The in vivo growing MC38 murine colon carcinoma from the Tumor Bank of the Radiobiological Institute TNO (Rijswijk, The Netherlands) was adapted to in vitro conditions as described by Pajtasz−Piasecka et al. (35). The culture of MC38/0 (named here MC38) cells was maintained in RPMI−1640 (Gibco) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 0.5% sodium pyruvate, 0.05 mM 2−mercaptoethanol (named here RPMI) and 5% fetal bovine serum (FBS; all reagents from Sigma−Aldrich). Lenti-X 293T cell line (Clontech) was maintained in high-glucose Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 1 mM sodium pyruvate and 10% FBS. Cultures were carried out under standard conditions (5% CO₂, 37°C).

The MC38 colon cancer cells were harvested and resuspended at a density of 5×10⁶ cells/mL in RPMI-1640 (Gibco) supplemented with 10% FBS (Sigma-Aldrich). All cells were subjected to 5 cycles of freezing in liquid nitrogen and quick thawing at 37°C. The cell suspension was then sonicated for 90 min. Tumor antigens (TAg) were used to stimulate dendritic cells.

Lentiviral vectors (LVs) were produced using a third-generation lentiviral system, which consisted of auxiliary vectors: pMDLg/pRRE, pRSV Rev, pMD2.G [the plasmids were a gift from Didier Trono (Addgene plasmids #12251, 12253, 12259)] and expression vector. The expression plasmids encoded the cytokine genes: il12, il15/il15ra or il18. In the vector carrying the il15 gene, the gene sequence of this cytokine was preceded by a signal sequence facilitating the release of the protein from the cell. In addition, after the gene encoding the cytokine and the linker, there was a gene sequence encoding the alpha subunit of the IL-15 receptor, in order to delay the process of intracellular degradation of this protein. A control vector was also created to check the effect of lentiviral transduction on dendritic cells. All expression vectors were purchased from VectorBuilder. Lentiviral vectors were produced by Lenti-X 293T cells (ClonTech) co-transfected with the above-mentioned plasmids and cultured for 48 hours. The supernatant containing lentiviral vectors was collected and concentrated by precipitation using PEG 6000 (Sigma-Aldrich). The pellet containing the lentiviral vectors was suspended in PBS and stored at -80°C. The procedure of lentiviral vectors production was described in our previous article (36). The titer of the lentiviral vector was determined by serial dilution method using MC38 cells and flow cytometry analysis.

Female C57BL/6 mice were obtained from the Center for Experimental Medicine of the Medical University of Białystok, Poland. All experiments were performed in accordance with Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes and were approved by the Local Ethic Committee for Experiments with the Use of Laboratory Animals, Wrocław, Poland (authorization number 077/2019). After the experiments, mice were sacrificed by cervical dislocation.

Dendritic cells were generated from bone marrow isolated from femurs and tibias of healthy female C57BL/6 mice according to the protocol described in our previous publication (37). Cells (named here DCs) were cultured in RPMI-1640 (Gibco) supplemented with 10% FBS (Sigma-Aldrich) in the presence of recombinant murine (rm)GM−CSF (ImmunoTools, 40 ng/ml) and rmIL−4 (ImmunoTools, 10 ng/ml). After 7 days of culture, loosely attached immature DCs were transduced with LVs (with the assumption: 4 viral infectious particles per 1 dendritic cell) in the presence of 8 µg/ml polybrene (Sigma−Aldrich). After 4 hours DCs, were stimulated with tumor antigen lysates (TAg, 10% v/v). Mature dendritic cells obtained on day 8 of DC culture were collected and applied to in vitro DC characteristics or used as cellular vaccines in in vivo experiments.

In order to estimate the influence of LV transduction on the differentiation level of DCs, the phenotype characteristic was performed on 8 and 10 days of DC culture by flow cytometry. Cells were labeled with 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 from BioLegend) and CD40 Brilliant Violet 605 (clone 3/23) (from BD Biosciences). In order to exclude dead cells, DAPI dye (Invitrogen) was added prior to analysis, which was performed using the LSRFortessa flow cytometer with Diva software (BD Biosciences).

The ability of the modified DCs to activate primary antigen-specific immune response was evaluated in a 5-day co-culture of mature DCs (0.18×10⁶ cells) and naive spleen cells (1.8×10⁶ cells) in the presence of recombinant human (rh)IL−2 (200 U/ml, Immunotools). In order to determine the percentage of CD107a⁺ cells, the degranulation assay was performed. Primarily stimulated spleen cells were incubated for 2 hours with MC38 cells in the presence of monoclonal anti−CD107a antibodies conjugated with APC (clone 1D4B, BioLegend) together with ionomycin (1 µg/ml, Sigma-Aldrich), phorbol-12-myristate-13-acetate (50 ng/ml, Sigma-Aldrich) and rhIL-2 (200 U/ml). Afterwards, cells were harvested 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 from BioLegend). For the elimination of the dead cells, DAPI dye was used. The flow cytometry analysis was performed using the LSRFortessa flow cytometer with Diva software (BD Biosciences).

Eight-to-ten-week-old female C57BL/6 mice were subcutaneously (s.c.) inoculated in the right flank with MC38 cells (1.1×10⁶ cells/0.2 ml NaCl 0.9%/mouse). When tumor nodules were palpable, mice were randomly divided on the basis of tumor volume into ten or eleven experimental groups in immuno- and chemoimmunotherapeutic schedule, respectively. In both experiments, cellular vaccines were inoculated peritumorally (p.t.) on the 17^th, 24^th and 31^st days of the experiment (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 (16^th, 23^rd and 30^th day of the experiment). The therapeutic schedule of chemoimmunotherapy was started on the 14^th day of the experiment with intravenously (i.v.) HES-MTX nanoconjugate injection in the tail vein (at a dose 20 mg/kg body weight). The HES-MTX preparation was described in detail in our previous papers (10, 12). In both experiments, three types of cellular vaccines were used – non-transduced DC/TAg, DC transduced with a control vector (DC/Vctrl/TAg) and DCs modified to overproduce cytokines. Mice received 2×10⁶ modified dendritic cells per 1 injection, regardless of whether the mice received cells overproducing only one cytokine or a mixture of cells producing 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 experiment, two or three times a week, tumors were measured by using an electronic caliper and tumor volume was calculated according to the formula: a / 2 × b 2 , where represents the largest and represents the smallest tumor diameter (38). MC38 tumor growth is shown as relative tumor volume. Relative tumor volume was defined as the ratio of tumor volume on the day of measurement to that tumor volume on the day of randomization (day 13). Tumor growth kinetics are presented as a non-linear least squares regression fits of the Gompertz function. The therapeutic efficacy was determined on the basis of the tumor growth inhibition (TGI) value calculated as follows: T G I   ( % ) = 100 − ( T V t / T V n t × 100 ) , where T V t refers to a median tumor volume in the treated group and TV_nt – median tumor volume in the non-treated (nt) group (37). Seven days after the last DC-based vaccine injection, spleen and tumor nodules were dissected from MC38-tumor bearing mice, homogenized, and stored in liquid nitrogen for further ex vivo analyses.

Single-cell suspensions of tumor tissue were thawed and stained for identification of myeloid and lymphoid cell subpopulations. Tumor suspensions were stained with the LIVE/DEAD Fixable Violet Dead Staining Kit (Thermo Fisher Scientific, Inc.) and then labeled with cocktails of fluorochrome−conjugated monoclonal antibodies: 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−CD45 Brilliant Violet 605 (clone 30-F11), 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−Ly6G APC−Cy7 (clone 1A8), 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 myeloid, or lymphocyte cocktail were additionally incubated with anti−CD206 APC (clone C068C2) (BioLegend) or anti−FoxP3 APC (clone FJK-16s) (eBioscience) monoclonal antibodies, respectively. The flow cytometry analysis was performed using the LSRFortessa flow cytometer with Diva software (BD Biosciences).

Spleen single-cell suspensions (2×10⁶ cells) were thawed and cocultured with mitomycin C-treated MC38 cells (0.1×10⁶ cells) in the presence of rhIL−2 (200 U/ml). After 5 days of restimulation, cells were harvested and the cytotoxic activity of effector splenocytes against target (MC38) cells stained with DiO lipophilic dye (Molecular Probes) was analyzed according to the previously described procedure (38). Two E:T (effector to target) ratios were investigated: 10:1 and 30:1. The dead target cells were distinguished with propidium iodide (PI, Life Technologies) solution and the percentage of DiO⁺PI⁺ MC38 cells was determined. The degranulation assay was used as described earlier (section: Primary stimulation of splenic cells by transduced dendritic cells). Supernatants were collected and stored at 4°C until ELISA was performed.

Production of cytokines was evaluated using commercially available ELISA kits (IL−10, IL−4; BD Biosciences and IL-12, IL-15/IL-15Rα, IL-18, IFN−γ; eBioscience) according to the manufacturer’s instructions.

All data were analyzed using GraphPad Prism 9 software (GraphPad Software, Inc.). The normality of the residuals was confirmed by the D’Agostino−Pearson omnibus test. When data were consistent with a Gaussian distribution and had equal SD values, the statistical significance was calculated using the parametric one−way ANOVA followed by Tukey’s multiple comparison post−hoc test. When data were consistent with a Gaussian distribution, but SD values were not equal, the Brown−Forsythe and Welch ANOVA test followed by Dunnett’s T3 multiple comparisons post−hoc test was performed. Data inconsistent with a Gaussian distribution were analyzed using the nonparametric Kruskal−Wallis test followed by Dunn’s multiple comparison post−hoc test. The statistical significance of tumor growth kinetics was calculated using the two−way ANOVA followed by Tukey’s multiple comparisons post−hoc test. The type of statistical analysis used is described in the captions under the figures. All statistically significant differences are presented in the graphs in the form of symbols described in Table 1 .

In the first stage of our research, we obtained mature bone marrow-derived dendritic cells (DCs) capable of increased production of interleukin: IL-12, IL-15 or IL-18 (DC/IL-12/TAg, DC/IL-15/IL-15Rα/TAg; DC/IL-18/TAg). The appropriate gene for each cytokine was introduced by transduction with lentiviral vectors. Dendritic cells were stimulated with a tumor lysate (tumor antigens, TAg) and harvested on the 8^th day of culture to determine their phenotypic and functional characteristics. DCs transduced with the control vector (DC/Vctrl/TAg) or non-transduced DCs (DC/TAg) stimulated with TAg were used as a control ( Figure 1A ). We observed a decrease in the percentage of CD11c⁺DAPI^- cells after transduction with vectors carrying genes of the particular cytokines in relation to the DC/TAg group ( Figure 1B ).

Phenotype analysis showed that the type of introduced gene considerably affected the maturation of DCs. The expression of CD40, CD80, CD86 costimulatory and MHC II molecules was determined on the surface of these cells ( Figures 1C–F ). DCs modified to cytokine production showed increased expression of CD40 and CD80 molecules compared to cells transduced with the control vector (DC/Vctrl/TAg) ( Figures 1C, D ). Compare to that control, a significantly increased expression of CD86 molecules was noticed after DC transduction with il15 and il18 genes ( Figure 1E ). Whereas only slight differences in the expression of MHC II molecules on the surface of these cells were observed ( Figure 1F ). The highest expression of MHC II was found on the surface of DC/IL-12/TAg and the lowest on DC/IL-18/TAg.

To assess the mutual influence of each overproduced cytokine on the level of DCs differentiation and activation of the antitumor response, all DC types were cultured for 48 hours in different configurations. We tested the simultaneous effect of two (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 three (DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg) cytokines on the DC phenotype and induction of a specific cellular response ( Figure 2 ). Based on our former experience with using DCs as anticancer vaccines, we have known that one of the essential parameters affecting the effectiveness of the therapy is the number of injected cells. Therefore, to determine the effect of DC modifications on their antitumor activity we decided to apply the same number of cells to every single injection regardless of the composition of the vaccines. Thus, we tested control vaccines (DC/TAg and DC/Vctrl/TAg) and 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 (DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg).

The highest concentration of introduced cytokines was noted in cultures with DCs producing one cytokine (groups III, IV and V) ( Figures 2A–C ). In two-component or three-component vaccines, a reduced concentration of interleukins was observed, which was related to the number of individual cytokine-producing cells in the mixture. Despite this, in mixed cultures (groups VI-IX), a greater influence of cytokines on changes in the phenotype of DCs than in monocultures was observed. The highest expression of the CD40 molecule was demonstrated on the surface of DC/IL-18/TAg cells and in a mixed culture of these cells with DC/IL-15/IL-15Rα/TAg and DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg ( Figure 2D ). No effect of cytokines on changes in the expression of the CD80 on the surface of vaccine cells was observed ( Figure 2E ). However, a slight increase in the expression of this molecule was shown in the culture of transduced DCs compared to DC/TAg. This may suggest that the presence of CD80 on the surface of transduced DCs is related to viral rather than cytokine stimulation. The expression of CD86 molecules was increased in the DCs mixed cultures, especially in relation to cells transduced with the control vector ( Figure 2F ). However, such an effect was not observed on the surface of one-component vaccines. An increase in the level of MHC II expression on transduced DCs in mixed cultures compared to the monoculture was determined ( Figure 2G ).

The DC ability to primary stimulate naïve T cells and induce a specific antitumor response was assessed in a 5-day co-culture of splenocytes with all types of vaccine cells. Changes in the percentage of CD8⁺, CD4⁺ and NK cells, as well as their ability for degranulation assessed by the expression of the CD107a molecule on their surface, were determined. Moreover, we investigated the ability of stimulated splenocytes to produce IFN-γ and IL-10.

The largest population of splenocytes in co-cultures were CD8⁺ cells, which accounted for approx. 50% of all spleen cells stimulated with DCs. An increase in the percentage of these cells was observed when splenocytes were cultured with the three-component vaccine in relation to co-culture with one-component vaccines and cells modified with the control vector ( Figure 2H ). A decisive effect of DC/IL-12/TAg or their mixture with other transductants was found to increase the percentage of CD4⁺ cells in the co-culture ( Figure 2I ), and oppositely – a decrease in NK cell percentage was revealed ( Figure 2J ). Among the CD8⁺ cell populations, the highest expression of the CD107a molecule was observed after splenocytes contact with DCs overproducing IL-12, regardless of whether it was produced alone or in combination with other cytokines ( Figure 2K ). No significant differences in the size of the CD107a cell population were observed among CD4⁺ cells ( Figure 2L ), while among NK cells ( Figure 2M ) a statistically significant increase was observed when splenocytes were co-cultured with DCs overproducing IL-12 (groups III, VI, VII and IX). Although increased production of IFN-γ ( Figure 2N ) and IL-10 ( Figure 2O ) was detected in all groups with DC/IL-12/TAg, the highest concentration of these cytokines was observed after splenocyte stimulation with DC/IL-12/TAg and DCs overproducing IL-15 or IL-18 (groups VI and VII).

The tested cellular vaccines were capable of secreting cytokines whose genes have been introduced using a third-generation lentiviral system. Although cellular vaccines consisting of dendritic cell mixtures produced correspondingly smaller amounts of particular cytokines, the effect of these combinations revealed stronger stimulation of DCs. Thus, a preliminary assessment of the effectiveness of DCs transduced with cytokine genes and stimulated with TAg showed their significant effect on inducing a specific cellular response in ex vivo conditions. Dendritic cells overproducing IL-12 alone or in combination with other cytokines could activate CD4⁺, CD8⁺, and NK cells, and consequently, increase their ability to produce IFN-γ and IL-10. This indicates that this way prepared vaccines can be potent inducers of the anticancer immune response.

In the next research stage, we determined whether, similarly to in vitro conditions, the combination of transductants will cause a better therapeutic effect manifesting the inhibition of tumor growth than one-component vaccines administered in the same final number of cells. In immunotherapeutic protocol, we decided to precede the administration of cell-based vaccines with an intraperitoneal injection of anti-IL-10R antibodies to make the immune system cells insensitive to the adverse effects of IL-10 produced in TME. This decision was based on our previous in vitro observations that vaccine dendritic cells are able to activate the splenocytes to produce the high IFN-γ amount accompanied by IL-10 secretion. Furthermore, after completing immunotherapy with cellular vaccines supported with anti-IL-10R antibodies, we conducted another chemoimmunotherapy experiment – in which the previous immunotherapy was supplemented with the administration of the HES-MTX nanoconjugate.

On the 16^th, 23^rd, and 30^th day, mice were inoculated intraperitoneally with anti-IL-10R antibodies (250 µg/mouse). Day after (17^th, 24^th, and 31^st day), cellular vaccines were administered peritumorally (p.t.) (2×10⁶ cells/mice). As control cells, DC/TAg, and DC/Vctrl/TAg were used. On the 38^th day, the therapeutic effect of the treatment was determined ( Figure 3A ). In the chemoimmunotherapy experiment, the protocol was supplemented with an intravenous injection of HES-MTX (20 mg/kg b.w.) on the 14^th day of the experiment ( Figure 3E ).

Based on tumor volume measurements carried out during the therapeutic experiments, tumor growth curves ( Figures 3B, F ), and violin plots ( Figures 3C, G ) were prepared to determine the kinetics of tumor growth. The efficacy of both therapies was estimated based on relative tumor volume - the ratio of tumor volume on the day of measurement to tumor volume on the day of randomization (day 13). This method of presentation depicted the tumor growth rate relative to their initial volume and unified the differences between individual therapeutic groups. Hence, on the 38^th day of the experiment, the degree of tumor growth inhibition (TGI) in the therapeutic groups compared to the non-treated group was calculated ( Figures 3D, H ).

The use of anti-IL10R antibodies in therapy inhibited the tumor growth rate in relation to the group of non-treated mice (TGI 30.2%). Moreover, the application of anti-IL10R antibodies and DC-based vaccines influenced the changes in tumor growth rate and this effect depended on the type of applied vaccine. It should be underlined that immunotherapy with one-component vaccines resulted in the greatest inhibition of tumor growth rate compared to the non-treated (nt) group ( Figure 3D ). The MC38 tumors growth inhibition in these groups ranged from 49.5% (for DC/IL-12/TAg) to 66% (for DC/IL-15/IL-15Rα/TAg). The effect of the two-component vaccines on TGI was weaker and an after the application of anti-IL-10R antibodies and the three-component vaccine, an ineffective treatment was shown.

In our previous studies, we found that using 20 mg/kg b.w. of the HES-MTX nanoconjugate leads to beneficial immunomodulation of the antitumor response (12). Therefore, this chemotherapeutic agent was administered to enhance the therapeutic effect of the herein used immunotherapy. The extension of the therapeutic scheme with the use of the HES-MTX prior to immunotherapy resulted in a strong increase in the value of TGI. The combined therapy resulted in the greatest inhibition of tumor growth when the vaccine containing DC/IL-15/IL-15Rα/TAg and DC/IL-18/TAg cells (72.4%, group VIII) was applied, whereas the use of the one-component vaccine (DC/IL-15/IL-15Rα/TAg or DC/IL-18/TAg, group IV and V, respectively) induced tumor growth inhibition on the level of 54.9% and 70.5% respectively ( Figure 3H ). Nevertheless, the most surprising result of combined chemoimmunotherapy was obtained after the administration of the three-component vaccine - supplementing the treatment schedule with the HES-MTX nanoconjugate administration resulted in an increase of TGI value to 69.9% in contrast to 24.0%, which was observed in this group but in the immunotherapeutic scheme of treatment.

Thus, our observations can suggest that the effect of the applied immunotherapy depends not only on the amount of delivered cytokines but also on their combination. Nonetheless, the administration of the HES-MTX nanoconjugate facilitated the enhancement of vaccine efficacy, even when the amount of produced cytokines turned out too weak to contribute to the therapeutic effect in the course of immunotherapy.

Interested in the changes in the tumor growth rate between the applied therapies, we decided to check their influence on leukocyte infiltration into tumors. For this purpose, MC38 tumor nodules were harvested on the 38^th day of therapy and multiparameter cytometric analyses were performed to identify myeloid and lymphoid cell influxes ( Figure 4A ). Briefly, among alive leukocytes (CD45⁺DAPI^-), we determined the effect of the applied therapies on changes in the percentage of lymphoid cells among leukocytes 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⁺). There were also distinguished populations of the myeloid cells (CD11b⁺) including DCs (CD11c⁺F4/80^intMHC II⁺), TAMs (CD11c⁺F4/80⁺), MDSCs (CD11c^-Ly6C⁺), M1 (CD206^-) and M2 (CD206⁺) macrophages. Despite extensive cytometric analysis of cells infiltrating tumor tissue, we did not observe significant differences in all populations. Therefore we decided to discuss only some of them in more detail.

After the application of immunotherapy, we observed a slight increase in the percentage of leukocytes in tumor tissue obtained from mice treated with DC-based vaccines, which was the highest when DC/IL-12/TAg was used. ( Figure 4B ). However, based on summary graphs presenting normalized data, we also visualized the differences in individual subpopulations of leukocytes infiltrating tumor tissue ( Figure 4C ).

The percentage of CD8⁺ cells infiltrating tumor tissue increased after the administration of cellular vaccines and anti-IL10R antibodies. However, a statistically significant increase was observed only after the use of DC/IL-12/TAg + DC/IL-18/TAg ( Figure 4D ). Application of DC-based vaccines generated an increase in the percentage of CD4⁺ cells relative to the non-treated (nt) and antibodies-treated (Ab) groups. A statistically significant increase in the percentage of CD4⁺ cells was observed after the use of DC/IL-12/TAg and DC/IL-15/IL-15Rα/TAg and two-component vaccines ( Figure 4E ). At the same time, there was a substantial reduction in the population size of regulatory T cells among CD4⁺ cells in tumor tissue. This decrease in the percentage of Tregs was rather related to the use of DC-based vaccines, regardless of the ability of DCs to overproduce cytokines ( Figure 4F ). We also observed a slight reduction in the percentage of NK cells after the administration of DC-based vaccines consisted of more than one type of produced cytokines (groups VI-IX) ( Figure 4G ).

After chemoimmunotherapy, a slight reduction in the leukocyte population size was noted in all therapeutic groups ( Figure 4H ). The lowest percentage of CD45⁺ cells was found after using DC/IL-12/TAg (group III). In addition, there were substantial differences between the effects of different types of vaccines on TAM, M1, M2, CD4+, CD8+, and NK cell population sizes ( Figure 4I ).

A statistically significant increase in the percentage of CD8⁺ cells was observed as a result of chemoimmunotherapy containing a mixture of DCs overproducing IL-15 and IL-18 (group VIII) or all three tested cytokines (group IX) in relation to control groups (groups nt, H-M) and one-component DC-based vaccines. Furthermore, a slight increase in the size of the CD8⁺ cell population was observed after the administration of DC/IL-12/TAg + DC/IL-18/TAg (group VII) ( Figure 4J ). The high percentage of CD4⁺ cells in tumor tissue was always observed when DC-based vaccines were applied ( Figure 4K ). In both T lymphocyte subpopulations, using the nanoconjugate prior to immunotherapy was the most conducive to the effect of two or three-component vaccines. The administration of the chemotherapeutic agent did not enhance the effect of the immunotherapy on changes in the size of Treg cell population ( Figure 4L ). Meanwhile, an increase in the percentage of NK cell population was visible, especially in groups I, II, III and VII ( Figure 4M ).

In the case of the immunotherapeutic treatment schedule, the applied DC-based vaccines did not cause significant changes in the percentage of myeloid cells in tumor tissue. Although there was no effect of this therapy on changes in the size of the TAM population ( Figure 5A ), we observed differences in their activation level. Based on the ratio of TAM MHC II^high to TAM MHC II^low, we found that in the groups receiving cellular vaccines, TAMs with high expression of MHC II were slightly dominated and the highest value of this ratio was observed after the administration of a two-component DC vaccine containing DC/IL-12/TAg and DC/IL-18/TAg ( Figure 5B ). Besides, analyzing the effect of therapy influences on macrophage influx, we observed an increased M1 to M2 macrophage ratio in the groups receiving two-component and three-component DC vaccines ( Figure 5C ).

The combined therapy with the use of the HES-MTX nanoconjugate induced statistically significant changes in the TAM percentage relative to the non-treated or the chemotherapeutic treated groups ( Figure 5D ). The highest value of the TAM MHC II^high/TAM MHC II^low ratio was shown after the administration of chemoimmunotherapy containing two or three-component vaccines comprising DC/IL-12/TAg ( Figure 5E ). On the other hand, the highest value of the M1/M2 ratio was demonstrated after therapy with HES-MTX, anti-IL-10R and DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg ( Figure 5F ).

The use of the HES-MTX nanoconjugate before immunotherapy created optimal conditions for the effective action of cell-based vaccines. The most favorable effect was observed in the groups receiving dendritic cell mixtures, especially the three-component vaccine.

To confirm the assumption that the enhancement of the effect of both multicomponent immunotherapy and chemoimmunotherapy depends mainly on the use of DCs mixtures, we assessed their effect on the activity of systemic antitumor response. For this purpose, on the 38^th day of both therapies’ spleens from treated tumor-bearing mice were harvested and restimulated with MC38 cells in a 5-day mixed culture. Next, multiparameter cytometric analyses were performed among alive splenocytes (CD45⁺DAPI^-), to identify changes in the percentage of CD8⁺, CD4⁺ and NK cells as well as their ability to secrete cytolytic granules based on the expression of the CD107a molecule ( Figure 6A ).

There was no significant change in the size of the CD8⁺ cell population among restimulated splenocytes obtained from mice after immunotherapy with cytokine-secreting DC-based vaccines ( Figure 6B ). Solely after the application of anti-IL-10R and DC/TAg (group I) the percentage of these cells was significantly lower than in the untreated group. An increase in the percentage of CD4⁺ cells was seen after treatment with cellular vaccines, especially after administration of DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg and DC/IL-12/TAg + DC/IL-18/TAg ( Figure 6C ). However, after the application of all cell-based vaccines, there was a reduction in the percentage of NK cell population ( Figure 6D ). The applied therapy did not significantly change the percentage of CD107a⁺ cells among CD8⁺ cells ( Figure 6E ) and NK cells ( Figure 6G ) in comparison to their controls. Meanwhile, the ability to release cytolytic granules increased among CD4⁺ cell populations, turn to be related to the anti-IL-10R antibodies administration ( Figure 6F ).

The supplementation of treatment with the HES-MTX nanoconjugate caused a reduction in the percentage of CD8⁺ cells, especially after DC/Vctrl/TAg (group II), DC/IL-12/TAg (group III), and DC/IL-15/IL-15Rα/TAg (group IV). However, the visible increase in the percentage of this population in relation to DC/Vctrl/TAg occurred after the administration of DC/IL-12/TAg + DC/IL-18/TAg (group VII) vaccines ( Figure 6H ). The administration of combined therapy with cytostatic caused an increase in the percentage of CD4⁺ cells, like immunotherapy, but the changes concerned other therapeutic groups. This cell population’s size increased after the DC/Vctrl/TAg (group II) and DC/IL-12/TAg (group III) vaccines. An enhanced effect of DC/IL-15/IL-15Rα/TAg and the three-component vaccine on the population of restimulated splenic CD4⁺ cells ( Figure 6I ) was also observed. The administration of the chemotherapeutic agent and DC-based vaccines supported with anti-IL-10R antibodies deepened the reduction in the percentage of NK cells in relation to immunotherapy alone ( Figure 6J ). The effect of the applied chemoimmunotherapy on the size of the cell subpopulations able to release cytolytic granules was also revealed. The lowest ability to secrete cytolytic granules was found in CD8⁺ cells obtained from mice treated with HES-MTX + anti-IL-10R + DC/Vctrl/TAg ( Figure 6K ). In contrast, among CD4⁺ cells, the highest cytolytic activity was observed in the group of mice receiving dendritic cells modified to overproduce IL-15/IL-15Rα ( Figure 6L ). The size of NK CD107a⁺ population increased in all groups receiving DC-based vaccines in relation to control groups (groups: nt, H-M and Ab) ( Figure 6M ).

Comparison of both therapies harnessing schedules, including treatment with antibodies, and/or additional supplementation with HES-MTX, resulted in marked differences compared to the non-treated group. The use of cytostatic agent deepened variations among particular groups and enhanced the mixture effect of the DC-based vaccines, especially these three-component. Consequently, such a multicomponent combination enhanced the systemic activity of main populations of immune cells.

As confirmation of the potential for antitumor activity, the ability of restimulated splenocytes to produce cytokines and their cytotoxic activity against MC38 cells was examined. In the case of immunotherapy, splenocytes obtained from mice treated with DC/IL-12/TAg were able to produce the highest amount of IFN-γ ( Figure 7A ). The highest production of IL-4 was noted after the application of the therapy consisted of DC/IL-15/IL-15Rα/TAg (group IV) and DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg (group VI) ( Figure 7B ). In contrast, increased production of IL-10 by splenocytes was shown in the groups of mice treated with DC-based vaccines, as well as anti-IL-10R antibodies alone ( Figure 7C ). The highest cytotoxic activity of splenocytes against MC38 cells ( Figure 7D ) was observed in all groups treated with DC-based vaccines, especially after the application of DC/TAg (group I), DC/IL-15/IL-15/Rα/TAg (group IV) and the three-component vaccine (group IX).

The use of the HES-MTX nanoconjugate prior to immunotherapy strongly changed the type and level of cytokine production by splenocytes causing an increase in their ability to produce IFN-γ ( Figure 7E ). Thus, while the production level of this cytokine did not change markedly after the administration of DC/IL-12/TAg alone, the splenocyte’s ability to produce IFN-γ increased after the use of DCs mixed cultures, especially in DC/IL-12/TAg + DC/IL-18/TAg group (group VII). The HES-MTX nanoconjugate also influenced changes in the production of IL-4 by spleen cells obtained from mice after therapy. The highest ability to produce IL-4 was characterized by splenocytes obtained from mice treated with combination therapy with DC/IL-15/IL-15Rα/TAg (group IV), DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg (group VIII) and three-component vaccine (group IX) ( Figure 7F ). Splenocytes obtained from mice which were treated with vaccines containing DCs showed a decrease in IL-10 production which suggest the influence of the HES-MTX nanoconjugate on the prolongation of the decrease of suppressor cell activity ( Figure 7G ). The addition of the nanoconjugate involved a slight increase in the splenocytes’ cytotoxic activity compared to immunotherapy. However, it depended on the type of vaccine and did not reveal which modified dendritic cell mixtures should be considered the most powerful ( Figure 7H ).

The observed changes in the systemic anticancer response confirm that the use of the HES-MTX chemotherapeutic agent has the most beneficial action on the cellular vaccines that are a mixture of two or three transductants. In these groups, an increase in IFN-γ and a decrease in IL-10 production, as well as an enhancement in the cytotoxic activity of restimulated splenocytes are particularly visible.

The use of the developed vaccines in anticancer therapy in combination with anti-IL-10R antibodies and/or a chemotherapeutic agent influenced both the changes in the tumor microenvironment and the activation of the systemic immune response. Both the antibodies and the chemotherapeutic administered prior to DC-based vaccines created a favorable microenvironment in which the vaccines were characterized by different mechanisms of action ( Figure 8 and Supplementary Tables 1 – 4 ) mainly representing the effect of overproduced cytokines. By virtue of the use of each of the developed vaccines, the percentage of Treg lymphocytes in both immuno- and chemoimmunotherapy decreased. The supplement of the cellular vaccines treatment with the HES-MTX nanoconjugate affected the enhanced tumor growth inhibition, with the exception of the DC/IL-15/IL-15Rα/TAg.

In the immunotherapy, the DC/IL-12/TAg mainly increased the influx of CD8⁺, CD4⁺ and NK cells to tumor nodules, which was accompanied by a decrease in the population of Treg and TAMs and an increase in IFN-γ production and cytotoxic activity of splenocytes. The addition of HES-MTX agent had a positive effect primarily on the systemic immune response. The use of DC/IL-15/IL-15Rα/TAg increased the influx of CD4⁺ and NK cells into tumors. The potential of splenocytes to release cytolytic granules by CD8⁺ and CD4⁺ cells, IL-10 production and cytotoxic activity also increased. Meanwhile, supplementation of the immunotherapy schedule with nanoconjugate did not favorably affect the effect of this vaccine in tumor tissue, resulting in a reduction of CD4⁺ and NK cell percentage. In addition, a decrease in the percentage of CD8⁺ cells and no change in the cytotoxic activity of this cell subpopulation among the restimulated splenocytes was observed. However, this treatment resulted in an increase in the percentage of CD4⁺CD107a⁺, NK CD107a⁺ cells, IL-4 production and cytotoxic activity. Dendritic cells overproducing IL-18 mainly affected the percentage reduction of Treg lymphocytes, NK cells, M1/M2 and TAM MHC II^high/TAM MHC II^low ratios in tumors, while the population of CD8⁺CD107a⁺ and NK CD107a⁺ cells and cytotoxic activity of splenocytes increased. The therapy combined with nanoconjugate caused the reduction of TAM percentage. Although among the restimulated splenocytes the percentage of cells capable of releasing cytolytic granules and production of IL-10 decreased, the production of IFN-γ increased.

In immunotherapy, all two-component vaccines caused a reduction of TAMs and NK cell populations in tumors, but a higher percentage of CD8⁺CD107a⁺ cells was observed among restimulated splenocytes. During immunotherapy, the DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg vaccine increased the percentage of CD4⁺ cells and the M1/M2 macrophages ratio in tumors, while the percentage of CD8⁺ cells, Treg, NK and TAMs decreased. Among the splenocytes, a higher percentage of CD4⁺, CD8⁺CD107a⁺ and NK CD107a⁺ cells, production of IFN-γ and IL-10 and cytotoxic activity were observed. During chemoimmunotherapy, only the M1/M2 ratio remained at an increased level and a high TAM MHC II^high/TAM MHC II^low ratio was observed. Among splenocytes, in addition to a decrease in the population of NK, CD8⁺CD107a⁺, CD4⁺CD107a⁺cells, an increase in the production of IFN-γ, IL-10 and cytotoxic activity was revealed. Administration of DC/IL-12/TAg + DC/IL-18/TAg resulted in an augmented influx of CD4⁺ and CD8⁺ cells into the tumors and growth of M1/M2 and TAM MHC II^high/TAM MHC II^low ratios. Among the splenocytes, an increase in the percentage of CD4⁺ cells and cytolytic activity of CD8⁺ and NK cells was observed, as well as higher production of IFN-γ and a decrease in the production of both IL-4 and IL-10, which could have enhanced the cytotoxic activity of splenocytes. The addition of the chemotherapeutic intensified the influx of NK cells, but the M1/M2 ratio decreased. In the spleens, an increase in the percentage of CD8⁺ cells was observed, with a simultaneous reduction of CD4⁺ cell percentage. During immunotherapy, a vaccine combining DC/IL-15/IL-15Rα/TAg and DC/IL-18/TAg induced tumor growth inhibition of 41.7%, which may be the result of the influx of CD4⁺ cells with a simultaneous reduction in the percentage of tumor suppressor Tregs and TAMs. However, this vaccine induced a decrease in the percentage of CD8⁺ and NK cells. Among the splenocytes, a decrease in the percentage of NK cells and a higher percentage of CD8⁺CD107a⁺ cells were observed. The addition of the chemotherapeutic agent resulted in this vaccine eliciting the highest tumor growth inhibition (72.4%), which may have been due to the additional influx of CD8 lymphocytes and the increase in the TAM MHC II^high to TAM MHC II^low ratio, while the overall TAM cell population, Treg, M1/M2 ratio decreased. In restimulated splenocytes, an increased percentage of CD8⁺ and NK cells capable of releasing cytolytic granules was observed, and in addition, these cells were capable of high IFN-γ and IL-10 production and were characterized by high cytotoxic activity.

The three-component vaccine DC/IL-12/TAg + DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg during immunotherapy showed a negligible therapeutic effect. Inhibition of tumor growth was only 24%, and we did not observe an increased influx of CD4⁺ and CD8⁺ cells in tumors, but a higher M1/M2 macrophage ratio and a decrease in the percentage of Treg, NK cells and TAMs. In the spleens, a reduction of CD4⁺, NK, NK CD107a⁺ cell percentage and IL-4 production was revealed, while an increase in the percentage of CD8⁺CD107a⁺ cells and cytotoxic activity was observed. However, the addition of the HES-MTX nanoconjugate to the therapy resulted in enhanced tumor inhibition (69.9%), an augmented influx of CD8⁺, CD4⁺ cells, and a higher TAM MHC II^high/TAM MHC II^low ratio in a tumor, at the same time reducing the percentage of Treg, M1/M2 ratio and TAMs. On the other hand, in the spleens, we observed an increase in the cytolytic activity of CD4⁺, CD8⁺ and NK cells, an increase in the production of IFN-γ and IL-4 and cytotoxic activity.