Lenalidomide and Programmed Death-1 Blockade Synergistically Enhances the Effects of Dendritic Cell Vaccination in a Model of Murine Myeloma

The therapeutic efficacy of dendritic cell (DC)-based immunotherapy may be potentiated in combination with other anticancer therapies that enhance DC function by modulating immune responses and the tumor microenvironment. In this study, we investigated the efficacy of DC vaccination in combination with lenalidomide and programmed death (PD)-1 blockade in a model of murine myeloma. MOPC-315 cell lines were injected subcutaneously to establish myeloma-bearing mice and the following five test groups were established: PBS control, DCs, DCs + lenalidomide, DCs + PD-1 blockade, and DCs + lenalidomide + PD-1 blockade. The combination of DCs plus lenalidomide and PD-1 blockade more potently inhibited tumor growth compared to the other groups. This effect was associated with a reduction in immune suppressor cells (such as myeloid-derived suppressor cells, M2 macrophages, and regulatory T cells) and an increase in immune effector cells [such as CD4+ and CD8+ T cells, natural killer (NK) cells, and M1 macrophages] in the spleen. Functional activities of cytotoxic T lymphocytes and NK cells were also enhanced by the triple combination. Levels of immunosuppressive cytokines, such as TGF-β and IL-10, were significantly reduced in the tumor microenvironment. These findings suggest that the combination of DCs plus lenalidomide and PD-1 blockade synergistically establishes a robust anti-myeloma immunity through a two-way mechanism, which inhibits immunosuppressive cells while activating effector cells with superior polarization of the Th1/Th2 balance in favor of the tumor immune response. This result should provide an experimental ground for incorporating check point inhibitors to existing immunotherapeutic modalities against multiple myeloma.

inTrODUcTiOn Multiple myeloma (MM) is characterized by the infiltration of clonal malignant plasma cells in the bone marrow (BM) (1,2). Despite advances in treating MM using novel therapies and hematopoietic stem cell transplantation, most patients experience relapses caused by immune evasion among the tumor, immune system, and tumor microenvironment (3). Thus, new therapeutic options with the potential to overcome impaired immune surveillance are needed.
Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) and play a key role in inducing and maintaining antitumor immunity. DCs are able to recognize, process, and present tumor antigens to generate antigen-specific cytotoxic T lymphocytes (CTLs) (4)(5)(6)(7)(8)(9). Immune cells in myeloma patients have quantitative and functional deficiencies that contribute to myeloma-associated immune tolerance (10,11). By contrast, the function of DCs from patients with MM can be recovered and enhanced by ex vivo culture (12)(13)(14). Lenalidomide is an immunomodulatory agent that targets tumor cells under immunosuppressive microenvironment (15)(16)(17)(18)(19)(20). Our previous studies demonstrated that the combination of DC vaccination and lenalidomide synergistically enhanced antitumor immune responses in mouse tumor models (21,22). Programmed cell death-1 (PD-1, CD279) and its ligands [either PD-L1 (B7-H1, CD274) or PD-L2 (B7-DC, CD273)] play a fundamental role in tumor immune escape by inhibiting effector functions (23)(24)(25)(26). The PD-1/PD-L1 blockade was recently found to effectively treat cancer by improving durable response rates and the survival profile with minimal toxicity, suggesting that blockade can be used as a cancer therapeutic agent (27)(28)(29)(30)(31). However, recent studies reported that PD-1 blockade alone is insufficient to stimulate anti-myeloma immunity in clinical treatment (32,33). Thus, combination approaches with immune-checkpoint blockade and therapies that stimulate myeloma-reactive T cells can be effective tools to treat myeloma. Such as with immunomodulatory drugs, cellular therapies are currently being applied in clinical trials. Previous studies demonstrated that lenalidomide reduces the expression of PD-1 on natural killer (NK) cells, helper cells, and CTLs, and inhibits PD-L1 expression on tumor cells and myeloid-derived suppressor cells (MDSCs) in patients with MM (20,34). Moreover, the combination of lenalidomide and PD-1 or PDL-1 blockade increased IFN-γ expression by BM-derived effector cells in myeloma and were associated with increased apoptosis of MM cells (35).
Thus, in this study, we investigated whether the combination of DCs plus lenalidomide and PD-1 blockade has a synergistic effect in a murine myeloma model. The results demonstrate that this combination enhanced antitumor immunity by inhibiting immunosuppressive cells and cytokines as well as activating and recovering effector cells with superior polarization toward Th1 immune response. This study provides a framework for developing a more advanced immunotherapeutic modality employing DCs, lenalidomide, and PD-1 blockade to inhibit tumor cell growth as well as restore immune functionin MM.

MaTerials anD MeThODs
Mice and Tumor cell lines 6-to 8-week-old female BALB/c (H-2 d ) mice were purchased from Orient Bio (Iksan, Republic of Korea) and maintained under specific pathogen-free conditions. All animal care, experiments, and euthanasia protocols were approved by the Chonnam National University Animal Research Committee. The murine MOPC-315 plasmacytoma cell line and the YAC-1 cell line were purchased from the American Type Culture Collection (Rockville, MD, USA). Cell lines were maintained in Dulbecco's Modified Eagle's Medium (Gibco-BRL, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco-BRL) and 1% (w/v) penicillin/streptomycin (PS).
immunomodulatory Drug (lenalidomide) and Programmed Death-1 (anti-PD-1) Lenalidomide (Revlimid ® ) was donated by Celgene Corporation (Summit, NJ, USA) and dissolved in dimethyl sulfoxide (DMSO) to 100 mg/mL immediately before use. For injection into mice, lenalidomide stock solutions were diluted in sterile 0.9% (v/v) normal saline to a final concentration of 10 mg/mL. The final concentration of DMSO in all experiments was <0.01% (v/v). Anti-PD-1 was purchased from BioXcell (West Lebanon, NH, USA).

animal Vaccination
The following five vaccination groups were established: (1) PBS control, (2) DC vaccination, (3) DC vaccination plus anti-PD-1, (4) DC vaccination plus lenalidomide injection, and (5) DC vaccination plus anti-PD-1 and lenalidomide injection. On day 0, mice were injected subcutaneously in the right flank with 5 × 10 5 MOPC-315 cells in a volume of 0.1 mL. After tumor growth, lenalidomide (0.5 mg/kg/day) was administrated orally once a day for 25 days with a 3-day break after the first 11-day dosing period. Each dose of DCs (1 × 10 6 /mouse) was injected subcutaneously into the left flank of BALB/c mice in a volume of 0.1 mL PBS on days 11, 15, 25, and 29; anti-PD-1 (250 μg/ mouse) was injected intraperitoneally in a 0.1-mL volume on the same days as DC vaccination. To assess the antitumor status of vaccinated mice, we measured the length, width, and height of each tumor every 3 to 4 days using a Vernier caliper, and we calculated tumor volume using the standard formula for calculating the volume of an ellipsoid: V = 4/3π(length × width × height/8).

In Vitro analysis of cytokine Production in Vaccinated Mice
We determined cytokine (IFN-γ, IL-10, and TGF-β) production in vaccinated mice using the BD OptEIA™ enzyme-linked immunosorbent assay (ELISA; BD Bioscience). Supernatants from restimulated splenocytes of vaccinated mice and from single tumor cells of all vaccinated mice were assayed to measure the production of Th1-and Th2-polarizing cytokines. Each sample was analyzed in triplicate, and the mean absorbance for each set of standards and samples was calculated.

intracellular staining assay of Tregs and Macrophages generated in the spleens of Vaccinated Mice
To evaluate the proportion of Tregs and macrophages, 1 × 10 6 splenocytes from vaccinated mice were harvested, washed, and stained with surface-staining antibodies of Tregs (CD4-PE and CD25-FITC) and macrophages (CD11b-FITC and F4/80-PE) for 30 min at 4°C. Fc block was added before incubation with surface-staining antibodies. Next, the cells were washed and permeabilized with Fixation/Permeabilization Solution 2 (eBioscience) for 30 min at room temperature. After washing twice, the cells were stained with an intracellular staining antibody, Tregs [Alexa Fluor-conjugated Foxp3 antibody (Miltenyi Biotec)] and macrophages (CD206-APC) for 30 min at room temperature. The samples were acquired on a FACSCalibur cell sorter (Becton Dickinson), and data were analyzed using WinMDI ver. 2.9.

statistical analyses
We performed statistical analyses using GraphPad Prism 4 (La Jolla, CA, USA). t-Tests, one-way analysis of variance (ANOVA), and two-way ANOVA were used as appropriate. We analyzed the survival of vaccinated mice using SigmaPlot 10.0 (Systat Software, San Jose, CA, USA). P < 0.05 was considered significant. Values are expressed as means ± SDs.

Dc Vaccination in combination With lenalidomide and anti-PD-1 Treatment induced a synergistic anti-Myeloma immunity effect
Our previous study (36) demonstrated that DCs maturated with GM-CSF, TNF-α, and IL-1β expressed higher levels of several molecules related to DC maturation and produced higher levels of IL-12p70 and lower levels of IL-10 compared to imDCs. In this study, we established myeloma-bearing mice to evaluate the antitumor efficacy of DC-based immunotherapies. Before treatment, we observed that high levels of PD-L1 were expressed on MOPC-315 cell lines ( Figure S1A in Supplementary Material). The established myeloma-bearing mice were initially treated with lenalidomide (0.25 or 0.5 mg/kg), PD-1 blockade (250 μg/ mouse), and dying myeloma cell-loaded DCs as a single therapy ( Figure 1A). All single treatment groups showed significant inhibition of tumor growth compared to the PBS control group (P < 0.05; Figures S1B,C in Supplementary Material). The combination therapy of DCs plus lenalidomide and PD-1 blockade was examined in an effort to more potently inhibit tumor growth in the murine myeloma model ( Figure 1A). All tumor-bearing mice vaccinated with PBS showed rapid tumor growth that led    led to a significant increase in IFN-γ-secreting splenocytes against MOPC-315 and YAC-1 cells (P < 0.05). The combination of DCs + lenalidomide + PD-1 blockade showed the highest number of IFN-γ-secreting splenocytes against MOPC-315 cells compared to the PBS control, DCs, DCs + lenalidomide, DCs + PD-1 blockade, and lenalidomide + PD-1 blockade (P < 0.05; Figure 2A; Figure S4A in Supplementary Material). In addition, cytotoxicity by NK cells, represented by the number of IFN-γ-secreting splenocytes against YAC-1 cells, was similar in all groups that received DCs. These results indicate that the tumor inhibitory effects of DCs + lenalidomide + PD-1 blockade treatment resulted from CTL rather than NK responses. In this study, vaccination with DCs + lenalidomide + PD-1 blockade led to the production of higher levels of IFN-γ compared to the PBS control, DCs, or lenalidomide + PD-1 blockade group (Figure 2B; Figure S4B in Supplementary Material). By contrast, TGF-β production in the DCs + lenalidomide + PD-1 blockade group was significantly lower compared to that in the PBS control, DCs, DCs + lenalidomide, or DCs + PD-1 blockade group (Figure 2C; Figure S4C in Supplementary Material). These results suggest that the combination of DCs + lenalidomide + PD-1 blockade induced tumor-specific CTL responses enhances through Th1 polarization. Additionally, the DCs + lenalidomide + PD-1 blockade regimen significantly increased percentages of effector CD4 + T cells (Figure 3A; Figure S4D in Supplementary Material), effector CD8 + T cells (Figure 3B), effector memory T cells (Figure 3C; Figure S4E in Supplementary Material), effector NK cells (Figure 3D; Figure S4F in Supplementary Material), and M1 macrophages (Figure 5A; Figure S6A in Supplementary Material) compared to the other groups.  Compared to the treatment groups, the PBS control group was significantly higher in the TGF-β production (P < 0.001). However, the production of TGF-β did not significantly differ among the treatment groups (Figure 5C; Figure S6C in Supplementary Material). In addition, the combination of DCs + lenalidomide + PD-1 blockade led to the production of the least IL-10 compared to other groups (P < 0.001; Figure 5D; Figure S6D in Supplementary Material), which suggests that the combination therapy of DC vaccination plus lenalidomide and PD-1 blockade changed the tumor microenvironment toward immunostimulatory by suppressing the production of inhibitory cytokines IL-10 and TGF-β.

DiscUssiOn
Dendritic cell-based vaccines serve a promising immunotherapeutic weapon with the potential to prolong the survival of patients with incurable MM (2,12). Several new tools have been developed and combined to improve clinical outcomes of DC vaccination against MM (17,18). Recent studies have defined immune checkpoint PD-1/PD-L1 signaling as a key pathway regulating the critical balance between immune activation and tolerance (24,(37)(38)(39)(40). The PD-1/PD-L1 pathway plays an important role in shaping the tumor-promoting, immunosuppressive microenvironment of MM. Rosenblatt et al. (41) reported that PD-L1 is highly expressed in plasma cells of MM patients but not in normal plasma cells. Our study confirmed that PD-L1 is also overexpressed on MOPC-315 cell lines (99%). Furthermore, significant PD-1 expression was observed in circulating T cells of advanced MM patients. Inhibition of the PD-1/PD-L1 signaling pathway induces an anti-MM immune response and can be a promising option for anti-myeloma therapy (42,43). The tumor microenvironment of MM promotes tumor cell growth and helps them escape from immune surveillance by actively suppressing anti-MM immune effector responses (1, 2). The production of (c) TGF-β and (D) IL-10 inhibitory cytokines in the tumors of tumor-bearing mice was evaluated by enzyme-linked immunosorbent assay. Compared to the treatments, PBS control led to the production of higher levels of TGF-β (***P < 0.001). However, the production of TGF-β did not differ significantly among the treatment groups. The production of IL-10 was significantly decreased in the DCs plus lenalidomide and anti-PD-1 combination therapy group compared to the other groups (***P < 0.001). Data are representative of at least three experiments. Lenalidomide, an immunomodulatory drug, inhibits the expression of PD-1 in NK cells, helper T cells, and CTLs of MM patients and downregulates the expression of PD-L1 in myeloma cell lines and primary myeloma cells (20,34). Lenalidomide was shown to reduce PD-1 expression in all effector cells (CD4 + T cells, CD8 + T cells, NK cells, and NKT cells), and PD-L1 expression in MM cells, MDSC, and monocyte/macrophages in an in vitro experiment (35). Additionally, Patients undergoing treatment of lenalidomide demonstrated reduced PD-1 expression in CD8 + T cells (44). Moreover, lenalidomide plus PD-1/PD-L1 checkpoint blockade suppressed MDSCs and stroma-mediated MM growth and enhanced MM-specific cytotoxicity of immune effector cells in BM environments (35). Our previous studies demonstrated that DC-based vaccines were safe and induced the expansion of circulating CD4 + T cells and CD8 + T cells that are specific for tumor antigens, which was synergistically enhanced by the combination of lenalidomide (21,22,36). Our expectation was that the therapeutic efficacy of DC vaccination will be far more Murine models of myeloma are critical tools to study the mechanisms of disease resistance, pathogenesis, and the development of new therapeutic strategies (45,46). This study has some limitation to interpret data due to subcutaneous injection of MOPC-315 cells for making plasmacytoma rather than BM involvement model for myeloma.
In conclusion, this study suggests that lenalidomide plus PD-1 blockade treatment synergistically enhances the efficacy of DC vaccination in a murine myeloma model by inhibiting the generation of immunosuppressive cells and the Th2 immune response and enhancing effector cells and the Th1 immune responses. We hereby propose a framework for a more efficacious DC-based vaccination strategy against MM with the combination of immunomodulatory drug lenalidomide and anti-PD-1 antibody.      (B) IFN-γ and (c) TGF-β production in the splenocytes of vaccinated mice was evaluated by enzyme-linked immunosorbent assay. The combination of DCs plus lenalidomide and anti-PD-1 led to the production of higher levels of IFN-γ compared to treatment with lenalidomide plus anti-PD-1 (***P < 0.001). The production of TGF-β did not differ significantly between the two groups. Data are shown as the mean (pg/mL) ± SD of triplicate cultures from three independent experiments. We measured proportions of (D) CD4 + T cells, (e) memory T cells, and (F) NK cells using flow cytometry (left panel) and compared them using quantitative bar graphs (right panel). The results revealed significant increases in CD4 + T cells and memory T cells in the DCs plus lenalidomide and anti-PD-1 group compared to the lenalidomide plus anti-PD-1 group (*P < 0.05; ***P < 0.001). Percentages of NK cells did not differ significantly between the two groups. Data are representative of at least three experiments.
FigUre s6 | Enhanced M1 and impaired M2 macrophage polarization and reduced inhibitory cytokine production by a combination of dendritic cells (DCs) plus lenalidomide and anti-PD-1. We measured proportions of (a) M1 macrophages (CD11b + F4/80 + CD206 − ) and (B) M2 macrophages (CD11b + F4/80 + CD206 + ) in the spleens of vaccinated tumor-bearing mice using flow cytometry (left panel) and compared them using quantitative bar graphs (right panel). The DCs plus lenalidomide and anti-PD-1 combination group showed significantly increased proportions of M1 macrophages compared to the lenalidomide plus anti-PD-1 group (*P < 0.05). Percentages of M2 macrophages did not differ significantly between the two groups. Data are representative of at least three experiments. The production of (c) TGF-β and (D) IL-10 inhibitory cytokines in the tumors of tumor-bearing mice was evaluated by enzyme-linked immunosorbent assay. The production of IL-10 was significantly decreased in the DCs plus lenalidomide and anti-PD-1 combination therapy group compared to the lenalidomide plus anti-PD-1 group (***P < 0.001). The production of TGF-β did not differ significantly between the two groups. Data are representative of at least three experiments.