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. Author manuscript; available in PMC: 2020 Dec 1.
Published in final edited form as: Cancer Immunol Res. 2020 Mar 24;8(6):743–755. doi: 10.1158/2326-6066.CIR-19-0910

Optimization of T-cell receptor–modified T cells for cancer therapy

Dylan J Drakes 1, Sarwish Rafiq 2, Terence J Purdon 3, Andrea V Lopez 3, Smita S Chandran 3,4,5, Christopher A Klebanoff 3,4,5,6, Renier J Brentjens 1,3,4,7,8,*
PMCID: PMC7269835  NIHMSID: NIHMS1579662  PMID: 32209638

Abstract

T-cell receptor (TCR)–modified T-cell gene therapy can target a variety of extracellular and intracellular tumor associated antigens, yet has had little clinical success. A potential explanation for limited antitumor efficacy is a lack of T-cell activation in vivo. We postulated that expression of pro-inflammatory cytokines in TCR-modified T cells would activate T cells and enhance antitumor efficacy. We demonstrate that expression of interleukin 18 (IL18) in tumor-directed TCR-modified T cells provides a superior pro-inflammatory signal than expression of interleukin 12 (IL12). Tumor-targeted T cells secreting IL18 promote persistent and functional effector T cells and a pro-inflammatory tumor microenvironment. Together, these effects augmented overall survival of mice in the pmel-1 syngeneic tumor model. When combined with sublethal irradiation, IL18-secreting pmel-1 T cells were able to eradicate tumors, whereas IL12-secreting pmel-1 T cells caused toxicity in mice through excessive cytokine secretion. In another xenograft tumor model, IL18 secretion enhanced the persistence and antitumor efficacy of NY-ESO-1–reactive TCR-modified human T cells as well as overall survival of tumor-bearing mice. These results demonstrate a rationale for optimizing the efficacy of TCR-modified T-cell cancer therapy through expression of IL18.

Introduction

T-cell activation is dependent upon T-cell receptor (TCR) engagement with peptides processed and presented in the context of a major histocompatibility complex (MHC) (signal 1) and co-stimulation (signal 2) [1,2]. Signal 2 is derived from CD28, 4–1BB, or OX-40 molecules [1,2]. T cells receiving both signals develop effector function and secrete pro-inflammatory cytokines. Without signal 2, T cells become anergic [3–5]. Pro-inflammatory cytokines interleukin-12 (IL12) or type I interferon can act as signal 3 to heighten the effector function of T cells [6–10]. Optimizing T-cell stimulation through this pro-inflammatory pathway may augment antitumor efficacy of tumor-targeted T cells.

Several clinical trials have tested autologous isolated tumor infiltrating lymphocytes (TIL) or TCR-modified T cells for cancer therapy [11]. Although these approaches can target extracellular and intracellular tumor-associated antigens, trial results have been modest [12–18]. Strategies to enhance the potency of these TCR T cells include increasing the affinity of the TCR to tumor-associated antigens, although this sometimes has adverse effects [12,19,20]. One promising method to enhance the efficacy of tumor-directed T cells is providing a stimulatory signal to TIL or TCR-modified T cells. Lack of T-cell activation may contribute to failure of T-cell therapies if tumor cells downregulate costimulatory molecule expression [21,22].

Chimeric antigen receptor (CAR) T cells carry an antigen-recognition domain fused to a costimulatory and CD3ζ domain, through which the cell receives both signals 1 and 2. This configuration eliminates the need for additional stimulation provided by APC or tumor cells [23,24]. With CAR T-cell therapy, the scFv in the CAR is directed towards extracellular antigens and not intracellular antigens that might be presented extracellularly within the context of an MHC. For certain tumor types, especially solid tumors, there are few extracellular antigens that can be distinguished from those of healthy tissues and specifically targeted by CARs, limiting potential targets. TCR-modified T cells, however, can be redirected to tumor-specific targets, including intracellular antigens, but are limited overall by a lack of T-cell activation [11,21,22]. We hypothesized that pro-inflammatory cytokine modifications could activate T cells and enhance the efficacy of tumor-directed TCR-modified T cells. Here we explore approaches to enhance TCR-modified T cells through genetic engineering with pro-inflammatory IL12 or IL18 cytokines.

Clinical trials of patients treated with systemic recombinant IL12 have shown modest efficacy, although results have been limited by toxicities [25]. Treatment with recombinant IL18 did not cause toxicities but showed limited clinical responses [26]. Directing cytokines to the tumor site may alleviate toxicity and enhance antitumor responses. IL18 dependent signaling occurs through a heterodimeric receptor (IL18Rα and IL18Rβ). Most immune cell types express IL18Rα. IL18Rβ is commonly expressed on T cells, dendritic cells, macrophages, and other myeloid cells [27,28]. Thus adoptive transfer of IL18-secreting T cells could enhance the activity of T cells while modulating the tumor microenvironment.

CAR T-cell function has been augmented by IL12 and IL18 cytokine secretion, with IL12 under the control of an IRES element to limit toxicities [29–31]. The pmel-1 TCR transgenic murine melanoma model has been used to show that IL12 enhances T-cell function when combined with a lymphodepleting preconditioning regimen [32–34]. Using a syngeneic and xenograft melanoma model, we show that expression of IL18 in TCR-modified T cells provides a potent and durable pro-inflammatory signal to activate T cells and enhance T-cell persistence and antitumor efficacy demonstrating that antitumor efficacy of TCR-modified T cells is optimized through engineering with IL18.

Materials and Methods

Contact for Reagent and Resource Sharing

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Renier J. Brentjens (brentjer@mskcc.org).

Experimental Model and Subject Details

Animal Models

Mice were bred and housed under SPF conditions in the animal facility of Memorial Sloan Kettering Cancer Center. Wild-type C57BL/6, pmel-1(B6.Cg-Thy1a/CyTg(TcraTcrb)8Rest/J), CD80/86–/– (B6.129S4-CD80tm1ShrCd86tm2Shr/J), IL18R–/– (B6.129P2-Il18r1tm1Aki/J), and NSG (NOD.Cg-PrkdcscidIL2rgtm1Wjl/SzJ) mice were purchased from Jackson laboratories. 6–8 week old gender-matched mice challenged with tumor were measured with calipers to confirm equal tumor load and randomized to different treatment groups one day before treatment. Mice were euthanized when tumor growth led to a volume greater than 1000mm3 by V=(π6)(ab2) where a is the longest length and b is the shorter length. The investigator was blinded when assessing the outcome.

Cell Lines

Phoenix-ECO packaging cells (catalog number CRL-3214) purchased from ATCC and 293 galv9 packaging cells (courtesy of the Sadelain lab) were maintained in DMEM supplemented with 10% heat-inactivated FBS nonessential amino acids, 2 mM L-glutamine, 1% penicillin/streptomycin. The B16F10 cell line, a kind gift from Dr. Jedd Wolchok in 2016, and A375 and SK-Mel5 human melanoma lines, a kind gift from Dr. David Scheinberg in 2018, were modified to express GFP-firefly luciferase for killing assays. Cell lines were kept in culture for up to two months. All tumor cell lines were maintained in RPMI-1640 supplemented with 10% heat-inactivated FBS, nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, 2 mM L-glutamine, 1% penicillin/streptomycin, 11 mM glucose, and 2 μM 2-mercaptoethanol. Cell lines were not re-authenticated in the past year but were routinely tested for potential mycoplasma contamination with Lonza MycoAlert Detection kit (LT07–318).

Generation of retroviral constructs

Plasmids encoding the mCD19t construct in the SFG γ-retroviral vector [35] were used to transfect gpg29 fibroblasts (H29) with the ProFection Mammalian Transfection System (Promega) according to the manufacturer’s instructions in order to generate vesicular stomatitis virus G-glycoprotein-pseudotyped retroviral supernatants. These retroviral supernatants were used to construct stable Moloney murine leukemia virus-pseudotyped retroviral particle-producing Phoenix-ECO cell lines or 293 galv9 cell lines. The SFG-mCD19t, SFG-mCD19tmIL12, and SFG-mCD19tmIL18 vector were constructed by restriction enzyme digest using the cDNA of mCD19 protein truncated to exclude the intracellular domain alone or with either the fusion gene encoding murine IL12 (mIL12) [29,30,36] (provided by Dr. Alan Houghton and Dr. Jedd Wolchok) or the mature form of murine IL18 (mIL18). mIL12 was preceded by an IRES element and mIL18 was preceded by a picornavirus 2a element (P2A) self-cleaving peptide. For xenograft experiments, an optimized NY-ESO-1 directed TCR, 1G4, α and β chain sequence was provided by the Klebanoff Lab [37–39] and cloned into the SFG γ-retroviral vector alone or in combination with an equine 2A element followed by a signal peptide and the mature, processed form of human IL18 (hIL18). Nucleotide sequences are provided in Supplementary Fig. S1.

T-cell isolation and retroviral transduction

Mice were euthanized, and their spleens were harvested. Following tissue dissociation and red blood cell lysis, T cells were activated with CD3/CD28 Dynabeads at a bead:cell ratio of 1:2 (Invitrogen). Cells were expanded in vitro by culturing in RPMI-1640 supplemented with 10% heat-inactivated FBS, nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, 2 mM L-glutamine, 1% penicillin/streptomycin, 11 mM glucose, 2 μM 2-mercaptoethanol, and 100 IU of recombinant human IL2 (Prometheus Therapeutics & Diagnostics). 24 and 48 hours after initial expansion, T cells were spinoculated with viral supernatant collected from Phoenix-ECO cells as described previously [40]. Human T cells were activated and transduced as described previously [41]. Peripheral blood mononuclear cells (PBMCs) were isolated from leukopacks (New York Blood Center, New York, NY, USA), activated with 2 μg/ml phytohemagglutinin and 100 IU/ml of IL2, and cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin for 2 days prior to transduction. 48 and 72 hours after initial expansion, murine and human T cells were transduced by centrifugation on RetroNectin (Takara Clontech) plates with retroviral supernatant from viral packaging cells.

Cytotoxicity assays

24 hour quantitative cytotoxicity assay

Cytolytic capacity of murine and human T cells was assessed through luciferase killing assay [42]. 5×104 target tumor cells expressing firefly luciferase were cocultured with adoptively transferred T cells at various effector-to-target ratios in triplicates in black-walled 96-well plates (Thermo Scientific) in a total volume of 200 μl of cell media. Target cells alone were plated at the same cell density to determine the maximal luciferase expression as a reference (max signal). 24 hrs later, 75 ng of D-Luciferin (Gold Biotechnology) dissolved in 5 μl of PBS was added to each well. Emitted luminescence of each sample (sample signal) was detected in a Spark plate reader (Tecan) and quantified using the SparkControl software (Tecan). Percent lysis was determined as (1 – (sample signal / max signal)) x 100.

Ex vivo cytotoxicity assay

5×104 B16F10 GFP/ffLuc were cocultured at a 1:1 ratio with 5×104 Thy1.1+ T cells excised from tumors on Day 6 and Day 13 post T-cell infusion and sorting via FACS, in a total volume of 200 μl of cell media. Target cells alone were plated at the same cell density to determine the maximal luciferase expression as a reference (max signal). 24 hrs later, 75 ng of D-Luciferin (Gold Biotechnology) dissolved in 50 μl of PBS was added to each well. Emitted luminescence of each sample (sample signal) was detected in a Spark plate reader (Tecan) and quantified using the SparkControl software (Tecan). Percent lysis was determined as [1 – (sample signal / max signal)] x 100.

Adoptive transfer of TCR-modified T cells

For syngeneic tumor studies, C57BL/6 mice were inoculated s.c. with 5×105 B16F10 melanoma tumor cells on day −10. On day −1, tumors were measured with calipers and mice with equal tumor burden were selected for treatment. Mice were then randomized into different treatment cohorts and on days 0, 7, and 14, mice were treated with 5×106 T cells intravenously. For irradiation studies, the above protocol was followed in addition to sublethally irradiating the tumor bearing mice with 5 Gy total body irradiation on D-1. For xenograft tumor studies, 5×106 A375 (NY-ESO-1+) tumor cells were injected s.c. into NSG mice on Day −10 and then treated with 2.5×106 adoptively transferred human T cells intravenously on Day 0.

Cell isolation for ex vivo analyses

5×106 armored or control modified pmel-1 T cells were infused and either six or thirteen days after T-cell infusion, tumor samples were excised on Days 6 and 13 post T-cell infusion and mechanically dissociated using a 150 μM metal mesh and glass pestle in 2.5% FBS/PBS and passed through a 100 μM cell strainer. The homogenate was spun down at 1500 RPM for 5 min at 4 °C to pellet the cells. Cells were counted, and an aliquot was stained for flow cytometry analysis. For the remaining sample, red blood cell lysis was achieved with an ACK (Ammonium-Chloride-Potassium) Lysing Buffer (Lonza). The cells were washed with PBS, FACS sorted, and used for subsequent analyses.

In vitro and ex vivo cytokine secretion analysis

To analyze in vitro and ex vivo T-cell cytokine production, T cells and antigen positive tumor cells were cocultured in a 1:1 ratio for 24 hours in a 96-well round-bottom plate in 200 μl of media. The supernatant was collected and analyzed for cytokines on a Luminex IS100 instrument. Luminex FlexMap3D system and Luminex Xponent 4.2 (Millipore Corporation) were used to detect cytokines.

Serum T cell and cytokine analysis

Whole blood was collected from mice and serum was prepared by allowing the blood to clot during centrifugation at 14,000 RPM for 30 min at 4 °C. Red blood cell lysis was achieved with an ACK (Ammonium-Chloride-Potassium) Lysing Buffer (Lonza). T cells were washed with PBS and used for subsequent flow cytometry analysis. Serum was analyzed for cytokines with Luminex.

In vitro tumor cell analysis

To assess CD80 and CD86 surface expression on tumor cells, the cells were collected, surface stained, and analyzed by flow cytometry.

Flow cytometry and FACS sorting

Flow cytometric analyses were performed using 10-color Gallios B43618 (Beckman Coulter, Indianapolis, IN) and 14-color Attune NxT (ThermoFisher Scientific) instruments to acquire data. Data were analyzed using FlowJo (Tree Star). DAPI (0.5 mg/ml, Sigma-Aldrich) or a LIVE/DEAD fixable yellow fluorescent dye (Thermo Fisher) were used to exclude dead cells in all experiments. Flow cytometry gating strategies are provided in Supplementary Fig. S2. Antibodies to the following mouse proteins were used for flow cytometry: CD3ε (eBioscience, clone 145–2C11), CD3 (BioLegend, 17A2), CD4 (eBioscience, GK1.5), CD4 (eBioscience, RM4–5), CD8α (eBioscience, 53–6.7), CD11b (eBioscience, M1/70), CD11c (eBioscience, N418), CD19 (eBioscience, eBio1D3), CD25 (eBioscience, PC61.5), CD44 (eBioscience, IM7), CD45 (BioLegend, 30-F11), CD62L (Invitrogen, MEL-14), CD80 (eBioscience, 16–10A1), CD86 (eBioscience, GL1), CD90.1 (Thy-1.1) (Invitrogen, HIS51), CD206 (Invitrogen, MR5D3), CD218a (IL18R) (eBioscience, P3TUNYA), CD223 (Lag-3) (eBioscience, C9B7W), CD279 (PD-1) (Invitrogen, J43), CD366 (TIM3) (Invitrogen, 8B.2C12), F4/80 (eBioscience, BM8), FoxP3 (eBioscience, FJK-16s), Ly-6C (eBioscience, HK1.4), Ly-6G/Ly-6C (Gr-1) (eBioscience, RB6–8C5), MHC Class II I-Ab (Invitrogen, AF6–120.1), and NK1.1 (eBioscience, PK136). Antibodies to the following human proteins were used for flow cytometry: CD3 (Invitrogen, clone S4.1 (7D6)), CD4 (eBioscience, OKT4), CD8 (BioLegend, SK1), CD80 (Invitrogen, MEM-233), CD86 (Invitrogen, BU63), HLA-A2 (Invitrogen, BB7.2), and HLA-A*02:01 NY-ESO-1 (SLLMWITQC) iTAg Tetramer (MBL). Quantification of total cell numbers by flow cytometry was done using 123count eBeads Counting Beads (Thermo Fisher). Staining of Foxp3 was done using the Foxp3/Transcription factor staining buffer set from eBioscience. All antibodies were purchased from BioLegend, BD Biosciences, eBioscience, Invitrogen, or MBL. Sorting of splenocytes after tissue processing was done using a BD FACSAria under sterile conditions.

Statistics

All statistical analyses were performed using GraphPad Prism software (GraphPad). Data points represent biological replicates and are shown as the mean +/− s.e.m. or mean +/− SD as indicated in the figure legends. Statistical significance was determined using an unpaired two-tailed Student t test, one-way ANOVA, or two-way ANOVA as indicated in the figure legends. Mann-Whitney test was used to determine statistical significance for tumor volume growth curves. The log-rank (Mantel-Cox) test was used to determine statistical significance for overall survival in mouse survival experiments. Significance was assumed with *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Study Approval

All experiments were performed in accordance with the MSKCC Institutional Animal Care and Use Committee (IACUC) approved protocol guidelines (MSKCC #00–05-065).

Results

IL18 and IL12 enhance the effector function of tumor-specific T cells in vitro

Retroviral constructs were generated to armor tumor-specific pmel-1 T cells with pro-inflammatory cytokines in a syngeneic murine system. Bicistronic constructs were created with a truncated version of murine CD19 (mCD19t) protein to assess transduction efficiency and either a murine IL12 α and β subunit fusion protein (mIL12) [36] or a bioreactive mature version of murine IL18 (mIL18) to provide constitutive IL12 or IL18 expression by the LTR promoter (Fig. 1A). Previous work has shown that IL12-secretion after an IRES element limits toxicities [30,43]. Gene transfer of each construct into murine T cells was equivalent (Supplementary Fig. S3A). mCD19tmIL12 and mCD19tmIL18 pmel-1 T cells secreted mIL12 and mIL18, respectively (Fig. 1B).

Figure 1. IL18 and IL12 enhance effector function of tumor-specific T cells in vitro.

Figure 1.

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(A) Schematic of retroviral constructs. (B) Secretion of mIL12 or mIL18 from transduced pmel-1 T cells (n=6, ****p<0.0001, ***p<0.001 by unpaired t tests). Data is mean +/− s.e.m of three independent experiments. (C) IFNγ secretion from IL18 or IL12-secreting pmel-1 T cells ( n=3, **p<0.01 by one-way ANOVA). Data is mean +/− standard deviation (SD) from three independent experiments. (D) Fold change in CD25 expression, determined by flow cytometry, of armored pmel-1 T cells compared to control following coculture with B16F10 tumor cells. (n=3, *p<0.05 by one-way ANOVA). Data shown is mean +/− SD from three independent experiments. (E) In vitro cytotoxicity of armored pmel-1 T cells against B16F10 GFP/luc (gp100+; CD80/86–) tumor cells ( n=3, ****p<0.0001, ***p<0.001, and *p<0.05 by one-way ANOVA). Data is mean +/− s.e.m. of three independent experiments.

We next investigated activation and effector function of cytokine-armored pmel-1 T cells through analysis of interferon-γ (IFNγ) secretion, expression of T-cell activation marker CD25, and cytolytic capacity of the transduced T cells after coculture with B16F10 murine melanoma cells. B16F10 cells were negative for costimulatory molecules CD80/CD86 (Supplementary Fig. S3B). Following coculture with B16F10 tumor cells, mIL12 and mIL18-armored pmel-1 T cells displayed enhanced secretion of IFNγ (Fig. 1C) and expression of CD25 (Fig. 1D). mIL12 secretion improved the in vitro cytolytic capacity of pmel-1 T cells against B16F10 tumor cells modified to express GFP/luciferase (B16F10 GFP/luc) relative to mIL18-secreting pmel-1 T cells and control pmel-1 T cells modified with mCD19t alone (Fig. 1E). mIL18-secreting pmel-1 T cells also enhanced in vitro antitumor lysis compared to control pmel-1 T cells (Fig. 1E). These results indicate that both mIL12 and mIL18 augment pmel-1 T-cell activation and effector function in vitro.

T cells secreting IL18 or IL12 modulate the tumor microenvironment and enhance survival

C57BL6 mice were inoculated subcutaneously with B16F10 tumor cells and treated weekly for three total infusions with either control or armored pmel-1 T cells to determine T-cell antitumor responses (Fig. 2A). Mice treated with mIL12 or mIL18-armored pmel-1 T cells had a significant delay in tumor growth in comparison to control pmel-1 T cell treated mice as assessed by tumor volume (Fig. 2B). Mice treated with mIL18-armored pmel-1 T cells were also able to delay tumor growth when compared to mIL12-secreting pmel-1 T cell–treated mice (Fig. 2B). mIL18-armored pmel-1 T cells enhanced overall survival of tumor-bearing mice without preconditioning therapy or exogenous cytokine treatments when compared to mice treated with either mIL12-armored or control pmel-1 T cells (Fig. 2C). The enhanced antitumor effect of the IL18-armored pmel-1 T cells was also evident in tumor bearing mice treated with a single infusion of pmel-1 T cells (Supplementary Fig. S4A). Tumor samples were isolated from each cohort of treated mice six days after a single T cell infusion for flow cytometric tumor microenvironment analysis. Although there was no difference in the total number of macrophages in the tumor samples among treatment groups (Supplementary Fig. S4B), tumors from mice that received either mIL18 or mIL12-secreting pmel-1 T cells had fewer immunosuppressive CD206+ M2 macrophages (Fig. 2D, Supplementary Fig. S4C). Only the mice treated with mIL18-secreting armored pmel-1 T cells had significantly fewer CD11b+, Ly6C+ myeloid-derived suppressor cells (MDSCs) in the tumor samples (Fig. 2E, Supplementary Fig. S4C). There was also a significant accumulation of activated MHCII+ dendritic cells in the tumors of mice treated with mIL18-armored pmel-1 T cells in comparison to the mIL12-armored or control pmel-1 T-cell groups (Fig. 2F, Supplementary Fig. S4C). The decrease in suppressive immune populations within the tumor microenvironment of mIL18-secreting pmel-1 T cells was associated with an enhanced accumulation of pmel-1 T cells (Fig. 2G, Supplementary Fig. S4C) and suggests an explanation for mIL18-armored pmel-1 T cell mediated control of B16F10 tumors.

Figure 2. Adoptively transferred IL18 and IL12-secreting T cells modulate the tumor microenvironment to enhance in vivo survival without preconditioning.

Figure 2.

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(A) In vivo experimental protocol. (B) Tumor regression and (C) survival of C57BL/6 mice bearing established B16F10 tumors and treated with armored or control pmel-1 T cells (n=15–17, ****p<0.0001 and **p<0.01). P-values for tumor growth determined by Mann Whitney test and survival by log-rank Mantel-Cox Test, with 95% confidence interval. Data are from three independent experiments. Phenotypic analysis of (D) CD206+, Gr1-, F4/80+ M2 macrophages, (E) CD11b+, Ly6C+ MDSCs, (F) CD11c+, MHC II+ dendritic cells, and (G) Thy1.1+ pmel-1 T cells from tumor samples of mice six days after T cell treatment (n=4–8, **p<0.01 and *p<0.05). Data is mean +/− s.e.m from two independent experiments and p-values determined by one-way ANOVA. (H) Tumor regression and (I) survival of CD80/CD86–/– and C57BL/6 hosts bearing B16F10 tumors and treated with armored or control pmel-1 T cells (n=4–8, ***p<0.001, **p<0.01, and *p<0.05). P-values for tumor growth determined by Mann Whitney test and survival by log-rank Mantel-Cox Test, with 95% confidence interval. Data shown are from two independent experiments.

To determine the dependency of the enhanced anti-tumor T cell response upon endogenous CD28 costimulation, we tested the same tumor model in CD80/CD86–/– host mice. Non-preconditioned, tumor-bearing CD80/CD86–/– mice treated with mIL18-armored pmel-1 T cells had a slower rate of tumor growth (Fig. 2H) and significantly enhanced survival (Fig. 2I) in comparison to the mIL12-armored and the control pmel-1 T cells. There was no difference in tumor growth or survival between wildtype C57BL6 or CD80/CD86–/– tumor-bearing host mice treated with mIL18-secreting pmel-1 T cells. Thus, IL18 enhanced the efficacy of T-cell therapy in a syngeneic in vivo system without preconditioning therapy and irrespective of CD28 T-cell costimulation.

IL18 supports tumor-specific T cells whereas IL12 drives T cells into dysfunction

We next investigated activation and ex vivo functionality of tumor-specific T cells. Thy1.1- host mice were treated with adoptively transferred Thy1.1+ pmel-1 T cells and tumors were dissected 6 days and 13 days post T-cell infusion (Fig. 3A). On day 6 we observed a significant increase in the ratio of Thy1.1+ pmel-1 to Thy1.1- endogenous T cells only in the mCD19tmIL18 treatment group, indicating an accumulation of mIL18-secreting pmel-1 T cells in the tumor not observed in the mice that were treated with mIL12 or the control pmel-1 T cells (Fig. 3B). There was no difference, however, in naïve/effector/central memory T-cell phenotype among the three treatment groups at the time of infusion or on days 6 or 13 after treatment (Supplementary Fig. S5A). There was no difference in the percentage of PD-1+, Thy1.1+ pmel-1 T cells, illustrating activation of both armored and control pmel-1 T cells (Fig. 3C, Supplementary Fig. S5B). On day 6 after infusion, tumor-infiltrating mIL18-armored pmel-1 T cells had a significantly lower percentage of cells that were triple-positive for the inhibitory markers PD-1, TIM-3, and LAG-3 than mIL12-armored pmel-1 T cells (Fig. 3D, Supplementary Fig. S5B). A triple positive phenotype is a marker of T-cell dysfunction [44]. Next we analyzed the CD8+ effector to CD4+ helper T-cell ratio in the tumors of mice six or thirteen days after T cell treatment. On day 6, there was no difference between the ratio of CD8+ to CD4+ T cells in the tumors of mIL12 and mIL18-secreting pmel-1 T cells. On day 13, the ratio of CD8+ to CD4+ T cells was higher in tumors of mIL18 pmel-1 T cell–treated mice (Fig. 3E, Supplementary Fig. S5C). Thus, mIL18-secreting CD8+ pmel-1 T cells retain a functional phenotype and persist at the tumor site after infusion. Conversely, mIL12-secreting pmel-1 T cells develop a dysfunctional phenotype in vivo when compared to control or mIL18-armored pmel-1 T cells.

Figure 3. IL18-secreting T cells functionally persist in vivo whereas IL12-secreting T cells become dysfunctional.

Figure 3.

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(A) Schematic of experimental setup. (B) Ratio of adoptively transferred pmel-1 Thy1.1+, CD8+ T cells to endogenous Thy1.1-, CD8+ T cells 6 days after T-cell infusion (n=15, ****p<0.0001 by one-way ANOVA). Phenotypic analysis of adoptively transferred pmel-1 T cells 6 days after infusion that were (C) single positive for PD-1 as a marker of activation or (D) triple positive for exhaustion markers, PD-1, LAG-3, and TIM-3 (n=15, ****p<0.0001 and ***p<0.001 by one-way ANOVA). (E) Ratio of CD8+ to CD4+ T cells in the tumors of mice 6 or 13 days after treatment with armored or control pmel-1 T cells (n=8–15, ****p<0.0001, **p<0.01, *p<0.05 by two-way ANOVA). Flow cytometry data from three independent experiments and shown mean +/− SD. Ex vivo IFNγ production of pmel-1 T cells sorted from tumor samples (F) six or (G) thirteen days after treatment with armored or control pmel-1 T cells (n= 4–10, **p<0.01 and *p<0.05 by unpaired t test). (H) Ex vivo cytotoxicity of pmel-1 T cells sorted from tumor samples of mice six or thirteen days after treatment with armored or control pmel-1 T cells (n=7–10, ****p<0.0001, **p<0.01, and *p<0.05 by two-way ANOVA). Ex vivo data obtained from two independent experiments and shown mean +/− s.e.m.

We analyzed the function of tumor-infiltrating pmel-1 T cells on days 6 and 13 by assessing the cytokine production and cytolytic capacity of sorted Thy1.1+ T cells ex vivo. On day 6 after T-cell therapy, both the mIL12 and mIL18 transduced pmel-1 T cells secreted IFNγ in comparison to the control pmel-1 T cells (Fig. 3F). On day 13, only the mIL18-secreting pmel-1 T cells were able to produce more IFNγ than the control or the mIL12-secreting pmel-1 T cells (Fig. 3G). Both mIL12 and mIL18 pmel-1 T cells were able to lyse B16F10 GFP/Luc tumor cells on day 6, but only the mIL18 pmel-1 T cells maintained this ability on day 13 (Fig. 3H, Supplementary Fig. S5D). These data align with the phenotypic results described in Figs. 3B–E and demonstrate that mIL18-secreting pmel-1 T cells functionally persist in the tumor longer than the mIL12-secreting pmel-1 T cells, which become dysfunctional.

IL18-secreting T cells plus sublethal irradiation eradicated established tumors

Although IL18 provides an activating signal and enhances function, persistence, and antitumor efficacy of tumor specific T cells, long-term survival of mice treated with mIL18-secreting pmel-1 T cells was modest. We hypothesized that combination with sublethal irradiation would have an additive effect and increase survival of tumor-bearing mice due to removal of suppressive cell populations. Sublethally irradiated B16F10 tumor-bearing mice received a single armored or control pmel-1 T-cell injection (Fig. 4A). All T-cell groups displayed equivalent mean fluorescence intensity of mCD19t prior to infusion, demonstrating similar vector expression (Supplementary Fig. S6A). The combination of sublethal irradiation and armored mIL18-secreting pmel-1 T cells regressed the tumors, which were eradicated completely in 8/9 mice (88.8%) (Fig. 4B). When sublethal irradiation of tumor-bearing mice was accompanied with three weekly injections of pmel-1 T cells, mIL18-secreting pmel-1 T cells eradicated established subcutaneous tumors (Supplementary Fig. S6B–D). In contrast, mice treated with mIL12-secreting pmel-1 T cells in combination with sublethal irradiation succumbed abruptly to toxicity even with mIL12 placed after an IRES element, a strategy meant to limit toxicity [29] (Fig. 4B). Thus, only mice sublethally irradiated and treated with mIL18-secreting pmel-1 T cells survived long term (Fig. 4C).

Figure 4. IL18-secreting pmel-1 T cells eradicate established tumors in sublethally irradiated tumor-bearing mice.

Figure 4.

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(A) In vivo experimental protocol. (B) Tumor regression and (C) survival of C57BL/6 mice bearing established B16F10 tumors and treated with armored or control pmel-1 T cells (n=9, ****p<0.0001 and **p<0.01). (D) Serum cytokine analysis 7 days after T cell infusion (n=8–9, ****p<0.0001, **p<0.001, and *p<0.05). Data shown is mean +/− SD from two independent experiments. Flow cytometry counts of (E) CD25+, FOXP3+ regulatory T cells, (F) CD11b+, Ly6G+ granulocytic MDSCs, and (G) Thy1.1+ T cells in tumors of both unconditioned mice (UC) and sublethally irradiated mice (IRR) 6 days after T-cell infusion (n=4–6, ****p<0.0001, **p<0.01, *p<0.05). Data shown is mean +/− s.e.m from two independent experiments. P-values for tumor growth determined by Mann Whitney test, survival determined by log-rank Mantel-Cox Test with 95% confidence interval, and cytokine and dissection data determined by one-way ANOVA.

To confirm that we were observing IL12 toxicity as previously described [25], we analyzed serum cytokines via retro-orbital bleeds from the sublethally irradiated, tumor-bearing mice one week after treatment with the armored or control pmel-1 T cells. The sublethally irradiated, tumor-bearing mice treated with mIL12-secreting pmel-1 T cells had significantly higher amounts of IL12, IFNγ, TNFα, IL6, and IL10 in serum in comparison to those mice treated with control mCD19t or mIL18-secreting pmel-1 T cells (Fig. 4D). Based upon this cytokine profile, we determined that these mice succumbed to toxicity related to IL12 administration. Taking into consideration both the phenotypic data suggesting that IL18 remains functional for a longer duration as well as the toxicity that developed in sublethally irradiated mice treated with IL12-secreting pmel-1 T cells, we focused further investigation on IL18-secreting T cells.

The enhanced antitumor effect from the combination of pmel-1 T cells with sublethal irradiation was hypothesized to be due to a decrease in suppressive immune cell populations. When compared to the tumors of unconditioned host mice, both CD25+, FOXP3+ regulatory T cells (Tregs) and CD11b+, Ly6G+ granulocytic MDSCs (gMDSCs) in the tumors of sublethally irradiated host mice treated with either control pmel-1 T cells or mIL18-secreting pmel-1 T cells were a significantly decreased (Fig. 4E, 4F, Supplementary Fig. S6E). Although changes in tumor microenvironment were similar in control and mIL18-secreting pmel-1 T cells, a greater number of Thy1.1+ pmel-1 T cells were detected within the tumors of unconditioned and sublethally irradiated mIL18 pmel-1 T cell–treated mice in comparison to control pmel-1 T cell–treated mice (Fig. 4G, Supplementary Fig. S6E). The elimination of suppressive immune populations in the tumors of the mIL18 pmel-1 T cell–treated mice with and without sublethal irradiation allowed for enhanced engraftment and persistence of pmel-1 T cells.

IL18-secreting pmel-1 T cells act in cis to enhance survival of tumor-bearing mice

Given the antitumor efficacy of mIL18-secreting pmel-1 T cells combined with sublethal irradiation, we postulated that IL18 was acting in cis on adoptively transferred T cells to mediate an immune response. We generated pmel+/+, IL18R–/– mice as a source of donor T cells by crossing pmel-1 and IL18R–/– mice (Fig. 5A). Next, we confirmed via flow cytometry that there was no IL18 receptor present on the naïve pmel+/+, IL18R–/– donor T cells, even after activation with supernatant from IL12-secreting pmel-1 T cells, which induces IL18 receptor expression (Fig. 5B). The lack of IL18R did not affect the retroviral transduction efficiency of either the control mCD19t or armored mCD19tmIL18 retroviral constructs in both pmel-1 and pmel+/+, IL18R–/– T cells (Fig. 5C). mIL18-secretion improved the in vitro cytolytic capacity of only pmel-1 T cells against B16F10 GFP/luc tumor cells relative to control pmel-1 T cells as well as pmel+/+, IL18R–/– T cells modified with either mIL18 or mCD19t (Supplementary Fig. S7A). This result demonstrated the cis effect of IL18 in augmenting in vitro T-cell antitumor efficacy.

Figure 5. IL18-secreting pmel-1 T cells act in cis to enhance in vivo survival of tumor-bearing mice.

Figure 5.

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(A) Schematic of breeding to generate donor pmel+/+, IL18R–/– mice. (B) Naïve C57BL6, pmel, and pmel+/+, IL18R–/– T cells were cultured with supernatant from either control mCD19t or mCD19tmIL12 pmel-1 T cells. Flow cytometry was used to detect the expression of IL18 receptor on splenocytes. Representative figure of three independent experiments. (C) Retroviral transduction efficiency by CD19 expression among the experimental groups utilizing either the pmel or pmel+/+, IL18R–/– T cells (n=6). (D) In vivo experimental protocol. (E) Tumor regression and (F) survival of C57BL/6 or IL18R–/– host mice bearing established B16F10 tumors and treated with armored or control pmel-1 T cells (n=6, ***p<0.001 and **p<0.01). P-values for tumor growth determined by Mann Whitney test and survival by log-rank Mantel-Cox Test, with 95% confidence interval. Data shown are from two independent experiments.

We asked whether the improved in vivo survival of tumor-bearing mice and enhanced antitumor function of mIL18-secreting pmel-1 T cells was dependent on donor T cell or host IL18R expression. In the B16F10 tumor model, both pmel-1 and pmel+/+, IL18R–/– transduced donor T cells were used to treat C57BL6 or IL18R–/– tumor bearing host mice (Fig. 5D). mIL18-secreting pmel-1 donor T cells delayed tumor progression and enhanced overall survival in both C57BL6 and IL18R–/– tumor-bearing mice (Fig. 5E–F). In contrast, the mIL18-secreting pmel+/+, IL18R–/– donor T cells did not cause tumorregression or prolong survival, similarly to control pmel and pmel+/+, IL18R–/– treated mice (Fig. 5E–F and Supplementary Fig. S7B–C). Antitumor activity is improved regardless of whether IL18R is expressed on the endogenous host immune cells when the mIL18-secreting donor T cell has an intact IL18 receptor. Thus, cis interactions between the secreted IL18 and the adoptively transferred T cells are necessary for enhancing antitumor activity.

Human T cells secreting IL18 enhance survival in a xenograft melanoma model

To demonstrate translational relevance of armoring TCR T cells with IL18, we transduced HLA-A2+ human T cells to express a NY-ESO-1–specific TCR, 1G4, or both the TCR and a mature, bioactive version of human IL18 (hIL18), 1G4hIL18 (Fig. 6A). 1G4 and 1G4hIL18 T cells had similar retroviral transduction efficiency (Supplementary Fig. S8A). 1G4hIL18 T cells significantly enhanced secretion of hIL18 in contrast to mock transduced and 1G4 T cells (Fig. 6B). After coculture with A375 (HLA-A2+, NY-ESO-1+) human melanoma cells, both 1G4 and 1G4hIL18 T cells showed enhanced secretion of IFNγ and IL2, demonstrating activation of the transduced T cells (Fig. 6C). Both 1G4 and 1G4hIL18 T cells enhanced cell lysis in comparison to mock transduced T cells when cocultured with A375 tumor cells but not against SK-Mel5 (HLA-A2+, NY-ESO-1-) tumor cells (Fig. 6D). To confirm this interaction in vivo, we utilized an A375 xenograft model of human melanoma and treated with transduced HLA-A2+ human T cells (Fig. 6E). A375 tumor cells lacked both CD80 and CD86 costimulatory molecules (Supplementary Fig. S8B). Seven days after T-cell infusion, more 1G4hIL18 T cells were present in peripheral blood of the tumor bearing mice in comparison to both the 1G4 or mock T cell–treated mice (Fig. 6F). 1G4hIL18 T cells delayed tumor progression in comparison to 1G4 and mock transduced T cells (Fig. 6G). Both 1G4 and 1G4hIL18 T cells enhanced the survival of tumor bearing mice (Fig. 6H), although the effect was stronger with 1G4hIL18 T cells, showing that IL18 provides a pro-inflammatory signal to activate human T cells and augment antitumor efficacy through increased persistence of TCR-modified T cells.

Figure 6. Human T cells secreting IL18 persist and enhance survival in a xenograft melanoma model.

Figure 6.

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(A) Schematic of retroviral constructs. (B) Luminex results demonstrating secretion of hIL18 by 1G4hIL18 T cells (n=9, ***p<0.0001, **p<0.001). (C) Luminex data demonstrating IFNγ and IL2 secretion from 1G4 and 1G4hIL18 (n=9, ****p<0.0001, ***p<0.0001, and *p<0.05). Luminex data shown as mean +/− s.e.m. of three independent experiments and p-values determined by one-way ANOVA. (D) A luciferase killing assay was conducted with 1G4, 1G4hIL18, and mock transduced human T cells against both A375 (HLA-A2+, NY-ESO-1+, luciferase+) and SK-Mel5 (HLA-A2+, NY-ESO-1-, luciferase+) tumor cells (n=9–15, ****p<0.0001 by two-way ANOVA). Data shown is mean +/− s.e.m of five independent experiments for A375 and three independent experiments for SK-Mel5 luciferase assay. (E) In vivo experimental protocol. (F) 1G4+ T-cell numbers in circulation on day 7 after T-cell infusion (n=5–10, ****p<0.0001,***p<0.001 by two-way ANOVA). (G) Tumor regression and (H) surival of NSG host mice bearing established A375 tumors and treated with armored or control 1G4 human T cells (n=10–15, ****p<0.0001 and *p<0.05). In vivo data shown from four independent experiments. P values for tumor growth determined by Mann Whitney test and survival by log-rank Mantel-Cox Test, with 95% confidence interval.

Discussion

Adoptive cellular therapy with TCR-modified T cells promises to expand the range of targetable tumor associated antigens, although this strategy has shown only modest success [12–17]. Because treatment failure correlates with a lack of T-cell activation, new methods to stimulate tumor-targeted T cells are necessary. In this study, we demonstrated that IL18 provides a pro-inflammatory signal to T cells. IL18-engineered TCR-modified T cells promote an enhanced and persistent effector T-cell response by augmenting in vitro activation, cytokine release, and tumor lysis of tumor-directed T cells. IL18 promotes a durable activating signal to tumor-specific T cells in both syngeneic and xenograft tumor models. TCR-modified T cells secreting IL18 exhibited enhanced persistence and mediated a delay in tumor growth corresponding to long-term survival of tumor-bearing mice in the absence of any preconditioning therapy by acting in cis to enhance the effector function of tumor-specific T cells and promote an immunostimulatory tumor microenvironment.

The survival benefit of mIL18-secreting pmel-1 T cells may be due to the reduction of repressive CD206+ M2 macrophages and MDSCs. The immune microenvironment was conducive to the accumulation of activated dendritic cells and expansion of tumor-specific pmel-1 T cells. This result mirrors that of IL18-secreting CAR T cells which promote a pro-inflammatory microenvironment with M1 macrophages and activated dendritic cells [31]. While IL18 secretion generates a stimulatory tumor microenvironment, the main contribution to the enhanced efficacy of the IL18-secreting TCR-modified T cells is conferred by IL18 acting in cis on adoptively transferred T cells. Tumor-specific pmel-1 T cells armored with mIL18 functionally persist at the tumor site longer than mIL12-armored pmel-1 T cells that become dysfunctional. Ex vivo cytokine secretion and cytolytic assays illustrate that mIL18-secreting pmel-1 T cells remain activated and retain effector function for at least a week longer than mIL12-secreting pmel-1 T cells.

Utilizing pmel+/+, IL18R–/– donor mice, we established that expression of IL18R on adoptively transferred T cells supports IL18-secreting pmel-1 T cells, regardless of IL18R expression on the endogenous host immune cells. This confirms our hypothesis that the cis interactions between the secreted IL18 and the adoptively transferred T cells are essential for enhancing antitumor activity. Although we believe that the cis effect of IL18 on the IL18R on adoptively transferred T cells confers the primary antitumor effect, effector cytokines upregulated in the periphery such as IL12, IFNγ, and TNFα may play a role in decreasing the suppressive M2 macrophage and MDSC populations. IL12 modulates the murine tumor microenvironment by reducing the number of MDSCs and suppressive macrophages at the tumor site [45]. Both IFNγ and TNFα pro-inflammatory cytokines polarize macrophages into a M1 activated phenotype [46–48]. From our studies, we have shown that IL18-secreting murine T cells increase the amount of IFNγ and TNFα in the periphery, which could be a mechanism by which IL18-secreting T cells polarize macrophages into M1 activated rather than a M2 suppressive phenotype. Any indirect mechanism of suppressive cell depletion may be complementary to the cis effect of IL18 on the IL18R on adoptively transferred T cells. The combination of in vitro, in vivo, and ex vivo data demonstrate that secretion of IL18 modulates the tumor microenvironment and provides a more durable pro-inflammatory signal than IL12 to enhance the antitumor effector function of tumor-specific T cells.

These results with IL18-armored pmel-1 T cells in unconditioned tumor-bearing mice are in contrast to those from other groups which observed modest efficacy of inducible IL18-secreting pmel-1 T cells following multiple methods of chemotherapeutic preconditioning [49] or enhanced toxicity of IL12-armored tumor specific pmel-1 T cells unless they are under the control of an inducible promoter [33,49–51]. Specifically, a study by Kunert et al., established that T cells transduced with a gp100 specific TCR in combination with an inducible IL18 gene led to antitumor responses in over half of the mice treated when provided in the context of both bisulfan and cyclophosphamide preconditioning regimens [49]. In our studies, pmel-1 T cells transduced to constitutively secrete IL18 combined with sublethal irradiation alone eradicated established tumors in a syngeneic tumor model. This antitumor response was correlated with tumor microenvironment modulation through the ablation of repressive MDSCs and regulatory T cells as well as enhanced engraftment and persistence of tumor-specific T cells within the tumor. In this manner, sublethal irradiation generated a tumor microenvironment devoid of immunosuppressive populations. This suppressive immune cell ablation may augment the stimulatory cis interactions of IL18-secreting T cells, leading to this antitumor response. Toxicity was not associated with the IL12-secreting pmel-1 T cells in immune-competent mice. However, after sublethal irradiation pre-conditioning, we observed fatal toxicity to mice in the form of elevated serum cytokine amounts. Increased IFNγ and IL6 has been attributed to cytokine release syndrome toxicities in patients [52] and may have been the effector cytokines that caused the death of the sublethally irradiated mice treated with IL12-secreting pmel-1 T cells.

The syngeneic tumor model data show that TCR-modified T-cell antitumor efficacy can be optimized safely through armoring with IL18 rather than with IL12 and establish IL18 as a superior and safer pro-inflammatory signal to tumor-directed T cells. These syngeneic studies were used as a proof-of-principle to engineer IL18-armored TCR-modified T cells in a clinically relevant human TCR system. Human T cells were transduced with an anti-NY-ESO-1 TCR. They were then validated and ultimately armored with human IL18. 1G4hIL18 TCR T cells were further shown to persist and enhance antitumor efficacy resulting in long-term survival of tumor-bearing mice in a xenograft model. The NY-ESO-1 directed TCR has been tested in various clinical trials with only modest success [14,53,54]. Our results provide a rationale for the clinical application of IL18-armored TCR-modified T cells to enhance the efficacy and potency of TCR-modified T-cell therapies.

Supplementary Material

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Acknowledgements

We thank A. Rookard, B. Qeriqi, and C. Bebernitz for technical assistance with in vivo experiments; the MSKCC Flow Cytometry Core; J.E. Jaspers, E.L. Smith, and C. Hackett for critically reading the manuscript; and the Klebanoff, Scheinberg, and Wolchok labs for reagents. Supported by US NIH grants 5 P01 CA190174-03 (R.J.B.), 5 P50 CA192937-02 (R.J.B.), and T32 GM073546-11 (D.J.D.), The Annual Terry Fox Run for Cancer Research (R.J.B.), Kate’s Team (R.J.B.), Carson Family Charitable Trust (R.J.B.), Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research (R.J.B. and C.A.K.); the Experimental Therapeutics Center of MSKCC (R.J.B., S.S.C., and C.A.K.). S.S.C. and C.A.K. are also supported by the Parker Institute for Cancer Immunotherapy, the Damon Runyon Cancer Research Foundation, NCI/NIH RFA-CA-18-003 R33, the Breast Cancer Alliance, The Manhasset Women’s Coalition Against Breast Cancer, and the MSKCC Core grant P30 CA008748.

Footnotes

Conflict-of-interest Statement: R.J.B. is a cofounder and receives royalties from Juno Therapeutics/Celgene and has submitted a patent related to this work.

References

  • [1].Huse M, The T-cell-receptor signaling network, J. Cell Sci, 2009. [DOI] [PubMed] [Google Scholar]
  • [2].Chen L and Flies DB, Molecular mechanisms of T cell co-stimulation and co-inhibition, Nature Reviews Immunology. 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [3].Schwartz RH, Models of T cell anergy: is there a common molecular mechanism?, J. Exp. Med, 2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].Mueller DL and Jenkins MK, Molecular mechanisms underlying functional T-cell unresponsiveness, Curr. Opin. Immunol, 1995. [DOI] [PubMed] [Google Scholar]
  • [5].Driessens G, Kline J, and Gajewski TF, Costimulatory and coinhibitory receptors in anti-tumor immunity, Immunological Reviews. 2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Valenzuela J, Schmidt C, and Mescher M, The roles of IL-12 in providing a third signal for clonal expansion of naive CD8 T cells., J. Immunol, vol. 169, no. 12, pp. 6842–6849, 2002. [DOI] [PubMed] [Google Scholar]
  • [7].Schmidt CS and Mescher MF, Peptide antigen priming of naive, but not memory, CD8 T cells requires a third signal that can be provided by IL-12., J. Immunol, vol. 168, no. 11, pp. 5521–5529, 2002. [DOI] [PubMed] [Google Scholar]
  • [8].Curtsinger JM et al. , Inflammatory cytokines provide a third signal for activation of naive CD4+ and CD8+ T cells., J. Immunol, vol. 162, no. 6, pp. 3256–62, 1999. [PubMed] [Google Scholar]
  • [9].Curtsinger JM and Mescher MF, Inflammatory cytokines as a third signal for T cell activation., Curr. Opin. Immunol, vol. 22, no. 3, pp. 333–40, 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Curtsinger JM, Gerner MY, Lins DC, and Mescher MF, signal 3 Availability Limits the CD8 T Cell Response to a Solid Tumor, J. Immunol, 2007. [DOI] [PubMed] [Google Scholar]
  • [11].Chandran SS and Klebanoff CA, T cell receptor‐based cancer immunotherapy: Emerging efficacy and pathways of resistance, Immunol. Rev, 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Morgan RA et al. , Cancer regression and neurologic toxicity following anti-MAGE- A3 TCR gene therapy, J. Immunother, vol. 36, no. 2, pp. 133–151, 2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Morgan RA et al. , Cancer regression in patients after transfer of genetically engineered lymphocytes., Science, vol. 314, no. 5796, pp. 126–129, 2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Phan GQ and Rosenberg S. a., Adoptive cell transfer for patients with metastatic melanoma: The potential and promise of cancer immunotherapy, Cancer Control, vol. 20, no. 4, pp. 289–297, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Zhang J and Wang L, The emerging world of TCR-T cell trials against cancer: A systematic review, Technology in Cancer Research and Treatment. 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Isao T et al. , Adoptive Transfer of WT1-Specific TCR Gene-Transduced Lymphocytes in Patients with Myelodysplastic Syndrome and Acute Myeloid Leukemia, Blood, 2015. [Google Scholar]
  • [17].Rohaan MW, van den Berg JH, Kvistborg P, and Haanen JBAG, Adoptive transfer of tumor-infiltrating lymphocytes in melanoma: a viable treatment option, J. Immunother. Cancer, 2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Stevanovic S et al. , A Phase II Study of Tumor-infiltrating Lymphocyte Therapy for Human Papillomavirus–associated Epithelial Cancers, Clin. Cancer Res, 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Stauss HJ et al. , WT1-specific T cell receptor gene therapy: Improving TCR function in transduced T cells, Blood Cells, Mol. Dis, vol. 40, no. 1, pp. 113–116, 2008. [DOI] [PubMed] [Google Scholar]
  • [20].Linette GP et al. , Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma, Blood, vol. 122, no. 6, pp. 863–871, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].de Charette M, Marabelle A, and Houot R, Turning tumour cells into antigen presenting cells: The next step to improve cancer immunotherapy?, European Journal of Cancer. 2016. [DOI] [PubMed] [Google Scholar]
  • [22].Seliger B, Maeurer MJ, and Ferrone S, Antigen-processing machinery breakdown and tumor growth, Immunology Today. 2000. [DOI] [PubMed] [Google Scholar]
  • [23].Brentjens RJ et al. , Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15, Nat. Med, vol. 9, no. 3, pp. 279–286, March 2003. [DOI] [PubMed] [Google Scholar]
  • [24].Brentjens RJ et al. , Genetically Targeted T Cells Eradicate Systemic Acute Lymphoblastic Leukemia Xenografts, Clin. Cancer Res, vol. 13, no. 18, pp. 5426–5435, September 2007. [DOI] [PubMed] [Google Scholar]
  • [25].Leonard JP et al. , Effects of single-dose interleukin-12 exposure on interleukin-12 associated toxicity and interferon-γ production, Blood, 1997. [PubMed] [Google Scholar]
  • [26].Tarhini AA et al. , A phase 2, randomized study of SB-485232, rhIL-18, in patients with previously untreated metastatic melanoma, Cancer, 2009. [DOI] [PubMed] [Google Scholar]
  • [27].Dinarello CA, Novick D, Kim S, and Kaplanski G, Interleukin-18 and IL-18 binding protein, Frontiers in Immunology. 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Wu C et al. , IL-18 Receptor β-Induced Changes in the Presentation of IL-18 Binding Sites Affect Ligand Binding and Signal Transduction, J. Immunol, 2003. [DOI] [PubMed] [Google Scholar]
  • [29].Pegram HJ et al. , Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning, Blood, vol. 119, no. 18, pp. 4133–4141, 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [30].Yeku OO, Purdon TJ, Koneru M, Spriggs D, and Brentjens RJ, Armored CAR T cells enhance antitumor efficacy and overcome the tumor microenvironment, Sci. Rep, vol. 7, no. 1, pp. 1–14, 2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Avanzi MP et al. , Engineered Tumor-Targeted T Cells Mediate Enhanced Anti-Tumor Efficacy Both Directly and through Activation of the Endogenous Immune System, Cell Rep, 2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Kerkar SP et al. , Tumor-specific CD8+ T cells expressing interleukin-12 eradicate established cancers in lymphodepleted hosts, Cancer Res, 2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [33].Zhang L et al. , Improving Adoptive T Cell Therapy by Targeting and Controlling IL-12 Expression to the Tumor Environment, Mol. Ther, vol. 19, no. 4, pp. 751–759, 2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Overwijk WW et al. , Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells, J. Exp. Med, 2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [35].Rivière I, Brose K, and Mulligan RC, Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells, Proc. Natl. Acad. Sci. U. S. A, 1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [36].Ferrone CR et al. , Adjuvanticity of plasmid DNA encoding cytokines fused to immunoglobulin Fc domains, Clin. Cancer Res, vol. 12, no. 18, pp. 5511–5519, 2006. [DOI] [PubMed] [Google Scholar]
  • [37].Wargo JA et al. , Recognition of NY-ESO-1+ tumor cells by engineered lymphocytes is enhanced by improved vector design and epigenetic modulation of tumor antigen expression, Cancer Immunol. Immunother, 2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [38].Cohen CJ, Enhanced Antitumor Activity of Murine-Human Hybrid T-Cell Receptor (TCR) in Human Lymphocytes Is Associated with Improved Pairing and TCR/CD3 Stability, Cancer Res, vol. 66, no. 17, pp. 8878–8886, 2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [39].Cohen CJ, Li YF, El-Gamil M, Robbins PF, a Rosenberg S, and Morgan RA, Enhanced antitumor activity of T cells engineered to express T-cell receptors with a second disulfide bond., Cancer Res, vol. 67, no. 8, pp. 3898–3903, 2007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].Lee J, Sadelain M, and Brentjens R, Genetic Modification of Hematopoietic Stem Cells, vol. 506, pp. 83–96, 2009. [Google Scholar]
  • [41].Rafiq S et al. , Optimized T-cell receptor-mimic chimeric antigen receptor T cells directed toward the intracellular Wilms Tumor 1 antigen., Leukemia, 2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [42].Smith EL et al. , Development and Evaluation of an Optimal Human Single-Chain Variable Fragment-Derived BCMA-Targeted CAR T Cell Vector, Mol. Ther, vol. 26, no. 6, pp. 1447–1456, 2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [43].Pegram HJ et al. , Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning, Blood, 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [44].Gros A et al. , PD-1 identifies the patient-specific CD8+ tumor-reactive repertoire infiltrating human tumors, J. Clin. Invest, 2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [45].Kerkar SP et al. , Tumor-specific CD8+ T cells expressing IL-12 eradicate established cancers in lymphodepleted hosts, vol. 70, no. 17, pp. 6725–6734, 2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [46].Martinez FO and Gordon S, The M1 and M2 paradigm of macrophage activation: Time for reassessment, F1000Prime Rep, 2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [47].Gordon S and Martinez FO, Alternative activation of macrophages: Mechanism and functions, Immunity. 2010. [DOI] [PubMed] [Google Scholar]
  • [48].Stein M, Keshav S, Harris N, and Gordon S, Interleukin 4 potently enhances murine macrophage mannose receptor activity: A marker of alternative immunologic macrophage activation, J. Exp. Med, 1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [49].Kunert A, Chmielewski M, Wijers R, Berrevoets C, Abken H, and Debets R, Intra-tumoral production of IL18, but not IL12, by TCR-engineered T cells is non-toxic and counteracts immune evasion of solid tumors, Oncoimmunology, 2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [50].Yamamoto TN et al. , T cells genetically engineered to overcome death signaling enhance adoptive cancer immunotherapy, J. Clin. Invest, 2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [51].Restifo NP, Dudley ME, and Rosenberg S. a., Adoptive immunotherapy for cancer: harnessing the T cell response, Nat. Rev. Immunol, vol. 12, no. 4, pp. 269–281, 2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [52].Grupp SA et al. , Chimeric antigen receptor-modified T cells for acute lymphoid leukemia, N. Engl. J. Med, 2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [53].Thomas R et al. , NY-ESO-1 based immunotherapy of cancer: Current perspectives, Frontiers in Immunology. 2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [54].D’angelo SP et al. , Antitumor activity associated with prolonged persistence of adoptively transferred NY-ESO-1c259T cells in synovial sarcoma, Cancer Discov, 2018. [DOI] [PMC free article] [PubMed] [Google Scholar]

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