Cytotoxic T Lymphocytes Block Tumor Growth Both by Lytic Activity And

Published OnlineFirst August 15, 2014; DOI: 10.1158/2326-6066.CIR-14-0098 Research Article Cancer Immunology Research Cytotoxic T Lymphocytes Block Tumor Growth Both by Lytic Activity and IFNg-Dependent Cell-Cycle Arrest Hirokazu Matsushita1, Akihiro Hosoi1,2, Satoshi Ueha3, Jun Abe3, Nao Fujieda1,2, Michio Tomura4, Ryuji Maekawa2, Kouji Matsushima3, Osamu Ohara5, and Kazuhiro Kakimi1 Abstract To understand global effector mechanisms of CTL therapy, we spotty apoptotic or necrotic areas. Thus, tumor growth suppres- performed microarray gene expression analysis in a murine model sion was largely dependent on G1 cell-cycle arrest rather using pmel-1 T-cell receptor (TCR) transgenic T cells as effectors than killing by CTLs. Neutralizing antibody to IFNg prevented and B16 melanoma cells as targets. In addition to upregulation of both tumor growth inhibition and G1 arrest. The mechanism of genes related to antigen presentation and the MHC class I path- G1 arrest involved the downregulation of S-phase kinase-associ- way, and cytotoxic effector molecules, cell-cycle–promoting genes ated protein 2 (Skp2) and the accumulation of its target were downregulated in the tumor on days 3 and 5 after CTL cyclin-dependent kinase inhibitor p27 in the B16-fucci tumor transfer. To investigate the impact of CTL therapy on the cell cycle cells. Because tumor-infiltrating CTLs are far fewer in number than of tumor cells in situ, we generated B16 cells expressing a the tumor cells, we propose that CTLs predominantly regulate fluorescent ubiquitination-based cell-cycle indicator (B16-fucci) tumor growth via IFNg-mediated profound cytostatic effects and performed CTL therapy in mice bearing B16-fucci tumors. rather than via cytotoxicity. This dominance of G1 arrest over Three days after CTL transfer, we observed diffuse infiltration other mechanisms may be widespread but not universal because of CTLs into the tumor with a large number of tumor cells IFNg sensitivity varied among tumors. Cancer Immunol Res; 1–11. arrested at the G1 phase of the cell cycle, and the presence of Ó2014 AACR. Introduction thought to be important for tumor elimination (9–11). None- theless, cytokines such as IFNg and TNFa produced by T cells are Adoptive T-cell immunotherapy (ACT) using autologous also likely to contribute to the prevention of tumor growth by ACT tumor-infiltrating lymphocytes (TIL) can be highly effective for via mechanisms other than cell lysis (12–14). treating melanoma (1). The recent development of genetically IFNg is a critical cytokine for antitumor immunity under natural engineered T cells stably expressing exogenous T-cell receptors and therapeutic conditions (15, 16). It enhances tumor immuno- (TCR) or chimeric antigen receptors (CAR) specific for tumor- genicity by upregulating components of the MHC antigen proces- associated antigens offers the possibility of testing the efficacy of sing and presentation pathway. It also induces the expression of ACT against a wide range of cancer types in addition to melanoma chemokines, including the angiostatic chemokines CXCL9 (MIG), (2, 3). Many clinical trials have now been conducted using CXCL10 (IP-10), and CXCL11 (I-TAC), that block neovasculariza- genetically engineered T cells specific for tumor antigens as well tion in the tumor and recruit effector immune cells (17–19). as TILs, and some objective responses have been achieved (4, 5). It Furthermore, IFNg has been reported to exert antiproliferative is clear from mouse models that adoptively transferred antigen- effects on the developing tumor (20, 21), and it triggers apoptosis specific T cells are capable of eradicating established cancer (6–8), of tumor cells by inducing proapoptotic molecules (22, 23). and the ability of CTLs to directly kill tumor and/or stromal cells is To understand the global antitumor effect mediated by ACT, we used the B16 melanoma pmel-1 TCR-transgenic T-cell model to perform a gene expression analysis of ACT-treated tumors. On the 1Department of Immunotherapeutics, The University of Tokyo Hospi- tal,Tokyo, Japan. 2Medinet Co Ltd.,Yokohama, Japan. 3Department of basis of these results, we focused on genes controlling the cell cycle Molecular Preventive Medicine, Graduate School of Medicine, The and arresting growth of B16 tumor cells in this model. We University of Tokyo,Tokyo, Japan. 4Center for Innovation in Immunor- examined the effects on tumor cells of the IFNg produced by egulative Technology and Therapeutics, Kyoto University Graduate in situ School of Medicine, Kyoto, Japan. 5Department of Human Genome the CTLs using cell-cycle status indicators and investigated Research, Kazusa DNA Research Institute, Chiba, Japan. the mechanism of cell-cycle arrest. Furthermore, we demonstrate Note: Supplementary data for this article are available at Cancer Immunology the importance of cell-cycle arrest induced by CTL-derived IFNg in Research Online (http://cancerimmunolres.aacrjournals.org/). the regulation of tumor growth. Corresponding Author: Kazuhiro Kakimi, Department of Immunotherapeutics, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-Ku, Tokyo 113-8655, Materials and Methods Japan. Phone: 81-3-5805-3161; Fax: 81-3-5805-3164; E-mail: Mice, tumor cells, and peptides [email protected] Six-week-old male C57BL/6 mice were purchased from Japan doi: 10.1158/2326-6066.CIR-14-0098 SLC. Mice transgenic for the pmel-1-TCR, which recognizes the H- b Ó2014 American Association for Cancer Research. 2D –restricted epitope EGSRNQDWL from gp100 (gp10025–33), www.aacrjournals.org OF1 Downloaded from cancerimmunolres.aacrjournals.org on September 27, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst August 15, 2014; DOI: 10.1158/2326-6066.CIR-14-0098 Matsushita et al. were obtained from The Jackson Laboratory. All mice were housed BioLegend: PerCP/Cy5.5-conjugated anti-CD45, Alexa Fluor647– in a pathogen-free environment, and all animal procedures were conjugated anti-CD90.1,and APC-Cy7–conjugated anti-CD8.Data conducted in accordance with institutional guidelines. All animal were analyzed with the Kaluza software (Beckman Coulter). experiments were approved by the University of Tokyo Ethics Committee for Animal Experiments (10-P-127). The H-2Db– Comprehensive gene expression analysis restricted peptide human gp100 (hgp10025–33, KVPRNQDWL) Gene expression profiling data of B16 tumor tissues on differ- was purchased from GenScript Japan at a purity of >90%, with free ent days were obtained by Agilent whole-mouse genome micro- amino and carboxyl terminals. B16F10, FBL3, and 3LL cell lines array. Total RNA was extracted with TRizol (Invitrogen) from B16 were maintained in culture medium consisting of DMEM with tumor tissues and fluorescently labeled using a One-Color Agilent 10% FCS, 100 U/mL penicillin, and 100 mg/mL streptomycin. Quick Amp Labeling Kit. The microarray slides were hybridized, EL4, P815, and CT26 were cultured in RPMI-1640 medium washed, and read on an Agilent Microarray scanner following the supplemented with 10% FCS, 100 U/mL penicillin, and 100 manufacturer's instructions, and raw fluorescence signal intensi- mg/mL streptomycin. All cell lines were tested for mycoplasma by ties were generated by Agilent Feature Extraction Software v9.5. the MycoAlert Mycoplasma Detection kit (Lonza). Cellular mor- The signals were normalized to align at 75th percentile, and then in vitro phology and growth curve were checked in all cell lines. turned into log2 ratio against day 1 in untreated and CTL-treated B16F10 and B16-fucci cells were authenticated by transplantation groups. We began with 45,018 probes, and removed probes if for assessing growth ability in vivo. their gIsWellAboveBG flag values were 0 at all samples, and then filtered out log2 ratio values that were unvarying (between À1 and Dendritic cell preparation and CTL stimulation 1) at all time points. We obtained 10,855 probes and ran hier- Dendritic cells (DC) were obtained by 8-day culture of archical clustering (standard correlation, UPGMA) on them. All C57BL/6-derived bone marrow cells with granulocyte-macro- data were analyzed with the Subio Platform and Basic Plug-in phage colony-stimulating factor (GM-CSF), as described pre- v1.16 (Subio Inc.). The microarray data are available from the viously (24). Briefly, bone marrow cells obtained from tibias Gene Expression Omnibus (GEO) database (series accession and femurs of C57BL/6 mice were cultured in RPMI-1640 number GSE57304; sample accession numbers GSM1379331– medium supplemented with 10% FCS, 10 mmol/L HEPES, 5 Â GSM1379344). À À 10 5 mol/L 2-mercaptoethanol, 1 Â 10 3 mol/L sodium pyru- For quantitative gene panel–based PCR, Cell Cycle RT2 Profiler vate, 1% nonessential amino acids, 100 U/mL penicillin, 100 PCR arrays (SABioscience; http://www.sabiosciences.com/ mg/mL streptomycin, and 20 ng/mL GM-CSF (PeproTech) for 8 rt_pcr_product/HTML/PAMM-020Z.html) were used to simulta- days. On days 3 and 6, half of the medium was replaced with neously examine the mRNA levels of 84 genes in 96-well PCR fresh medium containing GM-CSF. DCs were further incubated array plates. Total RNA was prepared using TRizol according to the with 1 mg/mL lipopolysaccharide for 16 hours and then pulsed manufacturer's instructions (Invitrogen), and reverse-transcribed with 1 mg/mL hgp100 peptide for 3 hours to obtain mature using RT2 First Strand kits (SABioscience). Real-time PCR was DCs. To prepare CTLs, 1 Â 107 spleen cells from pmel-1 TCR- performed as instructed by the supplier on an ABI PRISM 7900HT transgenic mice were cocultured with 2 Â 105 DCs in a medium Sequence Detection System (Life Technologies). Data were ana- containing 50 U/mL IL2 (Chiron Corporation). After 3 days of lyzed by a DD cycle threshold method to determine the fold in vitro stimulation, approximately 90% of the harvested cells changes of the mRNA levels (http://www.SABiosciences.com/ þ þ were CD3 CD8 CTLs. pcrarraydataanalysis.php). ACT and anti-IFNg mAb treatment Expression vectors C57BL/6 mice were inoculated subcutaneously with 1 Â 106 CSII-EF-MCS/mAG-hGeminin and CSII-EF-MCS/mKO-cdt1 B16 tumor cells followed by adoptive CTL transfer (1 Â 107 or 4 Â vectors were kindly provided by Dr.

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