Dendritic cell vaccination with a toll-like receptor agonist derived from mycobacteria enhances anti-tumor immunity.

Dendritic cell (DC)-based vaccines are considered useful in cancer immunotherapy, and the interaction of DC and adjuvants is important in the design of the next generation vaccines. In this study, whether DC combined with Rv2299c derived from mycobacteria could improve anti-tumor immune responses in a colon cancer mouse model was evaluated. MC38 cell lines were injected subcutaneously to establish colon-cancer-bearing mice and the following four groups were evaluated: PBS control, tumor antigen (TA) loaded-DC, Rv2299c, and a combination of TA-loaded-DC and Rv2299c. The combination treatment with TA-loaded-DC and Rv2299c exhibited greater inhibition of tumor growth compared to other groups. These effects were associated with the reduction of suppressor cells, such as myeloid-derived suppressor cells and regulatory T cells, and the induction of effector cells, such as CD4+ T cells and CD8+ T cells, in spleen, and with the activation of cytotoxic T Lymphocytes and NK cells. These results suggest that TA-loaded-DC vaccination with Rv2299c derived from mycobacteria enhanced anti-tumor immunity in a mouse colon cancer model by inhibiting the generation of immune-suppressive cells and recovering numbers of effector cells, and demonstrated superior polarization of the Th1/Th2 balance in favor of the Th1 immune response.


INTRODUCTION
Immune-based therapeutic options that use antigenpresenting cells (APCs) with increased potency are considered an attractive tool in cancer immunotherapy [1][2][3][4]. Dendritic cells (DC), the most potent APCs, play a central role in various immunotherapy protocols via the generation of cytotoxic T lymphocytes (CTLs) [5,6]. Tumor antigen (TA)-specific immunotherapy is an emerging approach in cancer treatment. Most TAs are self-proteins, thus, there is a need to employ vaccine adjuvants that can stimulate the efficient presentation of weakly immunogenic proteins in a manner that allows effective activation of T cells [7]. Potent adjuvants are prerequisites to the immunotherapy for overcoming low immunogenicity of TA [8]. Substantial progress has been made in identifying adjuvants that recruit and/or activate appropriate APCs to elicit tumor-specific immunity [9]. The use of a cocktail of toll-like receptor (TLR) ligand agonists as adjuvants in an anti-viral vaccine significantly enhanced the functional avidity of the T cells, rather than increasing their numbers, markedly improving the anti-viral response [10]. Intrinsic recognition of the TLR ligands induces maturation of APCs [11].
Heat shock protein 90 (HSP90) has adjuvant activity via association with TLR4 signaling in innate immune cells [12,13]. The interaction of HSP-peptide complexes with APCs leads to the presentation of antigenic www.impactjournals.com/oncotarget peptides to CD8 + and CD4 + T cells and to a cascade of non-antigen-specific events that activate APCs and promote immune responses [14]. Activation of the innate immune system by HSP90 induced various effects on in vivo tumor immunogenicity in experimental animal models [15][16][17][18]. Mycobacterium tuberculosis Rv2299c is a member of the HSP90 family [19,20]. Rv2299c is similar to the HSP high-temperature protein G (htpG) homologue from Bacillus subtilis [19,20]. It is possible that in a tuberculous granuloma both pathogens and host cells are stressed, leading to the production HSP.
In this study, whether the combination of DC with Rv2299c as an adjuvant resulted in enhanced activation of DC was evaluated. In theory, large numbers of activated DC would prime highly functional tumor-specific T cells to higher levels, enhancing the clinical efficacy of adjuvant vaccines. This study demonstrated that TA-loaded-DC vaccination with Rv2299c derived from mycobacteria enhanced anti-tumor immunity in a mouse colon cancer model by inhibiting immune-suppressive cells and recovering effector cells, and demonstrated superior polarization of the Th1/Th2 balance in favor of the Th1 immune response.

BM-derived CD11c + DC showed a fully mature phenotype and marked IL-12p70 secretion
BM-derived CD11c + DC were maturated with GM-CSF, TNF-α, and IL-1β, and then loaded with gamma-irradiated MC-38 cells, which constituted ~96% of cells that underwent apoptosis (Supplemental Figure 1). The DC expressed higher levels of several molecules related to DC maturation ( Figure 1A) than iDC and produced higher levels of IL-12p70 ( Figure 1B) and lower levels of IL-10 ( Figure 1C) after subsequent CD40L stimulation compared to iDC.

Rv2299c anti-tumor immunity effects in a colon cancer mouse model
To determine the optimal concentration of Rv2299c to achieve anti-tumor immunity against colon cancer, Rv2299c was intraperitoneally injected at doses of 1, 5, and 10 μg/mouse (Figure 2A). Treatments with 5 and 10 μg of Rv2299c showed significant inhibition of tumor growth compared to the PBS control or 1 μg of Rv2299c (P < 0.05) ( Figure 2B; Supplemental Figure 2). To examine the tumor-specific responses, the effects of Rv2299c on the proportions among splenocytes of CD4 + T cells ( Figure 2C), CD8 + T cells ( Figure 2D), CD4 + CD25 + Tregs ( Figure 3A), CD4 + FoxP3 + Tregs ( Figure 3B), and CD11b + Gr1 + myeloid-derived suppressor cells (MDSCs) ( Figure 3C) were evaluated in injected mice. The percentages of CD4 + T cells and CD8 + T cells increased in the 5-and 10-μg of Rv2299c treatment groups compared to the 1-μg Rv2299c group and the PBS control group. The percentages of MDSCs were not significantly different among the four groups. In contrast, the percentage of Tregs showed decreased in the 5-and 10-μg Rv2299c treatment groups compared to the 1-μg Rv2299c and PBS control groups. DC isolated from the splenocytes of mice injected with 5-and 10-μg Rv2299c showed increased expressions of MHC class I, class II, CD80, CD86, CD40, and CCR7 compared to those in the 1-μg Rv2299c and the PBS control groups ( Figure 3D).

TA-loaded DC vaccination in combination with Rv2299c treatment induced a synergistic antitumor immunity effect
In this study, 5-μg Rv2299c enhanced anti-tumor immunity in vivo more effectively than the other doses. Therefore, combination therapy of TA-loaded DC vaccine and Rv2299c was assessed for its ability to inhibit tumor growth in a colon cancer mouse model ( Figure 4A). All tumor-bearing mice vaccinated with PBS showed no inhibition of the rapid growth of the tumor and had to be euthanized within three weeks. In contrast, tumor-bearing mice vaccinated with TA-loaded-DC, Rv2299c, and TA-loaded-DC plus Rv2299c showed significantly greater inhibition of tumor growth compared with the control group. Treatment with the combination of TA-loaded-DC vaccination plus Rv2299c showed significantly greater inhibition of tumor growth (P < 0.05) compared to TA loaded-DC or Rv2299c alone ( Figure 4B; Supplemental Figure 3). Survival in mice that received a combination of TA-loaded-DC plus Rv2299c was prolonged compared to that of mice that received the TA-loaded-DC or Rv2299c alone (Supplemental Figure 4). These results indicated that TA-loaded DC vaccination plus Rv2299c induced a long-term systemic anti-colon cancer immune response in a mouse model.

Activation of CTLs and NK cells by vaccination with TA-loaded DC plus Rv2299c
To investigate the anti-tumor effect of CTLs produced by TA-loaded DC vaccination in a mouse MC-38 colon cancer model, splenocytes from each group of vaccinated mice were prepared for IFN-γ ELISPOT assays. MC-38 and YAC-1 cells were used as the target cells. In comparison with PBS control, vaccination with Rv2299c alone did not increase the number of IFN-γ-secreting splenocytes against MC-38 cells, whereas vaccination with TA-loaded DC or TA-loaded DC plus Rv2299c led to a significant increase in IFN-γ-secreting splenocytes against MC-38 cells (P < 0.05). In addition, TA-loaded DC plus Rv2299c vaccination showed the highest number of IFN-γ-secreting splenocytes against MC-38 cells among other groups.
The cytotoxicity of NK cells (shown by the number of IFN-γ-secreting splenocytes against YAC-1 cells) was significantly higher in the two groups of mice injected with Rv2299c than those groups not injected with Rv2299c. Compared to Rv2299c treatment alone, TA-loaded DC plus Rv2299c injection induced superior NK cell activity against the target cells (P < 0.05; Figure 5A). These results indicated that the tumor-inhibitory effects of TA-loaded DC plus Rv2299c injection were resulted from the CTLmediated responses induced by TA-loaded DC vaccination and the NK cells-mediated responses induced by the Rv2299c injection. In this study, vaccination with TAloaded DC plus Rv2299c led to production of lower levels of IL-10 compared to the PBS control, TA-loaded DC alone, or Rv2299c alone ( Figure 5B). In contrast, IFN-γ production by TA-loaded DC plus Rv2299c was higher compared to that by TA-loaded DC or Rv2299c alone ( Figure 5C). These results suggested that the combination of TA-loaded DC plus Rv2299c vaccination enhanced Th1 responses in addition to tumor-specific CTL responses. Regarding tumor-specific responses, the percentages of CD4 + T cells ( Figure 5D), CD8 + T cells ( Figure 5E), and memory T cells ( Figure 5F) were increase significantly in the TA-loaded-DC vaccination groups compared to the group administered Rv2299c alone.

Efficient inhibition of MDSCs and Tregs by the combination of TA-loaded DC vaccination plus Rv2299c treatment
To investigate the immunological mechanisms underlying the enhanced tumor-specific immune responses, the effects of the combination therapy on the proportion of MDSCs (CD11b + Gr1 + ) and Tregs (CD4 + FoxP3 + and CD4 + FoxP3 + cells) were analyzed. The percentages of MDSCs ( Figure 6A) and Tregs ( Figures 6B and 6C) were significantly increased in the PBS control group after tumor inoculation. Administration of Rv2299c alone increased the  I, MHC-II, CD80, CD86, CD40, and CCR7 using flow cytometry; mature DC (mDC) expressed higher levels of maturation molecules than immature DC (iDC). Representative histograms show marker expression levels (shaded) compared to those of the isotype controls (black line). B, C. An ELISA revealed that mDC produced higher levels of IL-12p70 and lower levels of IL-10 in culture supernatants after subsequent CD40L stimulation, compared to iDC (P < 0.05). The data are shown as the means (pg/mL) ± standard deviation (SD) of triplicate cultures from three independent experiments. generation of MDSCs compared to TA-loaded-DC alone or TA-loaded-DC plus Rv2299c, while the percentages of Tregs decreased in the Rv2299c adjuvant and TA-loaded-DC plus Rv2299c compared to TA-loaded-DC alone. These findings suggested that the combination of TA-loaded DC plus Rv2299c vaccination enhanced therapeutic anti-tumor immunity by inhibiting the immunosuppressive tumor microenvironment during the vaccination phases.

DISCUSSION
In this study, we demonstrated that the TLR-4 agonist Rv2299c could be used as a potential adjuvant with TA-loaded DC in a colon cancer vaccine. Local injection of Rv2299c together with a TA-loaded DC vaccine resulted in significantly greater protection from the progression of colon cancer than either therapy alone. Additionally, Rv2299c enhanced the priming of tumorspecific CD8 + T cells. Thus, we considered that the use of a TLR agonist, such as Rv2299c, may act as potent innate immune response modifier that can enhance the tumor microenvironment and "danger signals" critical for generating potent Th1-type immune response and effective antitumor immunity [21]. Our finding also highlights the delicate nature of immune responses in a mouse colon cancer model. Administration of Rv2299c resulted in a dramatic decrease in tumor growth, and we believe that the Rv2299c administration may still efficacious without inducing mortality in this model.
Recent evidence suggests that persistent TLR signaling may assist in bypassing regulatory T cell-induced immune tolerance [22], enhancing autoimmune T cell responsiveness [23], or even reversing regulatory T cell function [24]. These effects may represent an additional mechanism underlying the synergistic effect of the TLR-4 agonist with DC immunotherapy.
In conclusion, our data suggest that TA-loaded DC vaccination with Rv2299c derived from mycobacteria enhanced anti-tumor immunity in a mouse colon cancer model by inhibiting immunosuppressive cells during tumor progression. This, in turn, enhances the anticancer effects of vaccination, triggering direct co-stimulatory effects, including the proliferation and activation of T cells, and the subsequent generation of Th1-type immunity with enhancement of effector NK cell function.
Both properties of Rv2299c should prove useful in a clinical context; it is known that Rv2299c exerts general immunostimulatory effects in colon cancer patients. Future studies are needed to determine the exact mechanisms by which TLR agonists enhance DC immunotherapy, and to understand how various TLR agonists can be used safely in colon cancer immunotherapy.

Mice, tumor cell lines and Rv2299c
Six-to eight-week-old female C57BL6 mice were purchased from Orient Bio (Iksan, Republic of Korea), and

Generation of tumor antigen-loaded DC
Apoptotic cells were induced by γ-irradiation (100 Gy) (Gammacell-1000 Elite, MDS Nordion, Canada) followed by overnight culturing in RPMI-1640 without FBS. The apoptotic γ-irradiated cells were confirmed using an Annexin V-FITC Apoptosis Detection Kit (BD Bioscience, San Jose, CA, USA) and loaded on iDC at 2 h after the addition of maturating cytokines at a ratio of 2:1 (DC:apoptotic cells).

In vivo animal vaccination
Four vaccination groups were established: 1) PBS control, 2) TA-loaded DC vaccination, 3) Rv2299c injection, and 4) TA-loaded DC vaccination plus Rv2299c injection. On day 0, mice were injected subcutaneously with 5 × 10 5 MC38 cells in the right flank in a volume of 0.1 mL. TA-loaded DC vaccine (1 × 10 6 cells/mouse) was administered subcutaneously in the left flank of C57BL6 mice in a volume of 0.1 mL of PBS at 2-day interval on days 7, 9, 11 and 13, and Rv2299c (5 μg/mouse) was injected intraperitoneally in a volume of 0.1 mL on days 8, 10, 12, 14, 16 and 18. To assess the anti-tumor efficacy of the vaccinated mice, three perpendicular dimensions (length, width and height) of each tumor were measured individually every 2 days using a Vernier caliper, and the tumor volume was calculated using the following formula for the standard volume of an ellipsoid: V = 4/3 π (length × width × height/8).

In vitro analysis of cytokine production from vaccinated mice
Cytokine (IFN-γ and IL-10) production from vaccinated mice was determined by a BD OptEIA™ MDSCs and Tregs were significantly increased in the PBS control group after tumor inoculation. Rv2299c alone increased the generation of MDSCs compared to TA-loaded DC alone or TA-loaded DC plus Rv2299c, while the percentages of Tregs were lower in the Rv2299c adjuvant and TA-loaded DC plus Rv2299c groups compared to the TA-loaded DC alone group. And the data are representative of more than 3 experiments. Statistical comparisons were performed using the one-way nonparametric ANOVA (*P < 0.05). enzyme-linked immunosorbent (ELISA) assay (BD Bioscience). Supernatant from the stimulated splenocytes of all vaccinated mice was used for measurement of the production of Th1-and Th2polarizing cytokines. Each sample was analyzed in triplicate and the mean absorbance for each set of standards and samples was calculated.

Intracellular staining assay of regulatory T cell (Treg) generation in the spleens of vaccinated mice
To evaluate the proportion of Tregs, 1 × 10 6 splenocytes from vaccinated mice were harvested and stained with PE-conjugated CD4 and FITC-conjugated CD25 mAbs for 30 min at 4°C. Fc block was added before incubation with surface antibodies. The cells were then washed and permeabilized with FACSTM Permeabilizing Solution 2 (BD Bioscience) for 30 min at room temperature. After washing twice, the cells were stained with an Alexa Fluor -conjugated Foxp3 antibody (Miltenyi Biotec) for 30 min at 4°C. The samples were acquired on a FACS Calibur cell sorter (Becton Dickinson) and the data were analyzed using WinMDI Version 2.9 software (Biology software Net).

Statistical analysis
The Mann-Whitney U test was performed to assess the significance of non-parametric differences among the groups. Survival of the vaccinated mice was analyzed using SigmaPlot 10.0 (Systat Software Inc. San Jose, CA, USA). P values <0.05 were considered to indicate statistical significance.