Immunotherapy is defined as the treatment of disease by employing the host immune system or immune factors and may complement or substitute for conventional therapies in the treatment of intractable diseases such as cancer [1
]. To enable successful cancer immunotherapy, it is important to overcome the immunosuppressive environment [2
]. During the induction process of anti-tumor immune responses, tumor antigen (Ag) is taken up by antigen-presenting cells (APCs) and presented to Ag-specific T cells after processing. As a result, Ag-specific T cells are activated to exert their anti-tumor effects [3
]. However, the anti-tumor immune response is decreased by immune suppressors present in the tumor environment, such as myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), tumor-associated macrophages, and type II neutrophils [4
MDSCs account for approximately 2%–3% of cells under normal conditions but are multiplied by several orders of magnitude and accumulate under disease conditions such as cancer [5
]. These cells inhibit immune effectors through various mechanisms. Specifically, MDSCs can directly inhibit the growth and proliferation of T cells by inducing the differentiation of Treg cells through secretion of interleukin (IL)-10 and transforming growth factor-β [5
] or by breaking down L-arginine, which is required for the T cell cycle, using arginase 1 expressed in MDSCs [7
]. In addition, T cell activity can be inhibited by nitric oxide (NO) and reactive oxygen species (ROS) produced by inducible nitric oxide synthase (iNOS) and NADPH oxidase 2 (NOX2) expressed in MDSCs [8
MDSCs are a heterogeneous cell population typically classified into two subsets [9
]. In tumor-bearing mice, CD11b+
cells are classified as mononuclear (Mo)-MDSCs and CD11b+
cells as polymorphonuclear (PMN)-MDSCs [10
]. Recently, other MDSC subsets containing novel functional markers have been suggested [11
Overcoming MDSC-mediated immune suppression is important for successful cancer immunotherapy. This includes the use of various agents that can deplete MDSCs, mainly cytotoxic anti-cancer agents such as gemcitabine [13
], cisplatin [14
], docetaxel [15
], and 5-fluorouracil [16
]. In addition, beneficial effects of inhibitors of functional regulators such as NO [8
], arginase [18
], cyclooxygenase2 [19
], and ROS [9
] have been reported. There are also strategies that have been studied for reducing the immunosuppressive activity of MDSCs by using all-trans retinoic acid (ATRA) [20
], IL-12 [21
], and CpG [22
], which can differentiate MDSCs into macrophages or dendritic cells (DCs). Based on these studies, we investigated the role of antioxidants eliminating ROS (one of the main functional mediators of immune suppression of MDSCs) as a regulator of MDSCs.
ATX is a member of the carotenoid family with a strong antioxidant capacity. It modulates various signaling pathways, such as the extracellular-signal-regulated kinase (ERK), PI3K/Akt, and c-Jun N-terminal kinases pathways [23
]. It has been reported to increase the expression of nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated enzymes by inducing nuclear translocation of the transcription factor, Nrf2. This elicits anti-cancer effects by inhibiting cancer cell proliferation and apoptosis induction, by eliminating ROS, and through its anti-inflammatory activity [26
]. We predicted that the antioxidant activity of ATX would eliminate the major factors mediating immune suppression of MDSCs, such as ROS, and ATX might alter the viability or function of MDSCs by regulating cell signaling of MDSCs. In addition, ATX is expected to enable further differentiation of MDSCs into macrophages or DCs through the induction of glutathione (GSH) synthesis by activating the Nrf2 signaling pathway in MDSCs. This is because one of the targets of ATX, Nrf2, is a transcription factor of NQO-1, HO-1, GCLC
, and GCLM
]. Among these genes, GCLC
might contribute to antioxidant activity as well as cell differentiation through GSH synthesis.
Currently, there is insufficient information on the effect of ATX in immunosuppressive cells such as MDSCs. Through this study, we confirmed that treatment with ATX in vivo and in vitro changed the phenotype of MDSCs, similar to the immune effectors. In addition, the expression of functional mediators and Nrf2 target genes was significantly changed through ATX treatment. ATX not only acts as a direct antioxidant but also induces functional changes in MDSCs. The altered MDSCs are rather immunogenic APCs that activate the T cell response and mediate anti-cancer effects. Overall, the results of this study confirmed the direct and indirect actions of ATX as an antioxidant, as well as its maturation-inducing and function-regulating activity in immune cells. These data suggest the possibility of using ATX as an antioxidant with immunoregulatory functions in cancer therapy.
2. Materials and Methods
2.1. Mice and Tumor Model
Specific pathogen free-female BALB/c mice were purchased from Orient bio, Korea. All mice were kept at the Animal Resource Center of Inje University. Experiments were approved by the Institutional Animal Care and Use Committee of Inje University (Approval number: 2017-002).
Mouse colon tumor cell line, CT26 cells (Korean cell bank) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (all from Gibco, Germany). For solid tumor model, BALB/c mice were s.c. injected with 5 × 10⁵ cells/mouse of CT26 at the left flank. Tumor growth was monitored at 2- to 3-day intervals. Tumor size was measured by caliper and was calculated as follows: the longest length × the shortest width × height × π/6.
2.2. MDSC Isolation
CT26 tumor-bearing mice were sacrificed at about 40 days after tumor challenges. Splenocytes were prepared, and RBCs were removed using ammonium-chloride-potassium (ACK) lysis buffer (Gibco, USA). Cells were stained with anti-CD11b microbeads (Miltenyi Biotec, Germany), and CD11b+ cells were separated using MACS LS column (Miltenyi Biotec, Germany) according to the manufacturers’ recommendation.
2.3. Viability Assay
MDSCs were seeded at 1 × 10⁶ cells/wells in 96-well plate (SPL, Korea) and treated with 100 ng/mL of lipopolysaccharide (LPS, Sigma, USA) and the indicated concentration of ATX (Adipogen, Switzerland) or dimethyl sulfoxide (DMSO, Sigma, USA) as vehicle (veh). After 24 h incubation, 20 μL/well of thiazolyl blue tetrazolium bromide (MTT, Sigma, USA) was added to MDSCs. After 2 h in a humidified atmosphere, insoluble crystals were detected. After centrifugation of the plate, the media were removed and formazan crystals were solubilized in DMSO. Absorbance of samples at 570nm was measured using microplate reader Sunrise™ (Tecan, Austria).
2.4. Phenotype Analysis of MDSCs
CT26 tumor-bearing mice with about 100 mm³ of tumor size were administrated with 50 mg/kg of ATX or veh, olive oil (Sigma, USA) using sonde for 10 days daily. Splenocytes were obtained, and some cells were stained with anti-CD11b microbeads to MDSC isolation. After MACS separation, cells were stained with fluorescein isothiocyanate (FITC)-labeled anti-Ly-6G Abs and phycoerythrin (PE)-labeled anti-Ly6C Abs for MDSC gating. For analysis of MDSC phenotype, we used allophycocyanin-labeled anti-CD40 Abs, anti-CD80 Abs, anti-CD86 Abs, or anti-IA:IE Abs. Other splenocytes were stained with allophycocyanin-labeled anti-F4/80 Abs or anti-CD11c Abs. For T cell analysis, cells were stained with FITC-labeled anti-CD3 Abs and either PE-labeled anti-CD4 Abs or PE-labeled anti-CD8 Abs. For Treg staining, cells were fixed and permeabilized using fix/perm kit (ebioscience, CA) and stained with allophycocyanin-labeled anti-Foxp3 Abs (All from BioLegend, CA).
For in vitro ATX treatment, MDSCs were seeded at 2 × 10⁷ cells/well in 6-well cell culture dish (SPL, Korea) and incubated in the presence of 10 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF, BioLegend, USA) for 5 days. ATX (10 μM) or veh, DMSO was added to MDSCs on day 0 and day 3. After incubation, cells were harvested and stained with fluorescent-labeled Abs. Stained cells were analyzed by flow cytometry (FACSCalibur, BD Science, USA).
2.5. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR)
Isolated MDSCs were seeded at 10⁷ cells/well in 12-well cell culture plate (SPL, Korea) and treated with ATX or veh in the presence of 100 ng/mL of LPS for 24 h or 5 days. After incubation time, cells were harvested and RNA was purified using RNeasy mini kit (Qiagen, Germany). We used M-MLV cDNA Synthesis Kit for cDNA synthesis and TopReal™ qPCR Kit (Both from Enzynomics, Korea) for RT-qPCR. The following primers (All from Cosmogenetech, Korea) were used: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Forward 5′–CCT GGA GAA ACC TGC CAA GTA–3′, Reverse 5′–GGA AGA GTG GGA GTT GCT GTT G–3′, Arginase 1 (ARG1), Forward 5′–AAC ACG GCA GTG GCT TTA ACC T–3′, Reverse 5′–GTG ATG CCC CAG ATG GTT TTC–3′, NADPH oxidase 2 (NOX2), Forward 5′–GAC CCA GAT GCA GGA AAG GAA–3′, Reverse 5′–TCA TGG TGC ACA GCA AAG TGA T–3′, Inducible Nitric Oxide Synthase (INOS), Forward 5′–AGG AAG TGG GCC GAA GGA T–3′, Reverse 5′–GAA ACT ATG GAG CAC AGC CAC AT–3′, NAD(P)H: quinone oxidoreductase 1 (NQO1), Forward 5′–GCA TTG GCC ACA CTC CAC CAG–3′, Reverse 5′–AGT GCC CAC AGA GAG GCC AAA–3′, Hemox1 (HO-1), Forward 5′–CAC GCC AGC CAC ACA GCA CTA–3′, Reverse 5′–GGC TGT CGA TGT TCG GGA AGG–3′, Glutamate-Cysteine Ligase Catalytic Subunit (GCLC), Forward 5′–ACA TCT ACC ACG CAG TCA AGG ACC–3′, Reverse 5′–CTC AAG AAC ATC GCC TCC ATT CAG–3′, Glutamate-Cysteine Ligase Modifier Subunit (GCLM), Forward 5′–GGC TTC GCC TCC GAT TGA AGA–3′, Reverse 5′–TCA CAC AGC AGG AGG CCA GGT–3′.
2.6. ROS Detection
MDSCs were treated with 10 μM of ATX in the presence of LPS (100 ng/mL). After treatment, MDSCs were harvested via centrifugation and resuspended in 10 μM of CM-H2DCFDA (Invitrogen, USA) for 50 min according to the manufacturers’ recommendation. Cells were analyzed by flow cytometry.
2.7. In Vivo CTL Assay
MDSCs were treated with ATX or veh for 24 h. MDSCs were pulsed with Her-2/neu CTL epitope peptides p63 (Anygen, Korea) [29
] at a concentration of 5 μg/mL for an additional 90 min. Cells were harvested and transferred into naïve BALB/c mice via intravenous (i.v.) route. After 2 weeks, we performed in vivo CTL assay as previously described [30
]. For preparation of Her-2/neu-specific target cells, naïve splenocytes were pulsed or unpulsed with 5 μg/mL of p63 peptides for 90 min. Peptide pulsed cells were labeled with 20 μM of carboxyfluorescein diacetate succinimidylester (CFSE, Invitrogen, USA), whereas peptide unpulsed cells were labeled with 1.5 μM of CFSE. The same amount of peptide pulsed or unpulsed cells were mixed. Target cells (1 x 10⁷cells/mouse) were i.v. injected into mice. After 72 h, p63-specific target lysis in the splenocytes was analyzed by flow cytometry. The specific lysis was calculated as follows: r
= (% CFSElow
cells), % specific lysis = [1 − (runprimed
)] × 100.
2.8. Adoptive Transfer of MDSCs
Isolated MDSCs were seeded at 10⁷ cells/well in 12-well cell culture plate and treated with ATX or veh for 24 h. For maintenance of immunosuppressive MDSC function, MDSCs were incubated in Roswell Park Memorial Institute Medium 1640 (RPMI1640, Gibco BRL, Germany) supplemented with 10 ng/mL of GM-CSF, 20% of FBS, and 25% of tumor cell conditioned medium (TCCM) which were the supernatants of CT26 cell culture media [31
]. AXT-treated or veh-treated MDSCs (1 × 10⁷ cells/mouse) were i.v. transferred into CT26 tumor-bearing mice at the tumor size of about 100 mm³. Tumor size was monitored 3 times a week. We monitored tumor size until day 42 after tumor challenge.
2.9. Statistical Analysis
The student’s t-test was used to compare the differences between 2 groups. Values of p < 0.05 were considered significant at a 95% confidence interval.
Recently, various strategies targeting MDSCs have been verified in preclinical studies and clinical trials [12
]. One of the major immunosuppressive mechanisms of MDSCs is mediated by ROS, and therefore studies on the functional regulation of MDSCs using antioxidants have been conducted. The immunosuppressive activity of MDSCs on T cells was reduced when ROS levels were decreased using antioxidants [8
]. Furthermore, suppression of cancer metastasis through attenuation of ROS production using a synthetic Nrf2 inducer, 1-(2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl) imidazole (CDDO-Im), in MDSCs has been reported [35
]. On the contrary, MDSC expansion can be induced through Nrf2-dependent activation, and Nrf2 pathway contributes to defense mechanisms against oxidative stress exposed to MDSCs [36
]. Thus, the Nrf2 pathway can either positively or negatively regulate MDSC-mediated immune suppression.
In this study, ATX, a potent antioxidant with anti-cancer effects against various types of cancer cells, was investigated for MDSC targeting. It was hypothesized that the ROS level decreased in ATX-treated MDSCs because of the antioxidant activity of ATX, thereby regulating immune suppression and simultaneously inducing changes via Nrf2 signaling activation. In particular, we focused on GCLC
among the Nrf2 target genes [37
]. The synthesized GSH is involved in cell differentiation and antioxidant activity [38
]. Thus, we believed that ATX not only played a role as a direct/indirect antioxidant but may also induce maturation of MDSCs. This would be significant because differentiated MDSCs may function as immune effectors with reduced immunosuppressive activity [39
Previously, ATRA and CpG were used to study the differentiation of MDSCs into macrophages or DCs. ATRA induces the synthesis of GSH synthase through ERK signaling pathway in MDSCs, followed by their differentiation into macrophages or DCs with increased GSH [20
]. CpG induces MDSC maturation via the activity of interferon-α produced in plasmacytoid DCs [22
]. In this study, a significant decrease in the percentage of PMN-MDSCs, and an improved activation status of the remaining MDSCs, was observed in tumor-bearing mice treated with ATX, confirming a significant increment in CD11c+
cells. In addition, it was confirmed that in vitro ATX treatment increased the expression of GCLC
, a gene involved in GSH synthesis, in isolated MDSCs. As a result, the immunogenicity of ATX-treated MDSCs with altered functions was improved. Further, these cells had the characteristics of mature effector cells that exhibit a cancer growth inhibitory effect.