Carboxyamidotriazole combined with IDO1-Kyn-AhR pathway inhibitors profoundly enhances cancer immunotherapy
Cancer immunotherapy has generated significant excitement, mainly as a result of the development of immune checkpoint inhibitors. The blockade of PD-1 or its ligand with antibodies has resulted in impressive clinical efficacy. However, a subset of patients does not respond to biologic therapeutics, and another subset suffers from severe immune-related adverse events in certain cases. The modulation of the immune system with small molecules might yield surprising benefits.
CD8+ cells were obtained through a magnetic cell sorting system (MACS), and their capabilities for IFN-γ release and PD-1 expression were analyzed. The in vitro effects of drugs were studied in a coculture system of tumor cells and activated CD8+ cells. We further isolated the primary tumor cells in tumor-bearing mice treated with CAI, DMF, 1-MT or a combination (CAI and DMF/CAI and 1-MT) and analyzed the percentages of CD8+ T cells and PD-1+CD8+ T cells among TILs. The selective anti-tumor immune reactions of the two drug combinations were confirmed in a coculture system consisting of B16-OVA cells and OVA-specific CTLs derived from OT-1 transgenic mice. The anti-tumor effects of the single drugs or combined therapies were assessed according to their capability to slow tumor growth and extend the life span of tumor-bearing mice, and they were compared with the effects of PD-1 antibody.
CAI increased IFN-γ release from activated T cells, which might strengthen the anti-proliferative and anti-metastatic effects on cancer cells. However, CAI also stimulated IDO1-Kyn metabolic circuitry in the tumor microenvironment and facilitated tumor cell immune evasion. Combining CAI with 1-MT or DMF disrupted PD-1 expression and promoted IFN-γ production in CD8+ T cells, and it also increased T lymphocyte infiltration in the tumor microenvironment, inhibited tumor growth and prolonged the life spans of tumor-bearing mice.
Inhibitors of the IDO1-Kyn-AhR pathway could abolish the negative effects of CAI on CD8+ T cells and result in complementary and beneficial anti-tumor immune effects. The combination of CAI with 1-MT or DMF greatly augmented the ability of CD8+ T cells to kill malignant cells and showed a strong anti-cancer capability that was superior to that of either of the single agents was is comparable with that of anti-PD-1 antibody. The combinations of small molecules utilized in this study may serve as valuable new immunotherapy strategies for cancer treatment.
KeywordsCAI PD-1 IFN- IDO1 AhR
Aryl hydrocarbon receptor
Chimeric Antigen Receptor T-Cell Immunotherapy
Cytotoxic T lymphocytes
Myeloid-derived suppressor cells
Programmed cell death protein 1
Programmed cell death 1 ligand 1
Regulatory T cells
Cancer immunotherapy harnesses the immune system to fight cancer by either stimulating the functions of specific components of the immune system or counteracting the signals that protect tumor cells from immune defense . As one of the most important drug discoveries, specific inhibitors against programmed death 1 (PD-1) or its main ligand PD-L1 have achieved prominent clinical success [2, 3]. PD-1 is an inhibitory receptor expressed on T cells, and PD-L1, the ligand of PD-1, is upregulated by interferon γ (IFN-γ) and other cytokines produced after T cell activation . The binding of PD-L1 to PD-1 promotes T cell apoptosis, anergy, and functional exhaustion and serves as an important mechanism of cancer immune evasion . Therefore, antibodies that block PD-1 or PD-L1 provide a new benchmark for cancer immunotherapy, leading the way for developing new immunotherapeutic approaches .
Carboxyamidotriazole (CAI) exposure has been demonstrated to inhibit the growth of a variety of cancer cell lines [7, 8, 9, 10]. Despite the disease stabilization and improvement in performance status observed in patients with refractory cancers after CAI treatment [8, 11], CAI has failed to provide clinical benefit or improvement when used with other therapeutic modalities [12, 13]. Previously, we found that CAI results in anti-inflammatory activity in addition to its anti-tumor effect and is capable of regulating the secretion of a variety of cytokines [14, 15]. Recently, we focused on the increased level of IFN-γ production in T cells after CAI treatment. IFN-γ is a multipotent cytokine with cytostatic/cytotoxic activity during the cell-mediated adaptive immune response, which is produced mainly by cytotoxic T lymphocytes (CTLs) and NK cells. IFN-γ has also been reported to upregulate immunosuppressive molecules such as PD-L1 and IDO1, thus promoting tumor immune escape [4, 16]. Considering its IFN-γ-stimulating effects, CAI might play a unique role in anti-tumor immunity. We speculate that the mild anti-cancer effects of CAI might be due to some adverse factors that can impair its action. A prototypical integrative modifier, indoleamine 2,3-dioxygenase-1 (IDO1), which bridges inflammation, vascularization, and immune escape and can be enhanced by IFN-γ, has become our research focus.
IDO1 is the initial rate-limiting enzyme in tryptophan (Trp) catabolism within the Kyn pathway. The overexpression of IDO1 may lead to tryptophan depletion and metabolite (Kyn, kynurenic acid, 3-hydroxy-kynurenine, etc.) accumulation, which can actively suppress T-cell functioning . In addition, Kyn and Kyn derivatives can bind the aryl hydrocarbon receptor (AhR) , which has been shown to impair the proliferation and function of various immune effectors, including CD8+ T lymphocytes, and provide tumor cells with a means to evade anticancer immunosurveillance .
In the present study, we provide evidence that the effects that hamper the in vivo anti-tumor capability of CAI might occur through the IDO-Kyn-AhR cascade. 1-methyl-L-tryptophan (1-MT), a tryptophan derivative that disrupts IDO1 signaling , or 3′,4′-dimethoxyflavone (DMF), an antagonist of AhR that inhibits the Kyn-AhR pathway  were both used in combination with CAI. The two combinations (CAI + 1-MT and CAI + DMF) greatly enhanced PD-1 blockade in CD8+ T cells, enhancing the anti-cancer capacity of the anti-PD-1 antibody. This provides a valuable immunotherapy strategy for cancer by using low-cost small molecule drug combinations with a favorable toxicity profiles (Additional file 1: Figure S1).
Materials and methods
Cell lines and reagents
Mouse tumor cell lines B16 (melanoma), OVA-B16 (melanoma), C26 (colon cancer) and 4 T1 (breast cancer) were purchased from the China Center for Type Culture Collection (Beijing, China) and cultured in RPMI 1640 (Thermo Fisher, MA, USA) with 10% fetal bovine serum (FBS) (Gibco, MA, USA), with the exception of 4 T1 cells, which were grown in DMEM medium (Gibco, MA, USA) with 10% FBS.
Carboxyamidotriazole was synthesized by the Institute of Materia Medica, Chinese Academy of Medical Sciences (Beijing, China). Polyethylene glycol 400 (PEG400) was obtained from Sinopharm Chemical Reagent Beijing (Beijing, China). 1-Methyl-L-tryptophan, 3′, 4′-dimethoxyflavone and L-kynurenine sulfate salt were purchased from Sigma-Aldrich (Saint Louis, USA).
CD8+ T cell sorting
CD8+ T cells were isolated from the spleens of BALB/c mice using a negative magnetic cell separation kit (MACS, Mouse Naive CD8+ T Cell Isolation Kit, Miltenyi Biotec). The cell purity (above 95%) was confirmed by flow cytometry using an anti-mouse CD8 antibody (eBioscience, CA, USA). The isolated CD8+ T cells were cultured in RPMI 1640 medium containing 10% FBS and 10 ng/ml IL-2 (Peprotech, NJ, USA) and activated with 1 mg/ml anti-mouse CD3/CD28 microbeads (Thermo Fisher, MA, USA).
Human naive CD8+ T cells were isolated from human peripheral blood monocytes (PBMCs). Briefly, human blood samples were collected from 12 healthy donors, and then the samples were subjected to density gradient centrifugation to obtain the PBMCs. CD8+ T cells were isolated using positive MACS (Human Naive CD8+ T Cell Isolation Kit, Miltenyi Biotec). The cell purity was confirmed with the same method described above, and the same cell culture conditions were used, except that the antibody, recombinant protein and microbeads used were human-specific.
Animal experiments and treatment protocol
C57BL/6, BALB/c or RAG1 KO mice were subcutaneously injected with appropriate amounts of the indicated tumor cells (B16, C26, 4 T1 or B16-OVA) in the right flank. Three days after inoculation or after the tumor size reached 5 × 5 mm, the mice were randomized into different groups (n = 6~10). Then, the mice in each group were treated with the following drugs separately for the indicated time: CAI (intragastric injection of 20 mg/kg/day), anti-IFN-γ neutralizing antibody (250 mg per mouse),1-MT (5 mg/ml in drinking water, 3–4 ml/mouse/day), CAI + 1-MT (the same as that used for monotherapy), DMF (intragastric injection of 10 mg/kg once every 2 days), CAI + DMF (the same as that used for monotherapy), and anti-PD-1 neutralizing antibody (250 μg per mouse). Kyn was administered by intratumoral injection (20 mg/kg/day once every 2 days). The mice in the control group received an equal volume of saline as a mock treatment. Tumor growth and the survival of the mice were recorded daily. The tumor volume was calculated according to the following formula: tumor volume = length×width2/2.
Total RNA extraction and RT–PCR
Total RNA was isolated from CD8+ T cells using a Pure RNA Extraction Kit (BioTeke Corporation) and reverse-transcribed into cDNA with the TransScript First-Strand cDNA Synthesis Supermix (TransGen Biotech Co., Beijing, China). The primer sequences used were: IDO1, 5′-TGGCGTATGTGTGGAACCG-3′ (sense) and 5′-CTCGCAGTAGGGAACAGCAA-3′ (anti-sense); GAPDH, 5′AGGTCGGTGTGAACGGATTTG-3′ (sense) and 5′-TGTAGACCATGTAGTTGAGGTCA-3′ (anti-sense). Real-time PCR was performed using an IQ5 Real-Time System (BioRad, CA, USA). The values are the mean ± SEM of three independent experiments.
CD8+ T cell lysate containing 40 μg of protein was subjected to SDS/PAGE, and the separated proteins were transferred onto PVDF membranes. After being blocked with 5% nonfat dry milk in Tris-buffered saline containing Tween-20, the membrane was incubated with the following primary antibodies overnight: anti-mouse IDO1 (Cell Signaling, Cat No. 86630; 1:1000), anti-mouse β-actin (Cell Signaling, Cat No. 3700; 1:1000). Subsequently, the membrane was incubated with the appropriate secondary antibody, and the immunoreactive protein bands were visualized using a chemiluminescence kit (Millipore, MA, USA) followed by ECL-based autoradiography. The Western blots are representative of at least three independent experiments.
Cytokine release and Kyn production
Cytokine production in the supernatants was quantified by ELISA kits (BioVision, CA, USA) according to the manufacturer’s protocol. Kyn production was measured by ELISA (MYBioSource, CA, USA) according to the manufacturer’s instructions.
Cells cultured in the soft 90-Pa 3D fibrin gels were treated with dispase II (Roche, Swiss) for 10 min at 37 °C and then fixed with 4% paraformaldehyde, collected, embedded in paraffin, and sectioned. The sections were baked for 30 min at 60 °C, dewaxed, blocked in 2% BSA and stained with anti-AhR primary antibodies (Abcam, UK), followed by staining with Alexa 488-conjugated donkey anti-rabbit IgG secondary antibodies (Invitrogen, CA, USA). After 4,6-diamidino-2-phenylindole (DAPI) staining, the slides were mounted in Fluoromount G (Solarbio, Beijing, China) and stored at 4 °C in the dark. Images were collected by confocal microscopy.
Preparation of single-cell suspensions from implanted mouse tumors
Mice were killed at specific time points. The tumors were dissected, washed in PBS, digested with IV collagenase (Sigma, St. Louis, USA), and then transferred to RPMI 1640 medium (Gibco, USA) supplemented with 10% FBS and incubated at 37 °C for 1 h. Then, the digested tumor tissues were dispersed into ground glass, and the tissue suspensions were filtered through a 40 μm mesh (BD Falcon, CA, USA). Red blood cell lysis buffer (eBioscience, CA, USA) was added and incubated with the samples for 5 min at room temperature. The cells were washed three times with PBS and then resuspended in PBS for experiments.
For the flow cytometry analysis, CD8+ T cells were stained with APC-conjugated anti-mouse PD-1 Ab and FITC-conjugated anti-mouse IFN-γ Ab (eBioscience, CA, USA). To evaluate the tumor-infiltrating lymphocytes (TILs), a single-cell suspension from the implanted tumors was stained with the following Abs: APC-conjugated anti-mouse CD3, PE-conjugated anti-mouse CD8 and FITC-conjugated anti-mouse CD4 (eBioscience, CA, USA). Flow cytometry was performed on a BD Accuri C6 flow cytometer (BD Bioscience) and analyzed with BD Accuri C6 software.
In brief, complete CD8+ T cells for the ChIP assays were prepared according to the instructions for the ChIP-IT® Express Chromatin Immunoprecipitation Kit (Active Motif, CA, USA). Every group included 5 × 107 cells. Anti-mouse AhR antibody was used for chromatin immunoprecipitation (Cell Signaling, Cat No. 83200; 1:50). Control rabbit IgG was purchased from Cell Signaling. DNA was isolated and subjected to real-time PCR analysis. The following primers were used for promoter quantification: mouse PD-1 AhR 5′-GATGTGCTGACAGCCTGCTG-3′ (sense) and 5′-ATGCTCAGGGTAGCAAGACCC-3′ (anti-sense). All sequences were designed to produce amplicons that were < 200 bp. Real-time PCR amplification was carried out, and the amplification of each target gene is shown in terms of the fold enrichment compared to that of the relevant antibody control.
Adoptive T-cell transfer
C57BL/6 J CD45.1 mice were injected subcutaneously in the abdomen with 1 × 105 B16-OVA tumor cells per mouse. When the tumor size reached 5 mm in diameter, the mice were divided randomly and received one of the following treatments: vehicle, CTL (1 × 107 cells/mouse once every five days three times), CTL + CAI (intratumoral injection, 20 mg/kg/day once every 2 days), CTL + DMF (intragastric injection, 10 mg/kg once every 2 days) or CTL + 1-MT (5 mg/ml in drinking water, 3–4 ml/mouse/day), or CTL + CAI + DMF, CTL + CAI + 1-MT, or anti-PD-1 neutralizing antibody (250 μg per mouse). CD45.2+ CTLs were isolated from the spleens of OT-1 mice and cultured with OVA peptide for 48 h. For some experiments, the mice were sacrificed 5 days after adoptive T-cell transfer to obtain the TILs.
Data are presented as the mean ± SEM, and n represents the number of experiments or animals. The statistical significance of the differences between two groups was determined by Student’s t test or one-way ANOVA followed by Dunnett’s t-test. All statistical analyses were performed by using GraphPad Prism 6.0 software. P-values < 0.05 were considered statistically significant.
CAI improves the cell killing capability of CD8+ T cells by increasing IFN-γ levels
CAI stimulates IDO-Kyn metabolic circuitry and masks the underlying deficits of T cells via mechanisms involving Kyn-AhR activation
Combining CAI with 1-MT or DMF synergistically disrupts PD-1 expression and promotes IFN-γ production in CD8+ T cells
Combining CAI with DMF or 1-MT increased the number of cytotoxic CD8+ tumor-infiltrating T cells and downregulated PD-1 expression
Combining CAI with IDO1/AhR inhibitors affected the phenotype and function of transferred T cells in B16-OVA mice and showed beneficial anti-cancer effects
Interestingly, in RAG1 KO mice bearing B16 tumors, the tumor growth inhibition effect of the indicated treatment (monotherapy or combined therapy) was obviously weaker than that of the same treatment in the presence of transferred CTLs in RAG1 KO mice bearing B16-OVA tumors. Both combinations (CAI and DMF and CAI and 1-MT) had a stronger inhibitory effect on tumor growth than either single drug, but the difference was not significant until the transfer of CTLs was performed (Additional file 4: Figure S4). The results illustrate that T cell-mediated killing plays an important role in the enhanced anti-tumor activity of the two combinations.
IDO1 or AhR inhibitor enhanced the effect of CAI on xenograft tumors
The authors have been examining the multiple pharmacological effects of the noncytotoxic small molecule compound CAI, which has shown cancer-preventing, anti-angiogenesis and cancer cachexia-fighting properties by inducing cell apoptosis, blocking calcium entry and inhibiting cell oxidative phosphorylation in a variety of studies [22, 23, 24, 25, 26, 27]. CAI also increases tumor responses to other anticancer treatments . However, the in vivo anti-tumor activity of CAI and its performance in many clinical trials are barely satisfactory, which prevents it from being a first-line chemotherapy drug.
To abolish the negative effect of CAI on CD8+ T cells, 1-MT and DMF were separately combined with CAI, and both of them independently target the upstream and downstream effectors of the IDO-Kyn-AhR-PD-1 pathway (Fig. 7). Although IDO1 is a very important immune checkpoint controller, preclinical studies have noted that single-agent treatment with an IDO1 inhibitor has a negligible effect on decreasing the established cancer burden . Therefore, approaches combining IDO1 inhibitors and other complementary compounds or immune checkpoint inhibitors would probably produce synergistic benefits in terms of tumor growth and animal survival. The present study has confirmed that combining IDO1-Kyn-AhR inhibitors with CAI can greatly augment the activity of CD8+ T cells to enhance their killing malignant cells, and the reduced expression of PD-1 and the increase in interferon-γ production in CD8+ T cells both play key roles in this (Fig. 7). At the same time, rationally designed small-molecule combinations may also hold promise as adjunctive therapies for patients with other immune suppression-related diseases, such as tuberculosis and HIV.
Anti-PD-1 antibodies have achieved tremendous clinical success in cancer treatment; however, a significant fraction of patients remain unresponsive to these biologic modalities, including CAR-T therapy . Regulating the immune system through alternative pathways with small-molecule compounds may offer complementary benefits when used with biological immunotherapies, including improved feasibility, high oral bioavailability, greater exposure within the tumor microenvironment and lower costs . The two combinations used in the present study were precisely the types of potent approaches with enhanced anti-tumor activity comparable to that of anti-PD-1 antibody that are deserving of further study.
Inhibitors of the IDO1-Kyn-AhR pathway could abolish the potential negative effects of CAI in the tumor microenvironment. The combination of CAI with 1-MT or DMF greatly augments the activity of CD8+ T cells and enhances their killing of malignant cells as a result of the reduced expression of PD-1 and the increase in interferon-γ production. The anti-cancer capacity of the combination of CAI and DMF is superior to that of either single agent and comparable to that of anti-PD-1 antibody, and this is also true for the combination of CAI and 1-MT. The combinations of small molecules introduced in this study may become effective alternate immunotherapy strategies for the treatment of various cancer.
LG and CY conceived the project. JS, LG, and DZ participated in the research design. JS, CC and RJ conducted the experiments. JS, CY, CC and RJ contributed new methodology or analytic tools. CC, JL and QW provided technical or material support. JS and LG performed the data analysis. LG and JS wrote the manuscript. All authors read and approved the final manuscript.
This study as supported by the National Science Foundation of China grants 81872897, 81402943 and 81672966 and the CAMS Major Collaborative Innovation Project 2016-I2 M-1-011.
Ethics approval and consent to participate
All animal studies and procedures were approved by the Institutional Animal Care and Use Committee of Peking Union Medical College (registration number: ACUC-A02–2017-013).
Consent for publication
The authors declare that they have no competing interests.
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