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Research Article

Identification of Tumoricidal TCRs from Tumor-Infiltrating Lymphocytes by Single-Cell AnalysisKiyomi Shitaoka1, Hiroshi Hamana1, Hiroyuki Kishi1, Yoshihiro Hayakawa2,Eiji Kobayashi1, Kenta Sukegawa1,3, Xiuhong Piao1, Fulian Lyu1, Takuya Nagata3,Daisuke Sugiyama4, Hiroyoshi Nishikawa4,5, Atsushi Tanemura6, Ichiro Katayama6,Mutsunori Murahashi7, Yasushi Takamatsu8, Kenzaburo Tani9, Tatsuhiko Ozawa1,and Atsushi Muraguchi1

Abstract

T-cell receptor (TCR) gene therapy is a promising next-gener-ation antitumor treatment. We previously developed a single–T-cell analysis protocol that allows the rapid capture of paired TCRaand b cDNAs. Here, we applied the protocol to analyze the TCRrepertoire of tumor-infiltrating lymphocytes (TIL) of variouscancer patients. We found clonally expanded populations of Tcells that expressed the same clonotypic TCR in 50% to 70% ofCD137þCD8þ TILs, indicating that they responded to certainantigens in the tumor environment. To assess the tumor reactivityof the TCRs derived from those clonally expanded TILs in detail,we then analyzed the CD137þCD8þ TILs from the tumor ofB16F10 melanoma cells in six C57BL/6 mice and analyzed theirTCR repertoire.We also found clonally expanded T cells in 60% to

90% of CD137þCD8þ TILs. When the tumor reactivity ofdominant clonotypic TCRs in each mouse was analyzed, 9 of13 TCRs induced the secretion of IFNg in response to, andshowed killing of, B16F10 cells in vitro, and 2 of them showedstrong antitumor activity in vivo. Concerning their antigenspecificity, 7 of them reacted to p15E peptide of endogenousmurine leukemia virus-derived envelope glycoprotein 70, andthe rest reacted to tumor-associated antigens expressed on EL4lymphoma as well as B16 melanoma cells. These results showthat our strategy enables us to simply and rapidly obtain thetumor-specific TCR repertoire with high fidelity in an antigen-and MHC haplotype–independent manner from primary TILs.Cancer Immunol Res; 6(4); 378–88. �2018 AACR.

IntroductionSince the advent of immune checkpoint blockade therapies,

tumor immunotherapy has become a promising antitumorapproach (1). In this context, T-cell receptor (TCR) gene therapyand T-cell adoptive therapy are also promising next-generationtumor therapies (2, 3). To develop these T cell–related therapies,tumor-specific T cells or TCR genes are required. To obtain tumor-specific T cells or TCR genes, the establishment of tumor-specific

T-cell lines is conventionally required, which is time consumingand laborious. Additionally, treating patients using tumor-spe-cific T cells or TCRs derived from the patients themselves isdifficult because their cancer may progress rapidly. The subjectsof most studies on tumor immunotherapies were skewed towardpatients with major human leukocyte antigen (HLA) haplotypes(i.e., HLA-A�2402 for Asian patients and HLA-A�0201 for Cau-casian patients; see The Allele Frequency Net Database, http://allelefrequencies.net/default.asp; ref. 4), and thus, those patientswith minor HLA haplotypes cannot benefit from results fromthese studies.

Tumor-infiltrating lymphocytes (TIL) have been used as vehi-cles for adoptive T-cell therapies and TCR gene therapies (5–7).PD-1 and/or CD137 (4-1BB)-positive T cells in TILs express tumorreactive TCRs against shared tumor antigens and/or neoantigens(8–10). Thus, TILs are an attractive source of tumor-reactive TCRsfor individualized cancer immunotherapy.

Various protocols for TCR repertoire analysis have been devel-oped in many laboratories. Comprehensive analyses of the TCRrepertoire using next-generation sequencing (NGS)methods havebeen reported (11–13). However, it is difficult to use conven-tional NGS analysis to determine the unique TCRa and b pairsfrom single T cells, although several laboratories developed NGSanalyses that are able to determine TCRa and b pairs (14–16). Incontrast to NGS, TCR repertoire analyses at the single-cell level(17–19) and their functional determinations (20–23) can deter-mine the unique TCR a and b pairs quite simply. We alsoestablished a single–T-cell analysis method and an improvedversion that enables us to obtain the antigen-specific TCRs fromsingle primary T cells within 10 and 4 days, respectively (24, 25).

1Department of Immunology, Graduate School of Medicine and PharmaceuticalSciences (Medicine), Toyama, Japan. 2Division of Pathogenic Biochemistry,Institute of Natural Medicine, University of Toyama, Toyama, Japan. 3Departmentof Surgery and Science, Graduate School of Medicine and PharmaceuticalSciences (Medicine), Toyama, Japan. 4Department of Immunology, NagoyaUniversity Graduate School of Medicine, Nagoya, Japan. 5Division of CancerImmunology, Research Institute/Exploratory Oncology Research and ClinicalTrial Center, National Cancer Center, Kashiwa, Japan. 6Department ofDermatology, Osaka University Graduate School of Medicine, Suita, Japan.7Department of Advanced Cell and Molecular Therapy, Kyushu UniversityHospital, Fukuoka, Japan. 8Division of Medical Oncology, Hematology andInfectious Diseases, Department of Internal Medicine, Fukuoka University,Fukuoka, Japan. 9Project Division of ALA Advanced Medical Research, TheInstitute of Medical Science, The University of Tokyo, Tokyo, Japan.

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

Corresponding Author: Hiroyuki Kishi, Graduate School of Medicine andPharmaceutical Sciences (Medicine), University of Toyama, Toyama 930-0194, Japan. Phone: 81-76-434-7251; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-17-0489

�2018 American Association for Cancer Research.

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In this study, we have tried to obtain tumor-specific TCRs fromTILs using our single-cell analysis method in the absence ofinformation on antigen specificity or MHC haplotype. To thisend, we first investigated the TCR repertoire of TILs derived fromvarious cancer patients and then analyzed that of TILs derivedfrom tumor-bearing mice. We found clonally expanded popula-tions ofCD8þT cells in human andmouse TILs anddemonstratedthat many TCRs from the clonally expanded T cells were specificand cytotoxic to tumors both in vivo and in vitro in amousemodel.We show that our single–T cell analysis procedure can identifytumor-reactive TCRs from TILs without knowing their antigenspecificity or the haplotype of the MHC-presenting molecule andthat tumor-associated antigens can also be good targets for TCRswith tumoricidal activity.

Materials and MethodsReagents

The PerCP-Cy5.5–conjugated human CD3 monoclonal anti-body (mAb, 45-0037-41), allophycocyanin (APC)-conjugatedhuman CD8 mAb (17-0088-41), APC-Cy7 Fixable Viability Dye(65-0865-14), and APC-conjugated mouse PD-1 mAb (17-9985-82) were purchased from eBioscience. The phycoerythrin (PE)-conjugated human CD137 mAb (309803), fluorescein isothio-cyanate (FITC)-conjugated human PD-1 mAb (329903), and PE-conjugated mouse CD137 mAb (106105) were purchased fromBioLegend. FITC-conjugated mouse CD8 mAb (MAB 116) waspurchased from R&D Systems. APC-conjugated TRP2p/H-2Kb

tetramer (TS-M5004-2), APC-conjugated p15Ep/H-2Kb tetramer(TS-M507-2), and p15Ep (TS-M507-P) were purchased fromMBL. The pMXs-IRES-GFP retroviral expression vector (RTV-013) was acquired from Cell Biolabs.

CellsB16F10 cells (CRL-6475), B16F0 cells (CRL-6322), andC57BL/

6mouse-derived mouse embryonic fibroblast (SCRC-1008) wereprovided by the ATCC. 58a�b� T-cell hybridoma-derived cellswith no expression of endogenous TCRa and b (26) were gen-erously supplied by B. Malissen, INSERM, France. B16F10 cells,B16F0 cells, andmouse embryonic fibroblast were maintained inDulbecco's modified Eagle's medium (DMEM) containing 10%fetal calf serum (FCS), 50 mmol/L 2-mercaptoethanol, strepto-mycin (100 mg/mL), and penicillin (100 U/mL). EL4, MC38, and58a�b� cells were maintained in RPMI 1640 containing 10%FCS, 50 mmol/L 2-mercaptoethanol, streptomycin (100 mg/mL),and penicillin (100 U/mL). To prepare the luciferase-expressingB16F10 cells, luciferase cDNA was excised from pGL3 LuciferaseReporter Vectors (Promega, E1751) and transferred into a Piggy-Bac single promoter vector containing internal ribosome entry sitegreen fluorescent protein (IRES-GFP; System Biosciences,PB530A-2). The resultant vector was transfected into B16F10 cellsusing the Super PiggyBac transposase expression vector (SystemBiosciences, PB200PA-1). To select the luciferase-expressingB16F10 cells (B16F10-Luc), GFP-positive cells were sorted usinga FACSAria II (from Becton Dickinson).

Preparation of TILs from human cancer patientsHuman experiments were performed with the approval of

the Ethical Committee at Osaka University (melanoma),Fukuoka University (lymphoma), and University of Toyama.Informed consent was obtained from the subjects. After surgicalresection, the tumor specimens were minced and enzymatically

and mechanically digested using gentleMACS (Miltenyi Biotec,130-093-235) and a tumor dissociation kit according to themanufacturer's instructions. Single-cell suspensions of thetumor tissues were cryopreserved. To prepare the TILs fromlymphoma samples, the lymphoma tissue was minced with ascalpel and mashed on a 40-mm cell strainer (Falcon) to preparea single-cell suspension.

Mouse tumor models and the isolation of TILsThe experiments using mice were approved by the Committee

on Animal Experiments at the University of Toyama. Seven- to 8-week-old mice were inoculated subcutaneously in the lower rightflank with 5 � 105 B16F10 cells. Seven, 10, or 14 days afterinoculation, the mice were sacrificed, and the melanoma tissueswere obtained. The tumor specimens were minced and enzymat-ically and mechanically digested with gentleMACS and a tumordissociation kit according to the manufacturer's instructions. Thetumor single-cell suspensions were cryopreserved.

Single-cell sortingTo sort themouse TILs, theywere stainedwith FITC-conjugated

CD8mAb, and PE-conjugated CD137mAb for 20minutes on ice,followed by staining with APC-Cy7 fixable viability dye for 5minutes. After the staining, the cells were washed with PBS andanalyzed with a FACSAria II. CD137þCD8þ cells were single-cellsorted into a 96-well PCR plate. The data were analyzed usingFlowJo 8.4.7 software (Tree Star, Inc.).

Single-cell RT-PCR and sequencingThe TCR cDNAs were amplified from single T cells using one-

step multiplex RT-PCR as described previously (25). Briefly, 5 mLof the RT-PCR mix was added to each well containing a single Tcell, and one-step RT-PCR was performed. The program for theone-step RT-PCR was as follows: 40 minutes at 45�C for the RTreaction, 98�C for 1 minute and 30 cycles of 98�C for 10 seconds,52�C for 5 seconds, and 72�C for 1minute. The resultant one-stepRT-PCR products were diluted 10-fold with nuclease-free waterand used for a second cycle of PCR. In the second cycle, the cDNAsof TCRa and bwere amplified separately. To amplify the cDNA ofthe TCRa or TCRb, 2 mL of the diluted one-step RT-PCR productswas added to each well of a new 96-well PCR plate containing 18mL of the 2nd-PCR amix or the 2nd-PCR bmix, respectively. ThePCR program for the second PCR cycle for TCRa and b was asfollows: 98�C for 1 minute and 35 cycles of 98�C for 10 seconds,52�C for 5 seconds, and 72�C for 30 seconds. The DNA sequencesof the second PCR products were then determined by directsequencing.When T cells expressed dual TCRs, the direct sequenc-ing resulted in overlapping sequence signals. In that case, wecloned the PCR products into a plasmid vector using the MightyTA-cloning Reagent Set for PrimeSTAR (Takara Bio, 6019) andtransformed the vector into E. coli JM109 competent cells (Takara,9052). The dual TCR sequences were analyzed using colony directPCR and sequencing. The TCR repertoire was analyzed with theIMGT/V-Quest tool (http://www.imgt.org/).

Construction of the TCR expression vectorsThe vectors used to express the TCRs were constructed as

described previously (27). Briefly, the TCRb PCR fragment,the gene encoding the mouse TCRb constant-1 region conju-gated with the self-cleaving P2A peptide (mCb1-P2A-frag-ment), the TCRa PCR fragment and the codon-optimized

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mouse TCRa constant region gene (mCa-fragment) wereassembled together in a linearized pMXs-IRES-GFP retroviralvector using the Gibson Assembly Master Mix (New EnglandBiolabs, E2611) according to the manufacturer's instructions.The constructed plasmid vector, pMXs-TCRb-P2A-TCRa-IRES-GFP, was used for retrovirus production.

Retrovirus productionFirst, 1.25� 106 Plat-E Cells (generously provided by Professor

Toshio Kitamura, University of Tokyo) were cultured in a 6-cmdish with 4 mL of DMEM containing 10% FCS, 50 mmol/L2-mercaptoethanol, streptomycin (100 mg/mL), and penicillin(100 U/mL) 1 day before the transfection. The pMXs-TCRb-P2A-TCRa-IRES-GFP vectors were transfected into Plat-E cells usingthe FuGENE6 transfection reagent (Roche, E2692) accordingto the manufacturer's instruction. The cells were cultured at 37�Cin a 5%CO2 atmosphere. The following day, the cellmediumwasexchanged. After 2 days, the culture supernatant was harvested,filtered with a 0.45-mm filter (Millipore, SLHV033RS), aliquoted,and stored at �80�C until use.

Retroviral transduction of TCR into splenocytesFor this purpose, 1 � 106 splenocytes from C57BL/6 mice

were stimulated with 25 mL of Dynabeads Mouse T-ActivatorCD3/CD28 (Invitrogen, 11453D) and mouse interleukin (IL)-2(30 U/mL, PeproTech Inc. 212-12) in 1 mL of RPMI1640containing 10% FCS, 50 mmol/L 2-mercaptoethanol, strepto-mycin (100 mg/mL), and penicillin (100 U/mL, RPMI culturemedium). Two days later, the splenocytes were harvested andresuspended at 5� 105 cells/mL in RPMI culture medium in the

presence of mouse IL-2 (30 U/mL). To retrovirally transducethe TCRs into the splenocytes, wells in a non–tissue-culture-treated 24-well plate were coated with 0.5 mL of RetroNectin(50 mg/mL, Takara, T100B) at 4�C overnight, and the TCR-encoding retrovirus was spin-loaded into the wells by centrifu-gation for 2 hours at 1,900 � g at 32�C according to the manu-facturer's instructions. The suspended cells were then added tothe retrovirus-loaded wells and spun down at 1,000 � g at 32�Cfor 10 minutes and incubated overnight at 37�C in 5% CO2.The next day, the splenocytes were treated with the retrovirusas described above. The following day, the TCR-transducedcells were expanded in the presence of mouse IL-2 (30 U/mL)for 2 more days and used for the experiments.

IFNg secretion assessed by enzyme-linked immunosorbentassay (ELISA)

TCR cDNA-transduced spleen cells (1 � 105) were culturedtogether with 1 � 105 B16F10 cells that had been treated withIFNg (100 U/mL, PeproTech Inc. 315-05) for 48 hours in 0.2 mLof RPMI culture medium in a 96-well plate. After 16 hours ofculture, the supernatants were harvested, and the amount of IFNgin each supernatant was measured by ELISA (R&D Systems,DY485) according to the manufacturer's instruction.

T-lymphocyte cytotoxicity assessed by Luciferase assayWe analyzed the cytotoxicity against B16F10-Luc cells by

measuring the target cell viability based on the luciferaseactivity (28–30). B16F10-Luc cells (1 � 104) treated with100 U/mL IFNg and TCR-transduced spleen cells were culturedtogether in 96-well plates at the indicated effector-to-target

Figure 1.

The analysis of the surface phenotype and TCR repertoire of TILs from human cancer patients. A, Expression of CD137 and PD-1 in CD8þ TILs obtainedfrom various cancer patients. The percentage of cells in each quadrant is indicated. B, The TCR repertoire of CD137þCD8þ TILs of various cancerpatients. Each pie chart color indicates the T-cell populations in which T cells expressed the same clonotypic TCRa and b pair. The numbers of T cellsthat expressed the same clonotypic TCRa and b pair are indicated around or in the pie charts. An uncolored pie chart slice indicates the T-cell populations inwhich each T cells expressed a unique TCR. The numbers in the center of the pie charts indicate the total number of T cells with an identified TCRa and brepertoire. Representative data of 2 to 10 patients in each cancer are shown.

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(E/T) ratios and incubated for 48 hours. The viability of theB16F10-Luc cells was assessed by measuring the cell-associatedluciferase activity using the Steady-Glo Luciferase Assay System(Promega, E2520).

Analysis of the effect of TCR-transduced T cells on pulmonarymetastasis

The experiments using mice were approved by the Committeeon Animal Experiments at the University of Toyama. To assess

Figure 2.

The surface phenotype and TCRb repertoire of CD137þCD8þ T cells in TILs and regional lymph nodes (RLN). A, CD137 expression of TILs and RLN cells. TILsand RLN cells were prepared from B16F10 melanoma at 7, 10, and 14 days after inoculation into C57BL/6 mice, and their expression of CD8 and CD137 wasanalyzed using flow cytometry. Representative data of days 7, 10, and 14 are shown. The percentage of cells in each quadrant is indicated. B, The alteration ofCD8þCD137þ populations in TILs and RLN cells after the inoculation of tumor cells. The percentages of the CD8þCD137þ population in TILs (left) or RLN cells(right) are shown for each mouse on days 7, 10, and 14. �� , P < 0.001 and NS (not significant) by the Student t test. C, The TCRb repertoire of CD137þCD8þ T cellsin the TILs and RLN. Each pie chart color represents the T-cell populations in which T cells expressed the same clonotypic TCRb. The numbers of T cells thatexpressed the same clonotypic TCRb are indicated around the pie charts. An uncolored pie chart slice indicates those T-cell populations in which each T cellexpressed a unique TCRb. The numbers in the center of the pie charts indicate the total number of T cells with an identified TCRb repertoire. NA: not analyzed.The TCRs that were used for functional analysis are indicated with numbers in red outside of the pie charts, which correspond to the TCR numbers inSupplementary Table S1.






the antitumor effect of TCR-transduced T cells, we used anexperimental metastasis assay with B16F10-Luc cells. To thisend, 5 � 105 B16F10-Luc cells were i.v. injected into 8-week-oldC57BL/6 femalemice. On day 1, 2.5� 107mock-transduced cellsor tumor-specific TCR-transduced T cells were i.v. injected intothe mice. On day 4, the lungs were removed from the mice,and bioluminescence imaging was performed using the IVISImaging System (PerkinElmer) to monitor the tumor metastasis.The signal intensity of the tumor burdens was expressed intotal photons/s/cm2 (p/s/cm2/sr).

ResultsSingle-cell–based TCR repertoire analysis of "activated" TILsfrom cancer patients

There are several reports that showed the existence of tumor-specific T cells in CD137þ or PD-1þ TILs (8–10). Thus, we firstanalyzed the surface expression of CD137 or PD-1 on CD8þ TILsderived from patients with various cancers (melanoma, lympho-ma, breast cancer, thyroid cancer and colon cancer) and showdatafrom a representative patient (n¼ 2 to n¼ 10) with each cancer isshown in Fig. 1. Flow cytometry analysis revealed that a significantnumber of CD8þ cells expressed bothCD137 andPD-1 inmost oftumors (Fig. 1A).We single-cell sorted the CD137þPD-1þCD8þ Tcells and analyzed their TCR repertoire. The results showed thatthe CD137þPD-1þCD8þ TILs could be grouped into populationsof T cells expressing the same clonotypic TCRa and b pair (Fig.1B). The features of TILs in all analyzed tumors are summarized inSupplementary Table S1. Thus, the CD137þPD-1þCD8þ T cellswere clonally expanded in the human tumors. We also tried toanalyze the TCR repertoire ofCD137þCD8þPBMCs from2cancerpatients, but we could not because of the small number ofCD137þCD8þ T cells in PBMCs. Because tumor cell lines werenot available, we did not examine the specificity of the TCRs thatwere isolated from TILs.

Single-cell–based TCR repertoire analysis of activated TILs fromB16F10 tumors

Because we could not determine the human TIL-derived TCRspecificity, we next analyzed the TILs of tumor-bearing mousemodel. To this end, we subcutaneously inoculatedmouse B16F10melanoma cells in C57BL/6 mice, and after 7, 10, and 14 days,TILs were prepared from the tumors of each mouse. We firstanalyzed the expression of CD137 and CD8 on the TILs of tumorsbyflow cytometry. As shown in Fig. 2A andB, approximately 0.6%of TILs expressed CD8 and CD137 on day 7, and 4% and 5%of TILs expressedCD8andCD137ondays 10 and14, respectively.We also examined the expression of CD137 on CD8þ T cells inthe regional lymphnodes (RLN)of tumor-bearingmice ondays 7,10, and 14 and found that the RLN CD137þCD8þ lymphocytepopulation was not increased (Fig. 2A and B). The expressionof CD137 molecules on CD8þ cells in TILs was higher than thatof CD137þCD8þ RLN cells (Fig. 2A). We found virtually noexpression of CD137 on CD8þ T cells in the spleens of tumor-bearing mice (Supplementary Fig. S1A). These data show thatCD137highCD8þ T cells exist in the TILs of melanoma tumors.Additionally, we analyzed the expression of PD-1 onCD8þ T cellsin TILs and found thatmost CD137þCD8þ T cells expressed PD-1(Supplementary Fig. S1B).

To analyze the TCR repertoire of CD137þCD8þ T cells inTILs and the RLNs at the single-cell level, we single-cell sorted

CD137þCD8þ T cells in the TILs and RLNs of tumor-bearingmiceon days 10 and 14 after inoculation of mice with tumor (Sup-plementary Fig. S2A) and amplified the TCRb and TCRa cDNAfromsingle cells.Wedidnot sort the TILs onday7, because the cellnumber was too small to sort them. We then analyzed theirnucleotide sequences via direct sequencing of the TCRb ofCD137þCD8þ T cells in TILs and RLN cells (Fig. 2C).We observedmany populations of CD137þCD8þ TILs expressing the sameclonotypic TCR, which might be clonally expanded in the tumorbutnot in thepopulationofCD137þCD8þT cells in theRLNcells.Supplementary Table S2 shows the genes and amino acidsequences of CDR3 of the TCRb and a that were identified fromclonally expanded T cells in CD137þCD8þ TILs. These resultssuggest that tumor-specific T cells were clonally expanded in thetumor in response to tumor cells.

We also analyzed the TCR repertoire of CD137�CD8þ T cells inthe populations of the TILs, RLN cells, and spleen cells. Weobserved clonally expanded populations in the CD137�CD8þ

TILs ofmice #2, #3, and #6 (Supplementary Fig. S2B). Someof theTCR clonotypes were shared between CD137þ and CD137�

CD8þ TILs (Supplementary Table S3). These results indicate thatthe expression of CD137 on CD8þ, clonally expanded, TILsfluctuated in response to cell conditions. In this regard, Dawickiand Watts demonstrated the transient expression of CD137 onCD8þ and CD4þ T cells during antigen stimulation in vivo (31).We rarely observed clonally expanded populations inCD137�CD8þ T cells in the RLN and spleen (Supplementary Fig.S2B; Supplementary Table S3).

Cytokine secretion from TIL-derived TCR-transduced T cellsTo assess whether the TCRs of clonally expanded populations

are tumor reactive, we selected 13 clonotypic TCRs (Supplemen-tary Table S2; Fig. 2C) from clonally expanded populations withhigh frequencies in CD137þCD8þ T cells in TILs from eachmouse, constructed the vectors for their expression, and trans-duced them into splenic T cells. The transfection efficiency intoCD8þ T cells varied from 32% to 51% (Supplementary Fig. S3).We then cocultured TIL-derived TCR-transduced T cells withinterferon (IFN)-g–stimulated B16F10 cells and measured theIFNg production of the T cells. We stimulated B16F10 cells withIFNg prior to the coculture because major histocompatibilitycomplex (MHC) class I molecules (H-2Kb and H-2Db) were notexpressed on the cells; instead, their expression was induced

Figure 3.

The IFNg secretion of TIL-derived TCR-transduced spleen T cells. Spleen T cellswere transduced with a TIL-derived TCR and cocultured with IFNg-stimulatedB16F10 cells. The IFNg production by the TCR-transduced spleen T cells wasanalyzed by ELISA. OT-I TCR-expressing cells were used as a negative control.The statistical significance of the differences in IFNg-secretion from cellstransduced with TIL-derived TCR and OT-I TCR is indicated. � , P < 0.05 by theStudent t test.

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by stimulationwith IFNg (Supplementary Fig. S4).Nine out of the13 TIL-derived TCRs from mice #2, #4, #5, and #6 rendered theTCR-transduced T cells responsive to IFNg-stimulated B16F10cells and made them secrete IFNg (Fig. 3). OT-I TCR-expressingT cells that recognized ovalbumin-derived peptides in thecontext of H-2Kb (32) were used as a negative control. They didnot respond to IFNg-stimulated B16F10 cells. To confirm theTCR reactivity, we sorted TCR-transduced GFPþ T cells (Supple-mentary Fig. S5A) and examined their IFNg-secretion in responseto IFNg-stimulated B16F10 cells. As shown in Supplementary Fig.S5B, we obtained a result similar to that shown in Fig. 3.

Cytotoxicity of TIL TCR–transduced T cells in vitroWe next examined whether the TCRs that reacted with B16F10

cells killed B16F10 cells in vitro. To this end, we used the TCR-transduced T cells that had been used for the IFNg-secretion assay(Supplementary Fig. S3). The TCR-transduced T cells were cul-tured with luciferase-expressing B16F10-Luc cells for 48 hours,and the CTL activity was assessed by analyzing the viability of theB16F10-Luc cells using the luciferase activity as previouslydescribed (28–30). OT-I TCR-expressing T cells were used as a

negative control. T cells expressing the TCR that had reacted toB16F10 cells (Fig. 3) showed cytotoxicity against the IFNg-stim-ulated B16F10-Luc cells (Fig. 4) but not against the unstimulatedcells (Supplementary Fig. S6). Additionally, OT-I TCR-expressingT cells did not cause cytotoxicity to IFNg-stimulated B16F10-Luccells (Fig. 4). As expected, the cytotoxicity-inducing activity of theTCRs was correlated with their IFNg-inducing activity (Supple-mentary Fig. S7). These data show that TIL-derived TCR-trans-duced T cells that reacted to B16F10 melanoma cells exhibitedcytotoxicity toward the B16F10 melanoma cells in vitro.

Antitumor effect of TIL-derived TCR-transduced T cells in vivoWe then investigated whether TIL-derived TCR-transduced T

cells exhibit cytotoxicity toward B16F10 melanoma cells in vivo.To this end, we examined the antitumor effects of TCR 4-3 andTCR 6-1, which induced the highest IFNg secretion and cyto-toxicity against IFNg-stimulated B16F10 cells among the TCRsobtained from each mouse (Figs. 3 and 4). We first adminis-tered B16F10-Luc cells i.v. to five C57BL/6 mice for each TCR,and 1 day later, we i.v. injected the TCR-transduced T cells intothe mice (Fig. 5A). The transfection efficiency of the TIL-derived

Figure 4.

The cytotoxicity of TIL-derived, TCR-expressing T cells against B16F10 cells.Splenic T cellswere transducedwith theTIL-derived TCRs and cultured withIFNg-stimulated B16F10-Luc cells for 48hours. Viability of the B16F10-Luc cellswas analyzed by assessing luciferaseactivity. OT-I TCR-expressing T cellswere used as a negative control.






TCR in splenic CD8þ T cells varied from 44% to 56% (Sup-plementary Fig. S8). On day 4, we sacrificed the mice andanalyzed the pulmonary metastasis by monitoring with lucif-erase imaging. When TCR-untransduced or OT-I TCR-trans-duced T cells were administered as negative controls, a largenumber of B16F10 cells exhibited pulmonary metastasis (Fig.5B). In contrast, when TCR 4-3- and TCR 6-1-transduced T cellswere administered 1 day after B16F10-Luc cell administration,pulmonary metastasis was inhibited by 80% and 75%, respec-tively. These data revealed that the TIL-derived TCR-transducedT cells that had a cytotoxic effect in vitro also exhibited anantitumor effect in vivo on B16F10 melanoma cells, althoughtheir efficacy was limited, in spite of early tumor treatment withlarge numbers of T cells.

Specificity of TIL-derived TCRsTo examine the antigen specificity of TIL-derived TCRs, we first

determinedwhether TIL-derived TCRs were restricted byH-2Kb orDb molecules. We transfected either H-2Kb or H-2Db cDNA inconjunction with EGFP cDNA into B16F10 cells and prepared thecells that constitutively expressed either H-2Kb or H-2Db mole-cules (Supplementary Fig. S9A). H-2Kb cDNA-transfectantsexpressed only H-2Kb molecules on the cell surface. In contrast,some H-2Db cDNA transfectants expressed H-2Kb molecules, inaddition to H-2Dbmolecules, in the absence of IFNg stimulation.When TIL-derived TCR-transduced spleen cells were coculturedwith these B16F10 cells, all of them showed strong cytotoxicity toH-2Kb-expressing B16F10 cells, and weak cytotoxicity to H-2Db-expressing B16F10 cells (Supplementary Fig. S9B). The resultindicated that our cloned TIL-derived TCRs were specificallyrestricted to H-2Kb molecules.

We then analyzed the specificity of TIL-derived TCRs. We firstexamined the reactivity of the TCRs to various cells, includingB16F0 melanoma cells, EL4 lymphoma cells, MC38 colon cancer

cells, and normal mouse embryonic fibroblast (MEF) cells inaddition to B16F10 cells, all of which were derived fromC57BL/6mice. To this end, the cells were stimulated with IFNg . In theabsence of IFNg stimulation, B16F0 and MEF cells hardlyexpressed H-2Kb or H-2Db molecules on the cell surface, whereasEL4 cells or MC38 cells expressed them on the cell surface(Supplementary Fig. S10). Their expression on all cell lines wasenhanced by stimulation with IFNg . When the TIL-derived TCR-expressing spleen cells were cocultured with these cells, the cellsexpressing TCR4-3 or TCR6-1 responded tonot only B16F10 cellsbut also B16F0 cells and EL4 cells, and secreted IFNg (Fig. 6), butthey did not respond to MC38 cells or normal MEF cells. Incontrast, the spleen cells expressing TCR 4-1, 4-2, 5-1, or 5-2responded to IFNg-stimulated B16F0 cells as well as B16F10 cellsbut neither to EL4 cells, MC38 cells, nor MEF cells. TCR 2-1-expressing T cells, although weakly, responded to only B16F10cells, but not to the other cells.

We then examined whether those TCRs recognize antigensreported to be expressed in B16F10 cells. Because our clonedTIL-derived TCRs were restricted to H-2Kb molecules (Supple-mentary Fig. S9), we examined two antigenic peptides: one istyrosine-related protein 2-drived peptide (TRP2p; ref. 33) andthe other is p15E peptide (p15Ep) from envelope protein 70 ofendogenous murine-leukemia virus (34), both of which boundto H-2Kb molecules. We stained the TCR-expressing T cells withTRP2p/H-2Kb tetramer or p15Ep/H-2Kb tetramer. Unexpected-ly, p15Ep/H-2Kb tetramer, but not TRP2p/H-2Kb tetramer,bound to TCRs 4-1, 4-2, 5-1, 5-2, 5-3, and 5-4 (Fig. 7A). Thep15Ep/H-2Kb tetramer very weakly bound to TCR 2-1. Bothtetramers did not bind to TCR 4-3 and TCR 6-1. When the TCR-expressing splenic T cells were stimulated with p15E peptide-pulsed EL4 cells, T cells expressing TCRs 2-1, 4-1, 4-2, 5-1, 5-2,5-3, and 5-4 secreted IFNg (Fig. 7B). Taken together, theseresults showed that the TCRs 4-3 and 6-1 recognized certain

Figure 5.

The antitumor effect of TIL-derivedTCR-transduced T cells on B16F10melanoma cells in vivo. A,Experimental protocol. On day 0,the B16F10-Luc cells were i.v.administered to C57BL/6mice. Onday1, the TIL-derived TCR-transducedT cells were i.v. administered. On day4, the mice were sacrificed, and thelung metastasis was assessed bybioluminescence imaging with IVIS.B, The luminescence measurementsfrom the lungs metastasized byB16F10-Luc cells are shown on the left(n ¼ 5 for each TCR). The IVIS imagesof the lungs of five individual mice areshown on the right. Representativedata (mean � SD) from twoindependent experiments are shown.��, P < 0.01 by the Student t test.

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tumor-associated antigens whose expression was shared withB16 melanoma and EL4 lymphoma cells, whereas TCRs 2-1,4-1, 4-2, 5-1, 5-2, 5-3, and 5-4 recognized endogenous murineleukemia virus-derived antigens.

DiscussionIn this study, we first analyzed the TILs in various can-

cer patients. We observed clonally expanded populationsin which T cells expressed the same clonotypic TCR inPD-1þCD137þCD8þ TILs in most cancer patients examined.Because CD137 and PD-1 are expressed on activated T cells(35, 36), the results suggest that those clonally expandedpopulations responded to some tumor components, such astumor antigens. Because patients' primary culture tumor cellswere not fully available to examine the tumor reactivity of theobtained TCRs, we then examined the TILs from a melanoma-bearing mouse model. Similarly, we found clonally expandedpopulations in CD137þCD8þ TILs from mouse melanomatissues, yet the regional lymph nodes or spleen in the samemice had few or no clonally expanded populations among theCD137þ or CD137�CD8þ T cells. These results also indicatedthat tumor-specific T cells were activated and clonally expandedin the tumor. Indeed, T cells that expressed the TCRs obtainedfrom the clonally expanded TILs responded to B16F10 mela-noma cells by secretion of IFNg and exhibited cytotoxicity

against B16F10 melanoma cells both in vitro and in vivo. Inthis regard, Thompson and colleagues also reported thattumors support the infiltration, activation, and effector differ-entiation of naive CD8þ T cells (37).

We analyzed the TCR repertoire of TILs in six mice and foundclonal expansion of CD137þCD8þ T cells in all of the mice.However, some of the TCRs cloned from T cells that showedhigh clonal expansion in the tumor did not respond to B16F10cells. The possible reasons for this finding are that (i) thetumor-specific TCRa and b pairs were not efficiently expressedbecause of a mispairing between the transduced TCRs andendogenous TCRs in splenic T cells; (ii) because of the dualTCR expression in single T cells, a mismatched and tumor–nonreactive TCRa and b pair was cloned and used for func-tional analysis; and (iii) the tumor antigen–nonspecific T cellswere expanded in TILs.

With regard to the specificity of TCRs we obtained from TILs,TCRs 4-3 and 6-1 responded to more than one tumor line(B16F10, B16F0, and EL4 cells) but not to MC38 cells nornormal MEF cells, indicating that they recognized tumor-asso-ciated antigen(s) that were shared with melanoma and lym-phoma. To our surprise, the other seven TCRs reacted to p15Epeptide derived from endogenous murine leukemia virus enve-lope glycoprotein 70 (gp70; ref. 38). p15E (gp70) is a potentimmunogen (34) and highly expressed in B16F10 cells(approximately 105-fold higher than EL4 cells), whereas gp70

Figure 6.

Specificity of TIL-derived TCRs. SpleenT cells were transduced with theTIL-derived TCR and coculturedwith B16F10, B16F0, EL4, or MC38tumor cells and normal mouseembryonic fibroblast (MEF) cells thathad been stimulated with IFNg . TheIFNg secretion in the supernatant wasanalyzed by ELISA. OT-I TCR-expressing cells were used as anegative control. The statisticalsignificance of the differences inIFNg secretion from cells transducedwith TIL-derived TCR and OT-I TCRis indicated. � , P < 0.05 by theStudent t test.






expression on normal tissues is nearly undetectable (38). p15E-reactive TCRs induced weaker immune reactions than TCRs 4-3and 6-1 that recognized shared tumor-associated antigen(s).The results may reflect the central tolerance that depletes T-cellclones with strong reactivity against p15E.

When we analyzed the TCR repertoire of TILs obtained fromB16F10 tumors that had developed in C57BL/6mice, none of theTCRs obtained from different mice showed the same clonotypes,whereas the T cells that expressed the same clonotypic TCR wereexpanded in the tumor in the same mouse. These results seem tobe reasonable because TCR rearrangement occurs among the V, D,and J segments and between the V and J segments in the TCRb andTCRa genomes, respectively. N sequences are produced in thethymus irrespective of the genome, and this process occurs inde-pendently in each mouse. Consequently, T cells produced in thethymus of different mice expressed independent and distinctclonotypic TCRs.

Rosenberg and colleagues have shown that tumor- and neoan-tigen-reactive TCRs can be identified in TILs by NGS analysisfollowed by single-cell analysis (6, 37, 39). From their analysis,they discussed the usefulness of single-cell analysis for obtainingand determining TCR a and b pairs (7). In combination with ourobservations, the single-cell analysis of the TCR repertoire in TILsenabled us to efficiently obtain candidate TCRs for TCR-genetherapy.

In conclusion, the single–T-cell analysis of TILs enabled us toidentify tumor-specific T cells andobtain tumor-specific TCRs thatexert inhibitory effects against tumor growth, irrespective of theMHC haplotype and antigens. Because our single-cell RT-PCRprotocol can obtain TCR cDNAs from primary T cells in a simple,rapid, and high-fidelity manner, we can acquire TCRs from TILsfor the treatment of cancer patients. Thus, the single–T-cell anal-ysis of TILs will promote the personalized treatment of cancerpatients in the future.

Disclosure of Potential Conflicts of InterestK. Shitaoka was a senior researcher at SC World, Inc. H. Kishi is director

at SC World, Inc. and has ownership interest in patent JP 6126804. K. Tanireports receiving a commercial research grant from NeoPharma Japan Co. Ltd,Shinnihonseiyaku Co. Ltd., and Takara Bio Co. Ltd. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: K. Shitaoka, H. Hamana, H. Kishi, Y. Hayakawa,T. Ozawa, A. MuraguchiDevelopment of methodology: K. Shitaoka, H. Hamana, H. Kishi, Y. Hayakawa,E. Kobayashi1, D. SugiyamaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Shitaoka, H. Hamana, H. Kishi, Y. Hayakawa,K. Sukegawa, X. Piao, F. Lyu, T. Nagata, D. Sugiyama, H. Nishikawa,A. Tanemura, I. Katayama, Y. Takamatsu

Figure 7.

p15E reactivity of TIL-derived TCRs.A, Binding of p15Ep/H-2Kb tetramer.Endogenous TCR-negative 58a�b�

T-cell lines were transduced withTIL-derived TCRs, stained with CD3antibody and p15Ep/H-2Kb tetramerand analyzed with flow cytometry.TRP-2-specific TCR-expressing58a�b� T-cell line was used as anegative control. The percentage ofcells in each quadrant is indicated.B, p15E peptide–induced IFNg-secretion. TIL-derived TCR-transduced spleen cells were culturedin the presence of p15E peptide–pulsed EL4 cells, and IFNg secretionwas analyzed with ELISA on the nextday. � , P < 0.05 by the Student t test.

Shitaoka et al.





Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Shitaoka, H. Hamana, H. Kishi, Y. Hayakawa,A. MuraguchiWriting, review, and/or revision of the manuscript: K. Shitaoka, H. Hamana,H. Kishi, Y. Hayakawa, A. MuraguchiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. Shitaoka, Y. Hayakawa, X. Piao, F. Lyu,M. Murahashi, K. Tani, A. MuraguchiStudy supervision: H. Kishi, A. Muraguchi

AcknowledgmentsThis research was supported by MEXT KAKENHI grant numbers

JP15H04308 and JP16H06499 (H. Kishi) and JP15K06872 (H. Hamana).

The authors thank Sanae Hirota for providing technical assistance andKaoru Hata for performing secretarial work. We also thank Professor ToshioKitamura for generously providing the PLAT-E cells, Professor B. Malissenfor kindly suppling the 58a�b� cells and Keisuke Fujii for technical advice.

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 September 4, 2017; revised December 8, 2017; accepted February16, 2018; published OnlineFirst February 23, 2018.

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2018;6:378-388. Published OnlineFirst February 23, 2018.Cancer Immunol Res Kiyomi Shitaoka, Hiroshi Hamana, Hiroyuki Kishi, et al. Lymphocytes by Single-Cell AnalysisIdentification of Tumoricidal TCRs from Tumor-Infiltrating

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