The L929 murine fibroblast cell line was purchased from American Type Culture Collection and was cultured in medium consisting of DMEM (Gibco, Karlsruhe, Germany) supplemented with 2 mM glutamine (Sigma-Aldrich, Taufkirchen, Germany), 100 IU penicillin, 100 µg/mL streptomycin (Gibco), and 10% (v/v) fetal bovine serum (FBS; Gibco) in a humidified incubator at 37°C and 5% CO₂. B16 melanoma expressing ovalbumin (B16-OVA) cells were provided by Dr. Lieping Chen (Yale University, New Haven, Connecticut, USA) and Lewis lung carcinoma (LLC)-OVA were provided by Dr. Daniel Ajona (Cima Universidad de Navarra, Pamplona, Spain). These cells were cultured in medium RPMI 1640 (Gibco) supplemented like DMEM, but with addition of 50 µM β-mercaptoethanol (Sigma-Aldrich) and geneticin G418 400 µg/mL (InvivoGen, San Diego, California, USA).

MVA-BN was developed by Bavarian Nordic and is deposited at the European Collection of Cell Cultures (V00083008). All recombinants were generated from a cloned version of MVA-BN in a bacterial artificial chromosome. Infectious viruses were reconstituted from bacterial artificial chromosomes by transfecting bacterial artificial chromosome DNA into BHK-21 cells and superinfecting them with Shope fibroma virus as helper virus. After three additional passages of primary embryo fibroblasts, helper virus-free MVA recombinant viruses were obtained. All viruses used in animal experiments were purified twice through a sucrose cushion. Mouse IFN Alpha 1 protein was obtained from PBL Assay Science (Catalog No. 12 105–1; Piscataway, New Jersey, USA). Simvastatin (PHR1438-1G) and atorvastatin calcium (PHR1422-1G) were purchased from Sigma-Aldrich and were reconstituted in methanol. Bovine serum albumin (BSA)-fluorescein 5 (6)-isothiocyanate (FITC) was acquired from Sigma Chemical Co. (St. Louis, Missouri, USA). Trypan blue was obtained from Sigma-Aldrich.

Single-cell suspension of the indicated organs collected at the indicated times were stained as previously described.26 Fluorescence minus one or biological comparison controls were used for cell analysis.27 The following antibodies were used and acquired from BioLegend (San Diego, California, USA): PE anti-mouse IFNAR1 (MAR1-5A3), PE Mouse IgG₁ (MOPC-21), Zombie NIR, Brilliant Violet 650 anti-mouse CD19 (6D5), Brilliant Violet 510 anti-mouse CD45 (30-F11), Brilliant Violet 605 anti-mouse TCR β chain (H57-597), PERCPCy5.5 CD44 (IM7), APC CD62L (MEL-14), AF700 Ki67 (16A8), PE-Dazzel 594 Tim3 (B8.2C12), BV785 PD1 (29F.1A12), 7-AAD Viability Staining Solution. These were acquired from BD Biosciences: BUV395 Rat anti-mouse CD8a (53–6.7), BUV496 Rat anti-mouse CD4 (GK1.5), Purified Rat anti-mouse CD16/CD32 antibody (Mouse BD Fc Block), FITC Rat anti-mouse CD4 (RM4-4). These were acquired from eBioscience: PECy7 Tbet (eBio4B10), eFluor 450 Eomes (Dan11mag). PE-conjugated H-2K^b/OVA (257–264 complex was obtained from MBL (Nagoya, Japan). For evaluation of antigen presentation in DCs, CD11c⁺ cells from spleen were isolated using CD11c-coated magnetic beads (Miltenyi Biotec, Auburn, California, USA) and stained with Zombie NIR, Pacific Blue CD45 (RA3-6B2), BV605 CD11c (HL3) and PE OVA(257-264) (SIINFEKL) peptide bound to H-2Kb (25D1). Flow cytometry analysis was performed using a FACS Canto II flow cytometer (BD Bioscience) and a CytoFLEX LX apparatus (Beckman Coulter, Miami, Florida, USA). Data analysis was performed using FlowJo software (TreeStar).

RNA was isolated from the studied cell lines and tissues using the Maxwell 16 Total RNA Purification Kit (Promega, Madison, Wisconsin, USA), quantified in a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, Delaware, USA), and retrotranscribed (300 ng) to cDNA with Moloney murine leukemia virus reverse transcriptase from Promega, according to the manufacturer’s instructions.

Quantitative real-time PCR was performed with iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) using specific primers for each gene; they were purchased from Invitrogen (Thermo-FisherScientific, USA). As ribosomal protein lateral stalk subunit P0 (PRLP0) mRNA levels remained constant across different experimental conditions, this parameter was used to standardize gene expression. PRLP0 5’-aacatctcccccttctcctt-3’ 5’-gaaggccttgaccttttcag-3’; ubiquitin-specific peptidase 18 (USP18) sense 5’-ccaaaccttgaccattcacc-3’ USP18 as 5’-atgaccaaagtcagcccatcc-3’; interferon-stimulated genes gene 15 (ISG15) sense 5’-gattgcccagaagattggtg-3’ ISG15 as 5’-tctgcgtcagaaagacctca-3’; 2'-5'-oligoadenylate synthetase (2,5 OAS) sense 5’-actgtctgaagcagattgcg-3’ 2,5 OAS as 5’-tggaactgttggaagcagtc-3’; and the open reading frame 082 L of MVA as target for detection of MVA backbone DNA, MVA082L sense 5’-acgtttagccgcctttaatagag-3’ 2 MVA082L as 5’-tggtcagaactatcgtcgttgg-3’ .The amount of each transcript was expressed by the formula 2ΔCt, with Ct being the point at which the fluorescence rises significantly above background levels.

First, L929 cells (3×10⁴/ well) were seeded in 96-well plates in triplicate in DMEM 10% FBS culture media. After 24 hours, they were washed with phosphate-buffered saline (PBS) and treated for 3 hours with decreasing dilutions of the studied drugs or vehicle on 150 µL of DMEM without FBS. Simvastatin and atorvastatin two-fold dilutions curves were made from 600 µM (atorvastatin) and 50 µM (simvastatin) until 6.5 µM from a 10 mM stock. Then, cells were detached with 50 µL of trypsin-EDTA (Gibco), washed and stained for IFNAR1 detection with 50 µL of a cocktail of PE anti-mouse IFNAR1 (1:200), Zombie NIR (1:1000) and BD Fc Block (1:200) for 20 min at 4°C. The normalized geometric mean was calculated considering as 100% the geometric mean of the cells stained with the anti-IFNAR1 monoclonal antibody (mAb) and as 0% the geometric mean of the cells with isotype control. For the BSA-FITC endocytosis assay, cells were treated with atorvastatin (300 µM), simvastatin (37.5 µM) or vehicle methanol under the same conditions described above, but after 3 hours of incubation, they were washed with PBS and were incubated for 3 hours with DMEM 10% FBS. Then, the culture medium was removed and replaced by 150 µL of DMEM with BSA FITC (100 µg/mL), and cells were incubated for 1 hour for BSA endocytosis. After the last treatment, cells were detached, washed and stained with 7-AAD (1:75) in 50 µL of PBS for 15 min. Finally, they were resuspended in 200 µL of PBS with trypan blue (250 µL/mL) for cytometric analysis.

L929 cells (8×10⁴/ well) were seeded in 24-well plates in triplicate in DMEM 10% FBS culture media. After 24 hours, they were washed with PBS and treated for 3 hours with atorvastatin (300 µM), simvastatin (37.5 µM) or vehicle (methanol) in 250 µL of DMEM without FBS. Afterward, cells were washed with PBS and were kept for 0, 3, 6 and 9 hours with DMEM 10% FBS. Next, cells were stimulated with IFN alpha 1 protein (100 units/mL) for 3 hours in 250 µL of DMEM 10% FBS. Lastly, the culture medium was removed, and the cells collected for RNA extraction.

For Western blot analysis, L929 (1×10⁶/ well) cells were treated with atorvastatin (300 µM), simvastatin (37.5 µM) or vehicle (methanol) in 6-well plates with 2 mL DMEM culture media for 3 hours. Later, the supernatant was eliminated and IFNα alone (3000 units/ml) was added for 30 min and then Western blotting was accomplished using a rabbit polyclonal antibody to β-Actin (A2066, Sigma, Stockholm, Sweden), a rabbit monoclonal antibody to phopho-STAT1 (Tyr701) (58D6, Cell Signaling Technology, Danvers, MA) and a rabbit polyclonal antibody to STAT1 (ref. 9172, Cell Signaling Technology, Danvers, Massachusetts, USA).

For in vitro infection with MVA, L929 cells (6×10⁵) were seeded into 6-well plates. Following overnight incubation, cells were treated with atorvastatin (300 µM), simvastatin (37,5 µM) or vehicle (methanol) in 1 mL DMEM culture media for 3 hours. Supernatants were removed and cells were washed twice with PBS and were infected with MVA OVA at a multiplicity of infection of 5 in DMEM 2% FBS. The viral inoculum was removed 1-hour postinfection, cells were washed twice with PBS and replaced with fresh DMEM 2%. After 6 hours, cells were recollected to RNA analysis.

In vivo experiments were performed with 6 to 8 week-old female C57BL/6 mice purchased from Harlan Laboratories (Barcelona, Spain). The mice were kept under specific pathogen-free conditions.

2.5×10⁵ B16-OVA cells were inoculated subcutaneously on the right hind flank of C57BL/6 mice; the mice were randomized at day 7 and treated at days 7 and 14 after implantation. 20 µg of simvastatin per mouse were injected intraperitoneally or intra-muscularly in 100 µL of PBS. After 2 hours, the MVA vaccine was administered subcutaneously next to the tumor area (peritumorally) at a concentration of 5×10⁷ TCID₅₀ per mouse in 100 µL of PBS. In the case of intramuscular administration, the MVA vaccine was administered simultaneously. Tumor sizes were measured twice weekly using electronic calipers and mice were euthanized when tumors reached 17 mm on the longest axis. Experiments with MVA-gp70 were performed in C57BL/6 inoculated with 2×10⁶ LLC-OVA cells and treated at days 4 and 11.

The tumor model and treatment regimen were as in the antitumor activity assay but mice were depleted of CD4⁺ T cells using anti-mouse CD4 monoclonal antibody (clone GK1.5, BioXCell, L'Aigle, France), CD8⁺ T cells using anti-CD8β (clone H35-17.2, in house), NK cells using anti-mouse NK1.1 (clone PK136, BioXCell) or conventional CD4⁺CD25⁺Foxp3⁺ T regulatory cells using anti-CD25 (clone PC-61.5.3, BioXCell). InvivoMab rat IgG2b (clone LTF-2, BioXCell) was used as control. A total of 200 µg of each antibody per mouse were administered intraperitoneally 1 day before first therapeutic treatment administration and on days 2, 6, 9, and 13 after it to maintain immune cell depletion during the course of the experiment. B cells were depleted using 500 µg per mouse of InvivoMab anti-CD19 (clone BE0150, BioXCell) 1 day before first therapeutic treatment administration and on days +2, +7, +12, +17, +22 and +27. The effect of IL10 was neutralized using 250 µg/mice of InvivoMab anti-IL10R (CD210) (clone 1B1.3A, BioXCell) administered after the first therapeutic treatment administration and on days +7, and +14.

The tumor model was performed as in the antitumor activity assay, but, in this case, mice received a single dose of treatment. Tumor tissues, lymph nodes, and spleens were collected on day five after treatment and were processed for cytometric analysis as described in.28 Specific T-cell responses to OVA antigen were assessed ex vivo by a mouse IFN-γ Enzyme-linked Immunosorbent Spot (ELISpot) Assay kit (BD-Biosciences). Ninety-six-well Multiscreen IP Plates (Millipore, Bedford, Massachusetts, USA) were coated with 100 µL of assay diluent containing anti-IFN-γ monoclonal Ab and incubated overnight at 4°C. The plates were washed and then blocked with RPMI-1640 medium containing 10% FBS for 90 min at RT. Splenocytes depleted of erythrocytes were added to wells (4×10⁵) and stimulated with OVA (257-264) peptide (1 µg/mL) or 4×10⁴ irradiated (20 000 rads) B16-OVA, B16.F10 tumor cells in 200 µL/well. Prior to use, tumor cells as a stimulator were treated with 500 IU/mL of IFN-γ for 48 hours to increase MHC-I expression.

The tumor model was performed as in the antitumor activity assay, but, in this case, mice received a single dose of the treatment. Tumor tissues were collected in RNA homogenization buffer at 4 hours after treatment and were stored at - 80°C until they were processed.

RNA sequencing was performed by adapting the technology of SCRB-Seq29 to allow for the high cost-efficient multiplexed transcriptome characterization. Briefly, cells were collected in cell lysis buffer, and poly-(A)+RNA were purified using the Dynabeads mRNA DIRECT Purification Kit (ThermoFisher Scientific). poly-(A)+RNA were annealed to a custom primer containing a poly-(T) tract, a Unique Molecule Identifier (UMI), and a sample barcode. Retrotranscription using Template-switching oligonucleotides was then used to synthesize and amplify 3’ untranslated region (3’UTR) enriched cDNA, resulting in barcoded cDNA fragments. Library preparation was performed using the Nextera XT library preparation protocol, which introduces i5-P5 and i7-P7 structures for massive parallel sequencing. Quality control was performed following pre-amplification RT and library preparation to ensure quality and length accuracy and equilibrate sample pooling. Libraries were then circularized and sequenced using a DNBSeq-G400 sequencer (MGI), using the MGIEasy Circularization Kit (MGI). 5–10 million pair-end reads (100 bp) were sequenced for each sample. Raw sequences were called using Zebra caller (MGI) and demultiplexed using Cutadapt. RNAseq was carried out at the Genomics Unit of the CIMA Universidad de Navarra. Quality control was performed using FastQC. Bbduk from BBMap tools (V.38.90) was used to remove the adapter contamination, polyA read-through, and low-quality tails. Nf-core RNAseq pipeline was used to process the reads.30 UMI extraction was done using UMI-tools (V.1.1.1). STAR (V.2.7) was used to align the raw 3’RNA-Seq fastq reads to the mouse reference genome (GRCm38.p6). Read quantification was computed using featureCounts (Subread version 2.0.1). In order to identify viral reads in the 3’RNA-seq data, unmapped reads were mapped to the MVA viral genome (ASM645792v1) using Bowtie2, and read count was performed using featureCounts. Differential analysis was performed with the R package edgeR. False positive discovery <0.05 was set as cut-off for differentially expressed genes (DEGs). GO enrichment analysis was performed for the DEGs using the geneXplain platform (www.genexplain.com). Hierarchical clustering, volcano plots, and barplots were performed using ComplexHeatmap R package, R-base functions, and ggplot2 package, respectively.

GraphPad Prism V.8.2.1 software (GraphPad Software, San Diego, California, USA) was used for statistical analysis. Data were analyzed by one-way analysis of variance followed by Sidak’s multiple comparisons test. Longitudinal data were fitted to a third-order polynomial equation and compared with an extra sum-of-squares F test with Bonferroni adjustment for multiple comparisons. Survival analysis was performed in R using the pairwise_sruvdiff function of the package survminer with Benjamini-Hochberg adjustment for multiple comparisons. Values of p<0.05 were considered to be statistically significant.

We conducted a screening of clinically approved drugs to find novel inhibitors of the type I IFN. The selection was based on two assays. First, we searched for a reduction of the surface expression levels of the IFN-alpha/beta receptor 1. The second assay focused on interference with the endocytosis pathway. These assays were based on the differential intensity of type I IFN signaling depending on the cellular IFNAR density and on the fact that clathrin-dependent endocytosis of activated IFNAR complex is a requirement for type I IFN signaling.31 32 Several compounds such as terbinafine, procainamide, and quinidine successfully reduced both IFNAR1 levels and BSA endocytosis and increased the percentage of GFP positive cells after MVA-GFP infection (online supplemental figure 1). Based on these preliminary data, we focused our attention on statins. Simvastatin, and atorvastatin reduced the membrane levels of IFNAR1 in a dose-dependent manner. The simvastatin EC50 was 26.34 µM, and the atorvastatin EC50 was 68.91 µM. These EC50s are similar to those described for the inhibition of CYP2C8 (2.7 fold-higher in simvastatin and 1.7-fold higher in the case of atorvastatin) and in the same range as the in vivo estimated liver concentrations of both drugs.33 The maximum reduction was approximately 40% of baseline levels. The maximum effect was achieved at 37.5 µM for simvastatin and 300 µM for atorvastatin (figure 1A–D). Simvastatin was not toxic for the L929 cells at the dose range tested, and the level of viable cells analyzed by 7AAD incorporation was higher than 95% in all tested concentrations. Atorvastatin was slightly more toxic in culture, and at the dose of 300 µM, only 69% of cells remained alive. Of note, dead cells were excluded from the IFNAR1 flow cytometry expression analysis. Based on these assays, we selected the effective dose of simvastatin and atorvastatin. Using these doses, the internalization of BSA labeled with FITC was also inhibited (figure 1E,F). We had previously shown that BSA internalization is inhibited at 4°C and by treatment with chlorpromazine, as expected for a clathrin-mediated process.34 Importantly, both statins significantly inhibited fluorescent BSA uptake (figure 1E,F).

Once we verified that the compounds simvastatin and atorvastatin reduced the IFNAR1 membrane expression and endocytosis, we analyzed whether they were able to block recombinant IFNα-elicited signaling effectively. The signaling cascade initiated by the binding of rIFNα to the IFN-α/β receptor leads to the phosphorylation of STATs and the modulation of ISGs. Both simvastatin and atorvastatin reduced the IFNα-mediated STAT1 phosphorylation (figure 2A). To evaluate the timing of the statin effects on the IFNα activity, we analyzed the expression levels of three ISGs commonly used as markers of the activity of rIFNα: 2,5-OAS, ISG 15 (ISG15), and USP18. As expected, the expression of the three genes was markedly upregulated by the rIFNα. Preincubation with simvastatin or atorvastatin 3 hours before exposure to IFNα abrogated ISGs induction (figure 2B). The maximal inhibitory effect was observed when the IFNα exposure immediately followed the withdrawal of statins from the cultures and decreased if the resting period was prolonged. When time between the drug exposure and the IFNα treatment was 9 hours, we could not detect any effect of simvastatin and atorvastatin on the IFNα activity (figure 2C).

Transient reduction of IFN-α/β signaling by statins was hypothesized to enhance the effects of virotherapy. For experiments with MVA, we selected simvastatin due to the lower doses and reduced toxicity required for the IFNα inhibitory effects in cell cultures. The coincubation of simvastatin and MVA-OVA enhances viral expression as compared with MVA alone. Similar IFN beta expression levels were detected in both experimental conditions reflecting that simvastatin does not interfere with viral detection by of pathogen-associated molecular pattern receptor (figure 2D). However, simvastatin reduced the expression of several ISG such as IFNa, ISG15, or USP18 (figure 2D).

Simvastatin has been shown to exert an adjuvant activity when combined with recombinant proteins. This effect is mediated by prolonging antigen presentation in DCs due to the block of Rab5 lipidation.25 To evaluate whether simvastatin also promotes antigen presentation on MVA infection, mouse DCs were infected for 24 hour with MVA-OVA with or without simvastatin. Flow cytometry analysis revealed that simvastatin increased two-fold the number of DCs presenting the OVA 257–264 (SIINFEKL) bound to MHC class I, indicating that MVA therapeutic vaccination could also benefit of the adjuvant properties of simvastatin (online supplemental figure 2).

For in vivo experiments, we chose a dose of simvastatin based on the previous literature.25 Peritumoral injection of MVA-OVA in B16-OVA (figure 3A) induced the expression of MVA genes. The viral genes can be grouped in four clusters, depending on the expression level. The administration of simvastatin does not interfere with viral expression, and similar levels of the different clusters were observed (figure 3B and online supplemental figure 3). In contrast, simvastatin dampened the overall MVA-induced genes in the tumor microenvironment (figure 3C). Gene Ontology analysis of the simvastatin-inhibited pathways identified 20 involved in the viral immune response (figure 3D and online supplemental figure 4). The DEGs included a large number of cytokine-related genes and, specifically, several IFN-related genes (figure 3E and online supplemental figures 5 and 6), indicating that additional anti-inflammatory effects of simvastatin are involved in the mechanism of action of this combination in addition to the suppression of the type I IFN signaling. These results were validated by real-time PCR. Intraperitoneal administration of simvastatin slightly enhanced the expression of the viral gene 082 L while the induction of the ISGs was inhibited (figure 3F).

To evaluate the antitumor effect of the combination, we measured the tumor growth and survival after treatment with MVA-OVA or MVA-OVA combined with simvastatin. The administration of simvastatin alone had no impact on tumor growth (figure 4A). MVA-OVA was injected into the tumor and delayed tumor growth in all mice, showing increased survival but only completely eradicated the tumor in one mouse out of six (figure 4A,B). In contrast, the additional administration of simvastatin either intraperitoneally or intramuscularly markedly enhanced the percentage of cured mice at the end of the experiment. In our hands, the intramuscular administration was slightly more effective and significantly increased survival as compared with the mice treated with MVA-OVA alone (figure 4B). We also evaluated whether the intraperitoneal administration of the MVA synergized with simvastatin. In this case, no differences were observed in the antitumor effect of MVA alone or the MVA combined with simvastatin (online supplemental figure 7). Finally, we evaluated the combination of simvastatin with an MVA encoding an endogenous retroviral tumor-associated antigen such as gp70. The antitumor effect of intraperitoneal administration of simvastatin and the peritumoral administration of an MVA-gp70 exerted a potent antitumor effect in the LLC-OVA model. The antitumor effect of the combination was superior to the effect of MVA-gp70 alone (online supplemental figure 8). Altogether, these results indicate that simvastatin modulates the inflammation triggered by the peritumoral administration of MVA-OVA and promotes the MVA-mediated antitumor effect.

To further understand the impact of simvastatin on the antitumor effect of MVA-OVA, we used flow cytometry to evaluate the presence of different immune populations in cell suspensions from spleens, tumor-draining lymph nodes, and tumors (figure 5A) following the gating strategy depicted in online supplemental figure 9). B cells, CD4⁺ lymphocytes, CD8⁺ lymphocytes, and tumor-specific CD8⁺ lymphocytes were increased in the tumor-draining lymph node in those animals treated with MVA-OVA alone or with the combination (figure 5C). However, a marked difference between both experimental groups was observed both in the tumor tissue microenvironment and in the spleen (figure 5B,D). While MVA-OVA tended to decrease these immune populations as compared with PBS-treated mice, MVA-OVA combined with simvastatin dramatically increased all these immune populations in the spleen (figure 5B) and also increased the infiltration by B cells, CD8⁺ T lymphocytes, and, more importantly, tumor-specific CD8⁺ T lymphocytes into the tumor tissue (figure 5D). To study whether the immune response elicited by the combined treatment was restricted to the MVA-encoded tumor-associated antigens, we evaluated antigen spreading using an IFNγ ELISpot assay. We detected potent immune responses against an immunodominant OVA epitope and the melanoma cells expressing the OVA protein on treatment with MVA or MVA+ simvastatin. However, no response was detected against the parental melanoma cells that do not express the OVA proteins (online supplemental figure 10).

To evaluate the immune cells involved in the antitumor activity of the combination of MVA-OVA and simvastatin, we selectively depleted CD8⁺ T lymphocytes, CD4⁺ T lymphocytes, conventional CD4⁺CD25⁺FoxP3⁺ T regulatory cells or NK cells with monoclonal antibodies following administration of the combined systemic statin and peritumoral MVA-OVA treatment. The antitumor effect was dependent on CD8⁺ T lymphocytes as depletion of these effector immune cells completely abrogated the antitumor effect (figure 6A–C). Interestingly, CD4⁺ T lymphocytes and NK cells were detrimental for the antitumor effect and depletion of these immune cells led to the complete eradication of the implanted tumors in all cases (figure 6A–C). Depletion of CD4⁺ T lymphocytes further enhanced the tumor-infiltration of CD8 T cells and the activation of these intratumoral T lymphocytes. Depletion of NK cells maintained the tumor infiltration promoted by the combination of MVA and simvastatin (figure 6D and online supplemental figure 11). The main CD4⁺ T lymphocyte subset involved in the mechanism of action of the combined treatment was mainly conventional T regulatory cells as specific depletion of this cell population using anti-CD25 mimicked the effect observed with the anti-CD4 monoclonal antibody (figure 6A–C). IL10 is one of the mediators of the immunosuppressive activity of T regulatory cells.35 Frequent administration of a neutralizing antibody failed to enhance the antitumor activity of combined treatment, indicating that IL10 is not the main mediator of the T regulatory cells in this system (online supplemental figure 12). Finally, a B cell depleting antibody against CD19 was used to interrogate the relevance of the upsurge of B cells in the spleen and tumors treated with the simvastatin +MVA OVA combination. Depletion of this cell population slightly reduced the tumor growth rate but failed to enhance survival, excluding a predominant role of B cells in the mechanism of action of the combination (online supplemental figure 12).