0.01 survival proportions of anti–PD-1 therapy (28%) versus combination therapy (100%). Combination experiment was repeatedtwice, and anti-CD8 depletion condition was performed once.

Hurst et al.





data show that activationof ERO1a in rapidly dividing T effectors/TILs governs a cell-intrinsicmechanism that drives energy loss andoxidative stress. How the PERK-mediated transcription factorscontrol expression of ERO1a in T cells remains unknown. Thisquestion needs to be addressed due to potential implications forcancer immunotherapy (41, 42).

Based on the durability of antitumor immunity associatedwithmemory T cells in vivo (4, 21, 25, 43), we note that modulation ofthe PERK pathway could promote in vivo T-cell durability andcontribute to long-term efficacy of anti–PD-1 therapy. Multiplepotential benefits of targeting chronic ER stress have been iden-tified in modulation of pro- and antitumor immunity (35).Development of novel ER stress axis inhibitors that may circum-vent previous toxicities associated with inhibitors is on the hori-zon (44–48). Future research that defines the role of ER stress inT-cell biology is needed. Work that measures how tumor micro-environments drive cell stress signaling that affects T-cell antitu-mor function is essential.

Disclosure of Potential Conflicts of InterestL.R. Leddy reports receiving a commercial research grant from KCI. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: K.E. Hurst, K.A. Lawrence, L.R. Leddy, J.E. ThaxtonDevelopment of methodology: K.E. Hurst, K.A. Lawrence, Z.J. Walton, L.R.Leddy, J.E. Thaxton

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.E. Hurst, M.T. Essman, L.R. Leddy, J.E. ThaxtonAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.E. Hurst, J.E. ThaxtonWriting, review, and/or revision of the manuscript: K.E. Hurst, M.T. Essman,J.E. ThaxtonAdministrative, technical, or material support (i.e., reporting or organiz-ing data, constructing databases): K.E. Hurst, K.A. Lawrence, M.T. Essman,J.E. ThaxtonStudy supervision: Z.J. Walton, J.E. Thaxton

AcknowledgmentsFunding sources for this work were from NCI K12 CA157688, ACS IRG-97-

219-14, andACS IRG-16-185-17 to J.E. Thaxton. K12CA157688was granted viaHHS, NIH, NCI to L. Michael and ACS IRG-97-219-14 and ACS IRG-16-185-17to J. E. Thaxton.

We are thankful to Gyda Beeson and Dr. Craig C. Beeson for support withmetabolic assays, and to Dr. Jeffrey Axten for insight and thoughtful commen-tary on the manuscript. We thank Dr. B. Arman Aksoy for qualification ofprotein groups. We are grateful to Drs. Zihai Li, Chrystal Paulos, Michael Lilly,and Gustavo Leone for mentorship within the Hollings Cancer Center.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 28, 2018; revised September 23, 2018; accepted December12, 2018; published first January 18, 2019.

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Hurst et al.




2019;7:476-486. Published OnlineFirst January 18, 2019.Cancer Immunol Res Katie E. Hurst, Kiley A. Lawrence, Matthew T. Essman, et al.

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Endoplasmic Reticulum Stress Contributes to Mitochondrial ... · Endoplasmic Reticulum Stress Contributes to Mitochondrial Exhaustion of CD8þ T Cells Katie E. Hurst1, Kiley A. Lawrence1,