Monophosphoryl lipid A-induced activation of plasmacytoid dendritic cells enhances the anti-cancer effects of anti-PD-L1 antibodies

Abstract

Monophosphoryl lipid A (MPLA) is a toll-like receptor 4 ligand that promotes immune activation in mice and humans, without undesired inflammation. Immunotherapy by the combining immune checkpoint blockade and MPLA has shown promising anti-cancer effects in both mice and humans. In this study, we explored how MPLA enhanced the anti-cancer effects of anti-PD-L1 antibodies (Abs). Anti-cancer immunity induced by the combination of anti-PD-L1 Abs and MPLA failed in CD4 and CD8 cell-depleted mice. Moreover, the combination treatment of anti-PD-L1 Abs and MPLA synergistically enhanced the activation of plasmacytoid dendritic cells (pDCs) in the mouse in vivo, while conventional DCs were not. In addition, mice treated with anti-PD-L1 Abs and MPLA were not protected from B16 melanoma by blockade of interferon-alpha receptor (IFNAR). The combination of anti-PD-L1 Abs and MPLA also promoted human peripheral blood pDC activation and induced IFN-α-dependent T cell activation. Therefore, these results demonstrate that MPLA enhances anti-PD-L1 Ab-mediated anti-cancer immunity through the activation and IFN-α production of pDCs.

Introduction

Immunotherapies, including cancer vaccines [1–3], chimeric antigen receptor (CAR) T cell therapy [4, 5], and immune checkpoint blockades [6], are being developed to effectively treat cancer. Immune checkpoint proteins such as programmed cell death protein 1 (PD-1), PD ligand 1 (PD-L1), and cytotoxic T lymphocyte antigen-4 (CTLA-4) confer immune tolerance to healthy cells, thus preventing their destruction by immune cells [7–9]. It has previously been reported that cancer cells evade recognition and attack by cytotoxic T lymphocytes (CTLs) via the expression of immune checkpoint proteins [10, 11]. Many previous studies have shown that blockades of the immune checkpoint proteins by antibodies (Abs) elicit anti-cancer immunity in mice and humans [7–9].

PD-1 is expressed on the surface of activated T cells and binds to its ligands, including PD-L1 and PD-L2 [8, 12]. PD-L1 and PD-L2 are expressed in cancer cells, dendritic cells (DCs), and macrophages [13, 14]. Cancer cells expressing PD-L1 evade CTL attack [10, 11]; whereas, the expression of PD-L1 in DCs and macrophages promotes immune tolerance via regulatory T cell development and the suppression of effector T cells [13, 15]. Many studies have shown that PD-1 blockade by anti-PD-1 Abs elicits anti-cancer immunity while immune stimulatory molecules enhance the anti-cancer effect of anti-PD-1 Abs [15–17]. However, anti-PD-L1 Ab-mediated alteration of immunity against cancer and adjuvant effects has not been extensively studied.

Various immune adjuvants have been developed and studied to enhance the efficacy of vaccines and immunotherapies [18, 19]. Adjuvants boost the weak immunogenicity of antigens used in vaccines, leading to an improved immune response [20–22]. Due to the poor immunogenic of cancer antigen, an adjuvant is required for inducing cancer antigen-specific immune response. As DCs present antigens to T cells during maturation, they can be target cells for adjuvants to induce immune activation [23]. It has been proved that adjuvant-induced DC activation also showed enhancement of immune checkpoint blockade-induced anti-cancer immunity [17, 24].

As professional antigen presentation cells (APCs), DCs control T and B cell-mediated adaptive immunity [25]. There are two subtypes of DCs in human and mouse: conventional DCs (cDCs) and plasmacytoid DCs (pDCs) [26, 27]. Bacteria, fungi, and parasites can induce the activation of cDCs, that upregulate the co-stimulatory molecules in the cell surface and consequently present antigens to T cells [28, 29]. On the other hand, viral infections elicit the activation of pDCs, that release large amounts of interferon-alpha (IFN-α) [30, 31]. CpG-oligodeoxynucleotide (CpG-ODN) stimulates toll-like receptor (TLR)9 in pDCs; treatment with the combination of CpG-ODN and immune checkpoint blockade leads to effective anti-cancer immunity in mice, indicating that pDC activation by CpG-ODN contributes to the enhancement of immune checkpoint blockade-mediated anti-cancer effects [32–36].

Lipopolysaccharides (LPSs) are natural adjuvants that not only induce a strong immune response in humans and mice but also elicit cytokine storms and sepsis in humans [37]. Monophosphoryl lipid A (MPLA) has shown potential for inducing immune activation with low toxicity in humans and mice [3, 38]. MPLA acts as a TLR4 ligand and enhances humoral and cellular immune responses in humans and mice [39, 40]. In addition, MPLA enhances anti-PD-1 Ab-induced anti-cancer effects in mice [16]. However, the effect of MPLA on anti-PD-L1 Ab-induced anti-cancer effects has not yet, to the best of our knowledge, been studied. In the present study, we investigated the effects of MPLA and how it enhances the anti-cancer immunity of anti-PD-L1 Abs in mice and humans. We found that the MPLA enhances pDC-mediated activities of immune check point blockade, which suppressed tumor growth in the mice. It is the first report that MPLA induces pDC activation that may contribute to anti-cancer immunity by combination of immune check point blockade antibody treatment.

Materials and methods

Mice

BALB/c and C57BL/6 mice (female 6–8 weeks old) maintained in pathogen-free conditions were obtained from Shanghai Public Health Clinical Center (SPHCC). The mice were maintained under standard conditions at 20–24 °C with humidity of 40–60% and access to water and chow. The animal protocol used was 2019-A049-01, approved by the committee on the Ethics of Animal Experiments of the SPHCC. The study was conducted according to the guidelines of the Institutional Animal Care and Use committee at the SPHCC. In all experiments, efforts were made to minimize the suffering of the mice, which were euthanized by CO₂ inhalation.

Ethics statement

This study was conducted according to the principles of the Declaration of Helsinki. Peripheral blood samples were harvested from healthy donors at SPHCC. Written informed consent was obtained from all volunteers. The study was approved by the Institutional Review Board at SHAPHC Ethics Committee (IRB number: 2017-Y037).

Cancer cell lines and cultures

The murine carcinoma cell line CT26 (ATCC, CRL-2638) and murine melanoma cell line B16F10 (ATCC, CRL-6475) were cultured in RPMI-1640 consisting 10% fetal bovine serum (FBS), streptomycin (100 μg/ml), glutamine (2 mM), penicillin (100 U/ml), and HEPES (1 M) in an incubator maintained at 37 °C under a humidified atmosphere of 5% CO₂.

Preparation of MPLA

MPLA was purchased from Sigma Aldrich (St. Louis, Missouri, USA) and formulated by mixing with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at a 0.21:0.25 wt ratio. The mixture was dissolved using chloroform, which was then evaporated. The dried film formations were rehydrated in water and sonicated at 60 °C, and MPLA was stored at 4 °C.

Transmission electron microscopic (TEM) analysis

The structure and size of MPLA were determined by TEM (H-7600 transmission electron microscope; HITACHI, Japan). The sample preparation of TEM analysis was as follows: the MPLA was suspended in phosphate-buffered saline (PBS) and deposited onto the TEM grid.

Antibodies

Isotype control Abs (IgG1, IgG2a, and IgG2b), CD3 (17A2), CD8α (53-6.7), CD4 (GK1.5), CD11c (N418), CD40 (3/23), CD86 (GL-1), CD80 (16-10A1), CD317 (129C1), B220 (RA3-6B2), CD123 (5B11), anti-Granzyme B (GB11), anti-Perforin (S16009A), anti-IgG (Poly4060), anti-PD-L1 (10F.9G2), and anti-IFNAR (MAR1-5A3) were obtained from BioLegend (San Diego, CA, USA). Anti-IFN-α (7N41), anti-IFN-γ (B27), anti-MHC class I (28-8-6), and anti-MHC class II (M5/114.15.2) were purchased from eBioscience (San Diego, CA, USA).

Flow cytometry analysis

Cells were incubated for 15 min with unlabeled isotype control and Fc-block Abs (BioLegend, San Diego, CA, USA) to remove non-specific backgrounds before primary Ab staining. These cells were washed with PBS then incubated with fluorescence-conjugated Abs on ice for 20 min. After washing free Abs with PBS, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, Missouri, USA) to mark the dead cells and analyzed by FACS Fortessa (Becton–Dickinson, Franklin Lakes, New Jersey, USA) and FlowJo 8.6 software (Tree Star, San Diego, CA, USA).

Ex vivo T cell stimulation and intracellular cytokine staining

The spleens were digested with a digestion buffer (2% FBS and collagenase IV) and stimulated in vitro for 4 h with 10 μg of B16 self-antigens with the addition of Monensin solution (BioLegend, San Diego, CA, USA) for the final 2 h. For intracellular cytokine staining, the cells were initially stained to assess the surface molecules in the dark for 20 min at room temperature. Then, the cells were fixed and permeabilized with Cytofix/Cytoperm buffer (eBioscience, San Diego, CA, USA) for 20 min at room temperature and subsequently incubated with anti-cytokine Abs in Perm/Wash buffer (eBioscience, San Diego, CA, USA) for 30 min in the dark at room temperature. The staining of isotype control IgGs was performed in all experiments.

Mouse DC analysis

Tumor draining lymph node (tdLN) was homogenized and digested by collagenase for 20 min at room temperature. Cells were washed and re-suspended in 5 mL Histopaque-1077 (Sigma-Aldrich, St. Louis, Missouri, USA) medium. The cell suspensions were then carefully laid on 5 ml fresh Histopaque-1077 with additional 1 ml FBS on the top. The cells were centrifuged at 1700 × g for 10 min and the leukocyte fraction (< 1.077 g/cm³) was harvested and stained with fluorescence-labeled monoclonal Abs (mAbs) for 30 min. For pDC analysis, cells were stained with anti-CD317 (129C1) and anti-B220 (RA3-6B2), and the pDCs were defined as CD317⁺B220⁺ cells in live leukocytes. The cDCs were defined as lineage⁻CD11c⁺ cells [17]. Lineage staining included anti-B220 (RA3-6B2), anti-CD49b (DX5), anti-CD3 (17A2), anti-Gr1 (RB68C5), anti-TER-119 (TER-119), and anti-Thy1.1 (OX-7).

Enzyme-linked immunosorbent assay (ELISA)

The mouse IFN-α ELISA kit was purchased from eBioscience (San Diego, CA, USA). The human IFN-α2 ELISA kit was obtained from BioLegend (San Diego, CA, USA). The concentrations of IFN-α were measured by ELISA kits in triplicate.

Real-time polymerase chain reaction (PCR)

Total RNA was extracted from cells and reverse-transcribed into cDNA by Oligo (dT) and M-MLV reverse transcriptase (Promega, Madison, Wisconsin, USA). The cDNA was subjected to real-time PCR (Qiagen, Hilden, Germany) for 40 cycles with an annealing and extension temperature of 60 °C on a LightCycler 480 Real-Time PCR System (Roche, Basel, Switzerland). Primer sequences used were: mouse β-actin forward, 5ʹ-TGGATGACGATATCGCTGCG-3ʹ, reverse, 5ʹ-AGGGTCAGGATACCTCTCTT-3ʹ; IRF7 forward, 5ʹ-CCTCTGCTTTCTAGTGATGCCG-3ʹ, reverse, 5ʹ-CGTAAACACGGTCTTGCTCCTG-3ʹ

Human peripheral blood mononuclear cell (PBMC) analysis

PBMCs were isolated from whole blood via a density gradient centrifugation method using Ficoll Histopaque, then stained with fluorescence-conjugated lineage Abs, anti-CD11c (3.9), anti-CD123 (6H6), anti-CD80 (2D10), anti-CD83 (HB15e), anti-CD86 (BU63), anti-HLA-A, B, and C (W6/32), and anti-HLA-DR, DP, and DQ (Tü39). The CD11c^inter CD123⁺ cells and CD11c⁺ CD123^inter cells were gated, respectively, as pDC and cDC from live leukocytes by FACS Fortessa (Becton–Dickinson, Franklin Lakes, New Jersey, US).

Human T cell stimulation

PBMCs were stimulated in vitro for 4 h with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml) and ionomycin (1 μM; both from Merck, Kenilworth, New Jersey, USA), with the addition of Monensin solution for the final 2 h. The cells were processed via intracellular staining as indicated with ex vivo T cell stimulation and intracellular cytokine staining.

Statistical analysis

All data were expressed as the mean ± standard error of the mean (SEM). A one- or two-way ANOVA (Tukey’s multiple comparison test) and Mann–Whitney t test were used to analyze the data sets. P values <0.05 were considered statistically significant.

Results

MPLA enhances the anti-tumor effects of anti-PD-L1 Abs

Because the MPLA is not water soluble, it has formulated as liposome with DPPC (Fig. 1a, b). For evaluation of synergic effect of MPLA and anti-PD-L1 Ab, we chose CT-26 carcinoma and B16 melanoma cells. Both CT-26 and B16 cells expressed PD-L1 on surface (Fig. 1c). Next, we evaluated the effect of MPLA in enhancing anti-PD-L1 Ab-induced anti-cancer immune response. BALB/c and C57BL/6 mice were subcutaneously (s.c.) injected with CT-26 carcinoma and B16 melanoma, respectively. Five days after tumor injection, the mice were intraperitoneally (i.p.) treated with 10 mg/kg of anti-PD-L1 Abs, 0.5 mg/kg of MPLA, or combination of anti-PD-L1 Abs and MPLA every 5 days. Treatment with anti-PD-L1 Abs suppressed CT-26 tumor growth (Fig. 1d, e), while the combined treatment with anti-PD-L1 Abs and MPLA significantly increased the anti-PD-L1 Abs-induced anti-tumor effects in CT-26 tumor-bearing BALB/c mice (Fig. 1d, e). Moreover, the size of B16 melanoma masses substantially decreased as a result of anti-PD-L1 Abs treatment, indicating that the anti-tumor effect was strongly enhanced by the combination treatment with anti-PD-L1 Abs and MPLA in B16 tumor-bearing C57BL/6 mice (Fig. 1f, g). These data indicated that the additional treatment of MPLA enhances the anti-tumor effect of anti-PD-L1 Abs in mice in vivo.

Fig. 1.

MPLA enhances the anti-tumor effect of anti-PD-L1 Abs. a Transmission electron microscopy (TEM) image, b size distribution of MPLA was shown. c The expression levels of PD-L1 in CT-26 and B16F10 cell lines were analyzed by flow cytometry. d–g The BALB/c and C57BL/6 mice were s.c. injected with CT26 carcinoma and B16 melanoma cells, respectively. Five days after injection, the mice were i.p. injected with 10 mg/kg anti-PD-L1 Abs, 0.5 mg/kg MPLA, and a combination of anti-PD-L1 Abs and MPLA every 5 days. d The volumes of CT26 tumors in BALB/c mice are shown. Significance was determined by the log-rank test, mean ± SEM (n = 6 mice). e CT26 tumor mass on day 25 after tumor injection. f Tumor growth of B16 in C57BL/6 mice. Significance was determined by the log-rank test, mean ± SEM (n = 6 mice). g B16 tumor mass is shown 25 days after tumor injection

MPLA and the anti-PD-L1 Ab-induced anti-tumor effect was mediated by effector T cells

To evaluate the immune cell-mediated anti-tumor effect of the combination treatment with MPLA and anti-PD-L1 Abs, we examined the activation of T cells in the tdLN. The intracellular production levels of IFN-gamma (IFN-γ) in both CD4 and CD8 T cells were upregulated by MPLA in response to B16 antigen proteins, which were further increased by the combined treatment with anti-PD-L1 Ab and MPLA (Fig. 2a, b). In addition, intracellular levels of granzyme B and perforin, the inducer of programed cell death in target cells, were dramatically increased by the combination treatment with anti-PD-L1 Abs and MPLA in the tdLN in response to B16 antigen proteins (Fig. 2c, d). Although the combination of anti-PD-L1 Abs and MPLA promoted substantial upregulation of T cell immunity, anti-PD-L1 treatment alone did not induce the activation of T cells in tdLN (Fig. 2c, d).

Fig. 2.

The combination of MPLA and anti-PD-L1 Abs promotes T cell activation in tdLN. The C57BL/6 mice were s.c. injected with B16 melanoma. After 5 days of injection, the mice were injected i.p. with 0.5 mg/kg MPLA, 10 mg/kg anti-PD-L1 Abs, and a combination of MPLA and PD-L1 Abs every 5 days. After 25 days, tdLNs were harvested and incubated with B16 antigens for 4 h. a The intracellular production levels of IFN-γ on CD4 and CD8 T cells are shown in the response to B16 antigens. b The mean percentages of the IFN-γ producing cells. (n = 6 mice, one-way ANOVA, mean ± SEM). c The intracellular production levels of perforin and granzyme B in CD8 T cells are shown. d The means of producing cells of perforin and granzyme B in T cells are shown. (n = 6 mice, one-way ANOVA, mean ± SEM). e B16 tumor growth is shown for the duration of treatment with a combination of anti-PD-L1 Abs and MPLA in the depletion of CD4 or CD8 cells in the C57BL/6 mice. Significant difference was determined by the log-rank test, mean ± SEM (n = 6 mice)

Since T cell-mediated immunity was upregulated by the combination treatment with anti-PD-L1 Abs and MPLA, we next evaluated the anti-tumor effect of T cells in the mice treated with the combination of anti-PD-L1 Abs and MPLA. We found that the combined treatment did not inhibit B16 tumor growth in CD4 and CD8 cell-depleted mice (Fig. 2e). Therefore, these data suggested that this combined treatment promotes T cell-mediated anti-cancer immunity.

Anti-PD-L1 Abs promoted the enhancement of MPLA-induced pDC activation

T cell activation is controlled by DCs, thus, to evaluate the contribution of DCs in T cell activation and the anti-cancer effect due to the combination of anti-PD-L1 Abs and MPLA, we next examined whether co-stimulatory molecules are required for anti-cancer effects. The blockade of CD80 and CD86 inhibited the anti-tumor effects of the combined treatment in B16 tumor-bearing mice (Fig. 3a), which indicated that co-stimulatory molecules were required to induce anti-cancer immunity by the combined treatment of anti-PD-L1 Abs and MPLA.

Fig. 3.

Anti-PD-L1 Abs enhance MPLA-induced pDC activation. a B16 tumor growth is shown during the blockade of CD80 and CD86 in the C57BL/6 mice. Significance was determined by the log-rank test, mean ± SEM (n = 6 mice). b pDC and cDC analysis by flow cytometry. pDCs and cDCs were defined as B220⁺CD317⁺ and lineage⁻CD11c⁺ cells in live leukocytes, respectively. c The expression levels of PD-L1 in pDCs (left panel) and cDCs (right panel). d The expression levels of co-stimulatory molecules and MHC class I and II in pDCs. e The mean fluorescence intensities (MFI) of co-stimulatory molecules and MHC classes I and II are shown in pDCs (upper panel) and cDCs (lower panel). All data are representative of the average of six independent samples (two mice for three experiments, n = 6)

We next examined whether anti-PD-L1 Abs contribute to the upregulation of co-stimulatory molecules in DCs. The pDCs and cDCs were defined as CD317⁺B220⁺ cells and lineage⁻CD11c⁺ cells in live leukocytes in the tdLN, respectively (Fig. 3b). Both pDCs and cDCs express high levels of PD-L1 on the surface (Fig. 3c). MPLA induced increases in the co-stimulatory molecules, which were further upregulated by the combination treatment of anti-PD-L1 Abs and MPLA in pDCs (Fig. 3d, e). The treatment of MPLA promoted the upregulation of co-stimulatory molecules and major histocompatibility complex (MHC) class I and II in cDCs, while anti-PD-L1 Abs did not further increase the expression levels of co-stimulator and MHC molecules in the cDCs (Fig. 3e). Importantly, the blockade of PD-L1 by anti-PD-L1 Abs alone did not promote the activation of both pDCs and cDCs (Fig. 3d, e). In addition, MPLA did not induce the upregulation of MHC class I and II expression levels in pDCs (Fig. 3d, e), whereas the combination of anti-PD-L1 Abs and MPLA significantly increased MHC class I (Fig. 3d, e). Therefore, these data suggest that anti-PD-L1 Abs promote the enhancement of MPLA-induced pDC activation but not cDC activation.

IFN-α production in pDCs by the combination of anti-PD-L1 Abs and MPLA contributing toward anti-tumor immunity

As anti-PD-L1 Abs upregulated MPLA-induced pDC activation, we next examined IFN-α production in pDCs and found that MPLA-induced intracellular producing levels of IFN-α markedly increased upon combination treatment of anti-PD-L1 Abs and MPLA (Fig. 4a). The serum concentration of IFN-α was also elevated by the combination treatment compared to treatment with MPLA alone (Fig. 4b). Levels of Interferon regulatory factor (IRF)-7 mRNA, a transcription factor of IFN-α, also significantly increased and were maintained for 18 h after the treatment of anti-PD-L1 Abs and MPLA compared to MPLA alone (Fig. 4c). In addition, the blockade of the IFN-α receptor (IFNAR) substantially suppressed the anti-tumor effect by the combined treatment of anti-PD-L1 Abs and MPLA in B16 tumor-bearing mice (Fig. 4d). Moreover, the blockade of IFNAR inhibited the intracellular production of IFN-γ in both CD4 T cells and CD8 T cells by the combination treatment of anti-PD-L1 Abs and MPLA (Fig. 4e) The MPLA-induced upregulation of perforin and granzyme B production levels in CD8 T cells in response to B16 antigens was also significantly decreased by anti-IFNAR Abs in B16 tumor-bearing mice. Therefore, these data suggest that anti-PD-L1 Ab-induced upregulation of MPLA-mediated anti-cancer immunity was dependent on the production of IFN-α in pDC.

Fig. 4.

The pDCs produced IFN-α in response to combination treatment with MPLA and anti-PD-L1 Abs, which contributed toward anti-tumor immunity. a The intracellular production levels of IFN-α in pDCs were analyzed by flow cytometry (left panel). The mean intracellular IFN-α-producing pDCs are shown (right panel). b The concentrations of IFN-α in serum were measured by ELISA. c mRNA Levels of IRF-7 were analyzed. d The blockade of IFNAR was measured for combination treatment with anti-PD-L1 Abs and the MPLA-induced anti-cancer effect. Significance was determined by the log-rank test, mean ± SEM (n = 6 mice). e The intracellular production levels of IFN-γ in CD4 and CD8 T cells were analyzed. f The intracellular production levels of perforin and granzyme B in CD8 T cells are shown. Data are an average of six independent samples (two mice for three experiments, n = 6)

Anti-PD-L1 Abs enhanced MPLA-induced pDC and CD8 T cell activation in human peripheral blood

Anti-PD-L1 Abs enhancement of the MPLA-induced activation of pDC and CD8 T cells in the mice promoted us to examine the effect of anti-PD-L1 and MPLA on human immune cell activation. Human blood pDCs and cDCs were defined as CD11c^interCD123⁺ and CD11c⁺CD123^inter cells, respectively (Fig. 5a). The expression levels of PD-L1 in human pDCs and cDCs were almost similar (Fig. 5b). The treatment of anti-PD-L1 Abs enhanced the MPLA-induced upregulation of co-stimulatory molecules and MHC class I and II in pDCs (Fig. 5c), while the cDCs were not further activated by the combination treatment of anti-PD-L1 Abs and MPLA (Fig. 5c). Consistent with mouse data, anti-PD-L1 Ab elicited dramatic increases the MPLA-induced IFN-α production both in the intracellular expression levels of pDCs and cell culture medium (Fig. 5d, e).

Fig. 5.

Human pDCs were activated by a combination of anti-PD-L1 Abs and MPLA. Human PBMCs were treated with 500 ng/ml of MPLA, 10 μg/ml anti-PD-L1 Abs, and a combination of MPLA and anti-PD-L1 Abs. a Human pDCs and cDCs were defined by flow cytometry. b The expression levels of PD-L1 in human pDCs and cDCs were analyzed by flow cytometry (left panel). The MFI of PD-L1 in pDCs and cDCs was shown (right panel). c The levels of co-stimulatory molecules and MHC class molecules in human pDCs (upper panel) and cDCs (lower panel). d The percentages of IFN-α producing pDCs were shown. e The concentration of IFN-α in the cultured media was measured by ELISA. f Intracellular production of IFN-γ in CD4 and CD8 T cells. g Intracellular production levels of perforin and granzyme B in CD8 T cells. Data are an average of six independent samples (two donors for three experiments, n = 6)

To evaluate the anti-PD-L1 Ab effect on human T cell activation, PBMCs were treated with PBS, anti-PD-L1 Abs, MPLA, and the combination treatment. The production of IFN-γ-, perforin-, and granzyme B in CD8 T cells was increased by MPLA treatment, which was further upregulated by the combination treatment of anti-PD-L1 Ab and MPLA in CD8 T cells (Fig. 5f, g). These increased levels were due to combination treatment that may significantly inhibited by anti-IFNAR Abs, indicating that IFN-α is required to enhance T cell immunity. Therefore, these results demonstrate that anti-PD-L1 Abs enhance MPLA-induced pDC and CD8 T cell activation in human PBMCs.

In conclusion, we found that MPLA enhances anti-cancer immunity induced by anti-PD-L1 Abs in mice and humans. The combination of MPLA and the anti-PD-L1 Ab-induced anti-cancer effect contributed to the activation of pDCs. Therefore, these data demonstrate that MPLA could be a candidate as an adjuvant to enhance anti-PD-L1 Ab-mediated anti-cancer immunity in humans.

Discussion

In this study, we synthesized liposome formation of MPLA. The MPLA has removed toxic site from LPS; it still showed cytotoxicity in human and animal when used alone [41, 42]. However, liposome formation of MPLA has showed no toxicity [43]. In addition, typical MPLA-containing liposomes are commonly known as army liposome formulation (ALF) by addition of phospholipids such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, sodium salt (DMPG) and DPPC [16, 41, 44]. Therefore, the addition of DPPC in the MPLA-liposome enhances stability and safety in the human and animal.

Immune checkpoint proteins prevent normal cells from being destroyed by immune cells and cancer cells can use the same proteins to evade attack by immune cells such as CTLs [10, 11]. The majority of therapeutic trials have focused of the blockade of PD-1/PD-L1 interactions in T cells and cancer cells, which successfully elicit anti-cancer immunity in mice and humans [7, 45]. PD-1 is expressed in immune cells, such as CTL and DCs, and its blockade by anti-PD-1 Abs promotes immune cell activation and anti-cancer immunity [7]. PD-L1 is expressed in cancer cells as well as regulatory immune cells, which also elicit anti-cancer immunity by the blockade of PD-L1 [9, 14]. However, the mechanism of promoting immune cell activation due to the targeting of cancer cells by anti-PD-L1 Abs has not yet been determined. In this study, we found that both pDCs and cDCs expressed PD-L1; the treatment of anti-PD-L1 Abs synergistically enhanced MPLA-induced activation of pDCs, which consequently contributed toward anti-cancer immunity in mice. Therefore, these results suggest that the blockade of PD-L1 enhances the MPLA-mediated stimulation of pDCs.

Type I IFNs which consist of IFN-α and IFN-beta (IFN-β) are polypeptides released by infected cells [46, 47]. Type I IFNs regulate the expression of IFN-stimulated genes (ISGs) by binding to IFNAR, and ISGs trigger cellular anti-viral responses to prevent viral infection [48, 49]. In addition, type I IFNs also contribute toward the improvement of the antigen-presenting ability in APCs and the activation of natural killer (NK) cells, as well as the proliferation and activation of CTLs [50, 51]. Although many cell types produce IFN-β in response to viral infection, IFN-α is primarily produced by pDCs. It has been shown that the stimulation of endosomal TLRs expressed by pDCs, such as TLR7 and TLR9, promoted the activation of IRFs, the transcription factor of IFN-α [52, 53]. Although the expression of TLR4 in pDCs remains controversial, the pDCs produce IFN-α and activated IRF7 in response to LPS. In addition, LPS-mediated anti-cancer effects against a lung cancer model of B16 melanomas in mice were dependent on pDCs [54]. In this study, we also found that the TLR4 agonist MPLA induced pDC activation and IFN-α production, which was enhanced by the combination treatment with anti-PD-L1 Abs. Moreover, the blockade of IFNAR suppressed MPLA and anti-PD-L1 Ab-induced anti-cancer immunity. Therefore, these results support that the TLR4 agonist can stimulate pDCs and that the activated pDCs contribute toward anti-cancer immunity.

As the representative APCs, cDCs can be activated through TLR4 by LPS [37, 55]. MPLA which derived from LPS is also known as a TLR4 agonist and can enhance the activation of cDCs. We also showed that the co-stimulatory molecules and MHC molecules of cDCs were up-regulated by MPLA. Interestingly, the expression of MHC class I and II in pDCs was not increased by MPLA, whereas co-stimulatory molecules were upregulated. The role of MHC molecules is the presentation of antigens on the surface of APC, which are then recognized by T cells for antigen-specific immune responses [18, 22]. As the antigen presentation ability of pDCs is still controversial, MPLA may not contribute to antigen presentation on APCs. Moreover, we found that the combined treatment of anti-PD-L1 Abs and MPLA enhanced the activation of pDCs but not cDCs. There may be two possibilities; one is MPLA induces saturated activation of cDCs, which did not up-regulate the activation of cDCs by additional treatment of anti-PD-L1 Abs. The other is pDCs may have signaling pathway in the response to anti-PD-L1 but the cDCs may not. We will further investigate these two possibilities in pDCs and cDCs in the responses to combination of MPLA and anti-PD-L1 Abs.

Abbreviations

Ab

Antibody

DC

Dendritic cell

CTLA-4

Cytotoxic T lymphocyte antigen-4

CTL

Cytotoxic T lymphocyte

cDC

Conventional DC

IFN

Interferon

IRF

Interferon regulatory factor

MPLA

Monophosphoryl lipid A

PD-1

Programmed cell death protein

PD-L1

PD ligand 1

pDC

Plasmacytoid DC

tdLN

Tumor draining lymph node

TLR

Toll-like receptor

Author contributions

Conceptualization, JOJ; formal analysis, JOJ; data curation, WZ, SML, ST, MK and JOJ; original draft preparation, SML and JOJ; writing-review and editing, MK and JOJ. All authors approved the manuscript.

Funding

This study was supported by the Research fund of the National Research Foundation of Korea (NRF-2019R1C1C1003334 and NRF-2020R1A6A1A03044512).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.