Cell-culture and reagents

MD55A3 melanoma cells were derived from metastatic melanoma tumour resections6, collected with informed patient consent under a protocol approved by the National Institutes of Health (NIH) IRB Ethics Committee and approved by the MD Anderson IRB (protocol numbers 2012-0846, LAB00-063 and 2004-0069; NCT00338377). MD55A3 were cultured in Roswell Park Memorial Institute 1640 Medium (RPMI 1640, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 25 mM HEPES (Gibco) and 100 U ml−1 penicillin/streptomycin. All other cell lines were purchased for ATCC. hTERT RPE-1, MCF-7, MDA-MB-231, HEK 293T and HT29 were cultured in in Dulbecco’s modified Eagle’s medium (DMEM, Gibco), supplemented with 10% fetal bovine serum and 100 U ml−1 penicillin/streptomycin; MCF10A cells were cultured in DMEM/F12 containing HEPES (Gibco) supplemented with 5% horse serum (Gibco), EGF (10 ng ml−1; Millipore), insulin (10 μg ml−1; Sigma) and hydrocortisone (500 ng ml−1; Sigma). HCT116 cells were cultures in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum and 100 U ml−1 penicillin/streptomycin. All cell lines were maintained in a humidified atmosphere containing 5% of CO2 at 37 °C. All cell lines were tested regularly by PCR for mycoplasma contamination and were found to be negative.

Tryptophan-free DMEM/F12 medium was purchased from US Biologicals. Tyrosine-free and phenylalanine-free DMEM were custom-made (Cell Culture Technology). All these media were supplemented with 10% heat-inactivated dialysed fetal bovine serum (Gibco) and 100 U ml−1 penicillin/streptomycin. IFNγ (PeproTech) was used at 250 U ml−1 for 48 h. MG-132 (Selleckchem), dissolved in DMSO, was used at a final concentration of 10 µM. The IDO inhibitor 1-methyl-l-tryptophan (Sigma) was dissolved in 0.1N NaOH at a 20 mM concentration, adjusted to pH 7.5, filter sterilized and used at a final concentration of 300 µM. Polyethylenimine (PEI, Polysciences) was dissolved in water at a concentration of 1 mg ml−1.

Generation of reporter plasmids

TurboGFP was amplified by PCR using the primers listed in Supplementary Table 10 and the pLKO.1-tGFP plasmid (kind gift from R. Beijersbergen) as a template. The resulting PCR product was cloned into pCDH-blast vector by restriction–ligation cloning into the XbaI and NotI sites.

Mutagenesis was performed using the GeneArt site-directed mutagenesis system (Invitrogen) according to the manufacturer’s instructions. The primers used for generating tGFP F26W and F26A are listed in the Supplementary Table 10. Mutagenesis was performed on the pCDH-Blast-tGFP plasmid.

V5-ATF41–63/W93Y-tGFP was generated by PCR, where the codon for tryptophan (codon 93) was replaced by a codon for tyrosine in the original V5-ATF41–63-tGFP sequence. A first PCR was performed to amplify V5-ATF4 using the primers listed in Supplementary Table 10. This resulting PCR product was extended with tGFP by a second PCR with the V5-ATF41–63-tGFP plasmid as a template. The V5-ATF41–63/W93Y-tGFP gene was then inserted in the pCDH-Blast vector by restriction/ligation cloning in the XbaI and NotI sites.

A DNA sequence coding for the amino acid sequence LEQLESIINFEKL, or the mutated forms thereof, was cloned immediately downstream of the tGFP sequence in the pCDH-V5-ATF41–63/W93Y-tGFP reporter constructs. This was done by PCR on the V5-ATF41–63/W93Y-tGFP construct as template and using the primers listed in Supplementary Table 10. The resulting PCR products were then inserted by restriction–ligation cloning in the XbaI and NotI sites in the pCDH-Blast vector.

The H2-Kb gene was amplified from cDNA using the primers in Supplementary Table 10. The PCR product was cloned into the pCDH-puro backbone by restriction/ligation cloning by making use of the XbaI and EcoRI sites. Next, the puromycin selection cassette was replaced by a hygromycin cassette. This cassette and the PGK promoter were amplified by PCR using the primers in Supplementary Table 10 and the pLenti-Hygro plasmid as a template. The resulting DNA fragment was introduced between the BamHI and XhoI sites of the pCDH-H2-Kb plasmid by a restriction/ligation procedure.

All resulting plasmids were sequence verified by Sanger sequencing (Macrogen).

Lentiviral production and transduction

For lentivirus production, 4 × 106 HEK 293T cells were seeded per 100 mm dish, one day prior to transfection. For each transfection, 10 µg of the pCDH vector of interest, 5 µg of pMDL RRE, 3.5 µg pVSV-G AND 2.5 µg of pRSV-REV plasmids were mixed in 500 µl of serum-free DMEM. Next, 500 µl of serum-free DMEM containing 63 µl of a 1 mg ml−1 PEI solution was added. The entire mix was vortexed and left for 15 min at room temperature after which it was added to the HEK 293T cells to be transfected. The next day, the medium was replaced and the lentivirus-containing supernatants were collected 48 and 72 h post transfection, and snap frozen in liquid nitrogen. Target cells were transduced by supplementation of the lentiviral supernatant with 8 µg ml−1 polybrene (Sigma). One day after transduction, the transduced cells were selected by addition of 5 µg ml−1 blasticidin (Invivogen) or 50–1000 µg ml−1 hygromycin B (Gibco) to the medium.

Amino acid mass spectrometry

Cells were washed with cold PBS and lysed with lysis buffer composed of methanol/acetonitrile/H2O (2:2:1). The lysates were collected and centrifuged at 16,000g (4 °C) for 15 min and the supernatant was transferred to a new tube for liquid–chromatography mass spectrometry (LC–MS) analysis. For media samples, 10 µl of medium was mixed with 1 ml lysis buffer and processed as above.

LC–MS analysis was performed on an Exactive mass spectrometer (Thermo Scientific) coupled to a Dionex Ultimate 3000 autosampler and pump (Thermo Scientific). Metabolites were separated using a Sequant ZIC-pHILIC column (2.1 × 150 mm, 5 µm, guard column 2.1 × 20 mm, 5 µm; Merck) using a linear gradient of acetonitrile (A) and eluent B (20 mM (NH4)2CO3, 0.1% NH4OH in ULC/MS grade water (Biosolve), with a flow rate of 150 µl min−1. The mass spectrometer was operated in polarity-switching mode with spray voltages of 4.5 kV and −3.5 kV. Metabolites were identified on the basis of exact mass within 5 ppm and further validated by concordance with retention times of standards. Quantification was based on peak area using LCquan software (Thermo Scientific).

Public datasets

The following publicly available datasets were used for this study: Proteomics Data Commons PDC000234, PDC000270, PDC000198, PDC000221, PDC000173, PDC000204, PDC000110, PDC000116, PDC000153 and PDC000303; and PRIDE datasets (PXD020079, PXD020224 and PXD022707). The UNIPROT Database is sourced from UNIPROT.org with the identifier UP000005640.

Analysis of immunoprecipitation-based mass spectrometry data

Data generation

At the end of each experiment intended for V5-tag pulldown, cells were treated with 10 µM MG-132 for 4 h and subsequently collected by trypsinization and centrifugation. Next, cells were lysed in 300 µl ELB lysis buffer (50 mM HEPES, 125 mM NaCl, 0.5% (v/v) Tween-20 and 0.1% (v/v) Nonidet P40 substitute. Next, 3 µl mouse anti-V5 antibody solution (1.0 mg ml−1, Invitrogen) was added to the lysate and the samples were incubated on a rotating wheel at 4 °C overnight. Pulldowns were performed with Dynabeads protein G (Invitrogen) according to manufacturer’s protocol. All pulled down protein was eluted in 30 µl of 1× Laemmli buffer.

Next, the eluates were run briefly into a 4–12% Criterion XT Bis-Tris gel (Bio-Rad) and short, Coomassie-stained gel lanes were excised for each sample. Proteins were reduced with 6.5 mM DTT, alkylated with 54 mM iodoacetamide and digested in-gel with trypsin (Gold, mass spectrometry grade, Promega, 3 ng μl−1) overnight at 37 °C. Extracted peptides were vacuum dried, reconstituted in 10% formic acid and analysed by nanoLC–MS/MS on an Orbitrap Fusion Tribrid mass spectrometer equipped with a Proxeon nLC1000 system. Peptides were loaded directly on the analytical column and separated in a 90-min gradient containing a non-linear increase from 5% to 26% solvent B.

Generation of search database

Five search databases were generated, to cover all possibilities of aberrant protein production from the V5–ATF41–63–tGFP reporter protein. The first database consisted of the original ATF4 in-frame protein sequence, the ATF4 sequence until W93 and frame-shifted (+1) at W codon until the first stop codon (Fig. 1a), the in-frame ATF4 protein sequence with the tryptophan replaced by every other amino acid, and the ATF4 protein sequences where the tryptophan codon is skipped. The second database consisted of the tryptic peptide spanning the tryptophan codon in the in-frame ATF4 sequence, and was generated by replacing every amino acid in the sequence to every other possible amino acid. The third database was generated by replacing every phenylalanine in the in-frame ATF4 sequence to every other amino acid. The fourth database was generated by replacing every tyrosine in the in-frame ATF4 sequence to every other amino acid. Finally, Translational bypass was checked for with a final database consisting of the tryptic peptides that would originate from exclusion of W93. And additionally, the peptide that would arise from the event where a ribosome taking off with tRNA-Glu-TTC loaded in the P-site, and re-starting translation at the next GAA codon.

Searching of immunoprecipitation–mass spectrometry data against the databases

The search was performed using MaxQuant (version Peptide false discovery rate (FDR) threshold was set at 0.01. The parameters of the search were optimized for increasing sensitivity and were deposited in the PRIDE database49.

Analysis of 2D proteomics data

Data generation

MD55A3 and MCF10A expressing the V5–ATF41–63–tGFP+1 reporter were used for this purpose. On the first day, cells were seeded in 15cm dishes at around 60% confluency. The next day, cells were rinsed with PBS and were exposed to the appropriate treatment (IFNy or tryptophan-free medium). As control, tryptophan-free medium was supplemented with 5μg ml−1 l-tryptophan (Sigma). After 48 h of treatment, 10 μM MG-132 was added directly in the plates and cells were incubated for 4 h at 37 °C. Then, cells were washed once with PBS and collected by trypsinization and centrifugation. Cell pellets were washed once with PBS, after which the cell pellet was snap-frozen in liquid nitrogen.

Then, the samples were reduced and alkylated in heated guanidine (GuHCl) lysis buffer as described50. After dilution to 2M GuHCl, proteins were digested twice (4h and overnight) with trypsin (Sigma) at 37 °C, enzyme/substrate ratio 1:50. Digestion was quenched by the addition of TFA (final concentration 1%), after which the peptides were desalted on a Sep-Pak C18 cartridge (Waters). Samples were vacuum dried and stored at −80 °C until fractionation.

Dried digests were subjected to basic reversed-phase (HpH-RP) high-performance liquid chromatography for offline peptide fractionation. Two-hundred-and-fifty micrograms of peptides were reconstituted in 95% 10 mM ammonium hydroxide (NH4OH, solvent A)/5% (90% acetonitrile (ACN)/10mM NH4OH, solvent B) and loaded onto a Phenomenex Kinetex EVO C18 analytical column (150 mm × 2.1 mm, particle size 5 μm, 100 Å pores) coupled to an Agilent 1260 HPLC system equipped with a fraction collector. Peptides were eluted at a constant flow of 100 μl min−1 in a 90-min gradient containing a nonlinear increase from 5–30% solvent B. Fractions were collected and concatenated to 24 fractions per sample replicate. All fractions were analyzed by nanoLC–MS/MS on an Orbitrap Fusion Tribrid mass spectrometer equipped with an Easy-nLC1000 system (Thermo Scientific). Peptides were directly loaded onto the analytical column (ReproSil-Pur 120 C18-AQ, 1.9 μm, 75 μm × 500 mm, packed in-house). Solvent A was 0.1% formic acid/water and solvent B was 0.1% formic acid/80% acetonitrile. Samples were eluted from the analytical column at a constant flow of 250 nl min−1 in a 2 h gradient containing a linear increase from 8–32% solvent B. Mass spectrometry settings were as follows: full MS scans (375–1500 m/z) were acquired at 60,000 resolution with an automatic gain control (AGC) target of 3 × 106 charges and max injection time of 45 ms. Loop count was set to 20 and only precursors with charge state 2–7 were sampled for MS2 using 15,000 resolution, MS2 isolation window of 1.4 m/z, 1 × 105 AGC target, a max injection time of 22 ms and a normalized collision energy of 26.

Generation of mutant database

The human proteome was downloaded from UNIPROT51. All instances of tryptophan were replaced by other amino acids in a separate database (FASTA file).

Database Search and filtering

Each proteomics dataset was searched against the database. The parameters of the search are deposited in the PRIDE database49. Peptide FDR threshold was set at 0.01. After the search, only the tryptic peptides spanning the endogenous tryptophan codon were retained and used for further analysis. Further filtering was done to keep only the reproducibly detected peptides.

Analysis of immunopeptidomics data

Data acquisition

Immunopeptidomics data of colorectal cancer was sourced from the published study39. In addition, for glioblastoma (RA) datasets- HLA bound peptides were eluted as previously described52 from 200 million RA cells treatment or not with 500 IU ml−1 IFNγ for 48 h in 4 biological replicates each condition. W6/32 antibody cross linked to protein-A sepharose 4B beads was used for the immunoaffinity purification. HLA-bound peptides were measured on a LC-MS/MS system consisted of an Easy-nLC 1200 connected to a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific) as previously described52. Peptides were separated with a flow rate of 250 nl min−1 by a gradient of 0.1% formic acid (FA) in 95% ACN and 0.1% FA in water. Full MS spectra were acquired in the Orbitrap from m/z = 300–1,650 with a resolution of 60,000 (m/z = 200), ion accumulation time of 80 ms and AGC of 3 × 106 ions. MS/MS spectra were acquired in a data dependent manner on the twenty most abundant precursor ions with a resolution of 30,000 (m/z = 200), an ion accumulation time of 120 ms, isolation window of 1.2 m/z, AGC of 2 × 105 ions, dynamic exclusion of 20 s, and a normalized collision energy (NCE) of 27 was used for fragmentation. The peptide match option was disabled.

Generation of mutant database, search and filtering

The human proteome was downloaded from UNIPROT51 (release-2011_01, downloaded June 2019). All instances of tryptophan were replaced by phenylalanine and stored in a DB (FASTA file). The RAW MS data files were analysed using MaxQuant (version by performing a search against the generated database. The parameter file for the search is deposited in the PRIDE Database and basic parameters19 are provided as Supplementary Table 11. Briefly, we performed search scans with FDR 0.01, 0.05 and 0.1, and for lower thresholds (FDR < 0.1) controlled with other tryptophan substitutants. Additionally, we validated the peptides using targeted mass-spectrometry analysis (see methods, Supplementary Table 9). Only the fragmented peptides spanning the tryptophan codon were retained for further analysis of substitutant peptides. Further filtering was done to keep only the reproducibly detected peptides.

Validation with parallel reaction monitoring

Peptides were ordered from ThermoFisher Scientific as crude (PePotec grade 3) with amino acid where heavy stable isotope atoms were incorporated for parallel reaction monitoring. Synthetic peptides were spiked into the peptidomic samples at a concentration of 1 pmol μl−1. The mass spectrometer was operated at a resolution of 120,000 (at m/z = 200) for the MS1 full scan, with an ion injection time of 80 ms, AGC of 3 × 106 and scanning a mass range from 300 to 1,650 m/z. Each peptide was isolated with an isolation window of 1.2 m/z prior to ion activation by high-energy collision dissociation (HCD, NCE = 27). Targeted MS/MS spectra were acquired at a resolution of 30,000 (at m/z = 200) with 80 ms ion injection time and an AGC of 5 × 105.

The PRM data were processed and analysed as previously described52 using Skyline (v4.1.0.18169)53. Ion mass tolerance of 0.05 m/z was used to extract fragment ion chromatograms and peak lists for the heavy-labelled peptides and endogenous light counterparts were extracted. MS/MS matching assessment was performed by pLabel (v2.4.0.8, pFind studio, Sci. Ac.) and Skyline (MacCoss Lab, v21.1.0.146).

Structural analysis

The structural implications of the W>F substitutions were analysed using the HOPE meta-server54 which creates human-readable reports describing the structural and functional importance of the substituted residue, for example, from known variants and mutation data stored in the UniprotKB55 and sequence variability data from large-scale multiple sequence alignments in the HSSP databank56. If a suitable template structure model is available in the Protein Data Bank57, HOPE also creates homology models of the wildtype protein structures. All created homology models were visually inspected in Coot to assess whether the tryptophan residues made structurally important hydrogen bonds through their side-chains that are lost by W>F substitutions. It should be noted that possible hydrogen bonds with other proteins cannot be studied from these homology models.

Analysis of large-scale proteomics data of human cancer

LSCC, BC, LUAD, CCRCC, HCC, HNSCC, PDA, GBM, OV and BC-PDX data were download from the Proteomics Data Center22 in MZML file format. The human proteome was downloaded from UNIPROT51 (release-2011_01, downloaded June 2019), and all instances of tryptophan amino-acids in the proteome were changed to all other amino acids except Lysine and Arginine, in order to avoid creation of tryptic cleavage site in the scan. The resultant FASTA file was used as Philosopher pipeline58 was used to detect all peptides in mass-spectrometry datasets (MZML files), including the substitutant peptides. Briefly, MSFragger59 was used for peptide detection with the following parameters; Precursor mass lower: −20 ppm, Precursor mass upper: 20 ppm, precursor mass tolerance: 20 ppm, calibrate mass: True, Deisotoping: True, mass offset: False, isotope error: Standard, digestion: Strictly tryptic (Max. missed cleavage: 2), Variable modifications (For iTRAQ datasets): 15.99490 M 3, 42.01060 [^ 1, 229.162932 n^ 1, 229.162932 S 1, Variable modifications (For TMT datasets): 15.99490 M 3, 42.01060 [^ 1, 144.1021 n^ 1, 144.1021 S 1, Min Length: 7, Max Length: 50, digest mass range: 500:5000 Daltons, Max Charge: 2, remove precursor range: −1.5, 1.5, topN peaks: 300, minimum peaks: 15, precursor range: 1:6, add Cysteine: 57.021464, add Lysine (for ITRAQ datasets): 144.1021, add Lysine (for TMT datasets): 229.162932,among other basic parameters (PARAMETER.yml is submitted in the as Supplementary Table 12). PeptideProphet60 was then used for Peptide Validation with following parameters (accmass: TRUE, decoyprobs: TRUE, expectScore: TRUE, Glycosylation: FALSE, ICAT: FALSE, masswidth: 5, minimum probability after first pass of a peptide: 0.9, minimum number of NTT in a peptide: 2, among other parameters (Supplementary Table 12). Next, isobaric quantification was next undertaken separately for TMT and iTRAQ datasets with following parameters (bestPSM: TRUE, level: 2, minProb 0.7, ion purity cut-off: 0.5, tolerance: 20 ppm, among other parameters (Supplementary Table 12). Thereafter, FDR filtering was implemented to retain only confident peptides with following parameters (FDR < 0.01, peptideProbability: 0.7, among other parameters (Supplementary Table 12). Thereafter, TMT-integrator58 was used to integrate isobaric quantification with following parameters (retention time normalization: False, minimum peptide probability on top of FDR filtering (TMT datasets): 0.9, minimum peptide probability on top of FDR filtering (for iTRAQ dataset): 0.5, among other parameters). Substitutant peptides were fetched from the reports of TMT integrator command, and any detected peptide intensity score for a sample normalized to the reference channel above 0 (log scale) was considered as a positive peptide for that sample using a R-script. R was used to plot density plots as well as Barplots for number of peptide detections (Fig. 3f, Extended Data Fig. 3). For all intra-tumour type analysis, a filter for maximum number of samples (vertical lines in Extended Data Fig. 3) was applied to retain peptides with higher specificity in expression. Next, protein expression profiles for each cancer type were downloaded in already analysed format from PDC commons (https://pdc.cancer.gov). PERL scripts were designed to count number of substitutants when a gene is lowly expressed (intensity < 0) or highly expressed (intensity > 0). Gene ontology (GO)-term enrichment analysis was done using ToppGene61. Phosphoproteome data was downloaded from PDC commons (https://pdc.cancer.gov), and a similar analysis as to proteome analysis was undertaken using a customized PERL script.

Western blotting

Straight lysates from cells were made in 6 wells by addition of 200 µl of 1× Laemmli buffer. All protein samples were run on SDS-PAGE gels and blotted on 22 µm pore size nitrocellulose membranes (Santa Cruz). V5 stainings were performed using V5 tag monoclonal antibodies (Invitrogen, R960-25; 1:1,000), tGFP staining with rabbit anti TurboGFP (Invitrogen, PA5-22688; 1:1,000), IDO1 was visualized with rabbit anti-IDO D5J4E (Cell Signaling, 86630, 1:1,000) and tubulin with anti-tubulin (DM1A, Sigma, 1:10,000).

Subsequent stainings were performed with IRDye 680RD donkey anti-mouse (LI-COR, 926-68072, 1:10,000) and IRDye 800CW goat anti-rabbit (LI-COR, 926-32211, 1:10,000) secondary antibodies. Visualization was performed by use of an Odyssey infrared scanning device (LI-COR).

WARS1 activity assay

The human WARS1 gene was cloned in the LIC1_1 vector by PCR amplification and ligation independent cloning using the following primers: cagggacccggtATGCCCAACAGTGAGCCCGCATCTCTGC and cgaggagaagcccggttaCTGAAAGTCGAAGGACAGCTTCCGGGGAG. The inserted sequence was verified by Sanger sequencing and the recombinant protein was expressed in Rosetta2(DE3) cells. In short, cells were grown at 37 °C until OD600 of 0.7. Next, protein expression was induced by addition of 0.4 mM IPTG and the cells were grown overnight at 18 °C. After lysis, the recombinant WARS1 protein was purified using nickel beads, after which the protein was reconstituted in 25 mM Tris pH 8.0, 200 mM NaCl and 1 mM TCEP.

WARS1 aminoacylation activity toward different amino acids was estimated by measuring released phosphate. The assay was performed in a 50 µl reaction volume containing 20 µM purified WARS1 enzyme, 100 mM TRIS, 10 mM MgCl2, 40 mM KCL, 1 mM dithiothreitol, 0.25 U µl−1 pyrophosphatase (Sigma-Aldrich) and 0.5 mM of tryptophan, serine, glycine, phenylalanine or methionine. The reaction mixture was incubated at 37 °C for 30 min. Afterwards, 100 µl of BIOMOL Green TM (Enzo Life Sciences) was added and the samples were incubated at room temperature for 30 min. The released phosphate was quantified by measuring absorbance at 620 nm with an Infinite 200 microplate reader (Tecan).

Fluorescence-activated cell sorting

Measurement of tGFP fluorescent intensity

Cells expressing the tGFP reporters were seeded, and treatment was started the next day. 48 h after the start of treatment, the cells were collected by trypsinization and centrifugation. Next, the cells were analysed on an Attune NxT machine (Thermo Fisher Scientific) using Attune Nxt software version 4.2 and the data were analysed using FlowJo V10 software (FlowJo).

Measurement of H2-Kb-bound SIINFEKL levels

MD55A3 and HT29 cells were transduced with lentiviruses produced from pCDH-Hygro-H2-Kb and selected with hygromycin (Invitrogen)8. Next, the H2-Kb expressing cells were transduced with lentiviruses generated from the pCDH-V5-ATF41–63/W93Y-tGFP-SIINFEKL or the mutant versions thereof. Transduced cells were selected for using 5 µg ml−1 blasticidin (Invivogen).

For the detection of presented H2-Kb-bound SIINFEKL peptides, cells were treated for 48 h with 250 U ml−1 IFNγ (Peprotech), 1MT (IDOi, 300 μM, Sigma) and/or tryptophan-less DMEM/F12 (USBiologicals). Then, cells were washed with PBS and detached using PBS–EDTA (50 µM). Next, cells were pelleted and washed with PBS/0.5% BSA and incubated with APC anti-mouse H2-Kb-bound to SIINFEKL antibodies (Biolegend, clone 25-D1.16, 141606; 1:200 in PBS/0.1% BSA) for 30 min on ice, in the dark. The cells were then washed twice with PBS-BSA and analysed on an Attune NxT machine using Attune Nxt software version 4.2 (Thermo Fisher Scientific). Data were analysed using FlowJo V10 software (FlowJo). HT29 H2-Kb- and ATF41–63(W93Y)–tGFP–SIINwEKL-expressing cells contained a highly variable signal for H2-Kb-bound SIINFEKL after treatment, the highly positive cells were sorted out. First, these cells were treated for 48 h with IFNγ, after which they were stained for H2-Kb bound to SIINFEKL as described above. The top 7.5% positive cells were sorted out of the population using a BD FACSAria Fusion machine (BD biosciences).

OT-I T cell SIINFEKL recognition assays

OT-I (B6J) mice were originally from The Jackson Laboratory. Mice used for experiments were between 3 and 12 weeks old and of both sexes. All experiments involving animals were performed in accordance with Dutch and European regulations on care and protection of laboratory animals and have been approved by the local animal experiment committee at Netherlands Cancer Institute, DEC NKI (OZP ID 12051). Mice were bred and maintained in accordance with institutional, national and European guidelines for Animal Care and Use.

OT-I T cells were isolated using Dynabeads Untouched Mouse CD8 Cells Kit (Invitrogen) according to the manufacturer’s protocol. T cells were initially maintained in Roswell Park Memorial Institute 1640 Medium (Gibco) containing 10% fetal bovine serum (Sigma), 50 µM 2-mercaptoethanol (Sigma), 100 U ml−1 penicillin, 100 μg ml−1 streptomycin (both Gibco), 100µg/mL IL-2 (ImmunoTools),5 µg/mL IL-7 (ImmunoTools) and 10 µg ml−1 IL-15 (ImmunoTools).

MD55A3 cells expressing H2-Kb and V5–ATF41–63(W93Y)–tGFP–SIINFEKL or V5–ATF41–63(W93Y)–tGFP–SIINwEKL were treated for 2 days with the indicated treatments. To the IFN-treated samples, 7.2 × 102 µg ml−1 purified PEG–His–mpKynureninase38 and 2 µM pyridoxal 5′-phosphate hydrate (Sigma) were added. At the end of the treatment, the cancer cells were detached using PBS-EDTA and seeded at 100,000 cells per well in a 96 U-shaped-well plate. Next, 100,000 OT-I T cells were added to start the co-culture and the solution was supplemented with BD Golgiplug (BD Biosciences). The co-culture samples were then incubated for 12 h at 37 °C in a humidified CO2 incubator.

HT29 cells expressing H2-Kb or H2-Kb and V5–ATF41–63(W93Y)–tGFP–SIINwEKL were treated for two days with the indicated treatments. To the IFN-treated samples, 36 µg ml−1 purified His–mpKynureninase and 2 µM pyridoxal 5′-phosphate hydrate (Sigma) were added. In one of the IFN-treated samples 1MT (IDOi, 300 μM, Sigma) was added. At the end of the treatment, the cancer cells were detached using PBS/EDTA and seeded at 100,000 cells per well in a U-shaped 96 well plate. Next, 100,000 OT-I T cells were added to start the co-culture and the solution was supplemented with BD Golgiplug (BD Biosciences). The co-culture samples were then incubated for 4h at 37 °C in a humidified CO2 incubator.

Next, the cells were pelleted by centrifugation, blocked with 0.1% PBS-BSA and stained with anti-mouse CD8-VioBlue antibodies (Miltenyi, 130-111-638, 1:100) and Live/Dead Fixable near-IR dead cell stain kit (Invitrogen). Subsequently, the cells were fixed and permeabilized using the eBioscience Foxp3 Transcription Factor Staining Buffer Set (Invitrogen) according to manufacturer’s instructions. Next, the cells were stained with APC-conjugated anti-mouse IFNγ (Miltenyi, 130-109-723 and 130-120-805, 1:100) and PE-conjugated anti-mouse TNF (Miltenyi, 130-109-719 and 130-102-386, 1:100) antibodies. Cells were then washed and analysed on a BD LSR Fortessa (BD Biosciences). The data were analysed using FlowJo V10 software (FlowJo).

OT-I T cell-mediated killing assay

HT29 H2-Kb- or H2-Kb and ATF41–63(W93Y)–tGFP–SIINwEKL-expressing cells were mock treated for 48 h or treated with IFNγ in tryptophan-less DMEM/F12 medium in 12 well plates. To the IFNγ-treated samples, 36 µg ml−1 purified His–mpKynureninase and 2 µM pyridoxal 5′-phosphate hydrate (PLP, Sigma) were added. After this treatment, the medium was replaced with fresh DMEM supplemented with kynureninase and PLP for the corresponding samples. Then OT-I cells were added in ratios HT29:OT-I of 4:1, 2:1 and 1:1. The co-cultures were left for 24 h at 37 °C in a humidified CO2 incubator. After the co-culture, the cells were fixed using 4% formaldehyde (Merck) in PBS. Then the cells were stained using crystal violet (0.1% in water) for 30 min, after which the plates were washed thoroughly in water and left to dry. Bound crystal violet was extracted using a 10% acetic acid solution (in water). To quantify the bound crystal violet in each well, the solution from the well was diluted tenfold with water and the absorbance was measured at 590 nM using an Infinite 200 PRO reader (Tecan).

Kynureninase activity measurement

Kynurenine (l-kyn) analysis: Samples (50 µl) were mixed with 50 ul l-kyn-d4 (1 µM) in water and 10 µl of trifluoro acetic acid. Mixtures were centrifuged (10 min, 20,000g, 4’C). Supernatant (50 µl) was diluted with water (200 µl) and 10 µl was analysed by LC–MS using a API4000 (Sciex). Separation was achieved using a Zorbax Extend C18 column 100 × 2 mm ID) and an isokratic mobile phase comprising 0.1% formic acid in water: methanol (98:2 v/v). MS detection by multiple reaction monitoring using ion pairs 209.3/192.1 (l-kyn) and 213.3/196.1 (l-kyn-d4).

Induction of T cells reactive to substitutant peptides

PBMCs were isolated from buffy coats from previously HLA-typed healthy donor buffy coats from Oslo University Hospital Blood Bank. The study was approved by the Regional Ethics Committee (REC) and informed consent was obtained from healthy donors in accordance with the declaration of Helsinki and institutional guidelines (REC 2018/2006 and 2018/879). Isolation of T cells reactive to substitutant peptides was performed as previously described6,40 with modifications. In brief, on day −4 monocytes were isolated from PBMCs of HLA-A*24:02 positive healthy donors using CD14-reactive microbeads and an AutoMACS Pro Separator (Miltenyi Biotec). Cells were then cultured for three days in CellGro GMP DC medium (CellGenix) supplemented with 1% (v/v) human serum (HS, Trina Biotech) and 1% (v/v) penicillin–streptomycin containing 10 ng ml−1 IL-4 (PeproTec) and 800 IU ml−1 GM-CSF (Genzyme). Subsequently, monocyte-derived-dendritic cells were matured for 14–16 h by supplementing cultures with 800 IU ml−1 GM-CSF, 10 ng ml−1 IL-4, 10 ng ml−1 lipopolysaccharide (LPS; Sigma-Aldrich) and 5 ng/ml IFNγ (PeproTech). On day −1, autologous naive CD8+ T cells were isolated using a CD8+ T cell isolation kit and AutoMACS Pro Separator (Miltenyi Biotec). Naive CD8+ T cells were cultured overnight in TexMACS medium (Miltenyi Biotec) supplemented with 1% (v/v) penicillin/streptomycin and 5 ng ml−1 IL-7 (PeproTech). On day 0, monocyte-derived dendritic cells were peptide-pulsed with individual substitutant peptides for 2 h at a concentration of 1 μg ml−1, or incubated with DMSO vehicle. A total of six substitutant peptides (BI1, KLHL4, W2PPT5, F5GXS0, F8VXG7 and G5E9G0) were included. Individually peptide-loaded monocyte-derived dendritic cells were collected and pooled before combining with naive T cells for co-culture in CellGro GMP DC medium supplemented with 5% human serum and 30 ng ml−1 IL-21 (PeproTech) at a DC:T cell ratio of 1:2. In parallel control cultures, naive T cells were co-cultured with DMSO-vehicle-treated monocyte-derived dendritic cells. On days 3, 5 and 7, half of the medium was removed and replaced with fresh medium supplemented with 10 ng ml−1 of both IL-7 and IL-15 (PeproTech). On day 10, co-cultures were screened for the presence of substitutant pMHC multimer-reactive CD8+ T cells. pMHC multimers conjugated to four different streptavidin (SA)–fluorochrome conjugates were prepared in-house as previously described62,63. SA–phycoerythrin (SA–PE), SA–phycoerythrin-CF594 (SA-PE-CF594), SA–allophycocyanin (SA–APC) and SA–Brilliant Violet 605 (SA–BV605). Each pMHC multimer was labelled with two different fluorochromes for increased specificity. Positive T cells were identified by Boolean gating strategy in FlowJo (TreeStar) v10.6.2 software as live CD8+ T cells staining positively for two pMHC multimer fluorochromes and negatively for the two other pMHC multimer fluorochromes, shown in Supplementary Fig. 3 and as previously described64.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

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