Female C57BL/6 mice (8–10 weeks old at study initiation) were purchased from Charles River Laboratory (Wilmington, Massachusetts, USA) and housed in a specific pathogen-free facility.

Ad-empty, Ad-OVA and Ad-DCT are replication-deficient adenoviruses (E1/E3-deletion) based on the human serotype 5. VSV-DCT is a replication-competent oncolytic viral agent which derives from the wild-type Indiana strain of the vesicular stomatitis virus (VSV). Ad-empty has no transgene. Ad-DCT and VSV-DCT encode the human dopachrome tautomerase (DCT). The OVA transgene encodes the chicken ovalbumin and was used as a tumor-unrelated antigen control.

Murine melanoma B16-F10 cells (expressing the murine DCT antigen) were grown in F11-MEM containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate and vitamin solution, 0.01 mM non-essential amino acids, 50 mM β-mercaptoethanol, 100 U/mL penicillin, and 100 mg/mL streptomycin (all from Invitrogen, Grand Island, New York, USA).

For adenovirus injection, mice were anesthetized in a sealed chamber containing 5% inhalation isoflurane. Adenoviral vectors were administered intramuscularly at a total dose of 2×10⁸ pfu (1×10⁸ plaque forming units (pfu) in 50 µL phosphate-buffered saline (PBS) per thigh). VSV-DCT was injected intravenously in 200 µL PBS at a dose of 2×10⁹ pfu. CTX was administered through intraperitoneal (i.p.) injection at 50 mg/kg in 200 µL of saline solution 1 day prior to Ad prime (other groups received 200 µL of saline solution as control).

For lung tumor engraftment, 2.5×10⁵ B16-F10 melanoma cells were injected intravenously in 200 µL saline water. Mice were monitored daily and euthanized for tissue harvest or on signs of morbidity.

Blood sampling was performed through retro-orbital sinus bleeding using a heparinized capillary tube. Blood was collected into a heparinized saline solution (30 U/mL). Peripheral white blood cells were cleared of erythrocytes in ammonium chloride potassium (ACK) buffer at room temperature for 5 min then washed in Hanks medium; the procedure was repeated twice. After harvest, lymph nodes and spleen were crushed between glass slides to release leukocytes. Splenic red blood cells were lysed in ACK buffer as described for blood. Lungs bearing melanoma metastases were dissociated enzymatically using a cocktail of 0.05% collagenase, 0.002% hyaluronidase and 0.02% DNAse I. Lung-infiltrating leukocytes were isolated using the EasySep Mouse CD90.2 Positive Selection Kit II (Stemcell Technologies, Vancouver, British Columbia, Canada) following manufacturer’s recommendations.

Peptides corresponding to the immunodominant epitopes of DCT: (1) DCT_180–188 SVYDFFVWL/“SVY” that binds to H-2K^b; 100% conserved between human and murine DCT and (2) DCT_89–102 KFFHRTCKCTGNFA/“KFF” from human DCT that binds to I-A^b; differing from the murine epitope in position 92: His (human) → Asn (murine). Peptides corresponding to the immunodominant epitopes of OVA: (1) OVA_257–264 SIINFEKL/“SIIN” that binds to H-2K^b and (2) OVA_323–339 ISQAVHAAHAEINEAGR/“ISQ” that binds to I-A^b. Peptides were synthesized by Biomer Technologies (San Francisco, California, USA).

Monoclonal antibodies used in flow cytometry assays: anti-CD16/CD32 (clone 2.4G2) to block Fc receptors, anti-CD3 (clone 145-2 C11), anti-CD8α (clone 53-6.7), anti-CD4 (clone RM4-5), anti-CD25 (clone PC61) and anti-FoxP3 (clone FJK-16s) for detecting cell surface and intranuclear markers, and anti-interferon γ (IFNγ) (clone XMG1.2) and anti-tumor necrosis factor α (TNFα; clone MP6-XT22) for intracellular cytokine staining. All reagents were purchased from BD Pharmingen (Mississauga, Ontario, Canada) with the exception of anti-FoxP3 that was purchased from eBioscience/Thermo Fisher Scientific (Burlington, Ontario, Canada).

DCT-specific T-cell responses were measured 9 and 14 days post prime and 5 days post boost. Peripheral blood mononuclear cells (PBMCs) or splenocytes were incubated in complete Roswell Park Memorial Institute (RPMI) medium (ie supplemented with FBS 10%, penicillin–streptomycin 1% and L-glutamine 1%) with the SVY peptide (2 µg/mL) or KFF peptide (15 µg/mL) for DCT-specific CD8⁺ or CD4⁺ T-cell (re-)stimulation, respectively, or with the SIIN peptide (2 µg/mL) for OVA-specific CD8⁺ T-cell (re-)stimulation. Incubation was performed in an incubator (37°C, 5% CO₂, 95% humidity) for 5 hours with brefeldin A (1 µg/mL, GolgiPlug BD Pharmingen) during the last 4 hours. Leukocytes were labeled with LIVE/DEAD fixable near-IR dye (Life Technologies/Thermo Fisher Scientific) following supplier’s recommendations to identify live lymphocytes. Cells were incubated with antibodies targeting CD16/CD32 before staining with fluorescent-labeled antibodies targeting T-cell markers. Then, cells were permeabilized and fixed with Cytofix/Cytoperm (BD Pharmingen) and stained for intracellular cytokines. Data were acquired using a FACSCanto flow cytometer with FACSDiva software (BD Pharmingen) and analyzed with FlowJo v10 for Mac (Tree Star, Oregon, USA).

5-Bromo-2'-deoxyuridine (BrdU) was first administered i.p. at 50 mg/kg immediately after tumor challenge (ie, 4 days prior to CTX and 5 days prior to Ad) and maintained over the time until tissue harvest in the drinking water at 0.8 mg/mL. BrdU-containing water was refreshed every day. Tregs that incorporated BrdU into their DNA during the S phase of the cell cycle were stained using the APC BrdU Flow Kit (BD Pharmagen) following supplier’s recommendations.

GraphPad Prism was used for graphing and statistical analyses. Immune response data were plotted used mean±SEM or SD. Unpaired Student’s two-tailed t-test was used to compare two groups, ordinary one-way analysis of variance was used for multiple groups. Survival curves were plotted using Kaplan-Meier method, and compared using the log-rank test. Differences considered significant when p≤0.05 (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Mice bearing disseminated B16-F10 melanoma lung tumors were treated with varying combinations of vaccines encoding DCT, a well-characterized melanoma-associated antigen, with or without CTX preconditioning (figure 1A). Priming with Ad-DCT induced specific anti-DCT CD8⁺ T-cells and the magnitude of the circulating responses was significantly increased by a single low dose of CTX prior to priming (figure 1B, online supplementary figure S1). Boosting with systemic oncolytic VSV-DCT increased the magnitude of CD8⁺ T-cell responses compared with Ad alone and a further significant increase in both specific CD8⁺ Tc1 and CD4⁺ Th1 cell frequencies were seen when CTX was combined with this potent prime:boost platform (figure 1C, D, online supplementary figure S1) in a tumor-specific manner as no such effect was observed in tumor-free mice (figure 1E). CTX preconditioning also increased specific IFNγ production by both CD8⁺ and CD4⁺ T-cells when quantified using mean fluorescence intensities (figure 1F, G). Collectively, these data show that low-dose CTX improves the immunotherapeutic profile of this vaccine in a tumor-restricted manner.

In order to determine the effect of CTX pretreatment with relation to tumor-antigen specificity, secondary lymphoid organs were harvested from tumor-bearing mice 9 days after co-priming with Ad-DCT plus an Ad vector expressing the tumor-unrelated xenoantigen ovalbumin (Ad-OVA). DCT-targeting and OVA-targeting immune responses were quantified using intracellular cytokine staining and flow cytometry. Within the tumor-draining lymph nodes, CTX pretreatment failed to significantly alter the magnitude of OVA-specific single IFNγ⁺ and dual IFNγ⁺ TNFα⁺ CD8⁺ T-cell responses whereas a significant expansion was noted in both DCT-specific populations (figure 2A–D). These findings were mirrored by OVA-specific and DCT-specific CD8⁺ splenocytes (figure 2E–H). No significant effect of CTX preconditioning on the magnitude of OVA-specific CD4⁺ Th1-cell frequencies was observed (figure 2I). However, CTX significantly increased the corresponding DCT-specific CD4⁺ T-cell population (figure 2J). Taken together, low-dose CTX significantly improves vaccine-induced immunity against a well-defined melanocytic TAA expressed in B16-F10 cells but has no effect on the immune response against a xenoantigen that is irrelevant to the tumor.

As Tregs have been shown to constrain the expansion of CD8⁺ T cells,13 we hypothesized that the increased magnitude of vaccine-induced TAA-specific T-cell responses observed after low-dose CTX would be accompanied by decreased Treg proliferation. We interrogated the dynamics of various short-term circulating T-cell subsets in tumor-bearing mice following Ad-DCT±CTX. At 24 hours post-Ad, CTX preconditioning did not result in generalized lymphodepletion when CD4⁺ and CD8⁺ T-cells were quantified (online supplementary figure S2). A relative expansion of CD4⁺ and CD8⁺ T-cell frequencies within the circulating lymphoid compartment was documented (figure 3A, B). Co-staining for CD4, CD25 and FoxP3 revealed a significant decrease in circulating Treg frequency in the CTX group (figure 3C) and ratios of CD8⁺ T-cells/CD4⁺ Tregs were significantly increased after preconditioning (figure 3D). BrdU was administered in the drinking water to tumor-bearing mice to quantify Treg proliferation, and blood samples were taken 72 hours post-Ad. A significant decrease in the frequency of proliferating Tregs (figure 3E) and increase in the ratio of total CD8⁺ T cells/BrdU⁺ Tregs (figure 3F) in the CTX group was noted. Thus, prevaccination treatment with CTX reduced the population of proliferating Tregs resulting in a favorable proportion of effector CD8⁺ T cells over immunosuppressive CD4⁺ Tregs.

Further analyses were performed on mice bearing lung melanoma to ascertain the functional impact of CTX on the prime:boost vaccine. In long-term survivors, marked vitiligo was observed when animals were pretreated with CTX (figure 4A). As previously reported,7 this benign autoimmune manifestation resulted from a bystander elimination of melanocytes (which express the DCT antigen) by vaccine-induced CD8⁺ T-cells. Lungs were harvested from tumor-bearing mice 5 days after VSV-DCT boost, alongside untreated mice, and lymphocytes were isolated. Intracellular staining of CD8⁺ T-cells for IFNγ and TNFα revealed significant increases of DCT-specific, single and dual positive, populations within the TME in the CTX group, whereas no reactive T-cells were detected in untreated animals (figure 4B, C). At the same time point, lungs harvested were examined for gross lesions. Pretreatment with CTX resulted in an overt reduction in tumor burden (figure 4D). Ad-DCT:VSV-DCT resulted in long-term survival in a minority of mice. However, when this regimen was preceded by a single dose of CTX, the majority of mice were cured (figure 4E). Overall, low-dose CTX increases specific antitumor immunity resulting in a remarkable survival advantage in mice bearing aggressive lung disease. In conclusion, CTX preconditioning increased infiltration of the tissues harboring melanoma lesions by polyfunctional tumor-reactive effector T-cells which preceded durable tumor control.