Cell culture and chemicals
The cell lines MC38, LLC, B16F10, and EL4 (from the American type culture collection and identified by the Shanghai Yihe Biological Company) were cultured according to the supplier’s recommendations. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, HyClone, Logan, Utah) supplemented with 10% fetal bovine serum (FBS, Gibco, California, USA) and 1% penicillin/streptomycin (Gibco, California, USA). The second passage of the cells was used. All the cells were kept at 37 °C and cultured in a 5% CO2 cell incubator.
Wild-type C57BL/6 mice were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Shanghai, China). Females were 16 - 18 g and 6 - 8 weeks old. OT-I mice, CD45.1 mice, and CD45.2 mice were purchased from the Southern Model Biotechnology Co., Ltd. (Shanghai, China). Mice carrying the CD45.1 gene were mated with the OT-I mice to obtain CD45.1 OT-I double-positive mice. All mice were raised in Specific Pathogen Free (SPF) (license ID: SYXK(Shanghai)2020-0032). All the animal experiments were conducted in accordance with the U.K. Animals (Scientific Procedures) Act of 1986 and the associated guidelines, as well as the EU Directive 2010/63/EU for animal experiments. All animal studies complied with the ARRIVE guidelines.
Mouse type 17 cell differentiation
CD4+ CD25−CD62Lhigh cells were purified from C57BL/6 splenocytes using an EasySep Mouse Naïve CD4+ T cell isolation kit from STEMCELL Technologies (Vancouver, Canada), and they were differentiated into Th17 cells (TGF-β, 2 ng/ml; IL-6, 20 ng/ml; Anti-IFN-γ, 10 μg/ml; Anti-IL-4, 10 μg/ml, BioLegend, San Diego, CA) in the presence of plate-bound anti-CD3 (5 μg/ml, BioLegend, San Diego, CA) and anti-CD28 (2 μg/ml, BioLegend, San Diego, CA). The cells were harvested and processed for cytokine analysis at the RNA or protein level using real-time qPCR, flow cytometry, and ELISA on day five. Alternatively, splenocytes from OT-I mice were activated using OVA-derived peptides SIINFEKL (50 ng/ml, Sangon Biotech, Shanghai, China) and polarized to Tc17 cells using cytokine TGF-β (2 ng/ml, BioLegend, San Diego, CA) and IL-6 (20 ng/ml, BioLegend, San Diego, CA) for four or five days.
Human type 17 T cell differentiation
Human PBMCs were donated by Li Xia, who provided her written informed consent. All the cells were used in vitro only. The collection of human PBMCs was approved by the ethics committee of the Fudan Affiliated Minhang Hospital (2019-Pijian-010-01 K). Whole human blood was obtained from healthy volunteers, and peripheral blood mononuclear cells (PBMCs) were extracted from the whole blood using Ficoll (Fisher Scientific, Waltham, USA) centrifugation. CD3+ T cells purified from PBMCs were activated using anti-CD3/28 beads at a 1:1 ratio and polarized into type 17 T cells with human IL-1β (20 ng/ml, BioLegend, San Diego, CA), IL-6 (20 ng/ml, BioLegend, San Diego, CA), and IL-23 (50 ng/ml, BioLegend, San Diego, CA). After five days, the cytokine levels in the supernatant were determined using ELISA (Multisciences, Hangzhou, China). The cells were collected for flow cytometry analysis.
Ex vivo cytotoxicity assay
The EL4 cells were pulsed using 50 ng/ml OVA257-264 peptide (SIINFEKL) (Sangon Biotech, Shanghai, China) for 2 h at 37 °C and then labeled with 0.25 μM or 2.5 μM of CFSE (carboxyfluorescein succinimidyl ester; Thermo Fisher Scientific, Massachusetts, America) for 10 min at 37 °C. CFSEl°w (SIINFEKL loaded target) and CFSEhigh (irrelevant peptide control) EL4 cells were mixed at a 1:1 ratio and then co-cultured with Tc17 cells differentiated from OT-I T cells challenged (or not) with 8-074 at 30:1, 10:1, 3:1, and 1:1 effector to target cell ratios (E: T). The frequencies of the CFSEl°w and CFSEhigh EL4 cells in the CFSE positive fraction were determined using flow cytometric analysis 18 h after incubation, and the percent of the specific killing was calculated. Specific killing (%) = [1 − (Sample ratio) / (Negative control ratio)] × 100; Sample ratio = [CFSEl°w(target)/CFSEhigh(irrelevant)] value of each sample co-cultured with Tc17 cells; Negative control ratio = [CFSEl°w(target) / CFSEhigh (irrelevant)] value of EL4 cells not cultured with Tc17 cells.
Adoptive cell therapy tumor models
The B16-OVA tumor cells were implanted subcutaneously into the flank of C57BL/6 mice and allowed to grow. In parallel, splenocytes from OT-I mice were isolated and differentiated into Tc17 cells in vitro in the presence/absence of a 8-074 for five days. Once the tumor was measurable (normally between days seven and ten post-implant), the expanded T cells were injected intravenously. Antitumor responses were measured by assessing the tumor volume over time. The tumor volume was assessed once every two days using caliper measurement of the length and width of the tumor. The tumor volumes based on the caliper measurements were calculated using the modified ellipsoidal equation, where the tumor volume = 1/2 (length × width 2) . Mice were euthanized after the tumor volume reached three ethical endpoints of 2,000 mm.
All experiments were approved by the IACUC and performed with strict adherence to a series of documents and standards of procedures (SOPs) relative to animal ethics and welfare. The mice were housed in cages with controlled temperature (25 ± 2 °C) and humidity (65 ± 5%) under a 12 h light/dark cycle. After a one-week adaptation period, six to eight-week-old female mice were injected s.c. with LLC (5 × 105), B16F10 (2 × 105), or MC38 (2 × 106) cells into the lower right flank. Approximately seven days after the subcutaneous injection of tumor cells, the mice were randomly divided into four groups. RORγt agonist 8-074, LYC-55716 (BioChemPartner, Shanghai, China) and anti-PD-1 (BioXCell, New Hampshire, USA) treatment commenced when the average tumor size reached 50 mm3 for LLC and B16F10 and 150 mm3 for MC38. The 5 × 105 LLC cells were transplanted subcutaneously into the right flank of the C57BL/6 mice seven days after being transplanted, and the mice were randomly divided into four groups. Anti-PD-1 was administrated one day after the RORγt agonist was treated (Clone: PMP1-14; 200 μg via intraperitoneal injection on day 1, 4, 7, 10, 13 after treatment with 8-074). For the CD8+ T cell depletion, mice were injected intraperitoneally (i.p.) with 400 μg of anti-CD8α (YTS 169.4; BioXCell, New Hampshire, USA) one day before and dosed per week after the anti-PD-1 treatment. Mice with established tumors were treated using intraperitoneal injection of 8-074 (indicated dose), LYC-55716 (50 mg/kg) or DMSO (SIGMA, New Jersey, USA) every day. Anti-PD-1 was dosed at 10 mg/kg every three days by intraperitoneal injection. The tumor volume based on the caliper measurements was calculated using the modified ellipsoidal equation, where the tumor volume = 1/2 (length × width 2) and the length was the longer dimension . Two weeks after the 8-074 administration, the mice were sacrificed, and solid tumors were separated and photographed. TGI was calculated using the equation: [(Ct – C0) – (Tt – T0)] / (Ct – C0) × 100, where Ct = the mean tumor volume of the control group at the time (t); C0 = the mean tumor volume of the control group at t0; Tt = mean tumor volume of the treatment group at t; and T0 = mean tumor volume of the treatment group at t0.
Tumors were harvested and cut into small pieces after removing connective tissue and tissue stroma. To obtain a single-cell tumor suspension, the small tumor pieces were incubated in an enzyme mixture of collagenase A (2 mg/ml, SIGMA, New Jersey, USA) and DNase-I (1 mg/ml, Roche, Basel, Switzerland) in an incomplete RPMI medium (Hyclone, Logan, Utah) for 30-60 min at 37 °C on a rocking platform. After digestion, the single-cell suspension was obtained by passing the digested tissue through a 40 μm nylon mesh. The resultant cells were washed twice in phosphate buffer solution (PBS) before staining for flow cytometry.
Cells were stained with fluorochrome-labeled anti-mouse Ab such as CD45, CD3, CD4, CD8, Foxp3, IFN-γ, IL-17A, CD11b, CD11c, pAKT, pSTAT3, CCR6, or MHCII. For intracellular cytokine staining, single-cell suspensions from the tumor and TDLNs were stimulated using a cell stimulation cocktail (eBioscience, San Diego, California, USA, 500X used at 1X) consisting of PMA (40.5 μM, Cayman, Ann Arbor, Michigan, USA), ionomycin (670 μM, BioVision, San Francisco, USA), and protein transport inhibitors-brefeldin A (5.3 mM, Thermo, Massachusetts, America) and monensin (1 mM, Thermo, Massachusetts, America) for 6 h at 37 °C and 5% CO2. After 6 h, the cells were harvested and washed, surface stained with CD45, CD3, CD4, CD8, CD11b, CD11c, CCR6, and MHCII (FACS Buffer, Thermo, Massachusetts, America), fixed, permeabilized (IC fixation and Permeabilization buffer, Thermo, Massachusetts, America), and stained for pAKT, pSTAT3, IFN-γ, and IL-17A (Thermo, Massachusetts, America). Isotype controls with the same fluorochrome were used as controls. Cells were acquired using the FACS Aria II machine and analyzed using FlowJo software.
Measurement of cytokines by ELISA and real-time PCR
The intracellular cytokines by TILs or in vitro differentiated T helper cells were quantified after restimulation with PMA plus ionomycin in the presence of GolgiStop for 6 h. The total RNA was isolated using the improved TRizol-based (Sigma, Darmstadt, Germany) method for qPCR analysis, and the mRNA expression was analyzed using a StepOnePlus (Life Technologies, Carlsbad, USA) real-time PCR instrument using housekeeping gene β-actin and Gapdh internal standards. qPCR was performed using A Power SYBR Green PCR Master Mix (Accurate Biology, Hunan, China) and two-cycle amplification for 40 cycles followed by the melting curve. The sequence of primers is listed in the Additional file 6: Table S1. In addition, the cytokines were quantified in cell-free culture supernatants using enzymelinked immunosorbent assay (ELISA) kit (the optical density (OD) value was measured at 450 nm, using 570 nm or 630 nm as the reference wavelengths, Multisciences, Hangzhou, China). The kit was used according to the manufacturer’s instructions .
In vitro differentiation of the Mo-DC cells
Fluorescence staining panel for cell sorting of Mo-DC was assessed using flow cytometry. The Pan-DC (CD45+CD11c+) were enriched from C57BL/6 splenocyte lymphocytes with the EasySep™ Mouse Pan-DC Enrichment Kit from STEMCELL Technologies (Vancouver, Canada), then the cells were stained with CD45, CD11c, MHCII, CD11b, and Ly6c to obtain Mo-DC (CD45+CD11c+MHCII+CD11b+Ly6c+) through FACS.
Transwells with a 5-μm pore size (Costar, Corning, New York State, USA) were placed in a 24-well plate with 500 μl IMDM in the bottom chamber. 1) Different concentrations of recombinant murine CCL20 (PeproTech, Rocky Hill, USA) or 1 mg/ml neutralizing rat anti-CCL20 mAb (R&D Systems, Minn., USA) were added to the lower wells, and type 17 T cells were added to the upper wells. T cells were allowed to migrate through the Transwell membrane for 3 h at 37 °C. The migrated cells were then counted. 2) Different concentrations of recombinant murine CXCL10 (PeproTech, Rocky Hill, USA) or 1 mg/ml neutralizing rat anti-CXCL10 mAb (R&D Systems, Minn., USA) were added to the lower wells, and CD8+ T cells were added to the upper wells. The T cells were allowed to migrate through the Transwell membrane for 3 h and 6 h at 37 °C. The migrated cells were then counted. 3) Transwells with a 5 μm pore size (Costar, Corning, New York State, USA) were placed in a 24-well plate with 500 μL IMDM in the bottom chamber. 1 × 105 sorted Mo-DC cells were added in the upper chamber. The lower chamber contained medium alone (-), or medium with different concentrations of recombinant CCL20 with/without neutralizing anti-(α) CCL20 mAb and neutralizing anti-CCR6 mAb. Plates were incubated for 6 h at 37 °C in 5% CO2, and the migrated Mo-DC were counted. 4) Different concentrations of cell culture supernatant of the Th17 cells after treatments or 1 mg/ml of neutralizing rat anti-CCL20 mAb were added to the lower wells, and the Mo-DC cells were added to the upper wells. The T cells were allowed to migrate through the Transwell membrane for 3 h and 6 h at 37 °C. The migrated cells were then counted.
Male C57BL/6 mice were divided into two groups: 8-074 single intravenous injection group (2 mg/kg, n = 3) and 8-074 single gavage group (5 mg/kg, n = 3). After administration, blood samples were collected at 0.25, 0.5, 1, 2, 5, 7, and 24 h. Then the plasma samples were separated and stored at − 80 °C until the analysis. After being thawed at room temperature, 10 μL of plasma samples were added with 150 μL of precipitant containing the internal standard (verapamil 40 ng/mL) for the protein precipitation. The supernatant was mixed with a suitable volume of water and then analyzed using liquid chromatography in tandem with mass spectrometry (LC-MS/MS). The concentration of 8–074 in the plasma of the C57 mice after administration was determined using the inter-run standard curve samples (linear range of 3–10,000 ng/mL) and quality control samples.
Human nuclear receptor RORγt (residues 263–509)-GGG-SRC1 (SRC1 sequence: EKHKILHRLLQDS, Sangon Biotech, Shanghai). RORγt LBD was cloned in pET28a. Key residue mutations of RORγt LBD (such as PHE388, LEU391, CYS393, LEU396, ILE397, ILE400, CYS320, ALA321, LEU324, MET358, and PHE388) with LYC-55716 were cloned in pET28a. Key residue mutations of RORγt LBD (MET365, ALA368, PHE401, ILE400, ILE397, LEU396, TRP317, CYS320, LEU324, ALA327, TYR330, VAL331, MET358, and VAL361) with 8-074 were cloned in pET28a. The proteins were expressed in E. coli strain DE3. Then, the transformed E. coli culture was grown at 37 °C with 30 μg/mL Kanamycin LB (Luria–Bertani). When the OD600 of LB medium reached 0.6, the temperature was changed to 16 °C, and isopropyl-β-D-thiogalactopyranoside (IPTG, Beyotime, Shanghai, China) was added at a final concentration of 0.4 mM to induce protein expression for 16 h. After 16 h, the pellet was collected after centrifugation at 4,000 rpm for 15 min at 4 °C. RORγt protein was purified by Nickel Columns for Chromatography Nickel columns, and then the purified RORγt protein was concentrated in a 10 k enrichment tube (Millipore, Massachusetts, USA) and flash-frozen at − 80 °C. The RORγt LBD used in the binding assay was stored at − 80 °C in buffer containing 25 mM Hepes (Ph = 7.4), 200 mM NaCl, 5% glycerol.
The RORγt protein was immobilized on a CM5 chip (GE Health, Chicago, USA) using Biaocre 8 K. A sensogram was obtained using different serial concentrations of 8-074 (5000 nM, 2500 nM, 1250 nM, 625 nM, 312.5 nM, 156.25 nM, 78.125 nM, and 39.0625 nM). SPR sensorgrams have association time intervals of 40 s and dissociation time intervals of 60 s. Data were analyzed using Biacore Evaluation Software.
RORγt dual FRET assay
The assay was performed according to a previous study [15, 24]. The plates were incubated for 1 h at room temperature and then read on Envision in LANCE mode configured for the europeum-APC labels.
RORγt GAL4 cell-based reporter gene assay
The hRORγt LBD coding sequence was inserted into a pBIND expression vector (Promega, E1581) to express the ROR-GAL4 binding domain chimeric receptors. This expression vector and a reporter vector (pGL4.35, which carries a stably integrated GAL4 promoter-driven luciferase reporter gene [luc2P/9XGAL4 UAS/Hygro]) were co-transfected into the HEK293T host cells. The assay was performed according to a previous study . EC50 of the sigmoidal fits were analyzed using Prism 5 and a four-parameter logistic fit equation, Y = bottom + (top–bottom) / (1 + 10(logEC50 − X) × hill slope). "X" is the log of compound concentration, and "Y" is the response, which increases as X increases. Y starts at "bottom" and goes to "top" with a sigmoid shape.
Mouse Th17 differentiation assay
CD4+ T cells were purified from mouse splenocytes using a commercial CD4+ T cell negative selection kit (Invitrogen, California, USA). The 48-well plates were wrapped in the presence of anti-CD3 (0.25 mg/mL, Bioxcell, New Hampshire, USA) and anti-CD28 (1 mg/mL, Bioxcell, New Hampshire, USA) at 0 °C overnight. CD4+ T cells were skewed to Th17 cells by culturing cells in the presence of anti-IFNγ (10 mg/mL, Bioxcell, New Hampshire, USA), anti-IL-4 (10 mg/mL, Bioxcell, New Hampshire, USA), TGF-β (2 ng/mL, Peprotech, Rocky Hill, USA), and IL-6 (20 ng/mL, Peprotech, Rocky Hill, USA) for four days before analysis. Compounds or DMSO control were added to the culture on day 0 of Th17 differentiation at indicated concentrations. Percentage of IL-17 production from CD4+ T cells were analyzed by intracellular staining followed by flow cytometry. Dose–response curves were plotted to determine half-maximal inhibitory concentrations (EC50) for the compounds using the GraphPad Prism 5 (GraphPad Software, San Diego CA, USA).
The Cancer Genome Atlas (TCGA) datasets were downloaded from cBioPortal (http://www.cbioportal.org/). According to gene median expression level, samples were divided into high and low expression groups. For RORC and PDCD1 expression analysis, we downloaded log2-transformed, normalized mRNA expression values (RSEM, Illumina HiSeq_RNASeqV2) and clinicopathological data TCGA cohort from the Cell Index Database CELLX. For the analysis of TCGA dataset (LUAD, BC, EAC, KIRC, and LIHC), a Kaplan–Meier curve was constructed to compare the overall and disease-free survival rates of the two groups. The log-rank P value and HR were calculated using SPSS 22.0. A correlation analysis of the gene expression in the tumor-infiltrating immune cells was analyzed using the Tumor Immune Estimation Resource (TIMER). SPSS 22.0 for windows (Chicago, IL, USA) was used for the data analysis, and statistical significance was determined using a t test. P values were then calculated. A P < 0.05 was considered statistically significant.
In vitro experiments were done with biological replicates higher than or equal to three unless otherwise noted in the figure legends. Most critical experiments were conducted at least three times with similar results. Most data presented in the figures are mean ± SD of biological replicates. Statistics for in vitro data were done using Student t-test (two-tailed) by GraphPad Prism software. P-values < 0.0001, 0.001, 0.01 and 0.05 are represented as ****, ***, **and *, respectively.