Chimeric antigen receptor (CAR) T-cell therapy is an emerging immunotherapy against several malignancies including glioblastoma, the most common and most aggressive malignant primary brain tumor in adults. The challenges in solid tumor immunotherapy comprise heterogenously expressed tumor target antigens and restricted trafficking of CAR T cells to and impaired long-term persistence at the tumor site, as well as the unaddressed integration of CAR T-cell therapy into conventional anticancer treatments. We addressed these questions using a NKG2D-based chimeric antigen receptor construct (chNKG2D) in fully immunocompetent orthotopic glioblastoma mouse models. ChNKG2D T cells demonstrated high IFNγ production and cytolytic activity in vitro. Upon systemic administration in vivo, chNKG2D T cells migrated to the tumor site in the brain, did not induce adverse events, prolonged survival, and cured a fraction of glioma-bearing mice. Surviving mice were protected long-term against tumor rechallenge. Mechanistically, this was not solely the result of a classical immune memory response, but rather involved local persistence of chNKG2D T cells. A subtherapeutic dose of local radiotherapy in combination with chNKG2D T-cell treatment resulted in synergistic activity in two independent syngeneic mouse glioma models by promoting migration of CAR T cells to the tumor site and increased effector functions. We thus provide preclinical proof-of-concept of NKG2D CAR T-cell activity in mouse glioma models and demonstrate efficacy, long-term persistence, and synergistic activity in combination with radiotherapy, providing a rationale to translate this immunotherapeutic strategy to human glioma patients.

Significance: These findings provide evidence for synergy of conventional anticancer therapy and CAR T cells and heralds future studies for other treatment combinations. Cancer Res; 78(4); 1031–43. ©2017 AACR.

Glioblastoma is the most common malignant primary brain tumor in adults (1). It remains one of the most challenging cancers and has still a poor prognosis despite multimodal treatment regimens comprising surgery, radiotherapy, and chemotherapy with temozolomide (2, 3). Therefore, novel treatment modalities are urgently needed. Adoptive immunotherapy with genetically engineered T cells that express a chimeric antigen receptor (CAR) is an emerging treatment strategy that may also hold promise for neoplasms in the central nervous system (CNS; ref. 4).

The design of CARs, which consist of an extracellular tumor antigen–binding domain linked to hinge, transmembrane, and intracellular signaling domains (5, 6), allows customized T-cell engineering and an efficient antitumor response of bulk T cells in an MHC-independent manner (7). CAR T-cell therapy has led to encouraging clinical responses in hematologic malignancies (8, 9) and is also studied in solid tumors including glioblastoma (10). However, in solid tumors, there are several challenges that hamper the efficacy of CAR T-cell therapy, which need to be addressed, such as the identification of homogeneously expressed tumor-associated or tumor-specific target antigens, the migration of CAR T cells to the tumor site, and the immunosuppressive microenvironment that may impede the function and persistence of CAR T cells (11).

CAR T-cell strategies that are currently explored against glioblastoma target single tumor antigens such as EGFR variant III (EGFRvIII; ref. 12), erythropoietin-producing hepatocellular carcinoma A2 (EphA2; ref. 13), Her2 (14, 15), or IL13 receptor subunit alpha 2 (IL13Rα2; ref. 16). These targets are nonhomogeneously expressed and susceptible to antigen escape (17).

We and others have assessed the importance of the natural killer group 2-member D (NKG2D) system in glioblastoma (18–22), which has unique features such as the promiscuous binding properties of the NKG2D receptor to multiple tumor-associated NKG2D ligands and the inducibility of these ligands on the tumor cell surface by chemotherapy and radiotherapy (23). NKG2D-based CAR T cells elegantly use the favorable properties of the NKG2D system. The NKG2D CAR design comprises the full-length NKG2D protein fused to CD3ζ and it associates with DNAX-activation protein 10 (DAP10) at the cell surface. This NKG2D–CD3ζ–DAP10 complex functionally acts as a second-generation CAR, which provides a T-cell activation signal through CD3ζ and costimulation through DAP10 (24, 25). NKG2D CAR T cells have never been tested against intracranially growing tumors such as gliomas.

Furthermore, the combination of CAR T-cell therapy with conventional treatment regimens has never been examined, but the inducibility of NKG2D ligands by various stressors provides a strong rationale for this approach.

Here, we investigated NKG2D-based CAR T cells in orthotopic, syngeneic glioma models and addressed the questions of efficacy, trafficking, persistence, and combination with conventional anticancer therapy in fully immunocompetent hosts.

Cell lines

SMA glioma cell lines were obtained from Dr. D. Bigner (Duke University Medical Center, Durham, NC) and GL-261 cells were obtained from the National Cancer Institute (Frederick, MD). EL-4 cells were obtained from the ATCC. All cell lines were maintained in DMEM (Invitrogen), containing 2 mmol/L l-glutamine (Gibco Life Technologies), and 10% FCS (Biochrom KG) and regularly tested negative for mycoplasma by PCR. Cells were authenticated routinely at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures by short tandem repeat analysis, previously in 2013.

CAR design and generation of CAR T cells

The design of the murine NKG2D-based CAR (chNKG2D) and the corresponding control construct that overexpresses wild-type-NKG2D without the intracellular CD3ζ domain (wtNKG2D) has been described (25). CAR T cells were generated by retroviral transduction (25). In short, syngeneic splenocytes from C57BL/6 or VM/Dk mice were activated with 1 μg/mL Concanavalin A (Sigma-Aldrich) for 18–20 hours and retrovirally transduced with chNKG2D or wtNKG2D. The cells were maintained in RPMI1640 (Gibco Life Technologies) supplemented with 10% FCS, 10 mmol/L HEPES, 2 mmol/L l-glutamine, 1 mmol/L pyruvate, 0.1 mmol/L nonessential amino acids (all from Gibco), 50 μmol/L 2-mercaptoethanol (Sigma-Aldrich), and 25 IU/mL recombinant murine IL2 (PeproTech) for 6–8 days and subsequently used for experiments.

Antibodies and flow cytometry

The following mAbs were used for flow cytometry: anti-CD4-AF700, anti-CD8-APC, anti-IFNγ-BV421, anti-CD45.1-AF488, and anti-CD45.2-APC (Biolegend). For blocking of FC receptors, samples were preincubated with anti-mouse CD16/CD32 (Biolegend). As controls, we used isotype-matched antibodies from Sigma-Aldrich. Acquisition was performed on a BD FACSVerse Analyzer (BD Biosciences) and data were analyzed with FlowJo (TreeStar).

Cytotoxicity assay

Glioma cells as target cells were cocultured for 4 hours with chNKG2D or wtNKG2D T cells at different effector: target ratios. Glioma cell lysis was assessed by a flow cytometry–based assay (18). Specific lysis was expressed as percentage of death of labeled target cells. For blocking experiments, transduced T cells were preincubated for 1 hour at 4°C with anti-NKG2D or isotype control from eBioscience.

IHC

Staining of brain cryosections from tumor-bearing mice has been described in detail (26). Anti-CD45.1 and anti-CD45.2 antibodies for IHC were obtained from Novus Biologicals. The Histofine Simple Stain Mouse MAX PO was obtained from Nichirei and used as secondary antibody system.

Real-time PCR

RNA isolation and cDNA preparation were performed as described previously (27). Gene expression was measured in a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems) with SYBR Green from Thermo Fisher Scientific. The following primers (Microsynth AG) were used: chNKG2D (28): forward 5′-GGCGTCGACACCATGAGAGCAAAATTCAGCAGGAG-3′, reverse 5′-GGCGCTCGAGTTACACCGCCCTTTTCATGCAGAT-3′, mouse HPRT1: forward 5′- TTGCTGACCTGCTGGATTAC-3′, reverse 5′- TTTATGTCCCCCGTTGACTG-3′, respectively. The conditions were 40 cycles at 95°C/15 seconds and 60 °C/1 minute. Relative quantification of gene expression was calculated with the ΔΔCt method (29) for relative quantification compared with the housekeeping gene HPRT1.

IFNγ ELISPOT assay

Individual spleen samples (N = 3) were obtained from NKG2D CAR T-cell–treated long-term survivors 8 months after initial treatment or naïve control mice. Untouched T cells were isolated using Pan T Cell Isolation Kit II (Miltenyi Biotec) and 2 × 106 T cells were cocultured with GL-261 cells for 24 hours. The number of IFNγ-secreting cells was measured using mouse IFNγ ELISPOT Ready-SET-Go from eBioscience and spot forming cells (SFC) were counted using AID EliSpot Reader classic (AID GmbH).

IFNγ ELISA

NKG2D CAR T-cell–treated long-term survivors, 12 months after initial treatment, or naïve control mice were (re)challenged with intracranial implantation of GL-261 cells. Three days after tumor implantation, mice were euthanized, splenocytes were isolated, and brain-resident CD4+ or CD8+ T cells were FACS sorted. Subsequently, 5 × 104 splenocytes from long-term surviving mice or naïve control mice were cocultured with 2.5 × 104 GL-261 or EL-4 cells and 5 × 103 FACS-sorted brain-resident CD4+ or CD8+ T cells were cocultured with 2.5 × 103 GL-261 or EL-4 cells. After 72 hours, cell-free conditioned media was assessed for IFNγ by ELISA from Thermo Fisher Scientific.

Animal experiments

All experiments were done in accordance with the Institutional Animal Care and Use Committee of the cantonal veterinary office (ZH006/15) and according to guidelines of the Swiss federal law on animal protection. Wild-type C57BL/6 CD45.2 mice were purchased from Charles River Laboratories and C57BL/6 CD45.1 mice were obtained from the Jackson Laboratory. VM/Dk mice were bred in pathogen-free facilities at the University of Zurich. Mice of 6 to 12 weeks of age were used in all experiments. For intracranial tumor implantation, GL-261 cells (2 × 104) were stereotactically implanted into the right striatum at day 0. Mice were observed daily and sacrificed as indicated or when developing neurologic symptoms. Where indicated, mice received 5 × 106 chNKG2D or wtNKG2D T cells intravenously at days 5, 7, and 10 after tumor implantation. For local administration, up to 2 × 106 transduced T cells were injected intratumorally at day 5 after tumor implantation. If indicated, local cranial radiotherapy with a single dose of 4 Gy was applied at day 7 after tumor implantation using a Gulmay 200 kV X-ray unit at 1 Gy/min. Long-term surviving mice were rechallenged 5 to 8 months after the initial tumor inoculation with another tumor implantation in the contralateral hemisphere without any further treatment. For isolation of tumor-infiltrating immune cells at indicated time points, we perfused mice with ice-cold PBS to remove all circulating leukocytes from the CNS and isolated the brains. Subsequently, tumor cells were separated from myelin and red blood cells using a Percoll gradient suspension (Sigma-Aldrich). Cells were washed with PBS and stained with Zombie Aqua Fixable Viability Kit (Biolegend) and fluorochrome-conjugated antibodies specific to cell surface markers for flow cytometry as indicated.

MRI

MRI was performed with a 4.7 T imager (Bruker Biospin) at day 15 after tumor implantation. Coronal T2-weighted images were acquired using Paravision 6.0 (Bruker BioSpin). If indicated, mean and SD of the tumor volume in mm3 from 5 mice/group were calculated using the formula (length × width × depth)/2.

Fluorescence molecular tomography

Transduced T cells were labeled with CellBriteTM NIR790 (Biotium) and administered to tumor-bearing mice as indicated. For fluorescence molecular tomography (FMT) imaging at indicated time points after T-cell injection, mice were anesthetized by gas anesthesia, depilated at the tumor region, placed in a FMT4000 system (PerkinElmer) and scanned with the 790 nm laser channel. Image analysis was performed using TrueQuant 3.1 (PerkinElmer). A region of interest of equal size was placed above the tumor region and fluorescence intensity was automatically calculated by the software.

Statistical analysis

Data are presented as means and SD. Experiments were repeated at least three times, if not indicated differently. Statistical analyses were performed in GraphPad Prism using multiple two-tailed Student t tests and correction for multiple comparisons using the Holm–Sidak method. Kaplan–Meier survival analysis was performed to assess survival differences among the treatment groups and P values were calculated with the log-rank test. Significance was concluded at *P < 0.05 and **P < 0.01.

ChNKG2D-transduced T cells lyse glioma cells in a NKG2D-dependent manner

We generated murine chNKG2D CAR T cells or wtNKG2D overexpressing T cells by retroviral transduction of splenocytes derived from C57BL/6 or VM/Dk mice. NKG2D cell surface levels in splenocytes transduced with chNKG2D or wtNKG2D were equivalent (Supplementary Fig. S1). The differential biological effects exerted by these cells can therefore be attributed specifically to the chimeric construct. To determine the cytolytic activity of chNKG2D- or wtNKG2D-transduced T cells, we used these cells as effector cells and different syngeneic murine glioma cell lines as target cells in cytotoxicity assays. ChNKG2D T cells had a significantly higher specific cytolytic activity against all murine glioma cell lines than wtNKG2D T cells (Fig. 1A–D, top). Inhibition of NKG2D signaling using a blocking antibody abrogated the enhanced cytolysis, confirming the NKG2D dependency of chNKG2D T cells (Fig. 1A–D, bottom). Because cytokines are important effector molecules of CAR T-cell function, we assessed the T-cell–specific IFNγ production of chNKG2D or wtNKG2D T cells cocultured with syngeneic glioma cells by intracellular cytokine staining. Both CD4+ and CD8+ T cells produced more IFNγ after transduction with chNKG2D compared with wtNKG2D when the T cells were cocultured with syngeneic glioma cells (Fig. 2A–D).

Figure 1.

NKG2D CAR T cells lyse glioma cells in a NKG2D-dependent manner. The murine glioma cell lines GL-261 (A), SMA-497 (B), SMA-540 (C), or SMA-560 (D) were used as target cells in 4-hour cytolysis assays. T cells from C57BL/6 mice (for GL-261) or VM/Dk mice (for SMA-497, SMA-540, and SMA-560) were transduced with chNKG2D (CH) or wtNKG2D (WT) and used as effector cells at various effector:target (E:T) ratios (corresponding upper graphs for each cell line). Blocking anti-NKG2D or isotype control antibodies were used to preincubate chNKG2D or wtNKG2D T cells for 1 hour before using them as effector cells at an effector:target ratio of 40:1 (corresponding lower graphs for each cell line). Data are presented as mean ± SD (*, P < 0.05; **, P < 0.01).

Figure 1.

NKG2D CAR T cells lyse glioma cells in a NKG2D-dependent manner. The murine glioma cell lines GL-261 (A), SMA-497 (B), SMA-540 (C), or SMA-560 (D) were used as target cells in 4-hour cytolysis assays. T cells from C57BL/6 mice (for GL-261) or VM/Dk mice (for SMA-497, SMA-540, and SMA-560) were transduced with chNKG2D (CH) or wtNKG2D (WT) and used as effector cells at various effector:target (E:T) ratios (corresponding upper graphs for each cell line). Blocking anti-NKG2D or isotype control antibodies were used to preincubate chNKG2D or wtNKG2D T cells for 1 hour before using them as effector cells at an effector:target ratio of 40:1 (corresponding lower graphs for each cell line). Data are presented as mean ± SD (*, P < 0.05; **, P < 0.01).

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Figure 2.

NKG2D-based CAR T cells produce high levels of IFNγ upon coculture with glioma cells. The murine glioma cell lines GL-261 (A), SMA-497 (B), SMA-540 (C), or SMA-560 (D) were cocultured with chNKG2D- or wtNKG2D-expressing T cells. After 6 hours of coculture, IFNγ levels were determined in CD4+ and CD8+ T cells by flow cytometry. Bar plots show mean and SD from two independent experiments (*, P < 0.05; **, P < 0.01). FACS plots show data from one representative experiment and numbers indicate percentage of IFNγ-positive T cells.

Figure 2.

NKG2D-based CAR T cells produce high levels of IFNγ upon coculture with glioma cells. The murine glioma cell lines GL-261 (A), SMA-497 (B), SMA-540 (C), or SMA-560 (D) were cocultured with chNKG2D- or wtNKG2D-expressing T cells. After 6 hours of coculture, IFNγ levels were determined in CD4+ and CD8+ T cells by flow cytometry. Bar plots show mean and SD from two independent experiments (*, P < 0.05; **, P < 0.01). FACS plots show data from one representative experiment and numbers indicate percentage of IFNγ-positive T cells.

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NKG2D-based CAR T cells home to orthotopic gliomas after systemic administration

The concept of CNS immune privilege has been refined by the demonstration of an intact afferent arm of CNS-related immunity, which includes the discovery of classical lymphatic vessels in the meninges (30). However, the efferent arm is restricted, i.e., leukocyte migration to the brain (31) and the immunosuppressive microenvironment of solid tumors such as gliomas is another challenge for access of CAR T cells to the tumor site. To determine whether NKG2D-based CAR T cells reach orthotopically growing gliomas after systemic administration, we labeled chNKG2D T cells with a near-infrared dye that allows in vivo tracking by FMT. After a single intravenous injection of 5 × 106 chNKG2D T cells, we detected an FMT signal at the orthotopic tumor site that increased over several days (Fig. 3A). Labeled wtNKG2D T cells also reached the tumor site (Supplementary Fig. S2). To corroborate this finding, we injected chNKG2D-transduced T cells generated from splenocytes of CD45.1+ donor mice into CD45.2+ glioma-bearing animals. After tumor explantation and isolation of tumor-infiltrating immune cells, we detected a CD45.1+ chNKG2D T-cell population by flow cytometry (Fig. 3B; Supplementary Fig. S3). In addition, the infiltration of intracranial tumors with CD45.1+ chNKG2D T cells was verified by IHC (Fig. 3C). Compared with direct intratumoral injection, which served as a control, we detected fewer CD45.1+ cells after intravenous injection, indicating that only a fraction of the administered T cells migrates to and stays at the tumor site (Fig. 3B and C).

Figure 3.

NKG2D-based CAR T cells migrate to intracranially growing gliomas after systemic administration. A, ChNKG2D T cells were labeled with CellBrite NIR790. Subsequently, 106-labeled cells (+ctrl) or unlabeled cells (PBS, −ctrl, respectively) were injected intracranially in the brain of a nontumor-bearing mouse and the signal from labeled T cells was detected at the tumor site by FMT (left). Tumor-bearing mice were treated with a single intravenous injection of 5 × 106 chNKG2D T cells at day 5 after tumor cell implantation. The near-infrared signal was acquired at the tumor site by FMT at the indicated time points after injection (right). Color scales indicate signal intensities. B, A total of 5 x 106 CD45.1+ chNKG2D T cells were injected either intravenously or intratumorally at a single time point in CD45.2+ tumor-bearing mice at day 5 after tumor implantation. Three days later, tumor-infiltrating immune cells were isolated from the tumor-bearing hemisphere and CD45.1+ and CD45.2+ cells were detected by flow cytometry. C, The animals were treated as in B and CD45.1+ cells were detected by IHC after removal of the brains at day 10. CD45.1+ T cells are stained in brown. Spleen sections from CD45.1+ mice were used as positive control. One representative image out of three different mice per group is shown.

Figure 3.

NKG2D-based CAR T cells migrate to intracranially growing gliomas after systemic administration. A, ChNKG2D T cells were labeled with CellBrite NIR790. Subsequently, 106-labeled cells (+ctrl) or unlabeled cells (PBS, −ctrl, respectively) were injected intracranially in the brain of a nontumor-bearing mouse and the signal from labeled T cells was detected at the tumor site by FMT (left). Tumor-bearing mice were treated with a single intravenous injection of 5 × 106 chNKG2D T cells at day 5 after tumor cell implantation. The near-infrared signal was acquired at the tumor site by FMT at the indicated time points after injection (right). Color scales indicate signal intensities. B, A total of 5 x 106 CD45.1+ chNKG2D T cells were injected either intravenously or intratumorally at a single time point in CD45.2+ tumor-bearing mice at day 5 after tumor implantation. Three days later, tumor-infiltrating immune cells were isolated from the tumor-bearing hemisphere and CD45.1+ and CD45.2+ cells were detected by flow cytometry. C, The animals were treated as in B and CD45.1+ cells were detected by IHC after removal of the brains at day 10. CD45.1+ T cells are stained in brown. Spleen sections from CD45.1+ mice were used as positive control. One representative image out of three different mice per group is shown.

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NKG2D-based CAR T cells prolong survival of syngeneic orthotopic glioma-bearing mice

To explore the efficacy of NKG2D-based CAR T-cell therapy against experimental gliomas in vivo, we intravenously injected chNKG2D or wtNKG2D T cells on days 5, 7, and 10 after orthotopic tumor implantation. This resulted in a significantly prolonged survival of GL-261 glioma-bearing mice and cured 22% of the animals as confirmed by MRI and long-term follow-up (Fig. 4A). Systemic administration of CAR T cells may be associated with on-target off-tumor activity and thus local or systemic toxicity. We did not observe weight loss as an indirect indicator for toxicity following repetitive systemic administration of chNKG2D or wtNKG2D T cells (Fig. 4B). According to the mouse gene expression database and the BioGPS gene expression database, NKG2D ligands are generally expressed at low levels in normal tissues with the highest expression of RAE-1 in liver, hematopoietic system, and reproductive system (Supplementary Fig. S4A and S4B). We observed no changes in liver enzyme amounts in sera of mice treated repetitively with chNKG2D or wtNKG2D T cells. Furthermore, we did not observe differences in peripheral blood counts (Fig. 4B).

Figure 4.

NKG2D-based CAR T-cell treatment confers a survival benefit in syngeneic orthotopic glioma-bearing mice. A, GL-261 tumor-bearing mice were treated intravenously with 5 × 106 chNKG2D- or wtNKG2D-expressing T cells on days 5, 7, and 10 after tumor implantation. Survival data are presented as Kaplan–Meier plots (left). Representative T2w MRI images of mice receiving wtNKG2D (top) or chNKG2D T cells (bottom) at day 15 after tumor implantation are shown (right). White arrow, tumor. B, Body weight was determined every other day (top left), liver enzymes (upper right) were assessed at day 16, and peripheral blood counts were assessed at day 14 of GL-261 tumor-bearing mice treated intravenously with 5 × 106 chNKG2D or wtNKG2D T cells at days 5, 7, and 10 after tumor-implantation. C, GL-261 tumor-bearing mice were treated with a single intratumoral injection of 2 × 104, 2 × 105, or 2 × 106 chNKG2D T cells or PBS control. Survival data are presented as Kaplan–Meier plots (left). P values were calculated with log-rank test (*, P < 0.05; **, P < 0.01). Representative T2w MRI images of mice receiving wtNKG2D (top) or 2 × 106 chNKG2D T cells (bottom) at day 15 after tumor implantation are shown (right). White arrow, tumor.

Figure 4.

NKG2D-based CAR T-cell treatment confers a survival benefit in syngeneic orthotopic glioma-bearing mice. A, GL-261 tumor-bearing mice were treated intravenously with 5 × 106 chNKG2D- or wtNKG2D-expressing T cells on days 5, 7, and 10 after tumor implantation. Survival data are presented as Kaplan–Meier plots (left). Representative T2w MRI images of mice receiving wtNKG2D (top) or chNKG2D T cells (bottom) at day 15 after tumor implantation are shown (right). White arrow, tumor. B, Body weight was determined every other day (top left), liver enzymes (upper right) were assessed at day 16, and peripheral blood counts were assessed at day 14 of GL-261 tumor-bearing mice treated intravenously with 5 × 106 chNKG2D or wtNKG2D T cells at days 5, 7, and 10 after tumor-implantation. C, GL-261 tumor-bearing mice were treated with a single intratumoral injection of 2 × 104, 2 × 105, or 2 × 106 chNKG2D T cells or PBS control. Survival data are presented as Kaplan–Meier plots (left). P values were calculated with log-rank test (*, P < 0.05; **, P < 0.01). Representative T2w MRI images of mice receiving wtNKG2D (top) or 2 × 106 chNKG2D T cells (bottom) at day 15 after tumor implantation are shown (right). White arrow, tumor.

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To investigate the potential treatment effect and tolerability in the setting of an unrestricted migration to the tumor site, we injected chNKG2D T cells directly intratumorally in glioma-bearing mice. This application route significantly prolonged the survival after a single injection of chNKG2D T cells in a dose-dependent manner and increased the fraction of cured mice compared with intravenous CAR T-cell administration (Fig. 4C).

Glioma-bearing mice surviving after chNKG2D T-cell treatment are long-term protected against tumor rechallenge

An impressive feature of cancer immunotherapy is the potential for a long-lasting treatment effect (32). We therefore determined a potential long-term immune protection in glioma-bearing mice that had survived following chNKG2D CAR T-cell treatment. To this end, GL-261 glioma cells were implanted into the contralateral hemisphere 5 to 8 months after the first implantation. No additional treatment was administered. All of these mice that survived after the initial CAR T-cell treatment also survived the tumor rechallenge, and at day 18 after tumor implantation, no tumor mass was detectable by MRI (Fig. 5A and B). In contrast, all naïve mice had large tumors and finally had to be euthanized. To investigate the mechanism underlying this long-term protection, we rechallenged the long-term survivors a second time and isolated lymphocytes from the brain and from cervical, axillary, and inguinal lymph nodes 3 days after this second tumor rechallenge. A prominent CD8+ T-cell population was detected within the rechallenged hemisphere of chNKG2D T-cell–treated long-term survivors but not in naïve mice (Fig. 5C). The brain-resident CD8+ T-cell population predominantly secreted IFNγ in response to GL-261 glioma cells, as demonstrated by FACS-sorting of brain-resident CD4+ and CD8+ T cells from long-term survivors or naïve tumor-bearing mice 3 days after tumor rechallenge and ex vivo stimulation with GL-261 cells or MHC-matched but non-glioma EL-4 cells (Supplementary Fig. S5). There was no difference in intermediate and high CD44-expressing CD4+ and CD8+ T cells between lymph nodes of rechallenged chNKG2D T-cell–treated long-term survivors and those of naïve tumor-bearing mice (Fig. 5D). This suggests a local long-term protection independent from a peripheral memory response. In line with these findings, we noticed an equivalent tumor cell cytolysis and IFNγ production after coculture of GL-261 cells with T cells isolated from lymph nodes of rechallenged chNKG2D T-cell–treated long-term survivors or T cells from naïve tumor-bearing mice. We detected chNKG2D transcripts in brains from long-term survivors but not from naïve mice and not in peripheral lymph nodes or spleens from long-term survivors or naïve mice (Fig. 5D), which suggests local persistence of NKG2D CAR T cells in the CNS.

Figure 5.

Glioma-bearing mice surviving after NKG2D CAR T-cell treatment are long-term protected against tumor rechallenge. Long-term surviving mice cured by systemic (i.v.) or intratumoral (i.t.) administration of chNKG2D T cells were rechallenged 6 months after the initial tumor implantation with GL-261 cells in the contralateral hemisphere. As a control, GL-261 cells were inoculated into naïve mice. A, Kaplan–Meier survival curves for the three cohorts are indicated. B, T2w MRI scans at day 18 after tumor implantation are shown. White arrow, tumor. The top panel represents images from naïve control mice and the bottom panel represents images from rechallenged chNKG2D T-cell long-term survivors following tumor implantation. C, Long-term surviving mice received a second tumor rechallenge (2 months after the first rechallenge) and 3 days after tumor (re)implantation, tumor-infiltrating CD4+ and CD8+ T cells were isolated and analyzed by flow cytometry. An individual plot of tumor-infiltrating lymphocytes from one mouse is shown on the left and a diagram depicting the mean and SD of three mice is shown on the right (*, P < 0.05; **, P < 0.01). D, Same setup as in C and isolation of cervical, axillary, and inguinal lymph nodes at day 3 after the second tumor rechallenge. Effector (Teff) and memory (Tmem) T cells were separated using flow cytometry and CD44 expression levels. Populations with intermediate (effector T cells) or high (memory T cells) CD44 expression in the CD4+ or CD8+ T-cell compartments are indicated (left). The isolated lymph node–derived T cells were used as effector cells in a 4-hour immune cell lysis assay or IFNγ ELISPOT with fresh GL-261 cells as target cells (right top panel). Real-time PCR for chNKG2D was performed after RNA isolation and cDNA preparation from brains, spleens, and peripheral lymph nodes isolated 3 days after tumor challenge of naïve control mice or 3 days after second rechallenge of long-term surviving mice 8 months after initial NKG2D CAR T-cell treatment (right bottom panel; n.d., nondetectable). E:T, effector:target ratio.

Figure 5.

Glioma-bearing mice surviving after NKG2D CAR T-cell treatment are long-term protected against tumor rechallenge. Long-term surviving mice cured by systemic (i.v.) or intratumoral (i.t.) administration of chNKG2D T cells were rechallenged 6 months after the initial tumor implantation with GL-261 cells in the contralateral hemisphere. As a control, GL-261 cells were inoculated into naïve mice. A, Kaplan–Meier survival curves for the three cohorts are indicated. B, T2w MRI scans at day 18 after tumor implantation are shown. White arrow, tumor. The top panel represents images from naïve control mice and the bottom panel represents images from rechallenged chNKG2D T-cell long-term survivors following tumor implantation. C, Long-term surviving mice received a second tumor rechallenge (2 months after the first rechallenge) and 3 days after tumor (re)implantation, tumor-infiltrating CD4+ and CD8+ T cells were isolated and analyzed by flow cytometry. An individual plot of tumor-infiltrating lymphocytes from one mouse is shown on the left and a diagram depicting the mean and SD of three mice is shown on the right (*, P < 0.05; **, P < 0.01). D, Same setup as in C and isolation of cervical, axillary, and inguinal lymph nodes at day 3 after the second tumor rechallenge. Effector (Teff) and memory (Tmem) T cells were separated using flow cytometry and CD44 expression levels. Populations with intermediate (effector T cells) or high (memory T cells) CD44 expression in the CD4+ or CD8+ T-cell compartments are indicated (left). The isolated lymph node–derived T cells were used as effector cells in a 4-hour immune cell lysis assay or IFNγ ELISPOT with fresh GL-261 cells as target cells (right top panel). Real-time PCR for chNKG2D was performed after RNA isolation and cDNA preparation from brains, spleens, and peripheral lymph nodes isolated 3 days after tumor challenge of naïve control mice or 3 days after second rechallenge of long-term surviving mice 8 months after initial NKG2D CAR T-cell treatment (right bottom panel; n.d., nondetectable). E:T, effector:target ratio.

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Irradiation increases the therapeutic activity of NKG2D-based CAR T cells against gliomas

There is an intense discussion on the implementation of immunotherapy into conventional treatment regimens. Radiotherapy is part of the standard treatment of gliomas (33). We have previously observed an upregulation of NKG2D ligands on the glioma cell surface upon irradiation (23). This built the rationale to combine radiotherapy with NKG2D-based CAR T-cell therapy. A single cranial irradiation with 4 Gy at day 7 after tumor implantation had no effect on survival when given alone but further increased the activity of NKG2D-based CAR T cells against orthotopic gliomas as demonstrated by synergistically prolonged survival in two independent glioma models, reduced tumor volume measured by MRI, and an increased fraction of long-term surviving mice in the SMA-560 glioma model (Fig. 6A–D). We identified two underlying mechanisms for this synergistic activity. Low-dose preirradiation of glioma cells and subsequent coculture with chNKG2D- or wtNKG2D-expressing T cells resulted in an increased cytolysis and IFNγ production in vitro (Fig. 7A). Also in vivo, local tumor irradiation increased the IFNγ expression of tumor-infiltrating chNKG2D T cells (Fig. 7B; Supplementary Fig. S6A). This points towards a direct tumor-cell–related effect of irradiation that boosts NKG2D CAR T-cell activity, which can be attributed due to the induction of NKG2DL (Supplementary Fig. S6B; ref. 23). Moreover, we observed an indirect migration-related mechanism by tracking fluorescently labeled chNKG2D CAR T cells after intravenous injection. Here, we noticed an increased accumulation of CAR T cells upon irradiation (Fig. 7C), suggesting that local subtherapeutic irradiation promotes the migration of CAR T cells to the tumor site.

Figure 6.

Irradiation and systemic NKG2D-based CAR T-cell treatment act synergistically against experimental gliomas. GL-261 (A and C) or SMA-560 (B and D) tumor-bearing mice were treated intravenously with 5 × 106 chNKG2D (CH) or wtNKG2D (WT) T cells on days 5, 7, and 10 after tumor implantation either alone or with a single local irradiation with 4 Gy at day 7 after tumor implantation. A and B, Kaplan–Meier curves are shown and P values were calculated with log-rank test (**, P < 0.01 for WT vs. CH; ***, P < 0.001 for WT + IR vs. CH+IR). C, Representative MRI scans at day 15 of three animals of each of the four cohorts of GL-261 tumor-bearing mice are shown (left). White arrow, tumor. Tumor volumes (right) were calculated on the basis of MRI analyses using the formula H × W × L/2 (*, P < 0.05 and **, P < 0.01 for WT vs. CH or WT+IR vs. CH+ IR; +, P < 0.05 for CH vs. CH+IR). D, Same setting as in C but for SMA-560 tumor-bearing mice.

Figure 6.

Irradiation and systemic NKG2D-based CAR T-cell treatment act synergistically against experimental gliomas. GL-261 (A and C) or SMA-560 (B and D) tumor-bearing mice were treated intravenously with 5 × 106 chNKG2D (CH) or wtNKG2D (WT) T cells on days 5, 7, and 10 after tumor implantation either alone or with a single local irradiation with 4 Gy at day 7 after tumor implantation. A and B, Kaplan–Meier curves are shown and P values were calculated with log-rank test (**, P < 0.01 for WT vs. CH; ***, P < 0.001 for WT + IR vs. CH+IR). C, Representative MRI scans at day 15 of three animals of each of the four cohorts of GL-261 tumor-bearing mice are shown (left). White arrow, tumor. Tumor volumes (right) were calculated on the basis of MRI analyses using the formula H × W × L/2 (*, P < 0.05 and **, P < 0.01 for WT vs. CH or WT+IR vs. CH+ IR; +, P < 0.05 for CH vs. CH+IR). D, Same setting as in C but for SMA-560 tumor-bearing mice.

Close modal
Figure 7.

Irradiation-mediated boosting of CAR T-cell activity relies on direct effects on tumor cells as well as increased CAR T-cell migration. A, GL-261 cells, preirradiated (+IR) 24 hours prior to the assay with 4 Gy or not, were used as target cells in cytotoxicity assays using chNKG2D (CH) or wtNKG2D (WT) T cells as effector cells at the indicated effector:target (E:T) ratios (left). After 4 hours of coculture at a ratio of 40:1, IFNγ levels were determined by intracellular cytokine staining and flow cytometric assessment (right). Mean and SD are shown (*, P < 0.05; **, P < 0.01 for WT vs. CH and +, P < 0.05; ++, P < 0.01 for WT/CH vs. WT/CH + IR). B, 5 × 106 CD45.1+ wtNKG2D or chNKG2D were i.v. injected on days 5, 7, 10 after implantation of GL-261 tumors in CD45.2+ mice. In addition, mice were irradiated (IR) at day 7 with a single local dose of 4 Gy or not. At day 12 after tumor implantation, brain-infiltrating immune cells were isolated and IFNγ expression assessed in CD45.1+ cells by flow cytometry. Mean and SD from three mice are shown with **, P < 0.01 for WT vs. CH and WT + IR vs. CH+IR and +, P < 0.05; for WT/CH vs. WT/CH + IR. C, ChNKG2D T cells were labeled with CellBrite NIR790. Subsequently, 5 × 106 labeled cells were intravenously injected on days 5, 7, and 10 after tumor implantation. In addition, mice were irradiated at day 7 with a single local dose of 4 Gy or not. The near-infrared signal was acquired at the tumor-site by FMT at the following time points: T1 = 24 hours prior to irradiation, T2 = 24 hours post-irradiation, T3 = 144 hours post-irradiation. Representative images are shown on the left and a quantification of the detected signal is shown on the right (*, P < 0.05; **, P < 0.01).

Figure 7.

Irradiation-mediated boosting of CAR T-cell activity relies on direct effects on tumor cells as well as increased CAR T-cell migration. A, GL-261 cells, preirradiated (+IR) 24 hours prior to the assay with 4 Gy or not, were used as target cells in cytotoxicity assays using chNKG2D (CH) or wtNKG2D (WT) T cells as effector cells at the indicated effector:target (E:T) ratios (left). After 4 hours of coculture at a ratio of 40:1, IFNγ levels were determined by intracellular cytokine staining and flow cytometric assessment (right). Mean and SD are shown (*, P < 0.05; **, P < 0.01 for WT vs. CH and +, P < 0.05; ++, P < 0.01 for WT/CH vs. WT/CH + IR). B, 5 × 106 CD45.1+ wtNKG2D or chNKG2D were i.v. injected on days 5, 7, 10 after implantation of GL-261 tumors in CD45.2+ mice. In addition, mice were irradiated (IR) at day 7 with a single local dose of 4 Gy or not. At day 12 after tumor implantation, brain-infiltrating immune cells were isolated and IFNγ expression assessed in CD45.1+ cells by flow cytometry. Mean and SD from three mice are shown with **, P < 0.01 for WT vs. CH and WT + IR vs. CH+IR and +, P < 0.05; for WT/CH vs. WT/CH + IR. C, ChNKG2D T cells were labeled with CellBrite NIR790. Subsequently, 5 × 106 labeled cells were intravenously injected on days 5, 7, and 10 after tumor implantation. In addition, mice were irradiated at day 7 with a single local dose of 4 Gy or not. The near-infrared signal was acquired at the tumor-site by FMT at the following time points: T1 = 24 hours prior to irradiation, T2 = 24 hours post-irradiation, T3 = 144 hours post-irradiation. Representative images are shown on the left and a quantification of the detected signal is shown on the right (*, P < 0.05; **, P < 0.01).

Close modal

CAR T-cell therapy is an emerging immunotherapy that is under development against several malignancies including glioblastoma. However, the potentially immunosuppressive microenvironment and intraparenchymal location of solid tumors represent challenges that require a systematic development of strategies boosting the antitumor efficacy of CAR T cells against these tumors (34). Furthermore, it remains to be determined how immunotherapy with CAR T cells can be integrated at its best into conventional cancer therapies.

We addressed these questions in orthotopic, fully immunocompetent, preclinical glioblastoma models. Except for one report (35), all preclinical studies published so far have assessed CAR T cells against experimental gliomas in a xenograft setting. However, only a syngeneic setting, which involves an intact immune system and a physiologic tumor microenvironment, allows for appropriately assessing the challenges of CAR T-cell therapy against solid tumors, including potential off-tumor toxicities.

In contrast to other single target antigens for CAR T-cell therapy that are currently being investigated against glioblastoma, the NKG2D CAR elegantly targets multiple tumor-associated ligands. This may decrease the probability of tumor immune escape from CAR T-cell treatment due to antigen loss (17).

We demonstrate strong antitumor activity of NKG2D-based CAR T cells against glioma cells in vitro in a syngeneic setting (Figs. 1 and 2). Furthermore, in vivo tracking of CAR T cells verifies homing of CAR T cells to the intracerebral tumor site after systemic CAR T-cell administration (Fig. 3). The target antigens of the NKG2D CAR are not exclusively expressed on tumor cells but can be induced also in nontumoral tissues by unphysiologic cell stress or inflammation (36–38). Although NKG2D ligand expression by other tissues may result in on-target off-tumor toxicity, we did not observe any signs of toxicity upon systemic CAR T-cell administration (Fig. 4B) at the level of body weight, liver function tests or peripheral blood counts. In line with these findings, a first-in-human phase I trial assessing NKG2D-based CAR T cells against hematologic malignancies has completed enrollment of a first cohort of patients without treatment-related safety issues (39).

Treatment of glioma-bearing mice with NKG2D CAR T cells resulted in a significant proportion of surviving mice, which were long-term protected against tumor rechallenge in the contralateral hemisphere (Fig. 5A and B). In contrast to previous studies (28, 35), this may not only be the result of a classical memory T-cell response, but probably it may also involve long-term persistence of NKG2D CAR T cells in the brain as demonstrated by detection of the chNKG2D transcripts in the brain tissue of long-term surviving mice 8 months after initial CAR T-cell treatment (Fig. 5D). It remains an open question why NKG2D CAR T cells seem to persist in the CNS but not in extracerebral organs. Possibly, the CNS as an immune-privileged site provides a niche for homeostatic proliferation of tissue-resident T cells, which allows CAR T-cell persistence.

Future glioblastoma treatment regimens may rely on combination therapies and may implement novel immunotherapeutic strategies such as CAR T-cell administration. The combination of immunotherapy with conventional anticancer treatments such as radiotherapy may result in synergistic activity (40, 41). However, the combination of CAR T-cell therapy with radiotherapy has only been investigated with total body irradiation used as a myeloablative host-conditioning regimen before administration of CAR T cells (35). Our data demonstrate synergistic antitumor activity of local tumor irradiation and NKG2D CAR T-cell therapy (Fig. 6). Mechanistically, this combination produces stronger CAR T-cell activity upon recognition of irradiated tumor cells and an improved trafficking of intravenously injected CAR T cells to the tumor site (Fig. 7).

In summary, this study highlights the potential of NKG2D CAR T cells as a promising therapeutic approach against glioblastoma. Long-term tumor control due to persistence of these cells at the tumor site and synergistic activity in combination with radiotherapy suggest that NKG2D CAR T cells represent a novel therapeutic option that warrants clinical evaluation in glioma patients.

C.L. Sentman reports receiving a commercial research grant from Celyad and Celdara Medical, has ownership interest (including patents) in Celyad, is a consultant/advisory board member for Celyad and Celdara Medical, and has provided expert testimony for Celyad and Celdara Medical. P. Roth has received speakers bureau honoraria from BMS, Novartis, and Novocure and is a consultant/advisory board member for MSD, Virometix, Roche, Covagen, and Molecular Partners. No potential conflicts of interest were disclosed by the other authors.

Conception and design: T. Weiss, M. Weller, C.L. Sentman, P. Roth

Development of methodology: T. Weiss, P. Roth

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Weiss

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T. Weiss, M. Weller, C.L. Sentman, P. Roth

Writing, review, and/or revision of the manuscript: T. Weiss, M. Weller, M. Guckenberger, C.L. Sentman, P. Roth

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Weiss, M. Weller, M. Guckenberger, P. Roth

Study supervision: M. Weller, P. Roth

This study was supported by grants from the Swiss National Science Foundation (310030_170027 to P. Roth), the Gertrud-Hagmann Foundation, and the Swiss Cancer League (KFS-3478-08-2014 to P. Roth), and “Hochspezialisierte Medizin Zurich” (HSM-2 to P. Roth, M. Guckenberger, and M. Weller) as well as the Detas Foundation to M. Weller. We thank Professor Christian Münz for providing access to the ELISpot Reader.

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

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