Interleukin-7 loaded oncolytic adenovirus improves CAR-T cell therapy for glioblastoma

Background: T cell with chimeric antigen receptors (CAR-T) has presented remarkable ecacy for blood cancer as an emerging immunotherapy. However, for solid tumors, the therapeutic ecacy is much impaired due to the lack of inltration and persistence of CAR-T in tumor tissue. Thus, we constructed an interleukin-7-loaded oncolytic adenovirus and combined the use of oncolytic virus and CAR-T to improve the therapeutic outcome. Methods: We constructed an interleukin-7 loaded oncolytic adenovirus (oAD-IL7) and a B7H3-targeted CAR-T, and explored the ecacy of the single use of oAD-IL7, B7H3-CART or the combined therapy for glioblastoma in vitro and in vivo. The improved CAR-T anti-tumor ecacy was evaluated according to the proliferation, survival, persistence, exhaustion of T cells, and tumor regression. Results: Constructed oAD-IL7 and B7H3-CART presented moderate cytotoxicity during in vitro study, but failed to induce a thorough and persistent anti-tumor therapeutic ecacy in vivo. The combination of oAD-IL7 and B7H3-CART in vitro resulted in enhanced T cell proliferation and reduced T cell apoptosis. The joint ecacy was further conrmed using tumor-bearing xenograft mice. During in vivo study, the mice treated with both oAD-IL7 and B7H3-CART showed prolonged survival and reduced tumor burden. According to the ex vivo study, oAD-IL7 improved the proliferation and persistence of tumor-inltrating B7H3-CART, but failed to reverse the exhaustion. Conclusions: Our results indicated that oAD-IL7 is a promising auxiliary therapy to improve the therapeutic ecacy of B7H3-CART in glioblastoma by providing the activating signals for tumor-inltrating T cells. Our results also lay the basis for the future clinical trials for the combination of IL7-loaded oncolytic adenovirus and CAR-T therapy for glioblastoma.

CAR-T cells with self-secreting cytokines, or taking use of auxiliary therapy to reverse the immunosuppressive tumor microenvironment [5,6].
Oncolytic virus is a promising therapy in cancer treatment, especially as an auxiliary for tumor immunotherapy [7]. Different from traditional chemotherapeutic drugs or targeted therapy, in addition to the direct tumor lysis e cacy, the virus ingredients were recognized by innate immune system as pathogen-associated molecular patterns (PAMPs), which combines with damage-associated molecular patterns (DAMPs) and tumor-speci c antigens released by infected tumor cells and leads to a strong immuno-activating effect similar in virus infection diseases [8]. Moreover, after modifying the oncolytic virus with coding sequence of gene-therapy agents, the tumor restricted replication of oncolytic virus results in local release of loaded products [9]. This property of oncolytic virus makes it a safe and effective tool for the delivery of a series of powerful immuno-activators which are usually accompanied by serious toxicity when applied systematically, such as different types of interlukins [10,11].
Interleukin-7 (IL7) is a cytokine produced by marrow stromal cells and thymic epithelial cells, and has been supposed to be one of the most important cytokines for the long-term survival of tumor-in ltrating T cells [12]. Several pre-clinical studies have tried to equipped tumor-in ltrating CAR-T cells with IL7, such as modifying CAR-T cells with auto-secreting IL7, or taking use of IL7 engineered mesenchymal stem cells to provide IL7 signal for CAR-T [13,14]. Here, we constructed an oncolytic adenovirus loaded with IL7 (oAD-IL7) and combined it with B7H3-targeting CAR-T cells to explore their joint e cacy for glioblastoma treatment. Our results showed that the oAD-IL7 improved the therapeutic e cacy of CAR-T cells for glioblastoma in vitro and in vivo by improving the survival and proliferation of tumor-in ltrating CAR-T cells.

Materials And Methods
Cells 293T, 293A, and 4 types of glioblastoma (GBM) cell lines, including U87, U251, A172, T98G, were purchased from ATCC. Another GBM cell line H4 is a kind gift from Sheng Zenghua in our laboratory.
293T, 293A, U87, U251, A172, H4 and T98G were maintained in DMEM (Gibco), supplemented with 10% FBS (Gemini), penicillin (100 U/ml) and streptomycin (100 µg/ml). For in vivo imaging, patient-derived glioblastoma tissue were dissolved using 1‰ collagenase (Solarbio), and then cultured in an incubator at 37℃. 6 days later primary glioblastoma cells were modi ed to express luciferase by lentivirus transduction, and named GBM19-LUCF after puromycin selection. Primary tumor samples obtained from patients with GBM and blood samples from GBM patients and healthy donors were also approved by West China Hospital of Sichuan University Biomedical Ethics Committee (ethical approval document 2018-061). Written informed consent was obtained from patients with GBM and healthy donors.

Adenovirus Construction
The schematic representation of oAD-IL7) and its blank counterpart are shown in Fig. 1. Both oncolytic adenovirus were based on ADMAX system, which consists of an E1, E3-deleted serotype 5 human adenovirus backbone, pBHglox△E1,E3, and a shuttle plasmid, pDC316-EGFP. A cDNA encoding human IL7 purchased from Sinobiological (Beijing, China) was cloned and then inserted the E1 region, and an E1A gene under the control of hTERT, followed by a E1B gene under the control of TATA box was synthesized by General Biosystems (Chuzhou, Anhui province, China) and inserted into the E3 region. The two types of viruses were rescued from 293A cells 7 days after transduction of the modi ed backbone and shuttle plasmids for further experiment by three consecutive freeze-thaw cycles. The titration was conducted using plaque assay. Brie y, monolayer 293A cells were infected by gradient-diluted viral solutions. 4 hours later, the infection media were replaced by overlay media consisting of DMEM (Gibco), 0.8% agarose (Sangon Biotech), and 2% FBS (Gemini). The GFP-positive plaques were counted using a uorescence microscope (Zeiss, AXIO observer Z1) 3 days later. Finally, the viruses were puri ed by cesium chloride gradient centrifugation.

T-cell Generation
Primary lymphocytes were kindly donated by Dr. Zhang in our laboratory, and were isolated by gradient centrifugation at a speed of 800 g for 15 min. Puri ed T cells were then cultured in 24-well plate in X-vivo media (Lonza) with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin at a density of 1 × 106 cells/ml in a 37 °C incubator with an atmosphere containing 5% CO 2 . 100 U/ml interlukin 2 (Life Science), 5 ng/ml IL15 (Miltenyi), and CD3/CD28 T cell Transact (Miltenyi) were added for T cell activation and sustaining. 72 hours later, the activated T cells were transduced by lentivirus at an MOI of 10, with the assistance of 10 µg/well coated retronectin (Takara) in the presence of IL-2. After 12 hours incubation, the T cells were collected and maintained in daily refreshed media (X-vivo, Lonza) with 100 U/ml IL-2, until the following experiment 6-8 days later. The titration of the lentivirus is conducted using TCID50 assay.

Immuno uorescent Staining and Western Blot
Immuno uorescent staining and western blot analysis were conducted as previously described [15]. For western blot, 1 × 10 6 /well U87 cells were incubated in 6-well plate for 12 hours. Then 1 × 10 7 oncolytic adenovirus were added to each well. The cells were collected 24, 48 and 72 hours after viral infection and lysed for protein extraction and further experiment. For immuno uorescent staining, 1 × 10 5 /well GBM cells were incubated in 24-well plate with poly-L-lysine-treated coverlips for 12 hours, and then adenoviral infection was performed at an MOI of 1. The specimens were incubated in 4% paraformaldehyde followed by 0.1% Triton X-100 incubation for 20 min, and then stained with anti-IL7 and DAPI (Beyotime). The images were captured by confocal microscopy (Zeiss, AXIO observer Z1).
T-cell Activation, Cytokine Secretion and Cytotoxicity 2 × 10 5 U87 cells were seeded in 24-well plate overnight and cocultured with CAR-T cells for 48 hours. For activation analysis, T cells were collected and stained with CD3, CD4, CD8, CD25, and CD69 for ow cytometry. For cytokine secretion analysis, supernatants were harvested for TNF-α and IFN-γ quantitation by Elisa (R D). For cytotoxicity assessment, the U87 cells were incubated in culture media with 0.5 mg/ml MTT reagent (Sangon, E606334-0500) for 4 hours at 37℃. Then the media was carefully aspirated and 100 µl Formazan solubilization solution was added. 10 minutes later, the cytotoxicity was measured according to the absorbance at 570 nm.
CytoTel Blue Proliferation Assay 2 × 10 5 U87 cells were incubated in 24-well plate overnight, and then viral infection was conducted at an MOI of 1. 24 hours later the U87 cells were cocultured with 1 × 10 6 T cells for 3 days, and then the T cells were collected for another round of proliferation assay until the day of 7. T cells were labeled with CytoTel Blue (AAT Bioquest) according to manufacturer's instruction before coculturing. At day 3, 5, and 7, the T cells were collected for proliferation analysis by using ACEA NovoCyte (Agilent Biosciences).

Apoptosis Analysis
On the day 7 of coculture, Cells were collected and stained with Annexin V and 7-AAD and analyzed by ow cytometry. Anti-CD3 uorescent-conjugated antibody (Biolegend) was added to separate the T cells and U87 cells.

In Vivo Experiment
All animal experiments followed a protocol approved by the Institutional Animal Care and Use Committee of Sichuan University. All xenograft models were constructed based on six to eight weeks old NCG mice, which were purchased from Gempharmatech and were bred in the animal vivarium at the State Key Laboratory of Biotherapy, Sichuan University in a pathogen-free condition.

Xenograft Models
Xenograft models were established by intracranial injection of 2 × 10 5 GBM-LUCF cells suspended in 5 µl PBS solution for each mouse into the right frontal lobe of the brain located in 2 mm lateral and 1 mm anterior to bregma at a depth of 3 mm. 7 days later, xenografts were con rmed by bioluminescent imaging, and treated with an intratumoral injection of 1 × 10 8 pfu oAD-IL7 or oAD respectively into the same position aforementioned. B7H3-CART cells were intravenously administrated 2 days after viral injection. Tumors were captured twice a week by bioluminescent imaging, and the bioluminescent signals for each group were recorded using the IVIS system (Caliper Life Sciences). Mouse death served as the endpoint of the experiment. Bioluminescent Imaging GBM19 cells expressing luciferase were used to detect tumor growth during in vivo experiment. The mice were treated with intraperitoneal injection of 150 mg/kg D-Luciferin (Beyotime) dissolved in PBS.
Bioluminescent imaging was performed using the IVIS system (Caliper Life Sciences) 10 minutes after D-Luciferin injection, and the radiance of tumor region was recorded for each mouse.

Ex Vivo Experiment
Mice underwent delayed treatment were killed and the tumors were harvested 7 days after B7H3-CART administration. For H E stain, the specimens were xed in 4% formaldehyde for 24 hours. For cytometry analysis, the tumors were incubated in 1‰ collagenase for 1 hour and washed by PBS twice.

Statistics
During in vitro assay, three wells were set as a group, and the experiments were repeated for twice. Signi cance was determined by two-sided Student's unpaired t-tests. The survival curve was obtained by Kaplan-Meier plot and a two-sided log rank test was applied for mouse survival test. P < 0.05 was considered as signi cant (*p < 0.05, **p < 0.01, ***p < 0.001). All the statistics were performed using GraphPad Prism v6.04.

Modi ed oncolytic adenovirus successfully propagates in GBM cells and produces IL7
We generated a type V oncolytic adenovirus with a E1A gene under the control of an hTERT promoter and the coding sequence of IL7, together with its blank control ( Figure.1a) [16]. oAD-IL7 and blank oAD successfully infected and replicated in all the 5 types of GBM cell lines (Fig. 1b), during which the IL7carrying oncolytic virus presented similar replication capability compared with its blank control (supplementary Fig. 1a-e). We next titrated the exact virus copies by plaque assay for consecutive collection of infected GBM cells, in which the viral replication grew fastest in U87 and T98G while much attenuated in U251 (Fig. 1c). The cytotoxicity of oAD-IL7 was con rmed by the increased cleaved caspase-3 in infected tumor cells, which was parallel to the viral replication as presented by accumulated E1A protein (Fig. 1d). To con rm the expression of IL7 by infected GBM cells, immuno uorescence was performed 72 hours after the viral infection to capture the secretion of IL7 (Fig. 1e). Further, the soluble IL7 in the collected supernatant was quanti ed by Elisa assay (Fig. 1f).
B7H3-CART exerts selective cytotoxicity to B7H3-positive GBM cells B7H3 is a promising target for glioblastoma treatment, which is found to prevail on nearly 70% of the tumor tissue removed from patients. In the previous research, we constructed a B7H3-targeting CART, which was equipped with a B7H3-targeted chimeric-antigen-receptor (CAR) consisting of a JB7H3-scFv, a CD8α transmembrane domain, and a 4-1BB signaling intracellular domain (Fig. 2a, Supplementary table  1). This CART product has been proved to be effective and safe in our previous research for a series of B7H3 positive tumors. To measure the transduction e ciency of CART cells, we inserted a coding sequence of the extracellular domain of CD19 acting as a marker for ow cytometry analysis (Fig. 2b). The level of B7H3 expression varied among the 5 types of GBM cell lines, with the highest on A172 (Fig. 2c). To test the function of the B7H3-CART, we cocultured the CART cells with the GBM cells at an E:T rates of 1:1 and 1:5. After 48 hours coculturing, for CART cells, the activation of T cells was con rmed according to the upregulation of CD25 and CD69 measured by ow cytometry, together with elevated IFNγ and TNF-α levels detected by Elisa assay (Fig. 2d, e). For glioblastoma cells, MTT assay was performed to assess the cytotoxicity of the constructed B7H3-CART to the glioblastoma cell lines. (Fig. 2f) oAD-IL7 enhanced the survival and e cacy of B7H3-CART We hypothesized that the oncolytic virus might act as an ideal carrier for the IL7 to support the survival and e cacy of tumor-in ltrating CART cells. To test this, we conduct a serial coculture for untransduced T cells and CART cells with oAD or oAD-IL7 infected U87 cells for 7 days in total (Fig. 3a). The CART cells were stained with CytoTell Blue, and then cocultured with virus-infected U87 cells that were refreshed on day 4. According to cell number recorded for each day, the CAR-T cells exposed to oAD-IL7 outperformed their counterparts, especially in the second round of coculture that started at day 4 ( Figure.3b). We next assessed the promotion of oAD-IL7 to the proliferation and survival of CAR-T cells during coculture separately. For proliferation monitoring, we recorded the signals of CytoTell Blue by ow cytometry on day 3 and 7, and found that the CAR-T cells underwent increased division when exposed to oAD-IL7 ( Figure.3c). To assess the promotion of oAD-IL7 to the survival of CAR-T cells, we collected the CAR-T cells at the end of day 7, and performed an Annexin V /7-AAD staining, in which the CAR-T cells exposed to oAD-IL7 presented much lower rate of Annexin V + /7-AAD + , and higher rate of Annexin V − /7-AAD − staining, compared to the T cells cocultured with blank oAD infected U87 cells ( Figure.3d). Finally, the tumor cells alive after the second round of serial coculture were calculated by MTT to measure the in vitro improved long-term e cacy of CAR-T when exposed to oAD-IL7 ( Figure.3e, Supplementary Fig. 2). Combination of oAD-IL7 and B7H3-CART improved the antitumor effect in vivo We next combined the use of oAD-IL7 and B7H3-CART in GBM xenograft models to test their joint e cacy in vivo. A total of 15 mice were divided into 3 groups, and were separately treated with oAD-IL7, B7H3-CART, and both of them. To construct the GBM xenograft models, we modi ed a patient-derived GBM cells with luciferase gene by lentivirus transduction (GBM-LUCF), and inoculated 2 × 10 5 GBM-LUCF cells in each NOD prkdc-/-IL-2Rg-/-(NCG) mice by intracranial injection after 10 days culturing. Treatment started on day 7 after inoculation, with an intracranial injection of 1 × 10 8 PFU oncolytic adenovirus, and a dose of systematically applied 1 × 10 6 CART cells 2 days later (Fig. 4a). We monitored the growth of the tumors by in vivo imaging system twice a week, and the intensity of the bioluminescence signal for each mouse was recorded (Fig. 4b). In a parallel experiment, the tumors were removed from the xenograft models on day 30. Different from the in vitro results, the oAD-IL7 presented a comparatively weak therapeutic e cacy in vivo as the tumor-bearing mice did not show signi cant tumor relief after the orthotopic injection of oAD-IL7 (Fig. 4d). Meanwhile, the single use of CAR-T therapy led to a moderate but transient tumor reduction according to the radiance intensity, while the tumor soon relapsed since day 14, and nally led to the death of all the tumor-bearing mice (Fig. 4e). Finally, the combination of oAD-IL7 and B7H3-CART presented signi cantly improved therapeutic e cacy, as the tumor-bearing mice got a consistent tumor regression, and 4 out of 5 mice achieved long-term survival by the end of observation, day 60 (Fig. 4f,g). These results showed that the single use of either oncolytic adenovirus or CART only brought limited therapeutic e cacy to tumor treatment, and only the combined use of both of them induced a long-term and thorough anti-tumor effect. oAD-IL7 prolonged the survival of tumor in ltrating CART cells in xenograft models To certify the improved persistence and proliferation of the CAR-T cells when used together with oAD-IL7, we next conducted another set of xenograft model and delayed the treatment for the tumor-bearing mice to monitor the contributive effect of oAD-IL7 to the B7H3-CART. For mice receiving combined therapies, the local injection of oncolytic virus was conducted on day 21 after inoculation, with CART cells injected 3 days later for all mice. This delayed treatment leaded to reduced and incomplete tumor relieves. To evaluate the promotive e cacy of oAD-IL7 for in ltrating CART cells, tumors were captured using MRI, in which the tumor-bearing mice treated with combined therapy showed signi cantly improved tumor relief compared to their counterparts treated with single CAR-T therapy (Fig. 5a), and then removed from the mice for immunohistology stain, virus detection, and ow cytometry analysis 7 days after CART cell injection (Fig. 5b,c,d, supplementary Fig. 3). The number of the tumor-in ltrating CART was measured by ow cytometry according to the CD3 signal, in which the combined therapy led to a signi cant increase by approximately 4 times (19.30% vs 5.23%) as much as the single use of B7H3-CART (Fig. 5d,e). Moreover, these tumor-in ltrating CAR-T cells presented improved activation and proliferation according to the elevated PD1, LAG-3, and ki67 expression (Fig. 5f,g). These results con rmed that the use of oAD-IL7 indeed improved the persistence of tumor-in ltrating CAR-T cells, which nally led to elevated therapeutic e cacy of CAR-T against solid tumor.

Discussion
Apart from the activation of CD3 and CD28 pathway, the stimulation from interleukins have long been regarded as the third necessary signal for T cell homeostasis [17]. Considering the supplement of interleukins led to the prolonged survival of T cells during the in-vitro T cell cultivation, in recent years there were a series of attempts to provide tumor-in ltrating CAR-T with different kinds of interleukins to prolong the persistence and therapeutic e cacy of the CAR-T against solid tumor [6,18]. In this research, we constructed an IL7 carrying oncolytic virus and a B7H3-targeting CAR-T therapy, and veri ed the promotive e cacy of oAD-IL7 for B7H3-CART in an orthotopic glioblastoma model. Our results showed that oAD-IL7 promoted the proliferation and survival of B7H3-CART both in vitro and in vivo, and induced an improved anti-tumor e cacy compared with single B7H3-CART therapy according to the prolonged survival of mice undergoing combined therapy.
We constructed the oAD-IL7 in which the E1A gene was under the control of an hTERT promoter, and examined the anti-tumor e cacy of the constructed oAD-IL7 both in vitro and, as a control group, in vivo. Oncolytic adenovirus is a promising therapy for the treatment of solid tumor [19]. Compared with other types of oncolytic virus, its large capacity makes it an ideal vector to carry therapeutic agents [20]. However, the receptor of adenovirus, coxsackievirus-adenovirus receptor, is usually downregulated on the membrane of tumor cells, leading to reduced viral replication and infectivity for tumor tissue as con rmed in previous researches, which restrain its utility as monotherapy [21]. According to our results, the oAD-IL7 successfully infected all of the glioblastoma cell lines and resulted in the apoptosis of tumor cells in vitro, but failed to induce a signi cant and persistent anti-tumor e cacy when used alone in xenograft models as the mice treated with oAD-IL7 did not present signi cantly prolonged survival. This limited anti-tumor e cacy of oAD-IL7 may be attributed to the tumor heterogeneity which leads to the down-regulation of Coxsackievirus-Adenovirus receptor, and the innate anti-virus immunity of the mice which leads to increased viral eradication and reduced viral infection in tumor.
We constructed a B7H3-targeted CAR-T, and con rmed that the B7H3-CART underwent improved proliferation and survival in vitro with the existence of oAD-IL7. B7H3 is a promising target discovered in recent years prevailing in different types of tumors [22,23]. During our previous research, we certi ed the therapeutic e cacy of B7H3-targeted CAR-T in treating glioblastoma and hematological cancers [24]. Taking use of improved transduction protocols, we elevated the transduction e ciency of T cells to over 60% CAR + . When used with oAD-IL7, the enhanced B7H3-CART performance were attributed, at least partly, to the IL7 released from the oAD-IL7-infected tumor cells. The B7H3-CART in combined group didn't outperform the CAR-T used as single therapy on the third day of cocultivation, but at the end of the second round of cocultivation, the day 7, the former presented signi cantly improved proliferation according to the CytoTell analysis. This may be explained by the fact that CD3 and CD28 signals indeed induce a thorough activation of T cells, and IL7 is related to the long-term persistence of CAR-T cells, and is corresponsive to the results of previous study of auto-secreting IL7 equipped CAR-T cells [14].
We further explored the combined therapeutic e cacy of oAD-IL7 and B7H3-CART in xenograft models and the outcomes were consistent with the in vitro coculture results. During our research, the single use of B7H3-CART induced a signi cant tumor regression, but the tumor eventually revived and resulting in the death of the mice during the course of the treatment. This limited therapeutic e cacy of CAR-T therapy for solid tumors has been widely reported in previous researches that the CAR-T cells loses their antitumor capability due to the progress of tumor heterogeneity and the immunosuppressive tumor microenvironment [25,26]. While compared to its counterpart, the combined therapy led to a complete eradication of the tumors, and 4 out of 5 of the treated mice remain survived by the end of the experiment. Besides, according to the results of delayed treatment, when used together with oAD-IL7 the tumor-in ltrating B7H3-CART outnumbered its counterpart by 4 times. We also observed that when used with oAD-IL7, the tumor in ltrating T cells presented elevated PD1 and LAG-3 expression. This result has also been observed in previous study, such as using IL12 loaded oncolytic vaccinia, IL2 loaded oncolytic adenovirus, or bispeci c antibody loaded oncolytic adenovirus [27][28][29]. Other than T cells exhaustion, this result has been widely reported and was regarded as a sign of increased activation of tumorin ltrating T cells. Finally, the improved proliferation of tumor-in ltrating T cells was indicated by elevated ki67 expression. All of these results explained that the improved therapeutic e cacy of the combined use of oAD-IL7 and B7H3-CART were to some extent led by the improved activation and proliferation of the tumor-in ltrating T cells, but not simply because of the dual cytotoxicity of the oAD-IL7 and B7H3-CART.
To provide tumor-in ltrating CAR-T cells with different types of interleukins is an effective way to improve the therapeutic e cacy of CAR-T against solid tumor. During previous research, CAR-T cells equipped with auto-secreted IL7, or IL7R were proved to be signi cantly improved for the treatment of solid tumor [13,14]. However, when T cells are equipped with such consistent growth factor signal, there is a risk for the T cell to undergo malignant transformation, or intensi ed off-target side effect due to its overproliferation [30]. Meanwhile, the use of oncolytic virus has been proved to be a safe and effective way to carry immuno-activating agents into solid tumor, such as bi-speci c antibody, immune-checkpoint inhibitor, and interleukins. In our study, we used an oncolytic adenovirus to ensure that the IL7 mainly distributed in the tumor microenvironment, which also avoid extra manipulation of T cells. However, there are also two major limitations of our study. The rst is that the E1A gene of our oncolytic adenovirus is under the control of an hTERT promoter, which hinders the oAD-IL7 from infecting mouse-derived glioblastoma cells, such as GL261. The second one is that our B7H3-CART is based on a humanized anti-B7H3 scfv, which can only induce cytotoxicity to human-derived cancer cell lines. Both of these two limitations restrained our xenograft models to the immuno-de cient NCG mice, which failed to present intact immuno-activating pro le of the oAD-IL7, and prevented us from observing the bystander antitumor effect and the construction of long-term immune surveillance after the eradication of tumors reported during previous research.

Conclusion
To conclude, we have shown that the combined use of oAD-IL7 and B7H3-CART displayed synergistic antitumor effects in vitro and in vivo by enhancing T-cell persistence, which led to prolonged survival of the tumor-bearing mice. These data laid a solid foundation for the further clinical study of the combination therapy for glioblastoma treatment.

Declarations
Ethics approval and consent to participate: All animal experiments followed a protocol approved by the Institutional Animal Care and Use Committee of Sichuan University.

Patient Consent for publication:
Not required.
Availability of data and material: All of the data relevant to this research are included in the article and supplementary les. Materials are available from corresponding author under reasonable request.
Competing interests: All the authors declare no con ict of interest.  (C) Cells were collected 72 hours after infection for plaque assay. Each number of the virus tilter were calculated on average of three independent wells. (D) Oncolytic virus induced GBM apoptosis was con rmed by western blot. Scale bar, 100μm. (E) Cells were stained with IL7 antibody 72 hours after infection and imaged with a uorescence microscope. Green uorescence represents the oAD-IL7 infection; red uorescence represents the IL7 distribution; blue uorescence represents the cell nuclei stained with DAPI. Scale bar, 50nm. (F) Supernatants were collected for IL7 quanti cation. ***, P<0.001 (two-tailed paired t test). Figure 1 oAD-IL7 successfully replicate in glioma. (A) Schematic representation of genome of the oncolytic viruses used in this study. (B) Monolayers of glioblastoma cell lines were infected by oAD-IL7 at the MOI of 10.
Cells were imaged with a uorescence microscope 24, 48, and 72 hours after infection. Scale bar, 500μm. (C) Cells were collected 72 hours after infection for plaque assay. Each number of the virus tilter were calculated on average of three independent wells. (D) Oncolytic virus induced GBM apoptosis was con rmed by western blot. Scale bar, 100μm. (E) Cells were stained with IL7 antibody 72 hours after infection and imaged with a uorescence microscope. Green uorescence represents the oAD-IL7 infection; red uorescence represents the IL7 distribution; blue uorescence represents the cell nuclei stained with DAPI. Scale bar, 50nm. (F) Supernatants were collected for IL7 quanti cation. ***, P<0.001 (two-tailed paired t test).