Female C57BL/6 mice were obtained from the Center for Experimental Medicine of the Medical University of Bialystok (Białystok, Poland). Mice were housed in a standard light/dark cycle room with a constant temperature of 22 ± 2˚C, air humidity of 55 ± 10%, and unlimited access to food and water. All experimental procedures were by the guidelines set out in the EU Directive 2010/63/EU on animal testing and were approved by the Local Ethics Committee for Animal Experiments in Wrocław (permit number: 009/2021; 005/2022). Following the experiment, the mice were humanely sacrificed by cervical dislocation.

The non-metastatic murine melanoma B16 F0 cell line (ECACC 92101204) was cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM, ATCC) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS; Sigma−Aldrich). The cultures were maintained under typical conditions, which involving 5% CO₂ and a temperature of 37 °C.

To prepare a lysate from tumor cells, the B16 F0 melanoma cells were gathered and suspended at a density of 5 × 10⁶ cells/ml in RPMI-1640 (Gibco) supplemented with 10% FBS (Sigma-Aldrich). All cells underwent five freeze cycles in liquid nitrogen followed by rapid thawing at 37 °C. Subsequently, the cell suspension was sonicated for 90 minutes. The resulting B16 F0 tumor antigens (TAg) were utilized for stimulating dendritic cells.

Lentiviral vectors (LVs) were generated using a third-generation lentiviral system, which included auxiliary plasmids: pMDLg/pRRE, pRSV Rev, and pMD2.G (these plasmids were generously provided by Didier Trono, Addgene plasmids #12251, 12253, 12259), along with the expression vector. Various cytokine genes were encoded in the expression plasmids: il12, il15/il15ra, or il18. For the il15-carrying vector, the cytokine’s gene sequence was preceded by a signal sequence to facilitate its extracellular release. Furtheremore an alpha subunit of the il15 receptor gene sequence was incorporated to slow down intracellular protein degradation. The control vector (pLVctrl) was designed to assess the effect of lentiviral transduction on dendritic cells. All expression vectors were sourced from VectorBuilder.

Lentiviral vectors were obtained through co-transfection of Lenti-X 293T cells (ClonTech) with the aforementioned plasmids. The Lenti-X 293T cells were cultured with plasmids for 48 hours. The supernatant, enriched with lentiviral vectors, was harvested and subjected to concentration via precipitation utilizing PEG 6000 (Sigma-Aldrich). The pellet, containing the lentiviral vectors, was resuspended in PBS and preserved at -80 °C. The protocol for lentiviral vector production was delineated in a prior publication (27). To determine the lentiviral vector’s titer, a serial dilution method was employed, and flow cytometry analysis.

Dendritic cells were derived from bone marrow extracted from the femurs and tibias of healthy female C57BL/6 mice, following the methodology outlined in our prior publication (28). These cells, referred to as DCs hereafter, were cultured in RPMI-1640 (Gibco) with 10% FBS (Sigma-Aldrich). The culture was supplemented with recombinant murine (rm)GM-CSF (ImmunoTools, 40 ng/ml) and rmIL-4 (ImmunoTools, 10 ng/ml).

Following 7 days of cultivation, immature DCs were subjected to transduction with LVs, using the assumption of 4 viral infectious particles per 1 dendritic cell. This transduction was carried out in the presence of 8 µg/ml polybrene (Sigma-Aldrich). DCs were transduced by lentiviral vectors carring il12, il15/il15ra, il18 genes or by control vector and will be referred to as modified dendritic cells. Four hours after transduction the DCs were stimulated with tumor antigen lysates (TAg, 10% v/v). Mature dendritic cells, which were acquired on the eighth day of DC culture, were harvested and employed for examining in vitro DCs characteristics. Additionally, these mature dendritic cells were utilized as cellular vaccines in subsequent in vivo experiments.

Cytokine production was assessed by utilizing commercially accessible ELISA kits (IL-10, IL-4 (BD Biosciences); IL-12, IL-15/IL-15Rα, IL-18, IFN-γ (eBioscience)), following the guidelines provided by manufacturers.

To assess the impact of LV transduction on DC differentiation levels, we conducted phenotype characterization on the 10^th day of DC culture through flow cytometry. For this purpose, cells were labeled using a cocktail of monoclonal antibodies conjugated with fluorochromes: anti-CD11c Brilliant Violet 650 (clone N418), CD80 PerCP-Cy5.5 (clone 16-10A1), CD86 PE-Cy7 (clone GL-1), MHC II APC-Fire 750 (clone M5/114.15.2) – all sourced from BioLegend, and CD40 Brilliant Violet 605 (clone 3/23) from BD Biosciences. To exclude non-viable cells, DAPI dye (Invitrogen) was used before analysis. The flow cytometric analysis was executed using the LSRFortessa flow cytometer with Diva software (BD Biosciences).

The efficacy of the modified DCs in initiating a primary antigen-specific immune response was appraised through a 5-day co-culture involving mature DCs (0.18 × 10⁶ cells) and naive spleen cells (1.8 × 10⁶ cells). This co-culture was established on the 8^th day of DC cultivation and was conducted in the presence of recombinant human (rh)IL-2 (200 U/ml, ImmunoTools).

Restimulated or primary stimulated spleen cells obtained from 5-day co-culture were subjected to a 2-hour incubation with B16 F0 cells. This incubation occurred in the presence of monoclonal anti-CD107a antibodies conjugated with APC (clone 1D4B, BioLegend), ionomycin (1 µg/ml, Sigma-Aldrich), phorbol-12-myristate-13-acetate (50 ng/ml, Sigma-Aldrich), and rhIL-2 (200 U/ml). Post-incubation, the cells were gathered and stained with anti-CD45 Brilliant Violet 605 (clone 30-F11), anti-CD4 FITC (clone RM4-5), anti-CD8a APC-Fire (clone 53-6.7), and anti-NK1.1 PE-Dazzle 594 (clone PK136) – all obtained from BioLegend. DAPI dye was utilized to exclude non-viable cells. The flow cytometry analysis was conducted employing the LSRFortessa flow cytometer with Diva software (BD Biosciences).

After five days of primary stimulation, cells were harvested and the cytotoxic activity of effector splenocytes against DiO⁺ labeled (Molecular Probes) B16 F0 melanoma cells was analyzed, according to the previously described procedure (29). The dead target cells were distinguished with propidium iodide (PI, Thermo Fisher Scientific) solution and the percentage of DiO⁺PI⁺ B16 F0 cells was determined.

Female C57BL/6 mice aged eight to ten weeks were used in experiment. B16 F0 melanoma cells (3.0 × 10⁴ cells/0.1 ml Matrigel/mouse) were subcutaneously injected into the right flank of the mice. Once palpable tumor nodules were observed, the mice were divided into experimental groups based on tumor volume. Eleven groups were formed for immunotherapy, and twelve groups were formed for chemoimmunotherapy. In both experiments, cellular vaccines were administered peritumorally (p.t.) on the 11^th and 16^th days (2 × 10⁶ cells/0.2 ml NaCl 0.9%/mouse/injection). The anti-IL-10R antibodies (BioXCell) at a dose of 250 µg/0.2 ml/mouse/injection were administered intraperitoneally (i.p.) the day before injection of the cellular vaccines (10^th and 15^th day of the experiment). The therapeutic schedule of chemoimmunotherapy was started on the 8^th day of the experiment with intravenous (i.v.) HES-MTX nanoconjugate injection into the tail vein (at a dose of 20 mg/kg body weight). Detailed information about the HES-MTX preparation can be found in our previous papers (24). The cellular vaccines used in both experiments included three types: non-transduced DC/TAg, DC transduced with a control vector (DC/Vctrl/TAg), and DCs engineered to overproduce cytokines. Each mouse received 2 × 10⁶ modified dendritic cells per injection, regardless of whether the cells overproduced a single cytokine or a combination of two or three cytokines. Cellular vaccines named DC/IL-12/TAg, DC/IL-15/IL-15Rα/TAg and DC/IL-18/TAg consisted of 2 × 10⁶ cells of appropriate transductants, DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg, DC/IL-12/TAg + DC/IL-18/TAg and DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg consisted of 1 × 10⁶ cells of each listed transductants, while DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg was a mixture of three different transductants in the number of 0.667 × 10⁶ cells each.

During the in vivo experiment, tumor growth was regularly monitored and measured using an electronic caliper. The volume of tumors was calculated according to the formula: a2×b2, where a is the largest and b is the smallest diameter of the tumor. To analyze tumor growth kinetics, the nonlinear least squares regression fits of the Gompertz function were utilized. Therapeutic efficacy was determined by calculating the tumor growth inhibition (TGI) value as follows: TGI(%)=100−(TVt/TVnt×100), where TVt refers to a mean tumor volume in the treated group and TVnt – mean tumor volume in the non-treated (nt) group. Three days after the final injection of the DC-based vaccine (19^th day of therapy), spleens and tumor nodules from B16-tumor-bearing mice were dissected, homogenized, and preserved in liquid nitrogen for subsequent ex vivo analyses.

The cell suspension obtained from tumor tissue was thawed and labeled with monoclonal antibodies conjugated to fluorochromes. The following antibodies were used to determine the population of myeloid and lymphoid cells infiltrating the tumor tissue: anti−CD45 Brilliant Violet 605 (clone 30-F11), anti−CD3 PE−CF594 (clone 145-2C11), anti−CD19 PE−CF594 (clone 1D3), anti−NK1.1 PE−Dazzle 594 (clone PK136) (all from BD Biosciences), anti−CD11b PerCP−Cy5.5 (clone M1/70), anti−CD11c Brilliant Violet 650 (clone N418), anti−F4/80 Alexa Fluor 700 (clone BM8), anti−Ly6C PE (clone HK1.4), anti−MHC II FITC (clone M5/114.15.2) (all from BioLegend) for myeloid cell identification, and anti−CD45 Brilliant Violet 605 (clone 30-F11), anti−CD3 Brilliant Violet 650 (clone 17A2), anti−CD4 FITC (clone RM4-5), anti−CD8 APC/Fire 750 (clone 53-6.7), anti-CD19 Alexa Fluor 700 (clone 6D5), anti−CD25 PE (clone PC61) (all from BioLegend) for lymphocyte identification. Then, cells were fixed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Cells stained with lymphocyte cocktail were additionally incubated with anti−FoxP3 APC (clone FJK-16s) (eBioscience) monoclonal antibodies. The flow cytometry analysis was performed using the LSR Fortessa flow cytometer with Diva software (BD Biosciences).

Spleen single-cell suspensions (2.0 × 10⁶ cells) were thawed and cocultured with mitomycin C-treated B16 F0 cells (0.1 × 10⁶ cells) in the presence of rhIL−2 (200 U/ml). After 5 days of restimulation, cells were harvested and the degranulation assay was used according to the previously described procedure (section: Primary stimulation of splenic cells by transduced dendritic cells). Supernatants were collected and stored at 4 °C until ELISA was performed.

Statistical analysis was performed using the GraphPad Prism 9 software (GraphPad Software, Inc.). The analysis of tumor tissue-infiltrating cells and spleen cells was performed from 3–6 mice (nt - 6, Ab - 5, I - 4, II - 4, III - 4, IV - 5, V - 6, VI - 5, VII - 5, VIII - 3, IX - 5) in the case of immunotherapy and 5-9 (nt - 8, H-M - 7, Ab - 6, I - 8, II - 9, III - 8, IV - 6, V - 6, VI - 6, VII - 5, VIII - 6, IX - 7) in the case of chemoimmunotherapy. The normality of the data distribution was assessed using the Shapiro-Wilk test (Supplementary Tables 1-4). For data conforming to a Gaussian distribution (p > 0.05) with uniform standard deviation (SD) values, statistical significance was determined using the parametric one−way ANOVA, followed by Tukey’s multiple comparison post−hoc test. In cases where data aligned with a Gaussian distribution but exhibited unequal SD values, we employed the Brown−Forsythe and Welch ANOVA tests, followed by Dunnett’s T3 multiple comparisons post−hoc test. Data that didn’t adhere to a Gaussian distribution (p ≤ 0.05) underwent analysis using the nonparametric Kruskal−Wallis test, followed by Dunn’s multiple comparison post−hoc test. Number of mice considered for evaluating tumor growth kinetics in immunotherapy: nt - 6, Ab - 7, I - 4, II - 6, III - 5, IV - 6, V - 7, VI - 7, VII - 5, VIII - 3, IX - 7 and chemoimmunotherapy: nt - 8, H-M - 8, Ab - 8, I - 8, II - 10, III - 6, IV - 7, V - 8, VI - 8, VII - 6, VIII - 7, IX - 7. The statistical significance concerning tumor growth kinetics was assessed using the two−way ANOVA, followed by Tukey’s multiple comparisons post−hoc test. Specific details regarding the type of statistical analysis employed can be found in the figure captions. Graphs portray all statistically significant differences using symbols outlined in Table 1.

The study began with the characterization of the obtained cellular vaccines and the evaluation of their ability to trigger immune responses against B16 F0 melanoma. To achieve this, bone marrow-derived dendritic cells were transduced with lentiviral vectors carrying the genes il12, il15/il15Rα, or il18. Additionally, these genetically modified dendritic cells were stimulated with B16 F0 tumor cell lysate (tumor antigens, TAg). Control groups included DCs transduced with a control vector or non-transduced DCs stimulated with TAg (referred to as DC/Vctrl/TAg or DC/TAg, respectively). The vaccine preparation scheme has been detailed in our previous publications (26).

We tested the effectiveness of vaccines consisting of DCs producing one cytokine – one-component vaccine (DC/IL-12/TAg, DC/IL-15/IL-15Rα/TAg, DC/IL-18/TAg), a 1:1 mixture of two types of DCs – two-component vaccine (DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg; DC/IL-12/TAg + IL-18/TAg and DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg), or a 1:1:1 mixture of three types of DCs – three-component vaccine. To evaluate DC activity in in vitro and ex vivo assays, we ensured that the same number of cells was used for each well or single injection, regardless of the vaccine composition.

For this purpose, the dendritic cells were cultured for 48 hours, after which their ability to secrete cytokines was evaluated (Figures 1A–C). The highest concentrations of IL-12 (Figure 1A) and IL-15 (Figure 1B) were observed in cultures with a single type of dendritic cells. In two-component and three-component cell cultures, a proportional decrease in the concentrations of these cytokines was noted, corresponding to the number of cells of a particular cell type. However, all types of dendritic cells were capable of IL-18 secretion (Figure 1C). The highest concentration of IL-18 was recorded in the DC/IL-18/TAg group which confirms the effectiveness of the introduced genetic modification. At the same time, slightly lower levels were found in the mixed cultures containing DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg, as well as in the three-component mixed culture. Notably, the concentration of IL-18 in the mixed two-component culture containing DC/IL-12/TAg + IL-18/TAg was low.

We analyzed whether interleukins overexpression affects phenotypic and functional changes in dendritic cells on the 10^th day of culture. We found that the presence of cytokines in the culture resulted in a statistically significant increase in the percentage of DCs (CD11c⁺DAPI^-) compared to control cells. It was observed when IL-18 was present in the culture alone or when IL-15/IL-15Rα was combined with other factors (IL-12/TAg + IL-15/IL-15Rα/TAg or IL-15/IL-15Rα/TAg + IL-18/TAg) (Figure 1D). Additionally, the overproduction of these cytokines led to greater variation in the expression levels of costimulatory molecules CD40, CD80, CD86, as well as MHC II molecules (Figures 1E–H). The most substantial effect was observed in the presence of IL-12, while the smallest was in the culture of IL-18. Expression of CD40 on the DC cell surface, estimated by mean fluorescence intensity (MFI), was the highest when all three cytokines appeared together in the mixed culture (DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg). In contrast, the highest expression of CD80, CD86, and MHC II molecules occurred in a two-component mixed culture: DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg.

Based on the preliminary characterization of DCs genetically modified with the il12, il15 and il15Rα or il18 genes, we found that developed cells can be carriers of these cytokines and constitute their significant source in the tumor microenvironment. Moreover, these vaccine cells had a high potential to increase immune cell activation due to their higher maturity associated with the expression of costimulatory molecules.

To evaluate the ability of different dendritic cell types to induce a primary stimulation of naïve T cell activity, we conducted a 5-day co-culture with splenocytes (Figure 2). We analyzed changes in CD8⁺, CD4⁺ and NK splenocyte subpopulations. The presence of IL-12 produced by transduced DCs during the primary stimulation of spleen cells resulted in noticeable changes in specific lymphoid cell types. Whether IL-12 was administered alone or in combination with IL-15 or IL-18, we observed a decrease in the percentage of CD8⁺ cells and NK cells, while the population of CD4⁺ T cells increased (Figures 2A–C). The percentages of CD107a on the CD8⁺ and CD4⁺ cells were downregulated (Figures 2D, E), meanwhile, on the NK cells, increased regardless of whether IL-12 was produced by DCs alone (at the highest concentration) or in connection with other cytokines (Figure 2F).

It is noteworthy that the high percentage of active CD8⁺CD107a⁺ cells in mixed cultures lacking IL-12 or containing its low level (group IX) may be related to a slower rate of response compared to conditions with high IL-12 concentrations. Moreover, differences in the size of the CD4⁺CD107a⁺ cell subpopulation were more closely associated with the overproduction of IL-15/IL-15Rα. The presence of IL-12 (groups III, VI, VII, and IX) appeared to be necessary for a significant increase in the NK cell subpopulation.

We also examined the ability of stimulated splenocytes to produce IFN-γ and IL-10. Production of IFN-γ (Figure 2G) was strongly increased when cell stimulation was supported not only by IL-12, but also by IL-15 or IL-18 regardless of IL-12 concentration. This finding confirms previous results showing that a mixture of dendritic cells capable of producing different pro-inflammatory cytokines can be a better stimulator than a one-component vaccine.

A similar pattern was observed for the production of IL-10 (Figure 2H), which was enhanced when splenocytes were stimulated with the combination of DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg (group VI) or DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg (group IX). By analyzing the ratio of IFN-γ to IL-10 (Figure 2I), we indicated that IL-12 produced by transduced DCs significantly affected the primary stimulation of spleen cells. We also confirmed that its combination with IL-15 and/or IL-18 was substantially more effective in generating a strong response.

The cytotoxicity of splenocytes against B16 F0 melanoma cells confirmed the significant role of IL-12, which was overproduced by DC/IL-12/TAg. This effect was also noted at its lower concentrations when the cytokine was supported by DC/IL-15/IL-15Rα/TAg and DC/IL-18/TAg (Figure 2J).

Therefore, dendritic cells transduced with cytokine genes and stimulated with B16 F0 tumor antigens proved effective in inducing specific cellular responses under ex vivo conditions. By primarily overproducing IL-12, and especially in the presence of secreted IL-15 and IL-18, these cells could activate CD4⁺, CD8⁺, and NK cells, leading to high production levels of IFN-γ and IL-10. Additionally, based on the IFN-γ/IL-10 ratio, we propose that our cellular vaccine preparation method can effectively generate potent inducers of the anticancer immune response.

All types of cell vaccines characterized in in vitro experiments were used in the in vivo model.

One of the most critical factors influencing the effectiveness of therapy is the number of injected cells. In our previous studies, we confirmed that a dose of 2 million vaccine cells could induce an effective immune response, leading to significant inhibition of tumor growth (19, 26). Therefore, we decided to administer 2 million cells at a time, regardless of the combination of cytokines.

An above-mentioned in vitro study indicated that multi-component dendritic cell-based vaccines can activate splenocytes to produce significant amounts of IFN-γ and increase specific antitumor cytotoxicity, regardless of parallel IL-10 secretion. Therefore, at the outset of our therapeutic experiments using the B16 F0 melanoma model, we aimed to determine whether multi-component vaccines would be more effective in inhibiting tumor growth and initiating immune responses compared to one-component vaccines. Consequently, we applied cellular vaccines in a combined approach of immunotherapy and chemoimmunotherapy for B16 F0 melanoma (Figures 3A, E). The effectiveness of both treatments was compared based on the tumor growth rate (Figures 3B, F). Treatment outcomes were evaluated on the 19^th day of the experiments, where the median tumor volume was illustrated using violin plots (Figures 3C, G). Additionally, the tumor growth inhibition rate (TGI) for each treatment group was calculated relative to the untreated control group (Figures 3D, H).

The scheme of immunotherapy was presented in Figure 3A. To minimize the adverse effects of IL-10 present in the tumor microenvironment, the immunotherapeutic protocol involved administering cellular vaccines after the application of anti-IL-10R antibodies. Mice with subcutaneously growing B16 F0 tumors were treated intraperitoneally with anti-IL-10R antibodies (250 μg/mouse) on the 10^th and 15^th days of the experiment. One day after the administration of the antibodies, more precisely on the 11^th and 16^th days of the experiment, cellular vaccines (2 × 10⁶ cells/mouse) were applied peritumorally. DC/TAg (group I) and DC/Vctrl/TAg (group II) were used as control cells.

Analyzing the effects of various components of immunotherapy, we found that the use of anti-IL-10R antibodies alone was not efficient in inhibiting tumor growth. In contrast, the application of DC-based vaccines led to changes in tumor growth inhibition, which varied depending on the cell vaccine type. Notably, among the transduced DCs, the one-component vaccine DC/IL-12/TAg (group III) effectively reduced tumor nodules by 43.5%. These results were comparable to those obtained with the three-component vaccine (group IX), which also demonstrated a 43.5% tumor growth inhibition compared to the untreated group. However, the two-component vaccine combining DC/IL-12/TAg and DC/IL-18/TAg (group VII) achieved the most significant tumor growth inhibition, with a TGI of 62.3% (Figure 3D).

The scheme of chemoimmunotherapy was presented in Figure 3E. Our previous report demonstrated that administering the HES-MTX nanoconjugate at a dose of 20 mg/kg body weight resulted in beneficial immunomodulation of the antitumor response in a colon cancer model and B16 F0 melanoma model (24–26). Based on this evidence, the current study extended the therapeutic regimen to include the intravenous administration of the HES-MTX nanoconjugate (20 mg/kg body weight) on the 8^th day of the experiment.

The administration of HES-MTX prior to immunotherapy enhanced the therapeutic effect across most groups, resulting in higher tumor growth inhibition values compared to immunotherapy alone. The overall effectiveness was more influenced by the type of dendritic cell transduction than by high cytokine secretion. Therefore, the combined therapy, which included a three-component DCs vaccine (DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg – group IX), achieved the most significant reduction in tumor growth, with an inhibition rate of 59.1% (Figure 3H).

Our observation suggests that the effect of immunotherapy did not depend on the high capacity of DCs to produce one particular cytokine, but it was conditioned by the mutual beneficial effect of different cytokines, even if they were secreted by DCs at moderate levels. The use of HES-MTX nanoconjugate resulted in statistically significant differences in tumor growth rate between the untreated control group and the groups administered dendritic cells.

Tumor nodules were harvested on the 19^th day of therapy to identify leukocyte influx. In view of this, we used the extended protocol of a multicolor flow-cytometric analysis to determine the influence of the applied therapies on lymphoid and myeloid cell infiltration into tumors and creation of immune response inside the B16 F0 melanoma microenvironment (Figure 4A). Briefly, among alive leukocytes (CD45⁺DAPI^-), we examined changes in the percentage of particular leukocyte subpopulation such as lymphocytes T CD8 (CD3⁺CD8⁺), T CD4 (CD3⁺CD4⁺), Treg (CD3⁺CD4⁺CD25⁺FoxP3⁺), NK (CD3^-NK1.1⁺), NKT (CD3⁺NK1.1⁺) and B lymphocytes (CD19⁺) and also myeloid cells (CD11b⁺) including DCs (CD11c⁺F4/80^intMHC II⁺), TAMs (CD11c⁺F4/80⁺) and MDSCs (CD11c^-Ly6C⁺). Despite extensive cytometric analysis of cells infiltrating the tumor tissue, we did not notice significant differences in all analyzed immune cell populations. Therefore, we have limited discussion of our results to those immune cell subpopulations with the most meaningful alterations.

We started investigating the impact of immunotherapy by evaluating the changes in the percentage of infiltrating CD45⁺ cells due to various types of vaccines. The lowest percentages of leukocyte influx were observed in the anti-IL-10R Ab and DC/Vctrl/TAg groups. In contrast, the highest leukocyte influx was recorded with the combination of DC/IL-12/TAg and DC/IL-18/TAg (group VII) (Figure 4B). The summary plots displaying normalized data helped us visualize the differences among individual leukocyte subpopulations (Figure 4C). Analysis of these specific subpopulations confirmed that lymphocytes represented a small portion of the infiltrating leukocytes. A statistically significant increase in the size of this subpopulation was only observed when the DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg vaccine (group VI) or the DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg vaccine (group VIII) (Figure 4D) were used. The administration of DC-based vaccines led to an increase in the percentage of CD4⁺ cells compared to the non-treated (nt) or antibodies-treated (Ab) groups, however, these changes were not statistically significant (Figure 4E). Similarly, in the case of CD4⁺ cells, no significant changes were noted in the regulatory T cell subpopulations relative to either the nt group or the treated DC/TAg vaccine group (Figure 4F). Notable changes in the percentage of NK cells among the DC-treated groups were only observed after administering the three-component vaccine (group IX) (Figure 4G).

The use of chemoimmunotherapy reduced the percentage of CD45⁺ cells infiltrating tumor tissue, in relation to immunotherapeutic treatment, especially in groups V-VII, making the differences among them unsignificant (Figure 4H). Based on the summary plots with normalized data, we observed that the use of HES-MTX affected the increase in the percentage of CD19⁺ cells (Figure 4I). In comparison to control group DC/Vctrl/TAg the significant decrease in the percentage of CD8⁺ cells was observed when two-component vaccine DC/IL-12/TAg + DC/IL-18/TAg (group VII) was administered (Figure 4J). After the administration of HES-MTX nanoconjugate prior to immunotherapy, there was a strong increase in the percentage of CD4⁺ cells in tumor tissue and this was particularly evident in groups II - V and VIII (Figure 4K).

However, it should be highlighted that despite the HES-MTX-induced increased in the size of CD4⁺ T cells population, the percentage of Tregs among CD4⁺ cells was significantly diminished, especially in group receiving DC/IL-12/TAg (group III) and DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg (group IX) (Figure 4L). An increase in the percentage of NK cells was observed in groups I, VI and IX but was significant only after treatment with the DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg (group VIII) (Figure 4M).

An unexpected effect of immunotherapy with vaccines containing dendritic cell mixtures was observed regarding changes in the size of tumor-associated macrophage (TAM) subpopulations. Specifically, vaccines composed of two or three components increased the percentage of TAMs, whereas control cells (DC/Vctrl/TAg) resulted in a significant decrease in the percentage of TAMs within tumor tissue (Figure 5A). When examining the activation status of TAMs in tumors, as indicated by the level of MHC II expression on their surface, we found that TAMs expressing low levels of MHC II predominated. This suggests that the immune response induced by this type of immunotherapy was polarized more towards a Th2 than a Th1 type (Figure 5B). Additionally, a high percentage of MDSCs was observed, which exhibited only minor variations regardless of the cytokine present in the environment (Figure 5C).

The use of the HES-MTX nanoconjugate in combination with DC-based vaccines significantly reduced the percentage of tumor-associated macrophages compared to the untreated and chemotherapeutically treated groups (Figure 5D). The highest reduction of TAM subpopulation was observed after chemoimmunotherapy with DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg (group VI) or DC/IL-12/TAg + DC/IL-18/TAg (group VII). There was no significant difference between ratio of TAMs with high MHC II expression to cells with low expression (Figure 5E). Furthermore, the application of the nanoconjugate prior to most of the DC-based vaccines (except the group treated with DC/IL-12/TAg + DC/IL-18/TAg – group VII) led to an increase in the percentage of myeloid-derived suppressor cells (Figure 5F).

Overall, using the nanoconjugate prior to immunotherapy reduced the presence of immunosuppressive cells infiltrating the tumor tissue, specifically Treg and TAMs. The most significant effects were observed following the administration of the two-component vaccine (DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg – group VI) and the three-component vaccine.

In both therapies, we assessed the impact of DC-based vaccines on the activation of systemic antitumor responses. Specifically, spleens from B16 F0-bearing mice that had been treated were harvested on the 19^th day and restimulated ex vivo with B16 F0 cells in a 5-day mixed culture. Live splenocytes were then analyzed using multiparameter flow cytometry to determine the changes in the percentages of CD8⁺, CD4⁺, and NK cells, including the proportion of effector cells (identified by the expression of CD107a molecules) (Figure 6A).

Among restimulated splenocytes obtained from immunotherapy-treated mice, all types of DC-based vaccines resulted in a decrease in the percentage of CD8⁺ cells compared to the non-treated control group (Figure 6B). Notably, in the groups treated with DC/TAg, DC/IL-15/IL-15Rα/TAg, DC/IL-18/TAg, and DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg (groups I, IV, V, and VI, respectively), the percentage of CD8⁺ cells was significantly lower than that of the non-treated group.

However, it was observed that the use of DC-based vaccines, particularly DC/IL-18/TAg (group V) and DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg (group VIII), led to an increase in the percentage of CD4⁺ cells among all restimulated splenocytes compared to the untreated group (Figure 6C). Additionally, the percentage of NK cells among restimulated splenocytes reached almost 40% in the non-treated and antibody-treated groups. In contrast, all groups that underwent DC-based immunotherapy exhibited a significant reduction in the size of this subpopulation (Figure 6D).

It is important to note that each type of DC-based vaccine treatment significantly increased the percentage of CD107a⁺ cells compared to the non-treated control group. Among the treatments evaluated, a vaccine containing DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg (group VIII) was the most promising in activating CD8⁺ (Figure 6E) and NK cells (Figure 6F), both of which are capable of releasing cytolytic granules. Additionally, the restimulated CD4⁺ cells from B16 F0-bearing mice showed an increased percentage of CD107a⁺ cells following every type of treatment, indicating an enhancement in the cytotoxic capacity of these cells (Figure 6G).

Similar to the immunotherapy regimen, chemoimmunotherapy resulted in a decrease in the percentage of restimulated CD8⁺ cells in almost all DC-treated groups, especially after treatment with the DC/IL-12/TAg vaccine (group III) (Figure 6H). However, the three-component vaccine enhanced the growth of the CD8⁺ subpopulation compared to the DC/Vctrl/TAg control (group II). An opposing trend was observed regarding the CD4⁺ cell subpopulation after treatment with all vaccine types. This increase was particularly significant after the administration of DC/Vctrl/TAg (group II), DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg (group VI), and DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg (group VIII) (Figure 6I).

In all groups receiving cell vaccines, a decrease in the number of NK cells among splenocytes was observed compared to all control groups (groups: nt, H-M, Ab) (Figure 6J). The increase in the percentage of CD107a⁺ cells within the CD8⁺ cell population was observed following the administration of DC/IL-18/TAg (group V), the two-component vaccine DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg (group VIII) and the three-component vaccine (group IX) (Figure 6K). This suggests that primarily IL-18 may be responsible for the increased size of CD8⁺CD107a⁺ spleen cell subpopulation. In contrast to that subpopulation, the percentage of CD107a⁺CD4⁺ and CD107a⁺NK cells among splenocytes increased in groups receiving cell vaccines, regardless of their type (Figures 6L, M).

To confirm the effectiveness of the antitumor response activated by transduced DCs, we examined the ability of restimulated splenocytes to produce cytokines. In the context of immunotherapy, all restimulated splenocytes obtained from the treated groups (groups I-IX) demonstrated high production levels of IFN-γ, IL-10, and IL-4. Notably, splenocytes from mice treated with the combination of DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg (group VI) produced the highest amounts of IFN-γ (approx. 185 ng/ml) (Figure 7A). While the levels of IL-10 were similar across all groups, the vaccine consisting of two components, DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg (group VIII), induced the highest production of this cytokine (approx. 61 ng/ml) (Figure 7B). The highest levels of IL-4 were observed in the groups receiving either DC/TAg (group I, approx. 3.1 ng/ml), DC/Vctrl/TAg (group II, approx. 6.1 ng/ml) or the combination of DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg (group VIII) (Figure 7C, approx. 5.1 ng/ml). Overall, splenocytes from mice treated with dendritic cells that overproduce IL-12 were more efficient in producing IFN-γ, while the highest production of anti-inflammatory cytokines was observed following the administration of the vaccine DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg (group VIII).

The use of HES-MTX nanoconjugate before immunotherapy resulted in increased production of IFN-γ (Figure 7D) and IL-10 (Figure 7E) by restimulated splenocytes from all groups receiving cellular vaccines. However, there were no statistically significant differences between these groups. The HES-MTX nanoconjugate also influenced changes in the production of IL-4. The highest ability to produce this cytokine was characterized by splenocytes obtained from mice treated with combination therapy including DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg (group VIII, approx. 13.1 ng/ml) and three-component vaccine of DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg (group IX, approx. 17.2 ng/ml) (Figure 7F).