CD155T/TIGIT Signaling Regulates CD8þ T-cell Metabolism ...Microenvironment and Immunology CD155T/TIGIT Signaling Regulates CD8þ T-cell Metabolism and Promotes Tumor Progression

Microenvironment and Immunology

CD155T/TIGIT Signaling Regulates CD8þ T-cellMetabolism and Promotes Tumor Progression inHuman Gastric CancerWeiling He1, Hui Zhang2, Fei Han3, Xinlin Chen4, Run Lin5,Wei Wang6, Haibo Qiu7,Zhenhong Zhuang8, Qi Liao9,Weijing Zhang10, Qinbo Cai1, Yongmei Cui2,Wenting Jiang2, Han Wang2, and Zunfu Ke2

Abstract

The T-cell surface molecule TIGIT is an immune checkpointmolecule that inhibits T-cell responses, but its roles in cancerare little understood. In this study, we evaluated the role TIGITcheckpoint plays in the development and progression of gastriccancer. We show that the percentage of CD8 T cells that areTIGITþ was increased in gastric cancer patients compared withhealthy individuals. These cells showed functional exhaustionwith impaired activation, proliferation, cytokine production,and metabolism, all of which were rescued by glucose. Inaddition, gastric cancer tissue and cell lines expressed CD155,which bound TIGIT receptors and inactivated CD8 T cells. In aT cell–gastric cancer cell coculture system, gastric cancer cellsdeprived CD8 T cells of glucose and impaired CD8 T-celleffector functions; these effects were neutralized by the

additional glucose or by TIGIT blockade. In gastric cancer tumorcells, CD155 silencing increased T-cell metabolism and IFNgproduction, whereas CD155 overexpression inhibited T-cellmetabolism and IFNg production; this inhibition was neutral-ized by TIGIT blockade. Targeting CD155/TIGIT enhanced CD8T-cell reaction and improved survival in tumor-bearing mice.Combined targeting of TIGIT and PD-1 further enhanced CD8T-cell activation and improved survival in tumor-bearing mice.Our results suggest that gastric cancer cells inhibit CD8 T-cellmetabolism through CD155/TIGIT signaling, which inhibitsCD8 T-cell effector functions, resulting in hyporesponsive anti-tumor immunity. These findings support the candidacy ofCD155/TIGIT as a potential therapeutic target in gastric cancer.Cancer Res; 77(22); 6375–88. �2017 AACR.

IntroductionGastric cancer is one of themost commonmalignancies world-

wide (1). Five-year survival rate of gastric cancer is lower than30%, and the current therapeutic methods show little improve-ment for gastric cancer survival (2). Tumor-specific cytotoxic

T cells are present in gastric cancer tissue, but are unable to containtumors because of poor immune responses in the tumor micro-environment (3). Reversing this effect would be a potentialtherapeutic approach to enhance the effectiveness of currenttreatments. The mechanisms by which gastric cancer inhibitsantitumor immune responses are poorly understood that targetscannot be identified.

The balance of positive and negative signals is crucial tomaintain host immune tolerance and activation (4, 5). Immunecheckpoints are molecules in the immune system that either turnon a positive (costimulatory) signal or a negative (coinhibitory)signal. Antibody treatments that target immune checkpoints havesignificantly improved clinical outcomes of solid and hemato-logic malignancies (6, 7). Malignant tumors, including gastriccancer, escape antitumor immune responses by upregulatingcoinhibitory signals, such as PD-1/PD-L1, in the tumor microen-vironment (8, 9). Phase I clinical trial has provided promisingantitumor activity by targeting PD-1/PD-L1 signal (10), warrant-ing further investigation to the immune checkpoints tohavebetteroutcomes for gastric cancer.

The T-cell immunoreceptor with immunoglobulin and ITIMdomains (TIGIT) has emerged as an important immune check-point in recent years. TIGIT, which belongs to the CD28 family(11), and CD226 share the common ligand CD155; binding ofCD155 to TIGIT suppresses T-cell activation (12), whereas bind-ing to CD226 enhances T-cell activation (13). TIGIT expression isincreased in tumor-infiltrating lymphocytes (TIL) and tumorantigen-specific CD8 T cells in melanoma patients (14). BlockingTIGIT enhances CD8 T-cell effector functions in tumor-bearing

1Department of Gastrointestinal Surgery, The First Affiliated Hospital, Sun Yat-senUniversity, Guangzhou, China. 2Department of Pathology, The First AffiliatedHospital, Sun Yat-sen University, Guangzhou, China. 3Department of RadiationOncology, Sun Yat-sen University Cancer Center, Guangzhou, China. 4School ofBasic Medical Science, Guangzhou University of Chinese Medicine, Guangzhou,China. 5Department of Radiology, The First Affiliated Hospital, Sun Yat-senUniversity, Guangzhou, China. 6Department of Gastrointestinal Surgery, Guang-dong Provincial Hospital of Chinese Medicine, Guangzhou, China. 7Departmentof Gastric Surgery, Sun Yat-sen University Cancer Center, Guangzhou, China.8Department of Gastrointestinal Surgery, Eighth Affiliated Hospital, Sun Yat-senUniversity, Shenzhen, China. 9Department of Prevention Medicine, School ofMedicine, Ningbo University, Ningbo, China. 10State Key Laboratory of Oncologyin South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

W. He, H. Zhang, and F. Han contributed equally to this article.

Corresponding Author: Zunfu Ke, Department of Pathology, First AffiliatedHospital of Sun Yat-sen University, Guangzhou, Guangdong 510080, China.Phone/Fax: 8620-8733-1780; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-0381

�2017 American Association for Cancer Research.

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mice (15). In addition, CD155 expression is increased in mela-noma cells, and the T-cell response is inhibited via TIGIT�CD155interactions (16). However, the mechanisms of CD155/TIGIT–induced immune suppression and subversion in gastric cancerremain poorly understood.

Costimulatory and coinhibitory signals interact to activateT cells by regulatingmetabolic activity (17, 18). T-cellmetabolismis highly dynamic and controls T-cell activation, proliferation,and differentiation (19, 20). Upon initial antigenic stimulation,T cells increase in size and switch their metabolism to glycolysis,which permits proliferation and effector functions (21, 22).During T-cell clonal expansion, T cells preferentially metabolizeglucose to fulfill their increased energy requirements (23). Failureto fulfill the increased bioenergetic demands of cell growth resultsin deleted or unresponsive T cells (20).

T-cell activation, which is critical for the antitumor immuneresponse (24), depends on the AKT/mTOR signaling pathway.AKT promotes glucose metabolism by increasing glucose trans-porter 1 (Glut1) expression, which facilitates glucose uptake in Tcells (17), so mTOR signaling integrates immune signals andmetabolic cues in T cells (25). Previous studies have demonstratedthat AKT/mTOR signaling and T-cell metabolism are decreased inthe tumor microenvironment (26), and that limited nutrients inthe tumor microenvironment impair the T-cell antitumorimmune response (27).

Here, we report that TIGITþ T cells were significantly increasedin gastric cancer patients. TIGITþ CD8 T cells underwent meta-bolic reprogramming and exhibited functional exhaustion.CD155 expressed by gastric cancer cells interacted with TIGIT,resulting in the inhibition of glucose uptake and impaired T-celleffector functions. CD155/TIGIT pathway blockade enhancedT-cell effector functions and suppressed tumor progression. Thisstudy provides a potential treatment target for gastric cancer.

Materials and MethodsPatients

Peripheral blood and primary tumor tissues were collectedfrom 138 clinically and pathologically verified gastric cancerpatients from the First Affiliated Hospital, Sun Yat-sen Univer-sity, Guangzhou China; Eighth Affiliated Hospital, Sun Yat-senUniversity, Shenzhen China; Sun Yat-sen University CancerCenter; and Guangdong Provincial Hospital, Guangzhou, Chi-na. Infection and autoimmune diseases were excluded. A pre-vious study has shown that TIGIT is upregulated in CMVþ CD8T cells. To exclude the contaminated effects in CD8 T cellsinduced by CMV infection, CMV-infected cases were excludedfrom the current study. This study was approved by the Insti-tutional Review Board of First Affiliated Hospital, Sun Yat-senUniversity. Consent was informed and consent forms wereobtained from every patient. Patient studies were conductedin accordance with ethical guideline: Declaration of Helsinki.Clinical and pathologic characteristics of the included patientsare shown in Supplementary Table S1.

Cell isolationBlood samples were collected from gastric cancer patients or

age- and sex-matched healthy controls (HC). Peripheral bloodmononuclear cells (PBMC)were isolatedwith Ficoll–Hypaque bydensity gradient centrifugation within 2 hours of sample collec-tion. Fresh tumor tissues were obtained from gastric cancer

patients during surgical tumor resection. Samples were mincedand digested with type I collagenase (Sigma) in RPMI1640.Digested cells were filtered through a nylon mesh (70 mm) andwashed with PBS. CD8þTIGITþ, CD8þTIGIT�, CD4þTIGITþ, orCD4þTIGIT� cells were sorted using a BD FACS Influx. Total andna€�ve CD8 T cells were purified from PBMCs by negative selectionusing the EasySep human total or na€�ve CD8þ T Cell EnrichmentKits (STEMCELL Technologies Inc.). Cell purity was checked(>95%, Supplementary Fig. S1A–S1C).

Cell cultureHuman gastric cancer cell lines SGC7901, HGC27, and

BGC823 were obtained from the type Culture Collection ofChinese Academy of Sciences (Shanghai, China; ref. 28). Celllines were authenticated by cell viability analysis, short tandemrepeat profiling, and isoenzyme analysis. Cell lines were screenedfor mycoplasma contamination. Cells were grown in RPMI1640medium supplemented with 10% FBS, 50 U/mL penicillin, and50mg/mL streptomycin in a humidified atmosphere at 37�Cwith5% CO2.

Plasmids, retroviral infection, and transfectionCD155 constructs were generated by subcloning PCR-ampli-

fied full-length human CD155 cDNA into pcDNA3.1. To depleteCD155, siRNA sequences were cloned into GV248 to generateGV248-RNAi(s) targeting CD155. siRNA duplexes were synthe-sized and purified by RiboBio Inc. The CD155 siRNA sequenceswere as follows: sense, 50-GGUAUCCAUCUCUGGCUAUTT-30;antisense, 50-AUAGCCAGAGAUGGAUACCTT-30. siRNA transfec-tion was carried out using Lipofectamine 2000 reagent (Invitro-gen Co.) according to the manufacturer's instructions. Stable celllines expressing CD155 or CD155 RNAi(s) were selected viatreatment with 0.5 mg/mL puromycin for 10 days beginning48 hours after infection. Following selection, gastric cancer celllysates prepared from the pooled cell populations in samplingbuffer were fractionated by SDS–PAGE to detect protein levels viaWestern blotting (29).

Flow cytometryPBMCs isolated from gastric cancer patients or HCs were

stainedwith the following antibodies: FITC-conjugated anti-CD4,PE-conjugated anti-CD8, APC-conjugated anti-TIGIT, and FITC-conjugated CD226. Single-cell suspensions from gastric tumortissues were stained with FITC-conjugated anti-CD4, PE-conju-gated anti-CD8, and APC-conjugated anti-TIGIT antibodies.Sorted CD8þTIGITþ or CD8þTIGIT� cells were stained withPE-Cy7–conjugated CD69, FITC-conjugated anti-PD-1, andV450-conjugated anti–TIM-3 antibodies. Cultured T cells werestained with APC-conjugated p-AKT, PE-CY7–conjugated p-S6K,and Pe-Cy5.5–conjugated p-4EBP1 antibodies. Gastric cancertumor cell lines were stained with an APC-conjugated CD155antibody. A commercial kit from Roche was used to analyze cellapoptosis. Samples were analyzed using a BD FACS ARIA (BDBiosciences).

T-cell function assaysFor T-cell activation assays, cells were seeded in 96-well plates

and stimulated with anti-CD3/CD28 Dynabeads (aCD3/CD28)for 12 hours to measure CD69 expression by flow cytometry. Forthe proliferation assay, cells were labeled with carboxyfluorescein

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diacetate succinimidyl ester (CFSE) and stimulated with aCD3/CD28 at 37�C with 5% CO2 for 4 days. Cells were collected, andthe dilution of intracellular CFSE caused by proliferation wascalculated using a flow cytometer (30). For intracellular cytokinestimulation assays, cells were stimulated with 500 ng/mL PMAand 1 mg/mL ionomycin (Sigma-Aldrich) for 5 hours at 37�Cwith5% CO2. During the last 2 hours, 1 mg/mL brefeldin A wasincluded. Cells were collected and stained with V450-conjugatedanti-IFNg and PE-Cy7–conjugated anti-TNFa antibodies.

CocultureCD8þTIGITþ or CD8 T cells were sorted and cocultured with

gastric cancer SGC7901 cells in 48-well plates at a ratio of 5:1.Cells were stimulated with aCD3/CD28 in the presence of 5mg/mL anti-TIGIT blocking antibody (BPS Biosciences) or isotypecontrol. Alternatively, cells were treated with 10 mmol/L glucose.T cells were collected to determine the activation, proliferation,and cytokine production using the described T-cell functionassays. The phosphorylation of AKT, mTOR, S6K, and 4E-BP1 inCD8 T cells was measured by Western blotting or flow cytometry.

Glucose consumption assay2-Deoxyglucose (2-DG) is a glucose analogue taken up by

glucose transporters and metabolized to 2-DG-6-phosphate(2-DG6P). 2-DG6P cannot be further metabolized and accumu-lates in cells. 2-DG6P is oxidized to generate NADPH, which canbe measured by an enzymatic recycling amplification reaction.T cells (2 � 105/well) were stimulated with aCD3/CD28 for8 hours. Cells were washed with PBS three times and then glucosestarved by plating with 100 mL of Krebs-Ringer-Phosphate-HEPESbuffer containing 2% BSA for 40 minutes. Then, cells were stim-ulated with or without insulin (1 mmol/L) for 20 minutes. A totalof 10 mL 10 mmol/L 2-DG was added to the cells for 20 minutes.Glucose levels in the cells were analyzed using a Glucose AssayKit (Sigma-Aldrich) according to the manufacturer's instructions.

Lactate production assayCD8þTIGITþ or CD8þTIGIT� cells (2 � 105/well) were stim-

ulated with aCD3/CD28 for 8 hours and then cultured with freshcomplete medium containing glucose. Lactate concentrationswere analyzed in triplicate using a Lactate Assay Kit (Abcam)according to the manufacturer's instructions.

Humanized NOG mouse tumor modelNOD.Cg-PrkdcscidIl2rgtm1Sug/JicCrl (NOG) mice (Weitong

Lihua Experimental Animal Co., Ltd.) were immunodeficient sothat they could receive human immune cells. In the humanizedmice,we can study immune reactionof human cells against tumorand the underlyingmechanisms. PMBCs were isolated fromHCs,and 2 � 107 human PBMCs were injected into the mice perito-neally to reconstitute human immune system. Circulating humanT cells were evaluated by flow cytometry. To investigate theantitumor effects by target human T cells, mice were subcutane-ously inoculated with 2 � 106 SGC7901 or SGC7901-CD155RNAi. Mice were treated with an anti–PD-L1 antibody or isotypecontrol. The mice were monitored three times per week forevidence of morbidity and mortality associated with tumorgrowth and metastasis. In vivo bioluminescence imaging wasperformed by using the IVIS Imaging System. The Living Imageacquisition and analysis software (Caliper Life Sciences) wereused together as described before (31).

Statistical analysesData are expressed as means � SEM. Statistical analysis was

performed using SPSS version 13.0. Differences were assessedusing either the Student t test or one-way ANOVAwith or withoutrepeated measurements, followed by Bonferroni multiple com-parison posttest, as appropriate. Two-tailed P values <0.05 wereconsidered statistically significant.

ResultsTIGITþ T cells are associated with immune subversion inpatients with gastric cancer

TIGITþ T cells expand during malignancy (32). Here, to deter-mine whether TIGITþ T cells are expanded in gastric cancer, wecompared TIGIT expression in T cells from gastric cancer patientsor HCs by flow cytometry. The percentage of CD4þTIGITþ andCD8þTIGITþ T cells was increased significantly in gastric cancerpatients compared with age- and sex-matched HCs (Fig. 1A–C).The percentage of TIGITþ T cells decreased after surgical removalof the tumor tissue and increased again after tumor recurrence(Fig. 1D–F). In addition, TIGITwas strongly expressed in TILs (Fig.1G–I). TIGITþ T cells from PBMCs displayed a memory pheno-type. TIGITwas expressed inCD45RA�CD45ROþmemory T cells,but not in CD45RAþCD45RO� na€�ve T cells (Supplementary Fig.S2A and S2B). CD226 is the costimulatory molecule competingwith TIGIT for CD155 and the initiation of CD226 results in T-cellactivation (33). Compared with HCs, fewer CD226þ CD8 T cellshad been identified in gastric cancer patients (Supplementary Fig.S3A and S3B). CD4 and CD8 T-cell compartments were notsignificantly different in HCs and gastric cancer patients (Supple-mentary Fig. S4A and S4B), indicating that increased TIGITexpression on T cells is responsible for the higher number ofTIGITþ T cells in gastric cancer patients.

TIGIT is associatedwith T-cell exhaustion.Next, we investigatedthe effect of increased TIGIT expressiononT-cell effector functionsin patients with gastric cancer. CD8þTIGITþ or CD8þTIGIT� cellswere sorted from gastric cancer PBMCs by flow cytometry andstimulated with anti-CD3/CD28 Dynabeads (aCD3/CD28).More TIGIT� T cells were CD69þ than TIGITþ T cells (Fig. 1J andK). In addition, proliferation rate was significantly lower inTIGITþCD8 T than TIGIT�CD8 T cells (Fig. 1L andM). Cytokinesof IFNg and TNFa production (Fig. 1N andO) and cell migration(Fig. 1P) were impaired in TIGITþ CD8 T cells, while apoptosiswas higher in TIGITþCD8T cells (Supplementary Fig. S5A and B).Furthermore, TIGITþ CD4 T cells from gastric cancer patientsexhibited characteristic functional exhaustion that was similarwith CD8 T cells (Supplementary Fig. S6A–S6F).

In summary, the expansion of TIGITþ T cells was in accordancewith immune subversion in gastric cancer, implying immuneescape in gastric cancer through the upregulation of TIGIT.

Metabolic reprogramming of TIGITþ CD8 T cells in gastriccancer patients

Glucose uptake and glycolysis increase rapidly when T cells areactivated (20). To explore metabolic changes in TIGITþ CD8 Tcells, we evaluated the expression of genes involved in metabolicreprogramming by RT-PCR first. Expression of metabolism-asso-ciated genes was significantly reduced in TIGITþ CD8 T cellscompared with TIGIT� CD8 T cells (Fig. 2A). Glut1 has beenvery important for glucose uptake in T cells, and hexokinase 1 andhexokinase 2 (HK1/HK2) are the key kinases that initiate the

CD155/TIGIT Regulates T-cell Metabolism in Gastric Cancer

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Figure 1.

Increased TIGITþ T cells disrupt the immune response in gastric cancer patients. A, TIGIT expression in CD4 and CD8 T cells from PBMCs of gastric cancerpatients and HCs was analyzed by flow cytometry. B and C, Percentage of CD4þTIGITþ or CD8þTIGITþ cells in PBMCs [age (y): controls, 66.23 � 6.627 n ¼ 16;patients, 68.87 � 6.146 n ¼ 24]. (Continued on the following page.)

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process of glycolysis. The downregulation of Glut1 andHK1/HK2in TIGITþ T cells was confirmed by flow cytometry and Westernblotting, respectively (Fig. 2B–D). The AKT/mTOR pathway reg-ulates glycolysis and is important for cell growth and prolifera-tion. Western blot analysis revealed that phosphorylation of AKTandmTORwas significantly downregulated in TIGITþ T cells (Fig.2E). Furthermore, flow cytometric analysis showed decreasedexpression of p-S6K and p-4EBP1, which are downstream targetsof mTOR, in TIGITþ T cells (Fig. 2F–H).

To determine whether these changes were associated withchanges in T-cell metabolism, we measured glucose uptake andlactate production in TIGITþ and TIGIT� T cells. Glucose uptakewas impaired in TIGITþCD8 T cells comparedwith TIGIT�CD8 Tcells (Fig. 2I). In addition, lactate production was significantlylower in TIGITþ CD8 T cells (Fig. 2J).

Taken together, these findings indicated that TIGIT reducedglucose uptake and inhibited T-cell metabolism in gastric cancerpatients.

Figure 2.

Metabolic reprogramming in TIGITþ CD8 T cells from gastric cancer patients. CD8þTIGITþ and CD8þTIGIT� cells were sorted from PBMCs of gastric cancer patients.A, Cells were stimulated with aCD3/CD28 for 4 hours. Heatmap shows expression of metabolism-associated gene expression quantified by RT-PCR. B, CD8þTIGITþ

or CD8þTIGIT� cells were stimulated with aCD3/CD28 for 24 hours. Glucose transporter 1 (Glut1) expression in CD8þTIGITþ and CD8þTIGIT� cells wasdetermined by flow cytometry. Representative histograms are shown. C, Glut1 mean fluorescence intensity (MFI) is summarized from six independent samples.D, CD8þTIGITþ and CD8þTIGIT� cells were stimulated with aCD3/CD28 for 24 hours. Hexokinase 1 (HK1) and hexokinase 2 (HK2) expression was measured byWestern blotting. Representative blots are shown. E, CD8þTIGITþ and CD8þTIGIT� cells were stimulated with aCD3/CD28 for 5 minutes. AKT and mTORphosphorylationwas quantified byWestern blotting. F,Cellswere stimulatedwithaCD3/CD28 for 3minutes; p-S6K and p-4EBP1weremeasured by flow cytometry.Representative histograms are shown. Phosphorylation of S6K (G) and4EBP1 (H) is summarized. CD8þTIGITþor CD8þTIGIT� cellswere stimulatedwithaCD3/CD28.I,Glucose uptake by T cellswasmeasured indirectly using a 2-deoxyglucose (2-DG)–based assay. 2-DG is a glucose analogue that is taken up byglucose transportersand metabolized to 2-DG-6-phosphate (2-DG6P). 2-DG6P cannot be further metabolized and accumulates in cells. J, Lactate production was measured by acolorimetric assay 4 hours after stimulation. n ¼ 6; � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

(Continued.) D, The percentages of TIGITþ T cells were measured in PBMCs from postsurgery or recurrent gastric cancer patients by flow cytometry.Percentages of CD4þTIGITþ (E) and CD8þTIGITþ (F) cells in PBMCs (n ¼ 8). G, Single-cell suspensions were prepared from gastric tumor tissues. Percentagesof TIGITþ CD4 or CD8 T cells from tumor tissues were measured by flow cytometry. H and I, Individual percentages of TIGITþ and TIGIT� T cells from eightindependent samples. J–P, CD8þTIGITþ and CD8þTIGIT� cells in PBMCs from gastric cancer patients were sorted by flow cytometry. Cells were stimulated withanti-CD3/CD28 Dynabeads (aCD3/CD28). J, CD69 expression was determined by flow cytometry after 12 hours of stimulation. Representative flow histogramsare shown. K, CD69þ rates are summarized from 12 samples. L, Cells were stained with CFSE, and proliferation rates were measured by flow cytometry. M,Proliferation of CD8þTIGITþ versus CD8þTIGIT� cells in 12 independent samples. N, IFNg and TNFa production by CD8 T cells was measured by flow cytometry.Representative flow charts are shown.O, Percentages of IFNg- and TNFa-producing CD8þTIGITþ or CD8þTIGIT� cells. P, CD8þTIGITþ or CD8þTIGIT� cell migrationwas measured in a Transwell assay, and percentages of cells that migrated to the lower chamber were calculated by flow cytometry. Data were collectedfrom 12 samples. �� , P < 0.01; �� , P < 0.001.






Glucose reverses impaired TIGITþ T-cell metabolism andrescues T-cell functional exhaustion

As glucose is the major cellular fuel that promotes T-cellproliferation and survival, we investigated whether the additionof exogenous glucose could reverse TIGIT-associated T-cellexhaustion. To do this, TIGIT� CD8 T or TIGITþ CD8 T cells weresorted from gastric cancer PBMCs. Glucose treatment reversedmetabolic activities of TIGITþ CD8 T cells. Glucose uptake andlactate production were increased by glucose. Glucose alsoincreased glucose uptake and lactate production in TIGIT� CD8T cells (Supplementary Fig. S7A and S7B). Interestingly, fructose,another hexoses, could not reverse metabolic activities of TIGITþ

CD8 T cells (Fig. 3A and B). Next, we were to explore whetherglucose can reverse the exhaustion of TIGITþ CD8 T cells. Wefound that glut1 expressionwas increased by glucose treatment, asdetermined by flow cytometry (Fig. 3C). This increased metabo-lism in TIGITþ T cells was accompanied by enhanced T-celleffector functions, as the percentage of CD69þ T cells and T-cellproliferation were increased after glucose treatment (Fig. 3D–G).In addition, TIGITþ T-cell migration and cytokine productionwere restored by glucose (Fig. 3H–J). In addition, glucoseincreased IFNg production in a dose-dependent manner(Fig. 3K–L).

Taken together, these results indicate that TIGIT may havenegative regulatory effects on T-cell metabolism, which can bereversed by glucose.

Gastric cancer cells deprive CD8 T cells of glucoseTo investigate why T-cell metabolism is inhibited from the

gastric cancer, we used the coculture of CD8 T cells from HCPBMCs and gastric cancer SGC7901 cells. As shown in theheatmap in Fig. 4A, metabolism-associated gene expressionwas suppressed in CD8 T cells cocultured with SGC7901compared with CD8 T cells without coculture with gastriccancer cells. Glut1 expression was reduced and CD8þ T-cellexpression of the downstream molecules, HK2, was also down-regulated when compared with CD8 T cells without coculturewith gastric cancer cells (Fig. 4B–D). Glucose uptake by CD8 Tcells was significantly inhibited when CD8 T cells were cocul-tured with SGC7901 (Fig. 4E). In addition, lactate productionin CD8 T cells was much lower in SGC7901-T-cell cocultures(Fig. 4F). Furthermore, we found that the phosphorylationof mTOR and its downstream molecules, S6K and 4E-BP1, wasinhibited in T cells when cocultured with SGC7901 (Fig.4G–K). In addition, T-cell cytokine production (IL2, TNFa,and IFNg) was decreased significantly in SGC7901-T-cell cocul-tures (Fig. 4L–N). We confirmed these findings in anothergastric cancer cell line, HGC27 (Supplementary Fig. S8A–S8D). These data demonstrate that gastric cancer cells couldinhibit T-cell metabolism in tumor microenvironment to turn-over T-cell effector functions.

Next, we were to investigate whether glucose can affect theeffector function of CD8 T cells that are cocultured with gastriccancer cells. CD8 T cells from HC PBMCs were cocultured withSGC7901 with or without the additional glucose. We found thatthe inhibition T-cell function was neutralized by glucose whencocultured with SGC7901. The percentage of IFNg-producing Tcells increased after supplementation of the coculture systemwithglucose (Supplementary Fig. S9A and S9B). However, TIGITexpression was not affected by glucose supplementation (Sup-plementary Fig. S9C and S9D).

These findings suggest that gastric cancer cells impair T-cellfunction by depriving them of glucose and that this impairmentcan be reversed by the addition of exogenous glucose.

TIGIT blockade reverses the inhibition of T-cell metabolismand cytokine production by gastric cancer cells

As data show above that gastric cancer cells can inhibit T-cellmetabolism, we were to investigate how gastric cancer cells affectT-cell metabolism and effector functions. CD8 T cells were iso-lated fromHC PBMCs and cocultured with gastric cancer cell lineof SC7901. TIGIT expression was unregulated in T cells whencoculturedwith SGC7901 (Fig. 5A).Circulating tumor cells (CTC)were enumerated using a NanoVelcro system, as described pre-viously (34). The number of CTCs correlated closely with thepercentage of CD8þTIGITþ T cells (Fig. 5B), indicating CTCsmight contribute to the increased TIGITþ CD8 T cells in thecirculation of gastric cancer patients.

As we observed that gastric cancer cells could induce TIGITexpression in CD8 T cells, and TIGIT was associated with T-cellmetabolism and T-cell exhaustion, we next investigated wheth-er TIGIT blockade could reverse SGC7901-induced changes inT-cell metabolism and effector functions. CD8 T cells weresorted from HC PBMCs and cocultured with gastric cancer cellline of SGC7901. TIGIT-blocking antibody was used to blockTIGIT signal. Blocking TIGIT increased metabolism-associatedgene expression in CD8 T cells (Supplementary Fig. S10A).Increased Glut1 expression in CD8 T cells was confirmed at theprotein level by flow cytometry (Fig. 5C and D). AKT andmTOR phosphorylation was increased in CD8 T cells afterTIGIT blockade (Fig. 5E–G) compared with isotype control.Furthermore, blocking TIGIT increased p-S6K and p-4EBP1expression by CD8 T cells when cocultured with SGC7901(Fig. 5H–J). These metabolic changes were associated withincreased glucose uptake and lactate production in CD8 T cells(Fig. 5K and L). Consistent with this, gastric cancer cell-medi-ated inhibition of IFNg production by CD8 T cells was reversedby blocking TIGIT (Fig. 5M and N).

These observations demonstrate that gastric cancer cells induceTIGIT expression on CD8 T cells, through which gastric cancercells inhibit T-cell metabolism and impair T-cell effector function.

Gastric cancer cells inhibit T-cell metabolism through CD155/TIGIT signaling

Melanoma cells suppress T-cell responses through CD155–TIGIT interactions (16). To investigatewhetherCD155 is involvedin the inhibition on T cells mediated by gastric cancer cells, wetestedCD155 expressionon gastric cancer tissue andgastric cancercell lines. We found that CD155 expression was detected andsignificantly increased in gastric cancer tissue compared withnormal gastric tissue (Fig. 6A and B), and the gastric cancer celllines SGC7901, HGC27, and BGC823 all expressed CD155 (Fig.6C) as detected by flow cytometry. These data indicate that gastriccancer could interact with T cells through CD155-TIGIT and affectT-cell functions.

To determine whether CD155 expressed by gastric cancercells regulates T-cell metabolism and effector functions, wegenerated a SGC7901 cell line with stable downregulation ofCD155 expression, SGC7901-CD155 RNAi. The downregula-tion of CD155 was confirmed by flow cytometry (Fig. 6D). CD8T cells sorted from HC PBMCs were cocultured with SGC7901-CD155 RNAi or SGC7901-vector. AKT, S6K, and 4EBP1

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Figure 3.

Glucose reverses the effects of CD8þTIGITþ on T-cell metabolism and rescues T-cell exhaustion. CD8þTIGIT� or CD8þTIGITþ T cells were sorted fromPBMCs of gastric cancer patients. Cells were stimulated with aCD3/CD28 in the presence or absence of 10 mmol/L glucose (Glu) or 10 mmol/L fructose. Aand B, Cells were stimulated with aCD3/CD28 in the presence or absence of 10 mmol/L glucose for 24 hours. Cells were then collected to measure glucoseconsumption (A) and lactate production (B) as described above. C, Glut1 expression in T cells was determined by flow cytometry after stimulation for 24 hours.Representative histograms of 6 experiments were presented. D, CD69 expression was determined by flow cytometry after stimulation for 12 hours. E, Thepercentages of CD69þ CD8 T cells. F, CD8 T-cell proliferation was quantified by flow cytometric analysis of CFSE dilution after 4 days of stimulation. Representativehistograms of 6 experiments are shown. G, CD8 T-cell proliferation rates are summarized from 6 experiments. H, CD8 T-cell migration was measured using aTranswell system. Transmigrated cells were enumerated by flow cytometry. I, IFNg production was measured by flow cytometry after 24 hours of stimulation.J, Percentages of IFNg-producing CD8 T cells. K, CD8 T cells were stimulated with aCD3/CD28 for 48 hours and treated with different concentrations ofglucose. IFNg production was measured by flow cytometry. Representative flow charts are shown. L, Percentages of IFNg-producing CD8 T cells. n¼ 6; � , P < 0.05;�� , P < 0.01; �� , P < 0.001.






phosphorylation in CD8 T cells was decreased by coculturewith SGC7901-vector cells. Downregulation of CD155 inSGC7901 cells (SGC7901-CD155 RNAi) increased AKT, S6K,and 4EBP1 phosphorylation in CD8 T cells (Fig. 6E). Glucoseuptake and lactate production in CD8 T cells were both

decreased by SGC7901-vector cells, but this effect was reversedwhen CD155 was downregulated in SGC7901 cells (Fig. 6F andG). In addition, SGC7901-vector cells inhibited IFNg produc-tion in CD8 T cells, and this inhibition was reversed by CD155downregulation in SGC7901 cells (Fig. 6H and I).

Figure 4.

Gastric cancer cells deprive CD8 T cells of glucose. A–L, Na€�ve CD8 T cells isolated from HCs were stimulated with aCD3/CD28 and cocultured with gastriccancer cells (SGC7901) at a 5:1 ratio. A, Glycolytic gene expression levels in CD8 T cells were measured by RT-PCR after 12 hours of stimulation. Relativegene expression is shown as a heatmap. B, Glut1 expression in CD8 T cells was determined by flow cytometry after 48-hour stimulation. A representativehistogram is shown. C, Summary of Glut1 mean fluorescence intensity (MFI). D, HK1 and HK2 expression in CD8 T cells was determined byWestern blotting. Glucoseconsumption (E) and lactate production (F) in CD8 T cells weremeasured as described above after 24 hours of coculture.G, Phosphorylation of mTOR in CD8 T cellswasmeasured by flow cytometry.H, p-mTOR (S2448) MFI is summarized. I, Phosphorylation of SK6 and 4EBP1 wasmeasured by flow cytometry. J andK, Summaryof MFIs. L, CD8 T cells were stimulated with aCD3/CD28 and cocultured with SGC7901 for 48 hours. IFNg production by CD8 T cells was determined by flowcytometry. M, Percentages of IFNg-producing CD8 T cells. N, CD8 T-cell cytokine production in the supernatant was measured by ELISA. Data were analyzedrelative to the control group. n ¼ 6; �, P < 0.05; �� , P < 0.01; �� , P < 0.001.

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We also investigated the effect of CD155 overexpression inSGC7901 cells (SGC7901-CD155) on IFNg production in Tcells. CD8 T cells were cocultured with SGC7901-vector orSGC7901-CD155 cells. Overexpression of CD155 in SGC7901was confirmed by flow cytometry (Fig. 6J). SGC7901-vectorinhibited IFNg production compared with T cells stimulatedwith aCD3/CD28 alone. SGC7901-CD155 cells furtherdecreased IFNg production in CD8 T cells compared withSGC7901-vector cells, which could be neutralized by blockingTIGIT (Fig. 6K and L).

Taken together, these findings indicated that gastric cancer cellsinhibit T-cell metabolism through CD155/TIGIT signalingpathways.

TIGIT and PD-1 are coexpressed in CD8 T cells, and combinedinhibition of TIGIT and PD-1 signals has synergistic effects

A study has shown that exhausted CD8 T cells coexpressedTIGIT and PD-1, and combined blockade of these two signalsdemonstrates stronger effects in T-cell activation compared withblocking either. To study whether TIGITþCD8 T cells from gastric

Figure 5.

TIGIT blockade neutralizes gastric cancer cell–induced inhibition of T-cell metabolism and cytokine production. TIGITþ CD8 T cells were sorted from HCs byflow cytometry. Cells were stimulated with aCD3/CD28 and cocultured with SGC7901 at a ratio of 5:1. TIGIT was inhibited by an anti-TIGIT blocking antibody(aTIGIT). An isotype control was used as a control. A, TIGIT expression in CD8 T cells was measured by flow cytometry. Representative histograms are shown.B, Circulating tumor cells in gastric cancer patients were determined by NanoVelcro. Correlation of CTCs and CD8þTIGITþ cells are shown (n ¼ 25). C, Cellswere stimulated with aCD3/CD28 for 2 days, and Glut1 expression wasmeasured by flow cytometry.D, Summary of Glut1 mean fluorescence intensity (MFI). E,AKTand mTOR phosphorylation in CD8 T cells was determined by Western blotting. F and G, Semiquantification of p-AKT and p-mTOR. H, S6K and 4EBP1phosphorylation in CD8 T cells was measured by flow cytometry. The phosphorylation rates of S6K and 4EBP1 are summarized in I and J, respectively. Glucose (glu)consumption (K) and lactate production (L) in CD8 T cells. M, Total CD8 T cells were isolated from healthy PBMCs. Cells were cocultured with gastric cancercells at a ratio of 5:1 in the presence of aTIGIT or isotype control. IFNg production was measured by flow cytometry after 2 days of stimulation with aCD3/CD28.N, Percentages of IFNg-producing CD8 T cells. n ¼ 6; � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






cancer coexpress PD-1, we analyzed PD-1 expression inCD8þTIGITþ and CD8þTIGIT� cells by flow cytometry. PD-1expression was significantly higher in TIGITþ CD8 T cells thanin TIGIT� CD8 T cells (Fig. 7A and B). In addition, the expressionof another checkpointmolecule, TIM-3, was also higher in TIGITþ

CD8 T cells (Supplementary Fig. S11A–S11D). We next investi-gated whether TIGIT and PD-1 have synergistic effects on T-cellactivation when cocultured with gastric cancer cells. BlockingTIGIT or PD-1 increased IFNg production in CD8 T cells thatwere cocultured with SGC7901 cells. Blocking both TIGIT andPD-1 further enhanced IFNg production in CD8 T cells (Fig. 7C

and D). These findings indicate that TIGIT and PD-1 act syner-gistically to induce CD8 T-cell exhaustion.

Targeting CD155/TIGIT suppresses tumor progression in vivoTo assess the antitumor effects of targeting CD155/TIGIT sig-

naling, we used a humanized mouse tumor model using NOGmice. NOG mice are immunodeficient mice that can be used forreconstitution of human immune cells in themice.Mice were firstinjected with HC PBMCs. NOG mice were then subcutaneouslyinoculated with SGC7901-vector cells or SGC7901-CD155-RNAione week later when human immune system is established. Mice

Figure 6.

Gastric cancer cells inhibit T-cell metabolism through CD155/TIGIT. A, CD155 expression in normal gastric tissue or gastric cancer tissue was measured byWestern blotting. Representative blots are shown. B, Relative expression of CD155. C, CD155 expression in gastric cancer cell lines SGC7901, HGC27, andBGC823 determined by flow cytometry. Representative histograms are shown. D, CD155 knockdown efficiency was confirmed by flow cytometry. E–I, CD8T cells were stimulated with aCD3/CD28 and cocultured with SGC7901-vector or SGC7901-CD155-RNAi for 48 hours. E, Phosphorylation of AKT, S6K, and 4E-BP1in CD8 T cells was determined by flow cytometry. Representative histograms are shown. Glucose uptake (F) or lactate production (G) in CD8 T cells. H, IFNgproduction in CD8 T cells measured by flow cytometry. I, Percentages of IFNg-producing CD8 T cells. J, CD155 overexpression was confirmed by flow cytometry.K, CD8 T cells were cocultured with SGC7901-CD155 or SGC7901-vector cells. TIGIT was blocked using aTIGIT. IFNg production in CD8 T cells determined byflow cytometry. Representative flow charts are shown. L, Percentages of IFNg-producing CD8 T cells. n¼ 6; N, normal gastric tissue; P, gastric cancer tumor tissue.� , P < 0.05; �� , P < 0.01; �� , P < 0.001.

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Figure 7.

Combined inhibition of TIGIT and PD-1 signaling has synergistic effects in vitro and in vivo. A, PBMCs from gastric cancer patients were stained with anti-human CD8, anti-human TIGIT, and anti-human PD-1 antibodies. Representative flow chartswere gated on CD8þTIGITþ or CD8þTIGIT� cells.B, Percentages ormeanfluorescence intensity (MFI) of PD-1 in CD8þTIGITþ or CD8þTIGIT� cells. C and D, Total CD8 T cells were isolated from healthy PBMCs. Cells were stimulated withaCD3/CD28 and cocultured with or without SGC7901 for 2 days. Anti-TIGIT (aTIGIT), anti-PD-1 (aPD-1), isotype control, or a combination of aTIGIT and aPD-1 wasincluded. IFNg production in CD8 T cells was determined by flow cytometry. Percentages of IFNg-producing CD8 T cells were summarized from8 samples. E–H,NOGmicewere inoculated subcutaneouslywith Vector-SGC7901 or SGC7901-CD155-RNAi gastric carcinoma cells. At the same time,micewere reconstituted with humanPBMCs. When tumor sizes reached approximately 200 mm3, mice were treated with isotype control or anti–PD-1 antibody (aPD-L1) for 3 weeks. E, Representativeimages of showing CD8 T-cell infiltration in tumor microenvironment as detected by IHC. F, Mouse survival over time (n ¼ 12). G, Mean (top) or individuals oftumor volume (bottom 4 panels) over time (n ¼ 8). H, In vivo bioluminescence images of the tumor-bearing mice. � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






were treated with a PD-L1–blocking antibody or isotype control.CD8þ T-cell infiltration in the tumor was increased in mice thatreceived SGC7901-CD155-RNAi or mice that received anti–PD-1treatment, when compared with mice that received SGC7901-vector as measured by IHC (Fig. 7E). Anti–PD-1 treatment com-bined with SGC7901-CD155-RNAi further enhanced T-cell infil-tration to the tumor (Fig. 7E). In addition, TCR, IL2, and IFNgproduction in the tumor quantified by RT-PCR further confirmedthe increased T cell and enhanced immune response in the micethat received SGC7901-CD155-RNAi or anti–PD-1 treatment(Supplementary Fig. S12A–S12C). Mice that receivedSGC7901-CD155-RNAi showed inhibited tumor progression andimproved survival when compared with mice that receivedSGC7901-vector. Also, tumor progression was inhibited byanti–PD-1 treatment and survival was improved by anti–PD-1treatment. Tumor progression was further inhibited in mice thatreceived SGC7901-CD155-RNAi and anti–PD-1 treatment. Sur-vival was further improved in mice that received SGC7901-CD155-RNAi and anti–PD-1 treatment (Fig. 7F–H).

Taken together, these results suggest that downregulation ofCD155 in gastric cancer cells combined with PD-L1 blockademediated synergistic effects in terms of the inhibition of tumorprogression and increased survival compared with the effectsdownregulation of CD155 in gastric cancer cells or PD-L1 block-ade alone.

DiscussionImmunosurveillance is important for maintaining cellular

homeostasis and preventing carcinogenesis (35). Immune escapeis a defect of the immune system that facilitates carcinogenesis. Itis promoted by upregulation of immune checkpoints, such as PD-1, and induces T-cell exhaustion (36). CD8 T cells are the majoreffector cells in antitumor immunity. These cells are exhaustedand rendered dysfunctional by immune checkpoints in tumor-bearing hosts (37). In this study, we found that the percentage ofCD8þ T cells that are TIGITþ is dramatically increased in gastriccancer patients and that these cells exhibit functional exhaustionand reduced metabolic activity. We showed that gastric cancercells inhibited glucose uptake and reduced metabolic activity inCD8 T cells and that these effects were reversed by the addition ofglucose or blocking CD155/TIGIT. Targeting CD155 pathwaysuppressed tumor progression and improved survival in tumor-bearing mice.

We demonstrated that activation, proliferation, migration, andcytokine production are impaired in TIGITþ T cells from gastriccancer patients. These findings are in agreement with previousstudies that have shown that TIGIT deficiency causes T-cell hyper-proliferation and increased susceptibility to autoimmunity (12,33). Our results suggest that TIGITþ T cells in gastric cancerpatients contribute to immune dysfunction, leading to impairedantitumor immunity and accelerated tumor progression. TIGITmay represent a potential therapeutic target to enhance antitumorimmunity and control gastric cancer progression.

The immune system plays a key role in controlling tumorinitiation and progression (38). Activated T cells require adequateenergy supplies and changes in cellularmetabolism for antitumorimmune responses (26, 39). In the current study, we revealedabnormalmetabolic reprogramming and lowermetabolic activityin TIGITþ CD8 T cells than TIGIT� CD8 T cells. Glucose uptakeand lactate production were low in TIGITþ CD8 T cells than

TIGIT�CD8T cells fromgastric cancer patients. Strikingly, glucosecould reverse the hypometabolic profile of TIGITþ T cells. Glucosereversed the metabolic pathway of AKT/mTOR in TIGITþ CD8 Tcells. Cytokine production was increased in TIGITþ CD8 T cellsalong with reversed metabolism. These observations suggest thatTIGIT regulates T-cell metabolism and induces T-cell dysfunctionin gastric cancer patients. Harnessing T-cell metabolism may bethe potential method in reversing metabolic activity and effectorfunction of TIGITþ CD8 T cells.

T cells from leukemia patients aremetabolically impaired (40),and tumor cells outcompete T cells for glucose consumption andinduce T-cell exhaustion, (26), implying tumor cells may inhibitthe metabolism in T cells and damage T-cell antitumor effectssubsequently. In the current study, we found that CD8T cells weremetabolically impaired when cocultured with gastric cancer cells.Gastric cancer cells deprived CD8 T cells of glucose and down-regulated theAKT/mTORmetabolic pathway inCD8T cells. Thesefindings suggest that gastric cancer cells inhibit AKT/mTOR sig-naling pathway in CD8 T cells, which resulted in reduced glucoseuptake and lactate production. In agreement with our findings,extremely low levels of glucose and high lactate have beenreported in gastric tumor tissues (41). Furthermore, we foundthat gastric cancer cells induced TIGIT expression in CD8 T cells.TIGIT blockade activated metabolic pathway in CD8 T cells. Thephosphorylation of AKT/mTOR pathway was increased by TIGITblockade, resulting in increased metabolism and cytokine pro-duction in CD8 T cells. These findings demonstrate that TIGITsignaling in CD8 T cells leads to decreased phosphorylation ofAKT/mTOR pathway, resulting in the inhibition of metabolism.PD-1 inhibitsmTORpathway activation and suppresses glycolysisin T cells (42). The immune checkpoints may share some com-mon mechanism in the regulation of T-cell function. However,this signaling is different from the previous report that TIGITsignals through ZAP70 and ERK1/2 in NK cells (43). Together,gastric cancer cells inhibit AKT/mTOR pathway in CD8 T cellsthrough upregulating TIGIT expression on CD8 T cells.

Next, we demonstrated that CD155, ligand of TIGIT, is over-expressed in gastric cancer tumor tissue and cell lines, which wasin accordance with the previous report that soluble CD155 isincreased in gastric cancer serum (44). The expression of CD155on gastric cancer cells downregulated AKT/mTOR pathway andinhibited glucose uptake inCD8T cellswhen theywere coculturedtogether. Downregulation of CD155 in gastric cancer cellsincreased T-cell metabolism and cytokine production in thecoculture system.Moreover, mice inoculated with CD155-knock-down gastric cancer cells showed enhanced immune responsesand improved survival. Conversely, CD155 overexpression ingastric cancer cells decreased T-cell metabolism and inhibitedcytokine production. The effects of CD155 overexpression werereversed by inhibiting TIGIT. Together, gastric cancer cells upre-gulate CD155 expression and inhibit CD8 T-cell metabolismthrough CD155–TIGIT interaction.

Our finding that coexpression of TIGIT and PD-1 promoted cellexhaustion in CD8 T cells from gastric cancer patients indicatesthat combined blockade of these immune checkpoints is a poten-tial therapeutic option for gastric cancer. In the T cell–tumor cellcoculture, combined blockade of TIGIT and PD-1 showed stron-ger in cytokine production. In addition, combined blockadeof TIGIT and PD-1 further enhanced immune response in thetumor-bearing mice and had better tumor control and survivalcompared with targeting either one. These findings indicate the


He et al.




potential of combined immunotherapies to treat cancer, and thisis now receiving more attention from researchers (45, 46). Target-ing PD-1/PD-L1 signal has improved the clinical outcome incancer patients dramatically, but treatment responses vary, from24% in renal cell cancer and 87% in non-Hodgkin lymphoma (6,47, 48). A combination of PD-1 and CTLA-4 blockade has beenshown to exert synergistic antitumor effects on B16 melanomatumors (49), and the combined blockade of TIGIT and PD-1demonstrates further enhancement of immune activation asreported before (14). Together, TIGIT or TIGIT combined withPD-1 may be the potential therapy for gastric cancer.

In conclusion, our findings provide insights into the mecha-nism by which CD8 T-cell metabolism and function is impairedby TIGIT. Mediators that enhance CD8 T-cell metabolism andpromotemaximum antitumor immunitymay be novel therapeu-tic targets for the treatment of gastric cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: H. Zhang, Z. KeDevelopment of methodology: W. He, F. Han

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W. He, R. Lin, W. Zhang, H. WangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): W. He, F. Han, X. Chen, Q. Liao, Z. KeWriting, review, and/or revision of the manuscript:W. He, H. Zhang, F. Han,Z. KeAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): W. JiangStudy supervision: Y. CuiOther (patient sample collection): W. Wang, H.-B. Qiu, Z. ZhuangOther (collection and processing of human samples): Q. Cai

Grant SupportThis work was supported by grants from the National Natural Science

Foundation of China (30900650, 81372501, and 81572260), GuangdongNatural Science Foundation (2011B031800025, S2012010008378, and2015A030313036), and Guangdong/Guangzhou Science and TechnologyPlanning Program (2014J4100132, 2015A020214010, 2013B02180021,2016A020215055, 201704020094, 16ykjc08 and 2015ykzd07, 2012B03-1800115, and 2013B021800281).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 10, 2017; revised July 2, 2017; accepted September 1, 2017;published OnlineFirst September 7, 2017.

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2017;77:6375-6388. Published OnlineFirst September 7, 2017.Cancer Res Weiling He, Hui Zhang, Fei Han, et al. Promotes Tumor Progression in Human Gastric Cancer

T-cell Metabolism and+CD155T/TIGIT Signaling Regulates CD8

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http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-17-0381

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