Wild-type C57BL6, pMEL-1 (JAX stock no: 005023) and Nonobese diabetic/severe combined immunodeficiency (NOD-SCID) (JAX stock no: 005557) mice were maintained at animal facility of the Universidad de Chile, Faculty of Medicine. pMEL-1 mice have C57BL6 background and NOD-SCID mice were on NOD/ShiLtSz background. For all experiments, mice between 6 and 12 weeks of age were bred in specific pathogen-free conditions.

Tumor cell lines were maintained in RPMI-1640 (Corning) supplemented with 1% penicillin/streptomycin (Corning) and 10% heat-inactivated fetal bovine serum (FBS) (Corning). Bone marrow-derived DC (BM-DC) were cultured in RPMI-1640-GlutaMAX (Gibco) supplemented with 1% penicillin/streptomycin, 55 µM 2-mercaptoethanol (Gibco) and 10% FBS. Fluorescence-activated cell sorting (FACS) buffer was phosphate-buffered saline (PBS) (Corning), supplemented with 2% FBS and 2 mM EDTA (Ambion). CCH Inmunocyanin was provided by Biosonda. The gp100_25-33 peptide (KVPRNQDWL) was purchased from Genetel Laboratories. Dr Fabiola Osorio (Universidad de Chile) kindly provided FMS-like tyrosine kinasa 3-ligand (FLT3-L). Lipopolysaccharide (LPS), phorbol myristate acetate and ionomycin were purchased from Sigma-Aldrich. Brefeldin-A was from eBioscience.

Mel1, Mel2 and Mel3 are human melanoma cell lines isolated from metastatic lymph nodes (LNs) of three patients.9 B16F10 (ATCCCRL-6475) and MC38 (ATCCCRL-2639) cells were kindly provided by Dr Álvaro Lladser (Fundación Ciencia & Vida, Chile). FLT3L-expressing B16F10 cells (B16-FLT3L) were kindly provided by Dr María Rosa Bono (Universidad de Chile).

Cell lysates were made as previously described.9 TRIMEL is derived from a mix of equal amounts of Mel1, Mel2 and Mel3 cells, which were taken to a final concentration of 8×10⁶ cells/mL, HS treated at 42°C for 1 hour plus 2 hours at 37°C and then lyzed through three cycles of freeze/thaw in liquid nitrogen. The HS-conditioned lysate from B16F10 and MC38 cells were prepared using same approach (8×10⁶ cells/mL). Tumor cells not HS treated were incubated for 3 hours at 37°C before being lyzed.

For therapeutic assays, C57BL6 mice were subcutaneously inoculated with 2.5×10⁴ B16F10 or 1×10⁵ MC38 cells in lower right flanks. Mice were then immunized subcutaneously on lower left flanks on days 1, 6 and 12 post-tumor challenge with corresponding treatments: (1) lysates of B16F10 cells with or without CCH (25 µg/µL, total 200 µg CCH/doses), (2) lysates of HS-conditioned B16F10 cells±CCH, (3) 1:1 mixture of B16F10 cell lysate (preconditioned or not with HS) with TRIMEL±CCH, (4) 1:1 mixture of HS-conditioned MC38 cell lysate with TRIMEL±CCH, (5) CCH or (6) PBS. Each tumor cell lysate doses contained approximately 1.4×10⁶ cells in 172 µL. The mixture of HS-conditioned B16F10 cell lysate plus TRIMEL and CCH corresponds to TRIMELVax. For combination therapy assays, 200 µg/dose of anti-PD-1 (CD279) monoclonal antibody (RMP1-14 (BioXCell)) was intraperitoneally administered on days 4, 7 and 11 post-tumor challenges. For survival assays, mice were sacrificed when tumor growth exceeded 1500 mm³. On day 14 after challenge, mice were sacrificed, and tumors, tumor draining LNs (TdLNs) and serum were analyzed.

For prophylactic assays, C57BL6 or NOD-SCID mice were vaccinated subcutaneously at lower left flanks with TRIMELVax or PBS on days −19, −9 and −2 before tumor challenge. On day 0, 1.5×10⁵ B16F10 cells were subcutaneously inoculated at right flanks and tumor growth was measured every 2–3 days.

Antibodies for flow cytometry (BD Pharmigen, eBioscience, BioLegend or Miltenyi Biotec), and viability dye LIVE/DEAD (Thermo Fisher Scientific) were used for cell phenotyping. Depending on the experiment, cells were stained with following antibodies in presence of CD16/31 (Fc-Block): CD11b (M170), I-A/I-E (M5/114.15.2), XCR1 (ZET), CD8a (53.6.7), CD3 (17A2), B220 (RA3-6B2), CD103 (2E7), CD11c (N418), CD69 (IM7), CD24 (M1/49), CD172a (P84), CD45.1 (A20), NK1.1 (PK136), CD4 (GK1.5), CD64 (X54-5/7.1), F4/80 (BM8), CD86 (GL-1), CD24 (M1/69), CD44 (IM7) and PD-1 (29F.1A12). Acquisition and analysis of cell suspensions were performed on FACSVerse and LSR Fortessa X-20 (BD Biosciences) and subsequent analysis was made with FlowJo10 software (FlowJo). Cell sorting was performed on FACS Aria III (BD Biosciences).

BM-DCs differentiated in vitro using FLT3-L (FL-DCs) from BM precursor cells of C57BL6 mice femurs and tibias were cultured in BM-DC culture media in presence of 150 ng/mL of human recombinant FLT3-L (PeproTech) for 7–8 days before use.

Prior to HS treatment and lysis, mixtures of Mel1/Mel2/Mel3 and/or B16F10 cells were stained with 2 µM PKH26 (Sigma-Aldrich) following manufacturer’s instructions. HS-conditioned PKH26-prelabeled melanoma cell lysates were generated as previously described. 1×10⁵ FL-DCs were cocultured with the stained melanoma cell lysates in 1:1 tumor cell:DC ratio for 0 or 3 hours at 37°C or 4°C. After coculture, lysate capture was determined gating in each FL-DC population using flow cytometry. The phagocytic indexes were calculated as: (%phagocytosis at 37 °C/%phagocytosis at 4°C)×100.

1×10⁵ FL-DCs were stimulated in 1:1 ratio (taking into account the pre-lysis melanoma cell numbers) with TRIMELVax, TRIMEL, HS-conditioned lysates from B16F10 cells, TRIMEL plus HS-conditioned lysate of B16F10 cells, CCH (200 µg), LPS (100 ng/mL) or kept unstimulated for 24 hours. CD86 expression level was analyzed by flow cytometry.

For proliferation assays, CD8⁺ T cells were isolated from pMEL-1 splenocytes by negative selection, using a lineage depletion cocktail of biotinylated antibodies and antibiotin microbeads (Miltenyi Biotec) and labeled with 5 µM CellTrace Violet (CTV) (Thermo Fisher Scientific). For in vitro assays, FL-DCs were treated 1:1 with TRIMEL plus HS-conditioned B16F10 cell lysate and CCH (200 µg) for 5 hours at 37°C. For ex vivo assays, inguinal, axillary and brachial LNs were collected from B16-FLT3L tumor-bearing mice. Plasmacytoid DC (pDC), conventional type 1 DC (cDC1) and conventional type 2 DC (cDC2) were isolated by cell sorting. For T-cell activation assays, FL-DCs were collected, washed with FACS buffer and fixed with 1% paraformaldehyde for 10 min. Cells were washed with 0.2M glycine and culture media prior coculture with CD8⁺ T cells. 1×10⁵ fixed DCs were cultured with 1×10⁵ T cells at 37°C for 16 hours to evaluate CD69 or CD25 expression. For proliferation assays, FL-DCs or LN-DCs were collected and immediately cocultured in a 1:1 ratio with CTV-labeled T cells. After 3 days, proliferation was measured by flow cytometry.

On day 0, 2×10⁶ CTV-stained pMEL-1 CD8⁺ T cells were intravenously transferred into C57BL6 mice. On days 1, 4 and 7, mice were subcutaneously injected with 20 µg of hgp100 peptide plus CCH (200 µg), TRIMELVax, B16Vax, CCH or PBS. On day 10, inguinal, brachial and axillary LNs were processed and transferred T-cell proliferation was measured by flow cytometry.

Tumors were collected and fixed in 4% formalin. Three-micrometer-thick sections from paraffin-embedded tissues were mounted on glass slides, rehydrated and antigen retrieval was performed at 100°C (TRIS-Borate-EDTA (TBE), pH 8). Primary antibodies were used according to manufacturer’s instructions (Abcam, dilutions 1:200): anti-CD3 (SP7), anti-CD4 (EPR19514), anti-CD8 (ab203035). Slides were incubated with primary antibodies in moist chambers overnight at 4°C. Slides were washed with PBS before incubation with label-secondary goat antirabbit IgG for 1 hour at 4°C, washed three times, incubated with DAB substrate KIT (Abcam) solution for 10 min and washed with PBS. Background staining was performed with Mayer’s hematoxylin. Sections were dehydrated through ascending alcohols-to-xylene and mounted.

Indirect ELISA was used to detect antibodies against CCH in sera of treated mice.28 Antibodies against melanoma cells or peripheral blood leucocytes (PBLs) were analyzed by flow cytometry. In brief, 1×10⁵ B16F10 or Mel1, Mel2 and Mel3 cell mixtures were incubated with 1:20 dilutions of de-complemented sera. Cells were then incubated with FITC-conjugated anti-mouse IgG (Invitrogen). The binding of serum IgG to target cells was evaluated by flow cytometry.

Tumors were dissected, mechanically disaggregated and digested at 37°C for 30 min in Hanks’ balanced salt solution (5% FBS) containing collagenase-D (1 mg/mL) and DNase I (25 µg/mL) (Roche). The cell suspensions were filtered with 70 µm cell strainers (BD Falcon). Leucocytes were suspended in 40% Percoll (GE Healthcare) and gently layered over 70% Percoll. The gradient was centrifuged at 750 g for 20 min at room temperature. Mononuclear cells were collected, washed and resuspended in complemented RPMI-1640. TdLNs were mechanically disaggregated and cells incubated for 45 min at 37°C in a solution containing 100 µg/mL collagenase-D and 50 µg/mL DNaseI dissolved in 2% FBS-PBS. Single cell suspensions were washed in RPMI-1640 and depleted of erythrocytes using RBC lysis buffer (BioLegend) for 5 min at 4°C.

Differences between groups were analyzed by paired, two-tailed Student’s t-tests or Mann-Whitney test. Animal survival data were analyzed with the Kaplan-Meier method. Results with a p-value lower than 0.05 were considered significant. Mean values, SEM and statistics were calculated using GraphPad Prism Software.

Here, we investigated the effect of TRIMELVax vaccination using the aggressive murine B16F10 melanoma model (online supplementary figure 1). Taking into account the crucial role of DCs in antitumor responses induced by immunizations, first we asked whether mice BM-DCs were capable to sense TRIMEL-originated DAMPs and to phagocyte, process and present MAAs. Our results showed that BM-derived FLT3-L-differentiated DCs (FL-DCs) (online supplementary figure 2) effectively phagocytized both, TRIMEL and B16F10 HS-conditioned cell lysates. In particular, conventional FL-DCs successfully endocyted tumor lysates, while pDC FL-DCs showed lower phagocytic capacity (figure 1A,B). Competitive phagocytosis assays showed that HS-treated B16F10 cell lysates were effectively internalized by cDC1 FL-DCs when incubated in presence of TRIMEL and CCH, suggesting that they may be capable to acquire murine MAA from vaccine components (online supplementary figure 3). Additionally, HS treatment of B16F10 cells also induced ATP and HMGB1 release and calreticulin plasma membrane translocation, similarly to what was observed in HS-treated cell lines composing TRIMEL (online supplementary figure 4).9 10 All the FL-DC subtypes increased CD86 expression when pulsed with TRIMELVax or with each HS-conditioned cell lysate, separately or combined, while CCH failed to induce similar activation (figure 1C).

Optimal delivery of a wide-ranging pool of TAA coupled with factors promoting antigen cross-presentation seems to be critical for cancer vaccine success. TRIMELVax-stimulated FL-DCs induced cross-presentation of the MAA gp100 as indicated by augmented CD69 cell surface expression on pMEL-1 CD8⁺ T cells, in comparison with both CD8⁺ T cells cultured alone or stimulated with TRIMELVax in absence of DCs, an effect that was more clearly observed when using cDC1 FL-DCs (figure 1D). Furthermore, TRIMELVax-stimulated cDC1 FL-DCs triggered a threefold higher proliferation of CD8⁺ T cells compared with cDC2 and pDC FL-DCs (figure 1E). Altogether, our data indicate that TRIMELVax elicits efficient DC activation for MAA cross-presentation to CD8⁺ T cells, particularly mediated by cDC1 FL-DC.

Then, we assessed whether immune responses elicited by TRIMELVax in vitro translate in protective antitumor effects in vivo. Mice were immunized with TRIMELVax or control treatments (figure 2A). Two days after the last injection, mice were challenged with B16F10 cells and tumor growth was monitored. In this prophylactic setting, the tumor growth was significantly inhibited only in TRIMELVax-treated mice (figure 2B), indicating that the complete vaccine, but not lysates nor adjuvant alone, induces immune responses able to protect mice against tumor challenge. Moreover, no signals of autoimmunity damage in organs of vaccinated animals were observed (data not shown).

We next investigated whether TRIMELVax-mediated tumor control may be related to eliciting of adaptive antimelanoma immune responses in vivo. To test whether MAA present in TRIMELVax can be efficiently cross-presented to CD8⁺ T cells in vivo, CTV-preloaded pMEL-1 CD8⁺ T cells were transferred intravenously into C57BL6 mice. Mice were treated with TRIMELVax, HS-conditioned B16F10 cell lysate+CCH (B16Vax) or PBS. As a positive control, gp100_25-33 peptide+CCH were injected 7 days after T-cell transfer. Three days after treatment, dLNs were sampled and proliferation and activation of pMEL-1 CD8⁺ T cells were determined (figure 2C). Our results indicated that TRIMELVax, but not B16Vax, was able to induce potent pMEL-1 CD8⁺ T-cell proliferation and activation (figure 2D). These results strongly suggest that TRIMELVax induces cross-presentation of MAA in vivo promoting a cellular-mediated immune response.

cDC1 cells are the main DC subtype responsible for TAA cross-presentation and antitumor CD8⁺ T-cell activation.29 To test whether TRIMELVax was able to provide MAAs to cDC1 in vivo, we performed an ex vivo antigen cross-presentation assay using sorted cDC1, cDC2 and pDCs from TRIMELVax-vaccinated and control B16-FLT3L tumor-bearing animals (figure 2E and online supplementary figure 5). As predicted, only cDC1 cells isolated from dLNs of TRIMELVax-treated but not those isolated from control mice activated pMEL-1 CD8⁺ T cells (figure 2F and online supplementary figure 5).

TRIMELVax includes CCH, which is a strong adjuvant that induces both T-cell and humoral responses in different vaccine models.26 30 Therefore, we investigated the potential presence of antitumor antibodies in sera of vaccinated mice. Our results showed that sera from tumor-bearing mice vaccinated with either TRIMELVax or HS-melanoma cell lysates, but not CCH-treated mice, contains IgGs that bind B16F10 and Mel1/Mel2/Mel3 cells (figure 3A,B). Of note, we also observed a strong xeno-response induced by TRIMELVax, given that sera of vaccinated mice also reacts against human PBL (figure 3B). As expected, treatments with CCH alone or TRIMELVax, but not cell lysates alone, induced the generation of anti-CCH IgGs (figure 3C). These results suggest that TRIMELVax induce a strong humoral immune response against vaccine components, including MAAs.

We tested antitumor effects of TRIMELVax in therapeutic vaccination schemes (figure 4A). It was observed that despite DAMP induction in HS-treated B16F10 cells, vaccination with HS-conditioned B16F10 cell lysates was not able to protect against B16F10 tumor growth, even in combination with CCH (B16Vax) (figure 4B). In contrast, TRIMELVax treatment efficiently controlled B16F10 tumor growth (figure 4B). Notably, the effect observed by TRIMELVax was abrogated if untreated B16F10-derived lysate was used as antigen source (4B). As observed in the prophylactic approach, the use of CCH was required for the antitumor effects mediated by TRIMELVax in the B16F10 melanoma model (figure 4C), but it was dispensable for the control of a more immunogenic tumor as the colon cancer MC38 model (figure 4D). As expected, it was observed that full immune competence was required for TRIMELVax efficacy, given that it did not impact tumor growth in NOD-SCID mice (figure 4E). Moreover, depletion of CD4⁺ or, in a major grade, of CD8⁺ cells abrogated TRIMELVax antitumor effect (figure 4F,G), suggesting that the vaccine may promote both CD8⁺ and CD4⁺ T-cell-mediated immunity.

Our results indicated that all components of TRIMELVax were required for an efficient activation of cellular and humoral responses and for controlling tumor growth in a CD8⁺ and CD4⁺ T-cell-dependent manner.

As anti-PD-1 monotherapy have shown partial protective effects in B16F10 tumors,16 we investigated whether the combination of TRIMELVax with anti-PD-1 could improve their immunotherapeutic potentials. Thus, we challenged C57BL6 mice with B16F10 cells and then treated with TRIMELVax, anti-PD-1 monoclonal antibodies or its combination (figure 5A). We observed that both single treatments independently inhibited initial tumor growth with similar efficacy, while the combinatory regimen induced a slightly better inhibition of tumor growth than anti-PD-1 monotherapy (figure 5B,C). However, despite the initial tumor growth control, the survival of anti-PD-1-treated animals was similar to the mock-treated ones (figure 5). Remarkably, TRIMELVax treatments alone or in combination with anti-PD-1, induced a significant improvement in the survival of tumor-bearing mice (figure 5D).

Associated with its better antitumor activity, TRIMELVax induced a higher tumor infiltration of CD3⁺, CD4⁺ and CD8⁺ cells than anti-PD-1 monotherapy, while TRIMELVax/anti-PD-1 combination generated higher tumor infiltration of CD4⁺ cells than each treatment alone (figure 6A). Flow cytometry analysis of tumors and TdLNs from vaccinated mice, sampled 14 days after tumor challenge, was performed to evaluate whether immune cell compartments were differentially affected by treatments. At that timepoint, both TRIMELVax and anti-PD-1 treatments induced increased frequency of intratumor cDC1s but not cDC2s (figure 6B). Also, we observed a decrease of intratumor macrophages frequency in all treated groups as compared with the PBS-control group (figure 6B). Unlike anti-PD-1 treatment, TRIMELVax induced potent tumor infiltration of CD8⁺ T cells, being CD8⁺/CD4⁺ T-cell ratio significantly enhanced by TRIMELVax (figure 6B). TRIMELVax/anti-PD-1 combination but not TRIMELVax alone showed a significant increase in the percentage of intratumor B cells (figure 6B). Additionally, we observed significant positive correlations between cDC1 and CD8⁺ T-cell intratumor frequency, and between tumor macrophage frequency and tumor size. On the other hand, significant negative correlations between intratumor CD8⁺ T-cell or cDC1 frequency and tumor size were also observed (figure 6C). The TdLN immune cell profile of treated mice is shown in online supplementary figure 6.

Finally, we wanted to investigate the CD8⁺ T-cell lineage phenotypes at tumor microenvironment, comparing their frequencies in tumor-infiltrating lymphocyte (TIL) from animals treated with TRIMELVax versus B16Vax. Although B16Vax therapeutic treatments lead to higher numbers of CD8⁺ TILs than TRIMELVax (figure 6D), this non-effective vaccine (figure 4B) lead to the accumulation of a higher proportion of PD-1^hi CD8⁺ T cells, whereas TRIMELVax promoted major proportion of PD-1^lo CD8⁺ T cells in tumors, a phenotype associated with prototypic effector cells required for tumor growth control (figure 6E,F).