IL-7 coupled with IL-12 increases intratumoral T cell clonality, leading to complete regression of non-immunogenic tumors

All recombinant vaccinia viruses used in this study were developed based on the attenuated vaccine strain, LC16mO, as described previously [15]. Briefly, LC16mO was modified with functional deletion of VGF and O1L and partial deletion of the B5R glycoprotein. DNA coding human IL-7 protein and DNA coding murine IL-12 protein were inserted into the VGF and O1L locus, respectively, using a plasmid containing the human IL-7 gene and a plasmid containing the murine IL-12 gene to generate hIL-7/mIL-12-VV. Similarly, a plasmid encoding human IL-7 protein and a plasmid encoding LacZ were used to generate hIL-7-VV; a plasmid encoding murine IL-12 protein and a plasmid encoding luciferase were used to generate mIL-12-VV; a plasmid encoding Discosoma sp. Red fluorescent protein (DsRed) and a plasmid encoding LacZ were used to generate Cont-VV. RK13 or A549 cells were used for virus propagation. Infected cells were collected, and viruses were purified by tangential flow filtration or density gradient ultracentrifugation using OptiPrep (Axis-Shield) according to the manufacturer’s protocol. Purified virus solution was titrated using a plaque assay in a monolayer culture of RK13 cells, in which the number of plaque-forming units (pfu) was determined. Virus solution was stored at  − 80 °C until use.

Male C57BL/6 mice purchased from Charles River Laboratories Japan, Inc. were maintained on a standard diet and water ad libitum throughout the experiments under specific pathogen-free conditions. Rabbit kidney RK13 cells, human lung carcinoma A549 cells and murine Lewis lung carcinoma (LLC) cells were purchased from American Type Culture Collection (ATCC). RK13 was cultured in Eagle’s minimum essential medium (ATCC) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/v) penicillin–streptomycin (Thermo Fisher Scientific). A549 was cultured in F-12 K medium (ATCC) or Dulbecco’ s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/v) penicillin–streptomycin. LLC was cultured in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/v) penicillin–streptomycin. LLC-derived dendritic cells (DCs) were cultured in RPMI1640 (Sigma-Aldrich) supplemented with 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/v) penicillin–streptomycin. Culture conditions were maintained in a humidified atmosphere with 5% CO₂ at 37 °C. Cells were tested as mycoplasma-free.

Mice were subcutaneously inoculated with LLC cells into the right flank. Tumor diameter was measured using a digital caliper, and tumor size was calculated using the following formula: length × width² × 0.52. After establishment of tumor burden, mice were randomly allocated to experimental groups such that mean tumor volume was similar among the groups, and intratumoral treatment with 30 μL of vehicle or virus suspension was started. Tumor size and body weight of individual mice were continuously monitored. Mice with signs of deterioration or acute weight loss were euthanized. Complete tumor regression was defined as complete tumor disappearance.

LLC tumors were dissected immediately after euthanasia by cervical dislocation under isoflurane anesthesia. Tumor tissues were minced with scissors and further mechanically dissociated using Tumor Dissociation Kit, mouse (Miltenyi Biotec) and GentleMACS (Miltenyi Biotec). Cells were passed through a nylon mesh filter and prepared for immunofluorescence staining and flow cytometry analysis. Spleens were minced with scissors and dissociated using BioMasher (Nippi, Japan), and red cells were subsequently lysed with ACK Lysing Buffer (Thermo Fisher Scientific). The following monoclonal antibodies were used for immunostaining: mouse CD4 PE-Cy7, mouse CD8a V500, mouse CD366 (Tim3) PE, mouse MHC Class II (I-A/I-E) PerCP-Cy5.5 (BD Biosciences); mouse CD127 (IL-7Rα) Brilliant Violet 510, mouse Interferon-γ PerCP-Cy5.5, mouse Granzyme B Alexa Fluor 647, mouse CD45 Alexa Fluor 647, mouse CD45R (B220) FITC, mouse CD11c PE, mouse CD103 PE-Cy7, mouse CD80 APC, and mouse CD279 (PD-1) PE-Cy7 (BioLegend). BD fixation/permeabilization kit (BD Biosciences) was used for intracellular staining. Zombie Aqua (BioLegend) or Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific) was used to distinguish live and dead cells. Samples were acquired on MACSQuant Analyzer 10 (Miltenyi Biotec) and analyzed using FlowJo Ver.10 (BD Biosciences).

LLC tumors (approximately 90 mm³) were treated with 30 μL of PBS or virus suspension every other day for a total three times. Three days after the last treatment, tumor samples were collected in ISOGEN (Nippongene) immediately after euthanasia by cervical dislocation under isoflurane anesthesia. Total RNA was extracted using linear acrylamide and isopropanol, hybridized with the PanCancer Mouse Immune Profiling panel (NanoString Technologies) and analyzed using the nCounter DX Analysis System (NanoString). Data were processed using nSolver Analysis Software and the Advanced Analysis module (NanoString).

LLC tumors in the right flank of mice were intratumorally treated with 30 μL of PBS or virus suspension every other day for a total of three times. Eleven or twelve days after the last treatment, spleens and tumors were dissected. Spleens were minced with scissors and further mechanically dissociated using Spleen Dissociation Kit, mouse (Miltenyi Biotec) and GentleMACS. After removal of dead cells using the Dead Cell Removal Kit (Miltenyi Biotec), splenic CD4⁺ and CD8⁺ T cells were isolated using CD4⁺ T Cell Isolation kit, mouse (Miltenyi Biotec), and CD8a⁺ T Cell Isolation Kit, mouse (Miltenyi Biotec), respectively, according to the manufacturer’s protocols. Likewise, tumors were dissociated using Tumor Dissociation kit, mouse and dead cells were removed. Intratumoral CD4⁺ and CD8⁺ T cells were isolated using CD4 (TIL) MicroBeads, mouse (Miltenyi Biotec) and CD8a⁺ T Cell Isolation Kit, mouse, respectively. Isolated immune cells were collected in ISOGEN. RNA extracted using the RNeasy Plus Universal Mini kit (QIAGEN) was reverse transcribed into complementary DNA (cDNA) using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). Subsequently, double-stranded cDNA was synthesized, and TCR-β-chain genes were amplified using adaptor ligation-mediated PCR [19]. High-throughput sequencing was performed using MiSeq Reagent Kit v3 (600 Cycle) (Illumina, San Diego, CA, USA) and MiSeq (Illumina). Reads with a quality value (QV) score ≥ 20 were used for analyses [20]. TRBV and TRBJ segments in TCR genes were assigned based on the international ImMunoGeneTics information system (IMGT) database. A unique sequence read was defined as a sequence read having no identity in TRBV, TRBJ and deduced amino acid sequence of TCR-β-chain CDR3 with the other sequence reads. Data processing including calculation of the inverse Simpson's diversity index was performed using a repertoire analysis software (Repertoire Genesis, Osaka, Japan).

C57BL/6 mice that had been previously cured of subcutaneous LLC tumors by treatment with a mixture of hIL-7-VV and mIL-12-VV were subcutaneously inoculated with 4 × 10⁵ LLC cells 74 days after the last viral treatment. Growth of rechallenged tumors was continuously monitored. Age-matched treatment-naïve mice were used as a control group.

For RNA analysis of Cxcl3, Ccl3 and Clec4n in LLC-derived DCs, CD11c⁺ cells were isolated from mIL-12-VV-treated LLC tumors using EasySep Mouse CD11c Positive Selection Kit II (STEMCELL Technologies). Cells were plated at 5 × 10⁴ cells per well in a 96-well plate with serially diluted recombinant human IL-7 (PEPROTECH) or 1 μg/mL LPS (Sigma-Aldrich) and incubated for 5 h. Subsequently, total RNA was extracted using the RNeasy Plus Micro Kit (QIAGEN), and RNA was converted to cDNA using SuperScript IV VILO Master Mix (Thermo Fisher Scientific). Real-time PCR was performed using the TaqMan Gene Expression Master Mix and specific primers and probes from TaqMan Gene Expression Assays (Thermo Fisher Scientific). The mRNA expression level of each gene relative to β-actin was calculated using the ΔΔCt method.

First, we evaluated the antitumor activity of a recombinant oncolytic vaccinia virus carrying transgenes to express human IL-7 and murine IL-12 (hIL-7/mIL-12-VV) and compared it to a virus expressing human IL-7 (hIL-7-VV; human IL-7 is reactive for mouse immune cells [21]), murine IL-12 (mIL-12-VV) or neither cytokine (Cont-VV) (Supplementary Fig 1), against LLC tumors, which are known to be poorly immunogenic [22, 23]. Significant antitumor efficacy was observed in all viral treatment groups (p < 0.001 for all groups) on Day 17, with three of eleven mice treated with hIL-7/mIL-12-VV achieving CR on Day 28 compared to none in the other groups (Fig. 1a, b), which is consistent with our previous study [15]. Next, we examined the activation status of intratumoral immune cells. Whereas the number of total CD8⁺ T cells tended to be higher in tumors treated with hIL-7/mIL-12-VV than mIL-12-VV, the number of activated CD8⁺ T cells expressing granzyme B or IFNγ was significantly higher in tumors treated with hIL-7/mIL-12-VV (Fig. 1c, Supplementary Fig 2). There was no apparent difference in the number of PD-1⁺Tim3⁺ exhausted CD8⁺ T cells between tumors treated with hIL-7/mIL-12-VV and mIL-12-VV (Fig. 1d, Supplementary Fig 2). Furthermore, we performed multiplexed gene expression analysis of tumors treated with PBS, mIL-12-VV or hIL-7/mIL-12-VV using the NanoString nCounter system (NanoString Technologies). Treatment with hIL-7/mIL-12-VV upregulated various immune pathways, which were similarly upregulated following treatment with mIL-12-VV (Fig. 1e). Direct comparison of hIL-7/mIL-12-VV-treated tumors and mIL-12-VV-treated tumors showed significant changes in gene expression related to antigen-presenting cells (APCs): Clec4n, a C-type lectin receptor on dendritic cells (DCs) [24], and Cxcl3 and Ccl3, chemokines released from DCs and macrophages [25, 26] were upregulated, while Axl, a receptor tyrosine kinase which negatively regulates DCs to result in T cell inhibition [27], was downregulated in hIL-7/mIL-12-VV-treated tumors (Table 1). Little to no IL-7Rα expression was observed in intratumoral DCs regardless of the presence or absence of IL-12. Further, no upregulation of Cxcl3, Ccl3 or Clec4n was observed in isolated DCs following stimulation with IL-7 (Supplementary Fig 3), suggesting that IL-7 does not act directly on DCs to upregulate these genes. The number of tumor-infiltrating DCs, mature DCs expressing major histocompatibility complex (MHC) class II and activated CD103⁺ DCs with high expression of CD80 was identical between hIL-7/mIL-12-VV- and mIL-12-VV-treated tumors (Supplementary Fig 4).

Despite the minor molecular differences related to APCs between tumors treated with hIL-7/mIL-12-VV and mIL-12-VV, we hypothesized that this difference may have a lasting effect on intratumoral T cells, resulting in differences in antitumor efficacy. We isolated CD8⁺ and CD4⁺ T cells from tumors treated with PBS, Cont-VV, hIL-7-VV, mIL-12-VV or hIL-7/mIL-12-VV, sequenced the complementarity determining region (CDR) 3 of TCRβ, calculated the frequency of unique clones in each sample and ranked the top 50 clones from highest to lowest frequency (Fig. 2a, d). With respect to intratumoral CD8⁺ T cells, high-frequency (> 10%) clones were observed in seven out of nine mice treated with hIL-7/mIL-12-VV, compared to a lower proportion of mice treated with PBS, Cont-VV, hIL-7-VV and mIL-12-VV (two of five, one of five, none of four, and two of eight, respectively) (Fig. 2b). Furthermore, five out of nine mice treated with hIL-7/mIL-12-VV had multiple high-frequency clones, compared to only one out of eight mice treated with mIL-12-VV (Fig. 2b), resulting in higher cumulative frequencies of the top 3 clones in hIL-7/mIL-12-VV-treated mice than those in mIL-12-VV-treated mice (Fig. 2c). Likewise, high-frequency CD4⁺ T cell clones were observed in four out of nine mice treated with hIL-7/mIL-12-VV, compared to only two out of eight mice treated with mIL-12-VV and no mice treated with PBS, Cont-VV or hIL-7-VV (Fig. 2e). Four out of nine mice treated with hIL-7/mIL-12-VV, compared to no mice in the other experimental groups, had multiple high-frequency clones (Fig. 2e). Cumulative frequencies of the top 3 CD4⁺ T cell clones in hIL-7/mIL-12-VV-treated mice were expectedly higher than those in mice in the other groups (Fig. 2f). Next, to analyze the overall clonal distribution, we plotted the cumulative frequencies of the top 50 clones in each animal. The group median distribution of intratumoral CD8⁺ T cell clones revealed that Cont-VV induced minor changes, while hIL-7-VV comparatively increased diversity and mIL-12-VV increased clonality (Fig. 3a). Strikingly, hIL-7/mIL-12-VV increased the clonality of intratumoral CD8⁺ T cells and a higher proportion of mice (six out of nine) showed cumulative frequencies greater than 80% for the top 50 clones compared to the other groups, including mIL-12-VV (Fig. 3b). Changes in intratumoral CD8⁺ T cell diversity were also supported by trends observed in the inverse Simpson's diversity index (Supplementary Fig. 5). The effect of these viruses on intratumoral CD4⁺ T cell clonality differed from that on CD8⁺ T cells: Cont-VV and hIL-7-VV increased diversity, while mIL-12-VV most markedly increased clonality compared to hIL-7/mIL-12-VV (Fig. 3c, d, Supplementary Fig. 6). These results indicate completely different effects of IL-7 on CD8⁺ T cell diversity with and without IL-12.

We previously reported that intratumoral expression of IL-7 and IL-12 mediated by an oncolytic vaccinia virus led to the establishment of antitumor memory, resulting in rejection of rechallenged tumors [15]. In this study, we again demonstrated that mice treated with hIL-7-VV in combination with mIL-12-VV more than 90 days prior rejected the rechallenged LLC tumors (Supplementary Fig. 7), suggesting that T cell clones reactive to LLC tumors may migrate to, and be stored in, secondary organs. We examined splenic T cells and assessed whether changes in the intratumoral TCR repertoire after viral treatment systemically affects T cell clonality. The diversity of the TCR repertoire of CD8⁺ and CD4⁺ T cells in the spleen was much higher than in tumors (Fig. 4a, d). Compared to observing little to no CD8⁺ T cell clones that were identified in tumors in the spleen of mice treated with PBS, we found T cell clones with the same CDR3 sequence in the spleen of animals treated with Cont-VV, hIL-7-VV, mIL-12-VV and hIL-7/mIL-12-VV, indicating migration of intratumoral CD8⁺ T cells into secondary lymphoid organs, as expected (Table 2). The high cumulative frequency of the top 5 splenic clones in mIL-12-VV-treated and hIL-7/mIL-12-VV-treated mice (Fig. 4b) is consistent with the stronger antitumor efficacy of mIL-12-VV and hIL-7/mIL-12-VV compared to Cont-VV or hIL-7-VV. However, the cumulative frequency and overall clonality calculated for the top 50 clones were higher in mIL-12-VV-treated mice than hIL-7/mIL-12-VV-treated mice, which is in contrast to the proportion of mice that achieved CR (Fig. 4c). High-frequency T cell clones found in the spleen were not always highly frequent in tumors in the treatment groups. Furthermore, some T cell clones that were identified among the top 3 high-frequency clones in tumors were absent from the spleen. Unlike CD8⁺ T cells, there were no obvious differences in the cumulative frequency of the top 5 clones or the overall clonality of CD4⁺ T cells in the spleen among treatment groups (Fig. 4e, f, and Supplementary Table 1).