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Carboxyamidotriazole combined with IDO1-Kyn-AhR pathway inhibitors profoundly enhances cancer immunotherapy

Carboxyamidotriazole combined with IDO1-Kyn-AhR pathway inhibitors profoundly enhances cancer... Background: 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. Methods: 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. Results: 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. Conclusion: 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. Keywords: CAI, PD-1, IFN-, IDO1, AhR * Correspondence: leiguo@ibms.cams.cn Department of Pharmacology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, China © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 2 of 14 Introduction In the present study, we provide evidence that the Cancer immunotherapy harnesses the immune system to effects that hamper the in vivo anti-tumor capability of fight cancer by either stimulating the functions of spe- CAI might occur through the IDO-Kyn-AhR cascade. 1- cific components of the immune system or counteract- methyl-L-tryptophan (1-MT), a tryptophan derivative ing the signals that protect tumor cells from immune that disrupts IDO1 signaling [20], or 3′,4′-dimethoxyfla- defense [1]. As one of the most important drug discover- vone (DMF), an antagonist of AhR that inhibits the Kyn- ies, specific inhibitors against programmed death 1 (PD- AhR pathway [21] were both used in combination with 1) or its main ligand PD-L1 have achieved prominent CAI. The two combinations (CAI + 1-MT and CAI + clinical success [2, 3]. PD-1 is an inhibitory receptor DMF) greatly enhanced PD-1 blockade in CD8 T cells, expressed on T cells, and PD-L1, the ligand of PD-1, is enhancing the anti-cancer capacity of the anti-PD-1 anti- upregulated by interferon γ (IFN-γ) and other cytokines body. This provides a valuable immunotherapy strategy produced after T cell activation [4]. The binding of PD- for cancer by using low-cost small molecule drug combi- L1 to PD-1 promotes T cell apoptosis, anergy, and func- nations with a favorable toxicity profiles (Additional file 1: tional exhaustion and serves as an important mechanism Figure S1). of cancer immune evasion [5]. Therefore, antibodies that block PD-1 or PD-L1 provide a new benchmark for can- Materials and methods cer immunotherapy, leading the way for developing new Cell lines and reagents immunotherapeutic approaches [6]. Mouse tumor cell lines B16 (melanoma), OVA-B16 Carboxyamidotriazole (CAI) exposure has been dem- (melanoma), C26 (colon cancer) and 4 T1 (breast cancer) onstrated to inhibit the growth of a variety of cancer cell were purchased from the China Center for Type Culture lines [7–10]. Despite the disease stabilization and im- Collection (Beijing, China) and cultured in RPMI 1640 provement in performance status observed in patients (Thermo Fisher, MA, USA) with 10% fetal bovine serum with refractory cancers after CAI treatment [8, 11], CAI (FBS) (Gibco, MA, USA), with the exception of 4 T1 cells, has failed to provide clinical benefit or improvement which were grown in DMEM medium (Gibco, MA, USA) when used with other therapeutic modalities [12, 13]. with 10% FBS. Previously, we found that CAI results in anti-inflammatory Carboxyamidotriazole was synthesized by the Institute activity in addition to its anti-tumor effect and is capable of of Materia Medica, Chinese Academy of Medical Sciences regulating the secretion of a variety of cytokines [14, 15]. (Beijing, China). Polyethylene glycol 400 (PEG400) was Recently, we focused on the increased level of IFN-γ pro- obtained from Sinopharm Chemical Reagent Beijing duction in T cells after CAI treatment. IFN-γ is a multipo- (Beijing, China). 1-Methyl-L-tryptophan, 3′,4′-dimethox- tent cytokine with cytostatic/cytotoxic activity during the yflavone and L-kynurenine sulfate salt were purchased cell-mediated adaptive immune response, which is pro- from Sigma-Aldrich (Saint Louis, USA). duced mainly by cytotoxic T lymphocytes (CTLs) and NK cells. IFN-γ has also been reported to upregulate immuno- CD8 T cell sorting suppressive molecules such as PD-L1 and IDO1, thus CD8 T cells were isolated from the spleens of BALB/c promoting tumor immune escape [4, 16]. Considering its mice using a negative magnetic cell separation kit (MACS, IFN-γ-stimulating effects, CAI might play a unique role in Mouse Naive CD8 T Cell Isolation Kit, Miltenyi Biotec). anti-tumor immunity. We speculate that the mild anti-can- The cell purity (above 95%) was confirmed by flow cytome- cer effects of CAI might be due to some adverse factors try using an anti-mouse CD8 antibody (eBioscience, CA, that can impair its action. A prototypical integrative modi- USA). The isolated CD8 T cells were cultured in RPMI fier, indoleamine 2,3-dioxygenase-1 (IDO1), which bridges 1640 medium containing 10% FBS and 10 ng/ml IL-2 inflammation, vascularization, and immune escape and can (Peprotech, NJ, USA) and activated with 1 mg/ml anti- be enhanced by IFN-γ, has become our research focus. mouse CD3/CD28 microbeads (Thermo Fisher, MA, USA). IDO1 is the initial rate-limiting enzyme in tryptophan Human naive CD8 T cells were isolated from human (Trp) catabolism within the Kyn pathway. The overex- peripheral blood monocytes (PBMCs). Briefly, human pression of IDO1 may lead to tryptophan depletion and blood samples were collected from 12 healthy donors, metabolite (Kyn, kynurenic acid, 3-hydroxy-kynurenine, and then the samples were subjected to density gradient etc.) accumulation, which can actively suppress T-cell centrifugation to obtain the PBMCs. CD8 T cells were functioning [17]. In addition, Kyn and Kyn derivatives isolated using positive MACS (Human Naive CD8 T can bind the aryl hydrocarbon receptor (AhR) [18], Cell Isolation Kit, Miltenyi Biotec). The cell purity was which has been shown to impair the proliferation and confirmed with the same method described above, and function of various immune effectors, including CD8 T the same cell culture conditions were used, except that lymphocytes, and provide tumor cells with a means to the antibody, recombinant protein and microbeads used evade anticancer immunosurveillance [19]. were human-specific. Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 3 of 14 Animal experiments and treatment protocol Cytokine release and Kyn production C57BL/6, BALB/c or RAG1 KO mice were subcutane- Cytokine production in the supernatants was quantified ously injected with appropriate amounts of the indicated by ELISA kits (BioVision, CA, USA) according to the tumor cells (B16, C26, 4 T1 or B16-OVA) in the right manufacturer’s protocol. Kyn production was measured flank. Three days after inoculation or after the tumor by ELISA (MYBioSource, CA, USA) according to the size reached 5 × 5 mm, the mice were randomized into manufacturer’s instructions. different groups (n = 6~10). Then, the mice in each group were treated with the following drugs separately Immunofluorescence for the indicated time: CAI (intragastric injection of Cells cultured in the soft 90-Pa 3D fibrin gels were 20 mg/kg/day), anti-IFN-γ neutralizing antibody (250 mg treated with dispase II (Roche, Swiss) for 10 min at 37 °C per mouse),1-MT (5 mg/ml in drinking water, 3–4ml/ and then fixed with 4% paraformaldehyde, collected, em- mouse/day), CAI + 1-MT (the same as that used for bedded in paraffin, and sectioned. The sections were monotherapy), DMF (intragastric injection of 10 mg/kg baked for 30 min at 60 °C, dewaxed, blocked in 2% BSA once every 2 days), CAI + DMF (the same as that used and stained with anti-AhR primary antibodies (Abcam, for monotherapy), and anti-PD-1 neutralizing antibody UK), followed by staining with Alexa 488-conjugated (250 μg per mouse). Kyn was administered by intratu- donkey anti-rabbit IgG secondary antibodies (Invitrogen, moral injection (20 mg/kg/day once every 2 days). The CA, USA). After 4,6-diamidino-2-phenylindole (DAPI) mice in the control group received an equal volume of staining, the slides were mounted in Fluoromount G saline as a mock treatment. Tumor growth and the sur- (Solarbio, Beijing, China) and stored at 4 °C in the dark. vival of the mice were recorded daily. The tumor vol- Images were collected by confocal microscopy. ume was calculated according to the following formula: tumor volume = length×width /2. Preparation of single-cell suspensions from implanted mouse tumors Mice were killed at specific time points. The tumors Total RNA extraction and RT–PCR were dissected, washed in PBS, digested with IV collage- Total RNA was isolated from CD8 T cells using a Pure nase (Sigma, St. Louis, USA), and then transferred to RNA Extraction Kit (BioTeke Corporation) and reverse- RPMI 1640 medium (Gibco, USA) supplemented with transcribed into cDNA with the TransScript First-Strand 10% FBS and incubated at 37 °C for 1 h. Then, the cDNA Synthesis Supermix (TransGen Biotech Co., digested tumor tissues were dispersed into ground glass, Beijing, China). The primer sequences used were: IDO1, and the tissue suspensions were filtered through a 40 μm 5′-TGGCGTATGTGTGGAACCG-3′ (sense) and 5′- mesh (BD Falcon, CA, USA). Red blood cell lysis buffer CTCGCAGTAGGGAACAGCAA-3′ (anti-sense); GAP (eBioscience, CA, USA) was added and incubated with DH, 5′AGGTCGGTGTGAACGGATTTG-3′ (sense) and the samples for 5 min at room temperature. The cells 5′-TGTAGACCATGTAGTTGAGGTCA-3′ (anti-sense). were washed three times with PBS and then resuspended Real-time PCR was performed using an IQ5 Real-Time in PBS for experiments. System (BioRad, CA, USA). The values are the mean ± SEM of three independent experiments. Flow cytometry For the flow cytometry analysis, CD8 T cells were stained with APC-conjugated anti-mouse PD-1 Ab and Western blotting FITC-conjugated anti-mouse IFN-γ Ab (eBioscience, CD8 T cell lysate containing 40 μg of protein was sub- CA, USA). To evaluate the tumor-infiltrating lympho- jected to SDS/PAGE, and the separated proteins were cytes (TILs), a single-cell suspension from the implanted transferred onto PVDF membranes. After being blocked tumors was stained with the following Abs: APC-conju- with 5% nonfat dry milk in Tris-buffered saline containing gated anti-mouse CD3, PE-conjugated anti-mouse CD8 Tween-20, the membrane was incubated with the follow- and FITC-conjugated anti-mouse CD4 (eBioscience, CA, ing primary antibodies overnight: anti-mouse IDO1 (Cell USA). Flow cytometry was performed on a BD Accuri C6 Signaling, Cat No. 86630; 1:1000), anti-mouse β-actin flow cytometer (BD Bioscience) and analyzed with BD (Cell Signaling, Cat No. 3700; 1:1000). Subsequently, the Accuri C6 software. membrane was incubated with the appropriate secondary antibody, and the immunoreactive protein bands were ChIP-qPCR assay visualized using a chemiluminescence kit (Millipore, In brief, complete CD8 T cells for the ChIP assays were MA, USA) followed by ECL-based autoradiography. prepared according to the instructions for the ChIP-IT® The Western blots are representative of at least three Express Chromatin Immunoprecipitation Kit (Active independent experiments. Motif, CA, USA). Every group included 5 × 10 cells. Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 4 of 14 Anti-mouse AhR antibody was used for chromatin im- enhancement of the anti-tumor activity of CTLs by munoprecipitation (Cell Signaling, Cat No. 83200; 1:50). cotreatment with CAI was also observed when CTLs were Control rabbit IgG was purchased from Cell Signaling. cocultured with other types of tumor cells (Additional DNA was isolated and subjected to real-time PCR ana- file 2: Figure S2B). Given that cytokines play critical lysis. The following primers were used for promoter roles in the proper establishment of anti-tumor im- quantification: mouse PD-1 AhR 5′-GATGTGCTGA munity, we examined the levels of IFN-γ,IL-6 and IL-2 CAGCCTGCTG-3′ (sense) and 5′-ATGCTCAGGG in both murine- and human-derived CD8 T cells and TAGCAAGACCC-3′ (anti-sense). All sequences were in tumor cell coculture systems. IFN-γ production by CTLs designed to produce amplicons that were < 200 bp. Real- was greatly enhanced by CAI (Fig. 1b and Additional file 2: time PCR amplification was carried out, and the amplifi- Figure S2C). To gain further insight into the involvement cation of each target gene is shown in terms of the fold of IFN-γ,weadded IFN-γ neutralizing antibody to CAI- enrichment compared to that of the relevant antibody processed cocultured CTLs and B16 cells. The neutralizing control. antibody significantly counteracted the CAI-induced en- hancement of the cytotoxicity of CTLs (Fig. 1c). In addition, Adoptive T-cell transfer CAI could also promote IFN-γ release from activated C57BL/6 J CD45.1 mice were injected subcutaneously in spleen lymphocytes and TILs in tumor-bearing mice the abdomen with 1 × 10 B16-OVA tumor cells per (Fig. 1d, e, Additional file 2:FigureS2D), suggesting mouse. When the tumor size reached 5 mm in diameter, that there was a common phenomenon in terms of the the mice were divided randomly and received one of the effect of CAI on T cells. In B16 melanoma-bearing following treatments: vehicle, CTL (1 × 10 cells/mouse mice, CAI treatment could definitely delay tumor once every five days three times), CTL + CAI (intratumoral growth; however, the concurrent injection of anti-IFN- injection, 20 mg/kg/day once every 2 days), CTL + DMF γ antibody and CAI not only eliminated the beneficial (intragastric injection, 10 mg/kg once every 2 days) or effect of CAI but also promoted tumor growth (Fig. 1f). CTL + 1-MT (5 mg/ml in drinking water, 3–4ml/mouse/ These data suggest that in an environment where day), or CTL +CAI +DMF, CTL +CAI +1-MT, or anti- tumor cells and CD8 T cells coexist, the enhancement PD-1 neutralizing antibody (250 μg per mouse). CD45.2 of tumor anti-immunity by CAI is closely related to the CTLs were isolated from the spleens of OT-1 mice and cul- release of IFN-γ. tured with OVA peptide for 48 h. For some experiments, the mice were sacrificed 5 days after adoptive T-cell transfer CAI stimulates IDO-Kyn metabolic circuitry and masks the to obtain the TILs. underlying deficits of T cells via mechanisms involving Kyn-AhR activation Statistical analysis To determine the reason why CAI has a weaker anti- Data are presented as the mean ± SEM, and n represents cancer effect in vivo than expected, we tested the meta- the number of experiments or animals. The statistical bolic status of tryptophan (Trp). Intriguingly, increased significance of the differences between two groups was Kyn concentrations were found in the supernatant of determined by Student’s t test or one-way ANOVA B16 cells cocultured with CD8 T cells and in B16 followed by Dunnett’s t-test. All statistical analyses were tumor tissues from mice treated with CAI (Fig. 2a). 1-MT performed by using GraphPad Prism 6.0 software. P- reduced Kyn basal levels and CAI-induced Kyn produc- values < 0.05 were considered statistically significant. tion (Fig. 2a). CAI strongly induced mRNA and protein expression of a key Try-metabolizing enzyme, IDO1, in Results both CTLs and B16 tumor tissues (Fig. 2b, c). Kyn can CAI improves the cell killing capability of CD8 T cells by combine with aryl hydrocarbon receptor (AhR) to regulate increasing IFN-γ levels the expression of many genes. Here, the confocal data in- In this study, B16 melanoma tumor cells and CTLs were dicated that Kyn exposure increased the nuclear import of cocultured in the presence or absence of CAI for 24 h. AhR in CD8 T cells and that this effect was blocked by CTLs exposed to CAI showed stronger cytotoxic activity 3′,4′-dimethoxyflavone (DMF). To assess whether AhR against tumor cells than those not exposed to CAI, and could increase the expression of PD-1, ChIP-qPCR was the tumor-killing capacity was T cell number-dependent performed in CD8 T cells. Our data show that the AhR- + + (Fig. 1a). Furthermore, when CD8 T cells were pre- dependent expression of PD-1 in activated CD8 Tcells treated with CAI for 48 h and then cocultured with in the presence of Kyn tremendously enhanced the activity tumor cells, the cytotoxicity of the CD8 T cells was of the PD-1 transcriptional program (Fig. 2e). The number + + + similar to that of CD8 T cells exposed to CAI during of PD-1 CD8 T cells tended to increase over the time cell coculture, indicating that CAI might promote CTL during Kyn treatment. In addition, the combined use of activity directly (Additional file 2: Figure S2A). The Kyn and DMF resulted in a slight decline but did not Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 5 of 14 Fig. 1 | CAI improves the cytotoxicity of CD8 T cells and increases IFN-γ production. a B16 tumor cells and CTLs were cocultured at a ratio of 1:10 or 1:20 in the presence or absence of CAI (10 μM) for 24 h. The CTLs were preactivated with anti-CD3/CD28 beads for 48 h. The proportion of tumor cell apoptosis was determined by flow cytometry (quadrantal diagram), and the survival rate of the tumor cells in each group is shown in the bar chart. CM: culture medium (b) Contents of the cytokines in the supernatants of cocultured cells. c B16 cells were cocultured with activated CTLs at a ratio of 1:20 in the presence of vehicle (DMSO), CAI (10 μM) or IFN-γ antibody (10 mg/mL) for 24 h. The quadrantal diagrams show the proportions of tumor cell apoptosis, and the bar chart shows the survival rate of the tumor cells in each group. d, e and f) Mice were s.c. injected with 2 × 10 B16 (n = 10 per group). When the average tumor size reached approximately 3 × 3 mm, the following treatments were initiated: PBS or CAI (20 mg/kg) or a combination of CAI and anti-IFN-γ antibody (250 mg/day) every 2 days for 23 days. d IFN-γ production in TILs and spleen was analyzed by flow cytometry. e Interferon content in tumor tissue was detected by ELISA. f Tumor growth curves. The data represent the mean ± s.e.m. N.S., no significant difference; **p < 0.01, ***p < 0.001 by Student’s t test (a, b, d and e) or one-way ANOVA (c and f) Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 6 of 14 Fig. 2 | CAI stimulation of the IDO-Kyn metabolic circuitry and the effects of the metabolite Kyn on CD8 T cells. After CAI treatment (10 μM, 48 h) (a), the production of Kyn in the B16/T cell coculture system (left) and B16 tumor tissues (right) were determined. b and c The mRNA and protein expression of IDO1 determined by RT-PCR and Western blotting. d CTLs were treated with 200 mM Kyn for 2 days. The transfer of AhR from the cytosol to the nucleus determined by immunostaining assay. Bar, 2 μm. e ChIP-qPCR analysis of AhR-dependent PD-1 expression after Kyn treatment. The ChIP enrichment ratio relative to the control is shown. f CTLs were incubated with vehicle (DMSO), Kyn (200 mM) or DMF + + (20 μM) alone or a combination of Kyn and DMF for the indicated time spans, and the PD-1 CD8 T cells were analyzed by flow cytometry. Representative histograms (left) and the overall results (right) are shown. g B16 tumor-bearing mice received an intratumoral injection of Kyn + + with or without DMF treatment (10 mg/kg). Tumor-infiltrating lymphocytes (TILs) were then isolated from the tumor tissues, and the PD-1 CD8 T cells were analyzed by flow cytometry. Representative histogram (left) and the statistical histogram (right) are shown. h Intratumoral injection of Kyn reduced the proportion of IFN-γ-positive T cells in TILs isolated from B16 tumor tissues, and DMF treatment (10 mg/kg) rescued this inhibition. Representative histograms (left) and the statistical histograms (right) are shown. Data are from three independent experiments, and the error bars represent the mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA (a, g, f and h) or Student’s t test (b and e) Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 7 of 14 + + counterbalance the percentage of PD-1 CD8 Tcells were subcutaneously injected with tumor cells (C26, 4 (Fig. 2f). To further confirm the role of Kyn in the T1 or B16 cells, n = 6 in every group). The mice began tumor microenvironment, mice bearing tumors re- to receive drug treatment when the mean size of the tu- ceived intratumoral injections of Kyn, DMF or a com- mors reached 5 mm in diameter. Cells were isolated bination of Kyn and DMF. Similar to the above results, from solid tumor tissues and assayed with flow + + Kyn markedly increased the percentage of PD-1 CD8 cytometry. The combination of CAI and DMF showed T cells in TILs and inhibited the production of IFN-γ, consistent synergistic effects in the 3 tumor-bearing ani- while DMF showed a partial offsetting effect, which mal models, which elevated the percentage of CD8 T means that the excess production of Kyn may cause T cells in TILs in all 3 types of tumor tissues five- to eight- cell exhaustion and impair the immune surveillance fold compared with that in the control group (Fig. 4a, b). function of CD8 T cells in the tumor microenviron- Meanwhile, CAI, DMF or a combination significantly + + ment. These results also suggest that the CAI activation reduced the expression of PD-1 in CD8 T cells in the of the IDO-Kyn-AhR cascade might be the underlying combination group, demonstrating the strengthened in- mechanism that limits the anti-tumor efficacy of CAI. hibitory effect in comparison with that in both mono- therapy groups (Fig. 4c). When an alternative inhibitor Combining CAI with 1-MT or DMF synergistically disrupts of IDO1, 1-MT, was used instead of DMF in subsequent PD-1 expression and promotes IFN-γ production in CD8 independent experiments with the same types of tumor- T cells bearing mice, the abovementioned indicators showed To determine whether an IDO1 inhibitor or an AhR in- variations consistent with those observed in previous hibitor could improve the effects of CAI on T cells, both studies (involving DMF) for each monotherapy group mouse spleen-derived CD8 T cells and human PBMC- and combination group in all 3 types of tumor tissues derived CD8 T cells were treated with CAI, DMF, 1- (Fig. 4d~f). It was speculated that the augmentation of MT alone or a combination of CAI and DMF or 1-MT PD-1 blockade might promote CD8 T cell survival and for 48 h. There was a statistically significant difference enhance the cytotoxic activity of TILs in the tumor between the monotherapy group and the control group. microenvironment. In addition, the effects of the indi- However, the two combinations drastically decreased cated single drugs or combination drugs on other cell PD-1 expression and elevated IFN-γ production in CD8 types in the tumor microenvironment were comprehen- T cells (Fig. 3a~e). As for human PBMC-derived CD8 sively analyzed by flow cytometry. There were no signifi- T cells, the combination of CAI with DMF or 1-MT also cant differences in the number and typical function of resulted in obvious immune enhancement, as evidenced tumor-associated macrophages (TAMs) between the dif- by the enhancement of PD-1 blockade and an increase ferent treatment groups. The same was true for other in IFN-γ production (Fig. 3d, e). The ChIP-qPCR data cell types, including myeloid-derived suppressor cells showed that CAI facilitated the binding of AhR to the (MDSCs), regulatory T cells (Tregs) and CD4 T cells. It promoter of the PD-1 gene and caused an approximately is worth mentioning that the downregulation of PD-1 in 2.07-fold increase in PD-1 expression, while combining the combination groups was very obvious in CD8 T CAI with DMF or 1-MT obviously reduced the overex- cells but not in CD4 T cells (Additional file 3: Figure pression of PD-1 caused by AhR activation (Fig. 3h). S3). The results indicate that the enhanced anti-tumor Correspondingly, the immunostaining data showed that activity of the two combinations of drugs was mainly the nuclear translocation of AhR was significantly inhib- due to enhanced CD8 T cell function and number. ited by the combination of CAI with DMF or 1-MT. (Fig. 3i). The results suggest that the dampening effect Combining CAI with IDO1/AhR inhibitors affected the of CAI on T cells arising from the activation of the phenotype and function of transferred T cells in B16-OVA IDO/AhR axis could be overcome by combining CAI mice and showed beneficial anti-cancer effects with an IDO1/AhR inhibitor and that this combination Combining CAI with IDO1/AhR inhibitors could lead to might play a distinct role in promoting the antitumor a more selective anti-tumor immunoreaction, which was immunity of CD8 T cells. confirmed in a specialized coculture system consisting of B16 melanoma cells expressing ovalbumin (OVA) anti- Combining CAI with DMF or 1-MT increased the number gen (B16-OVA) and OVA-specific CTLs derived from of cytotoxic CD8 tumor-infiltrating T cells and OT-1 transgenic mice. Either combination resulted in downregulated PD-1 expression the lowest survival rate of B16-OVA cells in parallel ex- To study the effects of CAI, DMF, 1-MT and the two periments with single agents. (Fig. 5a, b). To further assess combinations (CAI with DMF/CAI or 1-MT) on T cells the immunotherapeutic effects of the two combinations within the tumor microenvironment, three tumor-bear- on T cells, adoptive cell transfer (ACT) was conducted. In + + ing mice models were used. BALB/c or C57BL/6 mice terms of the proportion of PD-1 CD45.2 TILs in B16- Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 8 of 14 + + Fig. 3 | Combining CAI with 1-MT or DMF synergistically disrupts PD-1 expression and promotes IFN-γ production in CD8 T cells. Sorted CD8 T cells were activated by CD3/CD28 beads for 48 h and treated with CAI (10 μM), DMF (20 μM) or a combination of CAI and DMF for 24 h. In another experiment, the sorted CD8 T cells were treated with CAI (10 μM), 1-MT (0.2 mM) or a combination of CAI and 1-MT for 24 h. a-b Flow + + cytometry plots showing the number of PD-1 CD8 T cells after various treatments. Representative histogram plots (left) and the statistical histogram plots (right) are shown. c The percentage of IFN-γ-producing T cells in each group was measured by flow cytometry (n = 3). d and e Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of 10 healthy volunteers, and PBMC-derived CD8 T cells were incubated with the indicated single drug or a combination of drugs for 48 h. The percentages of PD-1-positive cells and IFN-γ-producing cells in each group were measured by flow cytometry. f and g Activated CD8 T cells from the spleens of C57BL/6 mice were cocultured with B16 cells, or PBMC-derived CD8 T cells were cocultured with HCT116 cells at a ratio of 20:1 for 24 h. Tumor cell apoptosis was analyzed by flow cytometry. h ChIP-qPCR analysis of AhR-dependent PD-1 expression after various treatments. The ChIP enrichment ratio relative to the control is shown. i CD8 T cells isolated from mouse spleens were activated with anti-CD3/CD28 beads for 48 h. At the same time, activated T cells were treated with CAI (10 μM), DMF (20 μM), 1-MT (0.2 mM) or a combination of CAI and DMF/1-MT for 24 h. Then, the CD8 T cells were fixed and strained with an anti-AhR antibody and imaged by confocal microscopy. Bar, 2 μm. Data are from three independent experiments, and the error bars represent the mean ± s.e.m. **p < 0.01, ***p < 0.001 by one-way ANOVA (A, B, E-H) Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 9 of 14 Fig. 4 | Combining CAI with DMF or 1-MT increased the number of tumor-infiltrating CD8 T cells and downregulated PD-1 expression. BALB/c or C57BL/6 mice were subcutaneously injected with 1 × 10 tumor cells (C26 cells, B16 cells or 4 T1 cells, n = 6 in every group) and received the indicated drugs for 7 days after the day the tumor size reached 5 mm in diameter. T cells in the tumor microenvironment were sorted and + + analyzed by flow cytometry. a Representative flow cytometry plots showing the fraction of CD8 T cells within the CD3 TILs in the C26, B16- or + + + 4 T1- tumor microenvironment. b CD8 T cell numbers per gram of tumor in different groups. c The percentage of PD-1 CD8 T cells within TILs in the tumor microenvironment. d, e and f The same measurements from the evaluation of the effects of another drug combination (CAI and 1- MT) in C26-, B16- or 4 T1- tumor bearing mice. Data are from three independent experiments, and the error bars represent the mean ± s.e.m. **p < 0.01 by one-way ANOVA (a-f) OVA CD45.1 mice, monotherapy caused a slight decrease resulted in more encouraging effects that were comparable after 5 days of treatment. Noticeably, combined therapy with those of PD-1 antibody (Fig. 5e, f). We also assessed (CAI + DMF or CAI + 1-MT) resulted in a significant syn- the anti-tumor activity of the indicated therapeutics in ergistic inhibition compared with either single agent, with RAG1 KO mice bearing B16-OVA tumors. Each of the + + the proportion of PD-1 CD45.2 TILs decreasing to less three monotherapies, CAI, DMF, or 1-MT, slightly reduced than 1/3 of that in the control group (Fig. 5c, d). We then PD-1 expression on the surfaces of transferred CD45.1 T evaluated the anti-tumor effect in vivo using B16-OVA cells and showed little effect on tumor growth. In contrast, tumor-bearing mice who received T cell ACT. The com- combined therapy (CAI + DMF or CAI + 1-MT) signifi- + + bination of CAI and DMF and CAI and 1-MT obviously cantly reduced the expression of PD-1 in CD45.1 Tcells inhibited tumor growth compared with the control and clearly inhibited tumor growth (Fig. 5g~i). group or either single agent-treated group. In addition, Interestingly, in RAG1 KO mice bearing B16 tumors, prolonged treatment (> 30 days) with the combinations the tumor growth inhibition effect of the indicated Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 10 of 14 Fig. 5 | Phenotypic character of transferred CD45.2 T cells in B16-OVA mice and the in vivo anti-tumor activity of different treatments. a and b) Activated CD8 T cells from the spleens of OT-1 mice were cocultured with B16-OVA cells at a ratio of 2:1 for 5 h and 10 h and treated with CAI (10 μM), DMF (20 μM) or a combination of CAI and DMF. In another experiment, sorted CD8 T cells were treated with CAI (10 μM), 1-MT (0.2 mM) or a combination of CAI and 1-MT. B16-OVA cell apoptosis was analyzed by flow cytometry at 5 h and 10 h after drug treatment. c and d CD45.1 + + 7 C57BL/6 mice bearing 3 × 3 mm OVA-B16 melanomas were subject to the adoptive transfer of OVA-specific CD45.2 CD8 T cells (1 × 10 cells/ mouse) three times (every 5 days). At the same time, these mice were treated with PBS or CAI (20 mg/kg), 1-MT (5 mg/ml in drinking water), DMF (10 mg/kg), or CAI + 1-MT, CAI + DMF or anti-PD-1 neutralizing antibody (250 μg per mouse) for 20 days. Five days later, several of the mice (n =6) were sacrificed to obtain TILs for flow cytometry, and the remaining mice continued to receive drug treatment. Anti-CD45.2 antibody was used + + to distinguish donor CD45.2 T cells from host and competitor cells. c and d Flow cytometry plots showing the expression of PD-1 in CD45.2 TILs from B16-OVA CD45.1 mice after various treatments (left: representative histogram plot; right: statistical histogram plot). e and f Tumor growth was measured (left), and long-term survival was analyzed (right). RAG1 KO mice bearing 3 × 3 mm OVA-B16 melanomas were subject to the adoptive + + 7 transfer of OVA-specific CD45.1 CD8 T cells (1 × 10 cells/mouse) every 5 days. The mice were grouped and administered treatments as indicated above. g and h Tumors were harvested after 15 days of inoculation, dissociated into single-cell suspensions, and stained for flow cytometry (n =6/ + + group). The expression of PD-1 CD45.1 TILs in B16-OVA CD45.2 mice after various treatments was analyzed by flow cytometry (left: representative histogram plot; right: statistical histogram plot). i and j Tumor growth curves of RAG1 KO mice (n = 7/group). Data are from three independent experiments, and the error bars represent the mean ± s.e.m. **p < 0.01, ***p < 0.001 by one-way ANOVA and Kaplan-Meier survival analysis Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 11 of 14 treatment (monotherapy or combined therapy) was obvi- anti-PD-1 antibody. Regarding the survival time of ously weaker than that of the same treatment in the pres- tumor-bearing mice, all treatments prolonged the life ence of transferred CTLs in RAG1 KO mice bearing B16- span of tumor-bearing mice, with the exception that OVA tumors. Both combinations (CAI and DMF and CAI CAI monotherapy provided no improvement in life span and 1-MT) had a stronger inhibitory effect on tumor in 4 T1 tumor-bearing mice. The capacity of both com- growth than either single drug, but the difference was not bined therapeutics to prolong the survival of tumor- significant until the transfer of CTLs was performed (Add- bearing mice was similar to or even better than that of itional file 4: Figure S4). The results illustrate that T cell- anti-PD-1 antibody (Fig. 6a~e). For example, the median mediated killing plays an important role in the enhanced survival time of control 4 T1 tumor-bearing mice was anti-tumor activity of the two combinations. approximately 63.5 days. CAI in combination with DMF prolonged the survival time to 81 days, which surpassed IDO1 or AhR inhibitor enhanced the effect of CAI on that resulting from treatment with the positive control xenograft tumors anti-PD-1 antibody (71.5 days) (Fig. 6e). A similar advan- Given the important roles of activated T cells in the tage in terms of prolonging survival time was also ob- tumor microenvironment, three types of xenograft served in tumor-bearing mice treated with CAI plus 1- tumor models were developed to evaluate the in vivo ef- MT. fects. As shown in Fig. 6, CAI, DMF, or 1-MT alone was able to inhibit tumor growth to a certain extent. The Discussion combination of CAI and DMF and CAI and 1-MT led to The authors have been examining the multiple pharma- a dramatic reduction in tumor growth compared with cological effects of the noncytotoxic small molecule that in the control group or either single agent-treated compound CAI, which has shown cancer-preventing, group in all three types of tumor-bearing mice models, anti-angiogenesis and cancer cachexia-fighting proper- and the anti-tumor effects were comparable to those of ties by inducing cell apoptosis, blocking calcium entry Fig. 6 | In vivo anti-tumor effects of CAI, DMF, 1-MT and the combinations of CAI + DMF and CAI + 1-MT with PD-1 antibody as a positive control 5 5 5 drug. BALB/c or C57BL/6 mice were subcutaneously injected with B16 (2 × 10 cells/mouse), C26 (1 × 10 cells/mouse) or 4 T1 (1 × 10 cells/ mouse) tumor cells. When the tumor size was 5 × 5 mm, the mice were treated with PBS, CAI, 1-MT, CAI/1-MT, and anti-PD-1 neutralizing antibody or PBS, CAI, DMF, CAI/DMF, and anti-PD-1 neutralizing antibody for 28 days. The tumor growth curves and survival curves for tumor- bearing mice (n = 10) receiving various treatments are shown as indicated. a and b C26 colorectal cancer model. c and d B16 melanoma model. e and f 4 T1 breast cancer model. The data represent the mean ± s.e.m. ***p < 0.001 by one-way ANOVA (a-f, left panels) and Kaplan-Meier survival analysis (a-f, right panels) Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 12 of 14 and inhibiting cell oxidative phosphorylation in a variety negligible effect on decreasing the established cancer of studies [22–27]. CAI also increases tumor responses burden [30]. Therefore, approaches combining IDO1 in- to other anticancer treatments [28]. However, the in vivo hibitors and other complementary compounds or im- anti-tumor activity of CAI and its performance in many mune checkpoint inhibitors would probably produce clinical trials are barely satisfactory, which prevents it synergistic benefits in terms of tumor growth and animal from being a first-line chemotherapy drug. survival. The present study has confirmed that combin- Previously, we focused on synergistically blocking ing IDO1-Kyn-AhR inhibitors with CAI can greatly aug- oncogenic signaling pathways in tumor cells and inhibit- ment the activity of CD8 T cells to enhance their ing cell proliferation with CAI and other combinatorial killing malignant cells, and the reduced expression of agents, but we neglected the potential suppressive effect PD-1 and the increase in interferon-γ production in of CAI on immune cells in the tumor microenviron- CD8 T cells both play key roles in this (Fig. 7). At the ment. Changes in tumor-derived nutrient metabolites in same time, rationally designed small-molecule combina- the local microenvironment may affect T cell prolifera- tions may also hold promise as adjunctive therapies for tion and function [29]. For instance, IDO overexpression patients with other immune suppression-related dis- in tumor cells leads to the depletion of tryptophan, eases, such as tuberculosis and HIV. which contributes to an unfavorable environment for T- Anti-PD-1 antibodies have achieved tremendous clinical cell expansion. Instead, tumor-infiltrating lymphocytes success in cancer treatment; however, a significant frac- produce IFN-γ to suppress tumor cells, and a weakened tion of patients remain unresponsive to these biologic mo- immune response plays a pivotal role in tumor initiation, dalities, including CAR-T therapy [31]. Regulating the growth and metastasis. CAI boosts interferon-γ produc- immune system through alternative pathways with small- tion by CD8 T cells, which correlates with the abun- molecule compounds may offer complementary benefits dance and activation of T cells and plays a pivotal role when used with biological immunotherapies, including in antitumor host immunity. However, interferon-γ also improved feasibility, high oral bioavailability, greater ex- induces the expression of IDO1, and this might consti- posure within the tumor microenvironment and lower tute a naturally occurring negative feedback mechanism costs [32]. The two combinations used in the present that regulates the immune response to avoid cross-reac- study were precisely the types of potent approaches with tions with normal tissues (Fig. 7). enhanced anti-tumor activity comparable to that of anti- To abolish the negative effect of CAI on CD8 T cells, PD-1 antibody that are deserving of further study. 1-MT and DMF were separately combined with CAI, and both of them independently target the upstream and Conclusion downstream effectors of the IDO-Kyn-AhR-PD-1 path- Inhibitors of the IDO1-Kyn-AhR pathway could abolish way (Fig. 7). Although IDO1 is a very important immune the potential negative effects of CAI in the tumor micro- checkpoint controller, preclinical studies have noted that environment. The combination of CAI with 1-MT or single-agent treatment with an IDO1 inhibitor has a DMF greatly augments the activity of CD8 T cells and Fig. 7 | Schematic diagram illustrating the regulation of the IDO-Kyn-AhR pathway and IFN-γ production in T cells by CAI and the proposed signal modulation mediated by T cell activation Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 13 of 14 enhances their killing of malignant cells as a result of the 1: Programmed cell death protein 1; PD-L1: Programmed cell death 1 ligand 1; TAM: Tumor-associated macrophages; Tregs: Regulatory T cells reduced expression of PD-1 and the increase in interferon- γ production. The anti-cancer capacity of the combination Acknowledgements of CAI and DMF is superior to that of either single agent None. and comparable to that of anti-PD-1 antibody, and this is Authors’ contributions also true for the combination of CAI and 1-MT. The com- LG and CY conceived the project. JS, LG, and DZ participated in the research binations of small molecules introduced in this study may design. JS, CC and RJ conducted the experiments. JS, CY, CC and RJ contributed new methodology or analytic tools. CC, JL and QW provided become effective alternate immunotherapy strategies for technical or material support. JS and LG performed the data analysis. LG and the treatment of various cancer. JS wrote the manuscript. All authors read and approved the final manuscript. Funding Additional files 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. Additional file 1: Figure S1 | Safety evaluation of drugs. B16 tumor- bearing mice (n = 6 in every group) received the indicated drugs for 21 days Availability of data and materials after the day the tumors reached 5 mm in diameter. (A~E) All mice were All data are available in this article and the supplementary information files. sacrificed to detect the levels of blood aminotransferase (ALT), aminotransferase (AST), ALT/AST, urea nitrogen and serum creatinine (Cr E). (F) The tissues shown in the figure were subject to routine HE staining and Ethics approval and consent to participate morphological examination and were observed by a microscope. (DOCX 1420 All animal studies and procedures were approved by the Institutional Animal kb) Care and Use Committee of Peking Union Medical College (registration number: ACUC-A02–2017-013). Additional file 2: Figure S2 | CAI enhanced the anti-tumor activity of CTLs and promoted IFN-γ production. (A) B16 tumor cells and CTLs were Consent for publication cocultured at a ratio of 1:10 or 1:20 for 24 h. The CTLs were preactivated Not applicable with anti-CD3/CD28 beads in the presence or absence of CAI (10 μM) for 48 h. Tumor cell apoptosis was determined by flow cytometry (left quadrantal Competing interests diagram), and the tumor cell viability after coculture with CTL is shown in the The authors declare that they have no competing interests. bar chart. CM: culture medium. (B) HCT116 cells were individually cultured or cocultured with anti-CD3/CD28 bead-activated CTLs at a ratio of 1:10 or 1:20 Received: 4 May 2019 Accepted: 30 August 2019 for 48 h. Then, the cells were treated with vehicle (DMSO) or CAI (10 mM) for 24 h. Tumor cell apoptosis was determined by flow cytometry. (C) Cytokine level changes in the cocultured cell supernatants were detected by ELISA. (D) References The interferon content in C26 tumor tissue was detected by ELISA. (DOCX 356 1. Yousefi H, Yuan J, Keshavarz-Fathi M, Murphy JF, Rezaei N. Immunotherapy kb) (DOCX 357 kb) of cancers comes of age. Expert Rev Clin Immunol. 2017;13:1001–15. Additional file 3: Figure S3 | Effects of CAI, CAI + DMF, and CAI + 1-MT 2. 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Carboxyamidotriazole combined with IDO1-Kyn-AhR pathway inhibitors profoundly enhances cancer immunotherapy

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Springer Journals
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Copyright © 2019 by The Author(s).
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Medicine & Public Health; Oncology; Immunology
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10.1186/s40425-019-0725-7
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Abstract

Background: 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. Methods: 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. Results: 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. Conclusion: 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. Keywords: CAI, PD-1, IFN-, IDO1, AhR * Correspondence: leiguo@ibms.cams.cn Department of Pharmacology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, China © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 2 of 14 Introduction In the present study, we provide evidence that the Cancer immunotherapy harnesses the immune system to effects that hamper the in vivo anti-tumor capability of fight cancer by either stimulating the functions of spe- CAI might occur through the IDO-Kyn-AhR cascade. 1- cific components of the immune system or counteract- methyl-L-tryptophan (1-MT), a tryptophan derivative ing the signals that protect tumor cells from immune that disrupts IDO1 signaling [20], or 3′,4′-dimethoxyfla- defense [1]. As one of the most important drug discover- vone (DMF), an antagonist of AhR that inhibits the Kyn- ies, specific inhibitors against programmed death 1 (PD- AhR pathway [21] were both used in combination with 1) or its main ligand PD-L1 have achieved prominent CAI. The two combinations (CAI + 1-MT and CAI + clinical success [2, 3]. PD-1 is an inhibitory receptor DMF) greatly enhanced PD-1 blockade in CD8 T cells, expressed on T cells, and PD-L1, the ligand of PD-1, is enhancing the anti-cancer capacity of the anti-PD-1 anti- upregulated by interferon γ (IFN-γ) and other cytokines body. This provides a valuable immunotherapy strategy produced after T cell activation [4]. The binding of PD- for cancer by using low-cost small molecule drug combi- L1 to PD-1 promotes T cell apoptosis, anergy, and func- nations with a favorable toxicity profiles (Additional file 1: tional exhaustion and serves as an important mechanism Figure S1). of cancer immune evasion [5]. Therefore, antibodies that block PD-1 or PD-L1 provide a new benchmark for can- Materials and methods cer immunotherapy, leading the way for developing new Cell lines and reagents immunotherapeutic approaches [6]. Mouse tumor cell lines B16 (melanoma), OVA-B16 Carboxyamidotriazole (CAI) exposure has been dem- (melanoma), C26 (colon cancer) and 4 T1 (breast cancer) onstrated to inhibit the growth of a variety of cancer cell were purchased from the China Center for Type Culture lines [7–10]. Despite the disease stabilization and im- Collection (Beijing, China) and cultured in RPMI 1640 provement in performance status observed in patients (Thermo Fisher, MA, USA) with 10% fetal bovine serum with refractory cancers after CAI treatment [8, 11], CAI (FBS) (Gibco, MA, USA), with the exception of 4 T1 cells, has failed to provide clinical benefit or improvement which were grown in DMEM medium (Gibco, MA, USA) when used with other therapeutic modalities [12, 13]. with 10% FBS. Previously, we found that CAI results in anti-inflammatory Carboxyamidotriazole was synthesized by the Institute activity in addition to its anti-tumor effect and is capable of of Materia Medica, Chinese Academy of Medical Sciences regulating the secretion of a variety of cytokines [14, 15]. (Beijing, China). Polyethylene glycol 400 (PEG400) was Recently, we focused on the increased level of IFN-γ pro- obtained from Sinopharm Chemical Reagent Beijing duction in T cells after CAI treatment. IFN-γ is a multipo- (Beijing, China). 1-Methyl-L-tryptophan, 3′,4′-dimethox- tent cytokine with cytostatic/cytotoxic activity during the yflavone and L-kynurenine sulfate salt were purchased cell-mediated adaptive immune response, which is pro- from Sigma-Aldrich (Saint Louis, USA). duced mainly by cytotoxic T lymphocytes (CTLs) and NK cells. IFN-γ has also been reported to upregulate immuno- CD8 T cell sorting suppressive molecules such as PD-L1 and IDO1, thus CD8 T cells were isolated from the spleens of BALB/c promoting tumor immune escape [4, 16]. Considering its mice using a negative magnetic cell separation kit (MACS, IFN-γ-stimulating effects, CAI might play a unique role in Mouse Naive CD8 T Cell Isolation Kit, Miltenyi Biotec). anti-tumor immunity. We speculate that the mild anti-can- The cell purity (above 95%) was confirmed by flow cytome- cer effects of CAI might be due to some adverse factors try using an anti-mouse CD8 antibody (eBioscience, CA, that can impair its action. A prototypical integrative modi- USA). The isolated CD8 T cells were cultured in RPMI fier, indoleamine 2,3-dioxygenase-1 (IDO1), which bridges 1640 medium containing 10% FBS and 10 ng/ml IL-2 inflammation, vascularization, and immune escape and can (Peprotech, NJ, USA) and activated with 1 mg/ml anti- be enhanced by IFN-γ, has become our research focus. mouse CD3/CD28 microbeads (Thermo Fisher, MA, USA). IDO1 is the initial rate-limiting enzyme in tryptophan Human naive CD8 T cells were isolated from human (Trp) catabolism within the Kyn pathway. The overex- peripheral blood monocytes (PBMCs). Briefly, human pression of IDO1 may lead to tryptophan depletion and blood samples were collected from 12 healthy donors, metabolite (Kyn, kynurenic acid, 3-hydroxy-kynurenine, and then the samples were subjected to density gradient etc.) accumulation, which can actively suppress T-cell centrifugation to obtain the PBMCs. CD8 T cells were functioning [17]. In addition, Kyn and Kyn derivatives isolated using positive MACS (Human Naive CD8 T can bind the aryl hydrocarbon receptor (AhR) [18], Cell Isolation Kit, Miltenyi Biotec). The cell purity was which has been shown to impair the proliferation and confirmed with the same method described above, and function of various immune effectors, including CD8 T the same cell culture conditions were used, except that lymphocytes, and provide tumor cells with a means to the antibody, recombinant protein and microbeads used evade anticancer immunosurveillance [19]. were human-specific. Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 3 of 14 Animal experiments and treatment protocol Cytokine release and Kyn production C57BL/6, BALB/c or RAG1 KO mice were subcutane- Cytokine production in the supernatants was quantified ously injected with appropriate amounts of the indicated by ELISA kits (BioVision, CA, USA) according to the tumor cells (B16, C26, 4 T1 or B16-OVA) in the right manufacturer’s protocol. Kyn production was measured flank. Three days after inoculation or after the tumor by ELISA (MYBioSource, CA, USA) according to the size reached 5 × 5 mm, the mice were randomized into manufacturer’s instructions. different groups (n = 6~10). Then, the mice in each group were treated with the following drugs separately Immunofluorescence for the indicated time: CAI (intragastric injection of Cells cultured in the soft 90-Pa 3D fibrin gels were 20 mg/kg/day), anti-IFN-γ neutralizing antibody (250 mg treated with dispase II (Roche, Swiss) for 10 min at 37 °C per mouse),1-MT (5 mg/ml in drinking water, 3–4ml/ and then fixed with 4% paraformaldehyde, collected, em- mouse/day), CAI + 1-MT (the same as that used for bedded in paraffin, and sectioned. The sections were monotherapy), DMF (intragastric injection of 10 mg/kg baked for 30 min at 60 °C, dewaxed, blocked in 2% BSA once every 2 days), CAI + DMF (the same as that used and stained with anti-AhR primary antibodies (Abcam, for monotherapy), and anti-PD-1 neutralizing antibody UK), followed by staining with Alexa 488-conjugated (250 μg per mouse). Kyn was administered by intratu- donkey anti-rabbit IgG secondary antibodies (Invitrogen, moral injection (20 mg/kg/day once every 2 days). The CA, USA). After 4,6-diamidino-2-phenylindole (DAPI) mice in the control group received an equal volume of staining, the slides were mounted in Fluoromount G saline as a mock treatment. Tumor growth and the sur- (Solarbio, Beijing, China) and stored at 4 °C in the dark. vival of the mice were recorded daily. The tumor vol- Images were collected by confocal microscopy. ume was calculated according to the following formula: tumor volume = length×width /2. Preparation of single-cell suspensions from implanted mouse tumors Mice were killed at specific time points. The tumors Total RNA extraction and RT–PCR were dissected, washed in PBS, digested with IV collage- Total RNA was isolated from CD8 T cells using a Pure nase (Sigma, St. Louis, USA), and then transferred to RNA Extraction Kit (BioTeke Corporation) and reverse- RPMI 1640 medium (Gibco, USA) supplemented with transcribed into cDNA with the TransScript First-Strand 10% FBS and incubated at 37 °C for 1 h. Then, the cDNA Synthesis Supermix (TransGen Biotech Co., digested tumor tissues were dispersed into ground glass, Beijing, China). The primer sequences used were: IDO1, and the tissue suspensions were filtered through a 40 μm 5′-TGGCGTATGTGTGGAACCG-3′ (sense) and 5′- mesh (BD Falcon, CA, USA). Red blood cell lysis buffer CTCGCAGTAGGGAACAGCAA-3′ (anti-sense); GAP (eBioscience, CA, USA) was added and incubated with DH, 5′AGGTCGGTGTGAACGGATTTG-3′ (sense) and the samples for 5 min at room temperature. The cells 5′-TGTAGACCATGTAGTTGAGGTCA-3′ (anti-sense). were washed three times with PBS and then resuspended Real-time PCR was performed using an IQ5 Real-Time in PBS for experiments. System (BioRad, CA, USA). The values are the mean ± SEM of three independent experiments. Flow cytometry For the flow cytometry analysis, CD8 T cells were stained with APC-conjugated anti-mouse PD-1 Ab and Western blotting FITC-conjugated anti-mouse IFN-γ Ab (eBioscience, CD8 T cell lysate containing 40 μg of protein was sub- CA, USA). To evaluate the tumor-infiltrating lympho- jected to SDS/PAGE, and the separated proteins were cytes (TILs), a single-cell suspension from the implanted transferred onto PVDF membranes. After being blocked tumors was stained with the following Abs: APC-conju- with 5% nonfat dry milk in Tris-buffered saline containing gated anti-mouse CD3, PE-conjugated anti-mouse CD8 Tween-20, the membrane was incubated with the follow- and FITC-conjugated anti-mouse CD4 (eBioscience, CA, ing primary antibodies overnight: anti-mouse IDO1 (Cell USA). Flow cytometry was performed on a BD Accuri C6 Signaling, Cat No. 86630; 1:1000), anti-mouse β-actin flow cytometer (BD Bioscience) and analyzed with BD (Cell Signaling, Cat No. 3700; 1:1000). Subsequently, the Accuri C6 software. membrane was incubated with the appropriate secondary antibody, and the immunoreactive protein bands were ChIP-qPCR assay visualized using a chemiluminescence kit (Millipore, In brief, complete CD8 T cells for the ChIP assays were MA, USA) followed by ECL-based autoradiography. prepared according to the instructions for the ChIP-IT® The Western blots are representative of at least three Express Chromatin Immunoprecipitation Kit (Active independent experiments. Motif, CA, USA). Every group included 5 × 10 cells. Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 4 of 14 Anti-mouse AhR antibody was used for chromatin im- enhancement of the anti-tumor activity of CTLs by munoprecipitation (Cell Signaling, Cat No. 83200; 1:50). cotreatment with CAI was also observed when CTLs were Control rabbit IgG was purchased from Cell Signaling. cocultured with other types of tumor cells (Additional DNA was isolated and subjected to real-time PCR ana- file 2: Figure S2B). Given that cytokines play critical lysis. The following primers were used for promoter roles in the proper establishment of anti-tumor im- quantification: mouse PD-1 AhR 5′-GATGTGCTGA munity, we examined the levels of IFN-γ,IL-6 and IL-2 CAGCCTGCTG-3′ (sense) and 5′-ATGCTCAGGG in both murine- and human-derived CD8 T cells and TAGCAAGACCC-3′ (anti-sense). All sequences were in tumor cell coculture systems. IFN-γ production by CTLs designed to produce amplicons that were < 200 bp. Real- was greatly enhanced by CAI (Fig. 1b and Additional file 2: time PCR amplification was carried out, and the amplifi- Figure S2C). To gain further insight into the involvement cation of each target gene is shown in terms of the fold of IFN-γ,weadded IFN-γ neutralizing antibody to CAI- enrichment compared to that of the relevant antibody processed cocultured CTLs and B16 cells. The neutralizing control. antibody significantly counteracted the CAI-induced en- hancement of the cytotoxicity of CTLs (Fig. 1c). In addition, Adoptive T-cell transfer CAI could also promote IFN-γ release from activated C57BL/6 J CD45.1 mice were injected subcutaneously in spleen lymphocytes and TILs in tumor-bearing mice the abdomen with 1 × 10 B16-OVA tumor cells per (Fig. 1d, e, Additional file 2:FigureS2D), suggesting mouse. When the tumor size reached 5 mm in diameter, that there was a common phenomenon in terms of the the mice were divided randomly and received one of the effect of CAI on T cells. In B16 melanoma-bearing following treatments: vehicle, CTL (1 × 10 cells/mouse mice, CAI treatment could definitely delay tumor once every five days three times), CTL + CAI (intratumoral growth; however, the concurrent injection of anti-IFN- injection, 20 mg/kg/day once every 2 days), CTL + DMF γ antibody and CAI not only eliminated the beneficial (intragastric injection, 10 mg/kg once every 2 days) or effect of CAI but also promoted tumor growth (Fig. 1f). CTL + 1-MT (5 mg/ml in drinking water, 3–4ml/mouse/ These data suggest that in an environment where day), or CTL +CAI +DMF, CTL +CAI +1-MT, or anti- tumor cells and CD8 T cells coexist, the enhancement PD-1 neutralizing antibody (250 μg per mouse). CD45.2 of tumor anti-immunity by CAI is closely related to the CTLs were isolated from the spleens of OT-1 mice and cul- release of IFN-γ. tured with OVA peptide for 48 h. For some experiments, the mice were sacrificed 5 days after adoptive T-cell transfer CAI stimulates IDO-Kyn metabolic circuitry and masks the to obtain the TILs. underlying deficits of T cells via mechanisms involving Kyn-AhR activation Statistical analysis To determine the reason why CAI has a weaker anti- Data are presented as the mean ± SEM, and n represents cancer effect in vivo than expected, we tested the meta- the number of experiments or animals. The statistical bolic status of tryptophan (Trp). Intriguingly, increased significance of the differences between two groups was Kyn concentrations were found in the supernatant of determined by Student’s t test or one-way ANOVA B16 cells cocultured with CD8 T cells and in B16 followed by Dunnett’s t-test. All statistical analyses were tumor tissues from mice treated with CAI (Fig. 2a). 1-MT performed by using GraphPad Prism 6.0 software. P- reduced Kyn basal levels and CAI-induced Kyn produc- values < 0.05 were considered statistically significant. tion (Fig. 2a). CAI strongly induced mRNA and protein expression of a key Try-metabolizing enzyme, IDO1, in Results both CTLs and B16 tumor tissues (Fig. 2b, c). Kyn can CAI improves the cell killing capability of CD8 T cells by combine with aryl hydrocarbon receptor (AhR) to regulate increasing IFN-γ levels the expression of many genes. Here, the confocal data in- In this study, B16 melanoma tumor cells and CTLs were dicated that Kyn exposure increased the nuclear import of cocultured in the presence or absence of CAI for 24 h. AhR in CD8 T cells and that this effect was blocked by CTLs exposed to CAI showed stronger cytotoxic activity 3′,4′-dimethoxyflavone (DMF). To assess whether AhR against tumor cells than those not exposed to CAI, and could increase the expression of PD-1, ChIP-qPCR was the tumor-killing capacity was T cell number-dependent performed in CD8 T cells. Our data show that the AhR- + + (Fig. 1a). Furthermore, when CD8 T cells were pre- dependent expression of PD-1 in activated CD8 Tcells treated with CAI for 48 h and then cocultured with in the presence of Kyn tremendously enhanced the activity tumor cells, the cytotoxicity of the CD8 T cells was of the PD-1 transcriptional program (Fig. 2e). The number + + + similar to that of CD8 T cells exposed to CAI during of PD-1 CD8 T cells tended to increase over the time cell coculture, indicating that CAI might promote CTL during Kyn treatment. In addition, the combined use of activity directly (Additional file 2: Figure S2A). The Kyn and DMF resulted in a slight decline but did not Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 5 of 14 Fig. 1 | CAI improves the cytotoxicity of CD8 T cells and increases IFN-γ production. a B16 tumor cells and CTLs were cocultured at a ratio of 1:10 or 1:20 in the presence or absence of CAI (10 μM) for 24 h. The CTLs were preactivated with anti-CD3/CD28 beads for 48 h. The proportion of tumor cell apoptosis was determined by flow cytometry (quadrantal diagram), and the survival rate of the tumor cells in each group is shown in the bar chart. CM: culture medium (b) Contents of the cytokines in the supernatants of cocultured cells. c B16 cells were cocultured with activated CTLs at a ratio of 1:20 in the presence of vehicle (DMSO), CAI (10 μM) or IFN-γ antibody (10 mg/mL) for 24 h. The quadrantal diagrams show the proportions of tumor cell apoptosis, and the bar chart shows the survival rate of the tumor cells in each group. d, e and f) Mice were s.c. injected with 2 × 10 B16 (n = 10 per group). When the average tumor size reached approximately 3 × 3 mm, the following treatments were initiated: PBS or CAI (20 mg/kg) or a combination of CAI and anti-IFN-γ antibody (250 mg/day) every 2 days for 23 days. d IFN-γ production in TILs and spleen was analyzed by flow cytometry. e Interferon content in tumor tissue was detected by ELISA. f Tumor growth curves. The data represent the mean ± s.e.m. N.S., no significant difference; **p < 0.01, ***p < 0.001 by Student’s t test (a, b, d and e) or one-way ANOVA (c and f) Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 6 of 14 Fig. 2 | CAI stimulation of the IDO-Kyn metabolic circuitry and the effects of the metabolite Kyn on CD8 T cells. After CAI treatment (10 μM, 48 h) (a), the production of Kyn in the B16/T cell coculture system (left) and B16 tumor tissues (right) were determined. b and c The mRNA and protein expression of IDO1 determined by RT-PCR and Western blotting. d CTLs were treated with 200 mM Kyn for 2 days. The transfer of AhR from the cytosol to the nucleus determined by immunostaining assay. Bar, 2 μm. e ChIP-qPCR analysis of AhR-dependent PD-1 expression after Kyn treatment. The ChIP enrichment ratio relative to the control is shown. f CTLs were incubated with vehicle (DMSO), Kyn (200 mM) or DMF + + (20 μM) alone or a combination of Kyn and DMF for the indicated time spans, and the PD-1 CD8 T cells were analyzed by flow cytometry. Representative histograms (left) and the overall results (right) are shown. g B16 tumor-bearing mice received an intratumoral injection of Kyn + + with or without DMF treatment (10 mg/kg). Tumor-infiltrating lymphocytes (TILs) were then isolated from the tumor tissues, and the PD-1 CD8 T cells were analyzed by flow cytometry. Representative histogram (left) and the statistical histogram (right) are shown. h Intratumoral injection of Kyn reduced the proportion of IFN-γ-positive T cells in TILs isolated from B16 tumor tissues, and DMF treatment (10 mg/kg) rescued this inhibition. Representative histograms (left) and the statistical histograms (right) are shown. Data are from three independent experiments, and the error bars represent the mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA (a, g, f and h) or Student’s t test (b and e) Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 7 of 14 + + counterbalance the percentage of PD-1 CD8 Tcells were subcutaneously injected with tumor cells (C26, 4 (Fig. 2f). To further confirm the role of Kyn in the T1 or B16 cells, n = 6 in every group). The mice began tumor microenvironment, mice bearing tumors re- to receive drug treatment when the mean size of the tu- ceived intratumoral injections of Kyn, DMF or a com- mors reached 5 mm in diameter. Cells were isolated bination of Kyn and DMF. Similar to the above results, from solid tumor tissues and assayed with flow + + Kyn markedly increased the percentage of PD-1 CD8 cytometry. The combination of CAI and DMF showed T cells in TILs and inhibited the production of IFN-γ, consistent synergistic effects in the 3 tumor-bearing ani- while DMF showed a partial offsetting effect, which mal models, which elevated the percentage of CD8 T means that the excess production of Kyn may cause T cells in TILs in all 3 types of tumor tissues five- to eight- cell exhaustion and impair the immune surveillance fold compared with that in the control group (Fig. 4a, b). function of CD8 T cells in the tumor microenviron- Meanwhile, CAI, DMF or a combination significantly + + ment. These results also suggest that the CAI activation reduced the expression of PD-1 in CD8 T cells in the of the IDO-Kyn-AhR cascade might be the underlying combination group, demonstrating the strengthened in- mechanism that limits the anti-tumor efficacy of CAI. hibitory effect in comparison with that in both mono- therapy groups (Fig. 4c). When an alternative inhibitor Combining CAI with 1-MT or DMF synergistically disrupts of IDO1, 1-MT, was used instead of DMF in subsequent PD-1 expression and promotes IFN-γ production in CD8 independent experiments with the same types of tumor- T cells bearing mice, the abovementioned indicators showed To determine whether an IDO1 inhibitor or an AhR in- variations consistent with those observed in previous hibitor could improve the effects of CAI on T cells, both studies (involving DMF) for each monotherapy group mouse spleen-derived CD8 T cells and human PBMC- and combination group in all 3 types of tumor tissues derived CD8 T cells were treated with CAI, DMF, 1- (Fig. 4d~f). It was speculated that the augmentation of MT alone or a combination of CAI and DMF or 1-MT PD-1 blockade might promote CD8 T cell survival and for 48 h. There was a statistically significant difference enhance the cytotoxic activity of TILs in the tumor between the monotherapy group and the control group. microenvironment. In addition, the effects of the indi- However, the two combinations drastically decreased cated single drugs or combination drugs on other cell PD-1 expression and elevated IFN-γ production in CD8 types in the tumor microenvironment were comprehen- T cells (Fig. 3a~e). As for human PBMC-derived CD8 sively analyzed by flow cytometry. There were no signifi- T cells, the combination of CAI with DMF or 1-MT also cant differences in the number and typical function of resulted in obvious immune enhancement, as evidenced tumor-associated macrophages (TAMs) between the dif- by the enhancement of PD-1 blockade and an increase ferent treatment groups. The same was true for other in IFN-γ production (Fig. 3d, e). The ChIP-qPCR data cell types, including myeloid-derived suppressor cells showed that CAI facilitated the binding of AhR to the (MDSCs), regulatory T cells (Tregs) and CD4 T cells. It promoter of the PD-1 gene and caused an approximately is worth mentioning that the downregulation of PD-1 in 2.07-fold increase in PD-1 expression, while combining the combination groups was very obvious in CD8 T CAI with DMF or 1-MT obviously reduced the overex- cells but not in CD4 T cells (Additional file 3: Figure pression of PD-1 caused by AhR activation (Fig. 3h). S3). The results indicate that the enhanced anti-tumor Correspondingly, the immunostaining data showed that activity of the two combinations of drugs was mainly the nuclear translocation of AhR was significantly inhib- due to enhanced CD8 T cell function and number. ited by the combination of CAI with DMF or 1-MT. (Fig. 3i). The results suggest that the dampening effect Combining CAI with IDO1/AhR inhibitors affected the of CAI on T cells arising from the activation of the phenotype and function of transferred T cells in B16-OVA IDO/AhR axis could be overcome by combining CAI mice and showed beneficial anti-cancer effects with an IDO1/AhR inhibitor and that this combination Combining CAI with IDO1/AhR inhibitors could lead to might play a distinct role in promoting the antitumor a more selective anti-tumor immunoreaction, which was immunity of CD8 T cells. confirmed in a specialized coculture system consisting of B16 melanoma cells expressing ovalbumin (OVA) anti- Combining CAI with DMF or 1-MT increased the number gen (B16-OVA) and OVA-specific CTLs derived from of cytotoxic CD8 tumor-infiltrating T cells and OT-1 transgenic mice. Either combination resulted in downregulated PD-1 expression the lowest survival rate of B16-OVA cells in parallel ex- To study the effects of CAI, DMF, 1-MT and the two periments with single agents. (Fig. 5a, b). To further assess combinations (CAI with DMF/CAI or 1-MT) on T cells the immunotherapeutic effects of the two combinations within the tumor microenvironment, three tumor-bear- on T cells, adoptive cell transfer (ACT) was conducted. In + + ing mice models were used. BALB/c or C57BL/6 mice terms of the proportion of PD-1 CD45.2 TILs in B16- Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 8 of 14 + + Fig. 3 | Combining CAI with 1-MT or DMF synergistically disrupts PD-1 expression and promotes IFN-γ production in CD8 T cells. Sorted CD8 T cells were activated by CD3/CD28 beads for 48 h and treated with CAI (10 μM), DMF (20 μM) or a combination of CAI and DMF for 24 h. In another experiment, the sorted CD8 T cells were treated with CAI (10 μM), 1-MT (0.2 mM) or a combination of CAI and 1-MT for 24 h. a-b Flow + + cytometry plots showing the number of PD-1 CD8 T cells after various treatments. Representative histogram plots (left) and the statistical histogram plots (right) are shown. c The percentage of IFN-γ-producing T cells in each group was measured by flow cytometry (n = 3). d and e Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of 10 healthy volunteers, and PBMC-derived CD8 T cells were incubated with the indicated single drug or a combination of drugs for 48 h. The percentages of PD-1-positive cells and IFN-γ-producing cells in each group were measured by flow cytometry. f and g Activated CD8 T cells from the spleens of C57BL/6 mice were cocultured with B16 cells, or PBMC-derived CD8 T cells were cocultured with HCT116 cells at a ratio of 20:1 for 24 h. Tumor cell apoptosis was analyzed by flow cytometry. h ChIP-qPCR analysis of AhR-dependent PD-1 expression after various treatments. The ChIP enrichment ratio relative to the control is shown. i CD8 T cells isolated from mouse spleens were activated with anti-CD3/CD28 beads for 48 h. At the same time, activated T cells were treated with CAI (10 μM), DMF (20 μM), 1-MT (0.2 mM) or a combination of CAI and DMF/1-MT for 24 h. Then, the CD8 T cells were fixed and strained with an anti-AhR antibody and imaged by confocal microscopy. Bar, 2 μm. Data are from three independent experiments, and the error bars represent the mean ± s.e.m. **p < 0.01, ***p < 0.001 by one-way ANOVA (A, B, E-H) Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 9 of 14 Fig. 4 | Combining CAI with DMF or 1-MT increased the number of tumor-infiltrating CD8 T cells and downregulated PD-1 expression. BALB/c or C57BL/6 mice were subcutaneously injected with 1 × 10 tumor cells (C26 cells, B16 cells or 4 T1 cells, n = 6 in every group) and received the indicated drugs for 7 days after the day the tumor size reached 5 mm in diameter. T cells in the tumor microenvironment were sorted and + + analyzed by flow cytometry. a Representative flow cytometry plots showing the fraction of CD8 T cells within the CD3 TILs in the C26, B16- or + + + 4 T1- tumor microenvironment. b CD8 T cell numbers per gram of tumor in different groups. c The percentage of PD-1 CD8 T cells within TILs in the tumor microenvironment. d, e and f The same measurements from the evaluation of the effects of another drug combination (CAI and 1- MT) in C26-, B16- or 4 T1- tumor bearing mice. Data are from three independent experiments, and the error bars represent the mean ± s.e.m. **p < 0.01 by one-way ANOVA (a-f) OVA CD45.1 mice, monotherapy caused a slight decrease resulted in more encouraging effects that were comparable after 5 days of treatment. Noticeably, combined therapy with those of PD-1 antibody (Fig. 5e, f). We also assessed (CAI + DMF or CAI + 1-MT) resulted in a significant syn- the anti-tumor activity of the indicated therapeutics in ergistic inhibition compared with either single agent, with RAG1 KO mice bearing B16-OVA tumors. Each of the + + the proportion of PD-1 CD45.2 TILs decreasing to less three monotherapies, CAI, DMF, or 1-MT, slightly reduced than 1/3 of that in the control group (Fig. 5c, d). We then PD-1 expression on the surfaces of transferred CD45.1 T evaluated the anti-tumor effect in vivo using B16-OVA cells and showed little effect on tumor growth. In contrast, tumor-bearing mice who received T cell ACT. The com- combined therapy (CAI + DMF or CAI + 1-MT) signifi- + + bination of CAI and DMF and CAI and 1-MT obviously cantly reduced the expression of PD-1 in CD45.1 Tcells inhibited tumor growth compared with the control and clearly inhibited tumor growth (Fig. 5g~i). group or either single agent-treated group. In addition, Interestingly, in RAG1 KO mice bearing B16 tumors, prolonged treatment (> 30 days) with the combinations the tumor growth inhibition effect of the indicated Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 10 of 14 Fig. 5 | Phenotypic character of transferred CD45.2 T cells in B16-OVA mice and the in vivo anti-tumor activity of different treatments. a and b) Activated CD8 T cells from the spleens of OT-1 mice were cocultured with B16-OVA cells at a ratio of 2:1 for 5 h and 10 h and treated with CAI (10 μM), DMF (20 μM) or a combination of CAI and DMF. In another experiment, sorted CD8 T cells were treated with CAI (10 μM), 1-MT (0.2 mM) or a combination of CAI and 1-MT. B16-OVA cell apoptosis was analyzed by flow cytometry at 5 h and 10 h after drug treatment. c and d CD45.1 + + 7 C57BL/6 mice bearing 3 × 3 mm OVA-B16 melanomas were subject to the adoptive transfer of OVA-specific CD45.2 CD8 T cells (1 × 10 cells/ mouse) three times (every 5 days). At the same time, these mice were treated with PBS or CAI (20 mg/kg), 1-MT (5 mg/ml in drinking water), DMF (10 mg/kg), or CAI + 1-MT, CAI + DMF or anti-PD-1 neutralizing antibody (250 μg per mouse) for 20 days. Five days later, several of the mice (n =6) were sacrificed to obtain TILs for flow cytometry, and the remaining mice continued to receive drug treatment. Anti-CD45.2 antibody was used + + to distinguish donor CD45.2 T cells from host and competitor cells. c and d Flow cytometry plots showing the expression of PD-1 in CD45.2 TILs from B16-OVA CD45.1 mice after various treatments (left: representative histogram plot; right: statistical histogram plot). e and f Tumor growth was measured (left), and long-term survival was analyzed (right). RAG1 KO mice bearing 3 × 3 mm OVA-B16 melanomas were subject to the adoptive + + 7 transfer of OVA-specific CD45.1 CD8 T cells (1 × 10 cells/mouse) every 5 days. The mice were grouped and administered treatments as indicated above. g and h Tumors were harvested after 15 days of inoculation, dissociated into single-cell suspensions, and stained for flow cytometry (n =6/ + + group). The expression of PD-1 CD45.1 TILs in B16-OVA CD45.2 mice after various treatments was analyzed by flow cytometry (left: representative histogram plot; right: statistical histogram plot). i and j Tumor growth curves of RAG1 KO mice (n = 7/group). Data are from three independent experiments, and the error bars represent the mean ± s.e.m. **p < 0.01, ***p < 0.001 by one-way ANOVA and Kaplan-Meier survival analysis Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 11 of 14 treatment (monotherapy or combined therapy) was obvi- anti-PD-1 antibody. Regarding the survival time of ously weaker than that of the same treatment in the pres- tumor-bearing mice, all treatments prolonged the life ence of transferred CTLs in RAG1 KO mice bearing B16- span of tumor-bearing mice, with the exception that OVA tumors. Both combinations (CAI and DMF and CAI CAI monotherapy provided no improvement in life span and 1-MT) had a stronger inhibitory effect on tumor in 4 T1 tumor-bearing mice. The capacity of both com- growth than either single drug, but the difference was not bined therapeutics to prolong the survival of tumor- significant until the transfer of CTLs was performed (Add- bearing mice was similar to or even better than that of itional file 4: Figure S4). The results illustrate that T cell- anti-PD-1 antibody (Fig. 6a~e). For example, the median mediated killing plays an important role in the enhanced survival time of control 4 T1 tumor-bearing mice was anti-tumor activity of the two combinations. approximately 63.5 days. CAI in combination with DMF prolonged the survival time to 81 days, which surpassed IDO1 or AhR inhibitor enhanced the effect of CAI on that resulting from treatment with the positive control xenograft tumors anti-PD-1 antibody (71.5 days) (Fig. 6e). A similar advan- Given the important roles of activated T cells in the tage in terms of prolonging survival time was also ob- tumor microenvironment, three types of xenograft served in tumor-bearing mice treated with CAI plus 1- tumor models were developed to evaluate the in vivo ef- MT. fects. As shown in Fig. 6, CAI, DMF, or 1-MT alone was able to inhibit tumor growth to a certain extent. The Discussion combination of CAI and DMF and CAI and 1-MT led to The authors have been examining the multiple pharma- a dramatic reduction in tumor growth compared with cological effects of the noncytotoxic small molecule that in the control group or either single agent-treated compound CAI, which has shown cancer-preventing, group in all three types of tumor-bearing mice models, anti-angiogenesis and cancer cachexia-fighting proper- and the anti-tumor effects were comparable to those of ties by inducing cell apoptosis, blocking calcium entry Fig. 6 | In vivo anti-tumor effects of CAI, DMF, 1-MT and the combinations of CAI + DMF and CAI + 1-MT with PD-1 antibody as a positive control 5 5 5 drug. BALB/c or C57BL/6 mice were subcutaneously injected with B16 (2 × 10 cells/mouse), C26 (1 × 10 cells/mouse) or 4 T1 (1 × 10 cells/ mouse) tumor cells. When the tumor size was 5 × 5 mm, the mice were treated with PBS, CAI, 1-MT, CAI/1-MT, and anti-PD-1 neutralizing antibody or PBS, CAI, DMF, CAI/DMF, and anti-PD-1 neutralizing antibody for 28 days. The tumor growth curves and survival curves for tumor- bearing mice (n = 10) receiving various treatments are shown as indicated. a and b C26 colorectal cancer model. c and d B16 melanoma model. e and f 4 T1 breast cancer model. The data represent the mean ± s.e.m. ***p < 0.001 by one-way ANOVA (a-f, left panels) and Kaplan-Meier survival analysis (a-f, right panels) Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 12 of 14 and inhibiting cell oxidative phosphorylation in a variety negligible effect on decreasing the established cancer of studies [22–27]. CAI also increases tumor responses burden [30]. Therefore, approaches combining IDO1 in- to other anticancer treatments [28]. However, the in vivo hibitors and other complementary compounds or im- anti-tumor activity of CAI and its performance in many mune checkpoint inhibitors would probably produce clinical trials are barely satisfactory, which prevents it synergistic benefits in terms of tumor growth and animal from being a first-line chemotherapy drug. survival. The present study has confirmed that combin- Previously, we focused on synergistically blocking ing IDO1-Kyn-AhR inhibitors with CAI can greatly aug- oncogenic signaling pathways in tumor cells and inhibit- ment the activity of CD8 T cells to enhance their ing cell proliferation with CAI and other combinatorial killing malignant cells, and the reduced expression of agents, but we neglected the potential suppressive effect PD-1 and the increase in interferon-γ production in of CAI on immune cells in the tumor microenviron- CD8 T cells both play key roles in this (Fig. 7). At the ment. Changes in tumor-derived nutrient metabolites in same time, rationally designed small-molecule combina- the local microenvironment may affect T cell prolifera- tions may also hold promise as adjunctive therapies for tion and function [29]. For instance, IDO overexpression patients with other immune suppression-related dis- in tumor cells leads to the depletion of tryptophan, eases, such as tuberculosis and HIV. which contributes to an unfavorable environment for T- Anti-PD-1 antibodies have achieved tremendous clinical cell expansion. Instead, tumor-infiltrating lymphocytes success in cancer treatment; however, a significant frac- produce IFN-γ to suppress tumor cells, and a weakened tion of patients remain unresponsive to these biologic mo- immune response plays a pivotal role in tumor initiation, dalities, including CAR-T therapy [31]. Regulating the growth and metastasis. CAI boosts interferon-γ produc- immune system through alternative pathways with small- tion by CD8 T cells, which correlates with the abun- molecule compounds may offer complementary benefits dance and activation of T cells and plays a pivotal role when used with biological immunotherapies, including in antitumor host immunity. However, interferon-γ also improved feasibility, high oral bioavailability, greater ex- induces the expression of IDO1, and this might consti- posure within the tumor microenvironment and lower tute a naturally occurring negative feedback mechanism costs [32]. The two combinations used in the present that regulates the immune response to avoid cross-reac- study were precisely the types of potent approaches with tions with normal tissues (Fig. 7). enhanced anti-tumor activity comparable to that of anti- To abolish the negative effect of CAI on CD8 T cells, PD-1 antibody that are deserving of further study. 1-MT and DMF were separately combined with CAI, and both of them independently target the upstream and Conclusion downstream effectors of the IDO-Kyn-AhR-PD-1 path- Inhibitors of the IDO1-Kyn-AhR pathway could abolish way (Fig. 7). Although IDO1 is a very important immune the potential negative effects of CAI in the tumor micro- checkpoint controller, preclinical studies have noted that environment. The combination of CAI with 1-MT or single-agent treatment with an IDO1 inhibitor has a DMF greatly augments the activity of CD8 T cells and Fig. 7 | Schematic diagram illustrating the regulation of the IDO-Kyn-AhR pathway and IFN-γ production in T cells by CAI and the proposed signal modulation mediated by T cell activation Shi et al. Journal for ImmunoTherapy of Cancer (2019) 7:246 Page 13 of 14 enhances their killing of malignant cells as a result of the 1: Programmed cell death protein 1; PD-L1: Programmed cell death 1 ligand 1; TAM: Tumor-associated macrophages; Tregs: Regulatory T cells reduced expression of PD-1 and the increase in interferon- γ production. The anti-cancer capacity of the combination Acknowledgements of CAI and DMF is superior to that of either single agent None. and comparable to that of anti-PD-1 antibody, and this is Authors’ contributions also true for the combination of CAI and 1-MT. The com- LG and CY conceived the project. JS, LG, and DZ participated in the research binations of small molecules introduced in this study may design. JS, CC and RJ conducted the experiments. JS, CY, CC and RJ contributed new methodology or analytic tools. CC, JL and QW provided become effective alternate immunotherapy strategies for technical or material support. JS and LG performed the data analysis. LG and the treatment of various cancer. JS wrote the manuscript. All authors read and approved the final manuscript. Funding Additional files 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. Additional file 1: Figure S1 | Safety evaluation of drugs. B16 tumor- bearing mice (n = 6 in every group) received the indicated drugs for 21 days Availability of data and materials after the day the tumors reached 5 mm in diameter. (A~E) All mice were All data are available in this article and the supplementary information files. sacrificed to detect the levels of blood aminotransferase (ALT), aminotransferase (AST), ALT/AST, urea nitrogen and serum creatinine (Cr E). (F) The tissues shown in the figure were subject to routine HE staining and Ethics approval and consent to participate morphological examination and were observed by a microscope. (DOCX 1420 All animal studies and procedures were approved by the Institutional Animal kb) Care and Use Committee of Peking Union Medical College (registration number: ACUC-A02–2017-013). Additional file 2: Figure S2 | CAI enhanced the anti-tumor activity of CTLs and promoted IFN-γ production. (A) B16 tumor cells and CTLs were Consent for publication cocultured at a ratio of 1:10 or 1:20 for 24 h. The CTLs were preactivated Not applicable with anti-CD3/CD28 beads in the presence or absence of CAI (10 μM) for 48 h. Tumor cell apoptosis was determined by flow cytometry (left quadrantal Competing interests diagram), and the tumor cell viability after coculture with CTL is shown in the The authors declare that they have no competing interests. bar chart. CM: culture medium. (B) HCT116 cells were individually cultured or cocultured with anti-CD3/CD28 bead-activated CTLs at a ratio of 1:10 or 1:20 Received: 4 May 2019 Accepted: 30 August 2019 for 48 h. Then, the cells were treated with vehicle (DMSO) or CAI (10 mM) for 24 h. Tumor cell apoptosis was determined by flow cytometry. (C) Cytokine level changes in the cocultured cell supernatants were detected by ELISA. (D) References The interferon content in C26 tumor tissue was detected by ELISA. (DOCX 356 1. Yousefi H, Yuan J, Keshavarz-Fathi M, Murphy JF, Rezaei N. Immunotherapy kb) (DOCX 357 kb) of cancers comes of age. Expert Rev Clin Immunol. 2017;13:1001–15. Additional file 3: Figure S3 | Effects of CAI, CAI + DMF, and CAI + 1-MT 2. 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Published: Sep 11, 2019

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