Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jean-Ehrland Ricci ( ricci@unice.fr ).

Lymphoma-bearing C57BL/6 Eμ-Myc mice were killed by cervical dislocation as soon as they presented signs of illness. A single-cell suspension was prepared from lymph nodes by teasing them on a 70-μm nylon filter. Cells were either resuspended in DMEM (GIBCO) supplemented with 10% fetal bovine serum (FBS), 10 mM Hepes, 0.1 mM L-asparagine, and 50 μM β-mercaptoethanol for further ex vivo analysis or reimplantation in wild-type mice. B16 cells were obtained from the ATCC (#CRL-6323) and cultured in DMEM (GIBCO) supplemented with 10% fetal bovine serum (FBS). CT26 cells were obtained from the ATCC (#CRL-2638) and cultured in RPMI-1640 medium (GIBCO) supplemented with 10% FBS and 1% sodium pyruvate. When indicated CT26 cells were cultured for 24 hr in amino acid (AA)-deprived medium (USBiological, #D9800-13). All cell lines were incubated at 37°C in a 5% CO 2 atmosphere.

TUDCA (Sigma-Aldrich, #T0266) was intraperitoneally administered in PBS (250 μg/gram of mouse body weight). MKC4485 was administered by oral gavage at a dose of 10 mL/kg from a 30 mg/mL suspension in 1% microcrystalline cellulose in a simple sugar at 300 mg/kg daily (Provided by John B. Patterson). Both inhibitors were administered from day 11 until day 16 after subcutaneous tumor cell injection. Then, the mice were euthanized for further analysis. When described glycemia was measured after a few hours of fasting by using a freestyle Optium blood glucose monitoring device.

For antibody-mediated depletion experiments in vivo, mice were intraperitoneally injected with 100 μg of an anti-CD8-depleting antibody (Bioxcell, clone53-6.7, #BE0004-1) or vehicle (PBS) every second day for seven doses during 2 weeks after tumor cell injection. For antibody-mediated blockade experiments in vivo, mice were intraperitoneally injected with 100 μg of anti-CD86-blocking antibody (Bioxcell, clone GL-1, #BE0025) or vehicle (PBS) every second day for seven doses during 2 weeks after tumor cell injection. For dendritic cell and macrophage depletion in vivo, mice were injected intraperitoneally with 200 μL of a 5mg/mL clodronate-loaded liposome suspension (Liposoma B.V., #PBS-02) every second day for seven doses during 2 weeks after tumor cell injection. Control mice were injected with 200 μL PBS-loaded liposomes using the same schedule.

Syngeneic C57BL/6 mice and NSG mice injected with Eμ-Myc cells were monitored for lymphoma development and systemic signs of illness, including apathy, breathing problems, precipitous weight loss, and limited ability to reach food or water. Animals were euthanized as soon as they exhibited any signs of illness. After subcutaneous B16 and CT26 tumor cell injection syngeneic C57BL/6 and BALB/c mice were inspected daily for tumor development. Increase in tumor size was measured with a caliper. Tumor volume was calculated as follows: (Length x width to the power of 2)/2, where L is the longer of the 2 measurements.

WT syngeneic C57BL/6 mice were intravenously injected with 0.1 × 10 6 Eμ-Myc cells. At day four after injection, the food was replaced with isocaloric diets generated by ENVIGO for 2 weeks: Ctl, Low CHO or Low PROT. BALB/c and C57BL/6 mice were fed with isocaloric Ctl and Low PROT diets (-12.5%, -25%, -40%) one week before subcutaneous injection with 0.5 x 10 6 CT26 cells or 0.25 x 10 6 B16 cells. NOD scid gamma c -/- (NSG) mice were fed with Ctl, Low CHO or Low PROT isocaloric diets generated by ENVIGO one week before intravenous injection with 0.1×10 6 Eμ-Myc cells. For the groups with splenocyte co-injection, splenocytes from wild-type C57BL/6 mice were freshly isolated and washed in PBS. Then, 2×10 6 splenocytes were co-injected with 0.1×10 6 Eμ-Myc cells.

Mice were fed with isocaloric diets generated by ENVIGO: Control (Ctl: TD.130931), low carbohydrates (Low CHO: TD.130932) and low protein diet (Low PROT or Low PROT -25%: TD.130933). When specified, two other low protein diets were used (Low PROT -12.5%: TD.170630 and Low PROT -40%: TD.170631). % energy CHO:PROT:FAT: Ctl - (70.9%:19.5%:9.6%); Low CHO - (54%:26.9%:19.2%); Low PROT -25% - (73.7%:14.9%:11.5%); Low PROT -12.5% - (72.2%:17%:10.8%); Low PROT -40% - (76.4%:12.2%:11.4%).

All animal experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee and the regional ethics committee (approval reference PEA-232 and PEA-233). All experiments used age-matched female littermates. Eμ-Myc/wild-type (WT) mice were obtained from The Jackson Laboratory (#002728). Five-week-old WT syngeneic C57BL/6 mice and BALB/c mice were obtained from ENVIGO. NOD scid gamma c -/- (NSG) mice were obtained from The Jackson Laboratory (#005557) and housed in our animal facilities (C3M-Nice, France).

Method Details

Cytotoxicity Assay CD3+ cells were negatively sorted from mice spleens using autoMACS (Miltenyi Biotec) with FITC antibodies against CD19 (Miltenyi Biotec, #130-102-494), CD45R (Miltenyi Biotec, #130-110-845), CD49b (BD Biosciences, #553857), CD11b (BD Biosciences, #553310) and Ter-119 (BD Biosciences, #557915). The resulting purified cells were co-incubated with CT26 or B16 cells at a ratio 1:5 in the presence of IL-2 (0.1 ng/mL, AbD Serotech, #PMP38) for 4 hr or 48 hr at 37 °C. Flow cytometry (MACS-Quant Analyzer, Miltenyi Biotec) was used to analyze the cell viability of CT26 and B16 cells. CD3-negative population and back gating was used to confirm the difference in forward scatter and side scatter parameters between cells. Cell death was evaluated by looking at plasma membrane permeabilization of CT26 and B16 cells using 4',6-diamidino-2-phenylindole staining (DAPI, Sigma-Aldrich #D9542).

Flow Cytometry Analysis To obtain a single-cell suspension from tumors, lymph nodes, and spleens were filtered through a 70-μm strainer, stained, and analyzed on MACS-Quant Analyzer (Miltenyi Biotec). The following fluorochrome-conjugated anti-mouse antibodies were used: CD4 (VioBlue, #558107), CD8 (PE, #553032), NK1.1 (PE, #557391), CD11c (PE, #557401), CD86 (PECy7, #560582), CD127 (APC, #564175), CD25 (APCCy7, #557658) (BD Biosciences). CD3 (FITC, #11-0031-85), F4/80 (VioBlue, #48-4801-82) (eBioscience). Percentage of CD4+ and CD8+ cells are calculated within CD3+ cells. T regs were defined as the CD3+CD4+CD25+CD127- T-cell population. NK cells were defined as CD3-NK1.1+ cells. DCs were defined as F4/80-CD11c+ cells and macrophages were defined as F4/80+ cells. Percentage of CD86+ cells was calculated within CD11c+ cells.

Confocal Laser Scanning Microscopy Tumoral tissue was snap-frozen in O.C.T. compound (Tissue-Tek, #4583). Then, 5-μm cryosections were prepared and fixed in acetone. Purified anti-mouse CD8a (Biolegend, clone 53-6.7, #100701) was used for CD8 staining and was visualized using Alexa Fluor 594 anti-rat secondary antibody (Molecular Probes, #A11007). All sections were stained with DAPI. For each condition at least three measurements were performed. The number of CD8-positive cells was determined in optical fields of 40× on individual sections. Samples were imaged using a Nikon A1R confocal microscope and processed with ImageJ software analysis.

Western Blot Analysis Tissue samples were collected and lysed using a Precellys 24 (Bertin Instruments) homogenizer (3 × 30 s, 6500 × g) in Laemmli buffer. Proteins were immunoblotted with the indicated antibodies. Immunoblots were visualized (FUJIFILM LAS4000) using the ECL Western Blotting Detection Reagents (GE Healthcare, #RPN2106), and quantification was performed using ImageJ software. anti-XBP1 (#sc-8015), and anti-ERK2 (#sc-1647) antibodies were purchased from Santa Cruz Biotechnology. Anti-IRE1α (#3294), Anti-pS6K (#9234), anti-S6K (#9202), anti-pAKT (#9271), anti-AKT (#2967), anti-LC3B (#3868), anti-eIF2α (#9722), anti-peIF2α (#9721), anti-RIG1 (#3743), anti-ATF-4 (#11815), anti-CHOP (#2895) and GCN2 (#3302) antibodies were purchased from Cell Signaling. Anti-ATF6 (#NBP1-40256) and anti-pIRE1α (#NB100-2323) were purchased from Novus Biologicals. Anti-GRP78 (#ab21685) and anti-pGCN2 (#ab75836) was purchased from Abcam.

Reverse Transcriptase Quantitative-PCR (RT-qPCR) Analysis CT26 tumors were dissociated with the mouse Tumor Dissociation Kit (Miltenyi Biotec, #130-096-730) and CD4+/CD8+ TILs were sorted from the obtained single cell suspension using mouse CD4/CD8 (TIL) MicroBeads (Miltenyi Biotec, #130-116-480) in an autoMACS (Miltenyi Biotec). CD11c+ DC were sorted from spleens of tumor-bearing BALB/c mice using mouse CD11c Microbeads ultrapure in an autoMACS (Miltenyi Biotec, #130-108-338). For total tumor tissue samples were collected and lysed using a Precellys 24 (Bertin Instruments) homogenizer (3 × 30 s, 6500 × g) and total RNA was isolated from cells and tissue using the RNAeasy minikit (Qiagen, # 74104) according to the manufacturer’s protocol. Reverse transcription was performed using the Omniscript RT Kit (Qiagen, #205113). Quantitative-PCR was performed with Fast SYBR Green (Applied Biosystems, Life Technologies, # 4385616) or TaqMan Fast Universal PCR Master Mix (Applied Biosystems, #4352042) using the 7500 Fast and the Step One real-time PCR systems (Applied Biosystems) following the manufacturers’ instructions. Villeneuve et al., 2010 Villeneuve J.

Lepreux S.

Mulot A.

Bérard A.M.

Higa-Nishiyama A.

Costet P.

De Ledinghen V.

Bioulac-Sage P.

Balabaud C.

Nurden A.T.

et al. A protective role for CD154 in hepatic steatosis in mice. The following primers were used for SYBR Green qPCR: Scara3 Forward 5′-TGCATGGATACTGACCCTGA-3′ and Reverse 5′-GCCGTGTTACCAGCTTCTTC-3′; Blos1 Forward 5′-CAAGGAGCTGCAGGAGAAGA-3′ and Reverse 5′-GCCTGGTTGAAGTTCTCCAC-3′ Col6 Forward 5′-TGCTCAACATGAAGCAGACC-3′ and Reverse 5′-TTGAGGGAGAAAGCTCTGGA-3′ IFNγ Forward; 5′-TCAAGTGGCATAGATGTGGAAGAA-3′ and Reverse 5′-TGGCTCTGCAGGATTTTCATG-3′; CXCL10 Forward 5′-GCTGATGCAGGTACAGCGT-3′ and Reverse 5′-CACCATGAATCAAACTGCGA-3′; βactin Forward 5′-TGGAATCCTGTGGCATCCATGAAA-3′ and Reverse 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′. sXBP1 Forward 5′-GCTGAGTCCGCAGCAGGTG-3′; uXBP1 Forward 5′-GAGTCCGCAGCACTCAGACT-3′ and XBP1 Reverse 5′-GTGTCAGAGTCCATGGGAAGA-3′ (). The housekeeping gene β-actin was used as a control for RNA quality and for normalization. The following Taqman assay primer sets from Applied Biosystems were used: Atf3 Mm00476033_m1; Ero1lb Mm00470754_m1; Sars Mm00803379_m1; Trib3 Mm00454879_m1; Hsp90b1 Mm00441926; Hyou1 Mm00491279_m1. The housekeeping gene Rn18s was used as a control for RNA quality, and used for normalization: Mm03928990_g1. All analyses were performed in triplicate, and melting curve analysis was performed for SYBR Green to control product quality and specificity.

Generation of shIFNγ- and shIRE1α-Transduced Cells Frecha et al., 2011 Frecha C.

Fusil F.

Cosset F.L.

Verhoeyen E. In vivo gene delivery into hCD34+ cells in a humanized mouse model. Self-inactivating viruses were generated by transient transfection of 293T cells (ATCC, #CRL-1573) and tittered as described previously (). Briefly For VSV-G preparation, 3 microgram of envelope plasmid was co-transfected using the classical calcium phosphate method with a 8,6 microgram Gag-Pol packaging plasmid (psPAX2, Adgene, #12260) and 8,6 microgram of a plasmid encoding a control shRNA plasmid (Santa Cruz, #sc-108060) and a self-inactivating mouse lentiviral shIFNγ plasmid (Santa Cruz, #sc-39607-SH). Eighteen hours after transfection, the medium was replaced by Opti-MEM supplemented with HEPES (Invitrogen). Viral supernatants were harvested 48 hr after transfection and filtered. The vectors were concentrated at low speed by overnight centrifugation of the viral supernatants at 3000g at 4°C. For the generation of stable CT26 with silenced IRE1α, we used the pSUPER retroviral vector with neo+GFP (Oligoengine, #VEC-PRT-0005/0006). The target sequences were as follows: sh#1= 5′-CCAAGATGCTGGAGAGATT-3′and sh#2= 5′-GCTCGTGAATTGATAGAGA-3′. Oligonucleotides were cloned into the pSUPER vector following the manufacturer’s protocol. Double-stranded DNA templates encoding siRNA oligonucleotides for IRE1α were synthesized. The specific oligonucleotide sequence contained a sense strand of 19 nucleotides followed by a short spacer (TTCAAGAGA) and the reverse complement of the sense strand. Five thymidines were added at the end of the synthesized oligonucleotide as an RNA polymerase III transcriptional stop signal. Oligonucleotides were annealed and ligated into the pSUPER vector digested by BglII and HindIII, and insertion was confirmed by EcoRI–HindIII digestion via migration in an agarose gel. For VSV-G preparation, 3 microgram of envelope plasmid was co-transfected using the classical calcium phosphate method with a 8,6 microgram MLV-Gag-Pol packaging plasmid and 8,6 microgram of the empty pSUPER plasmid or the shIRE1α containing pSUPER plasmids. CT26 cells were transduced and sorted using a SONY sorter SH800 based on GFP expression, resulting in >95% purity.

Generation of CRISPR/Cas9 Cells For the generation of stable CT26 with invalidated IRE1α or RIG1 cells were transfected with 3 μg of CRISPR–Cas9-expressing knockout plasmids (control, sc-418922; IRE1α, sc-429758; RIG1, sc-432915; all from Santa Cruz) using the jetPEI DNA transfection Reagent (PolyPlus Transfection, #POL101-10N) as described by the manufacturer. The knockout plasmids are a mixture of three plasmids, each carrying a different guide RNA specific for the target gene, as well as the Cas- and GFP-coding regions. GFP+ cells were selected by sorting on a SH800S Cell Sorter (Sony Biotechnology) 24 hr after transfection, and depletion of target proteins was verified by immunoblotting.

Sampling of Intracellular Metabolites Tumor samples were resuspended in 170 μL of ultrapure water, manually crushed with a micro potter, vortexed, and then sonicated 5 times for 10 s using a sonication probe (vibra cell, Bioblock Scientific). At this step, 20μL of each sample were withdrawn for further determining the total protein concentration (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific, #23225). Then, we added 350 μL of methanol to the remaining 150 μL of lysate and we sonicated again twice for 10 s each using a sonication probe. Cell debris were then removed by centrifugation for 30 min at 4°C and 20,000g. Supernatant was recovered and incubated 1h30 on ice before a second centrifugation step for 15 min at 4°C and 20,000g. The resulting metabolic extracts were dried under a stream of nitrogen using a TurboVap instrument (Thermo Fisher Scientific) and stored at −80°C until analysis. Dried extracts were dissolved using a given volume of 95 % mobile phase A / 5% mobile phase B to give in a 1000 ng/mL total protein concentration for alanine, arginine, proline methionine, tyrosine, isoleucine, leucine, phenylalanine, and tryptophan quantification. The extract was then diluted 2-fold for aspartic acid, glutamine, glycine, and valine quantification, while another 4-fold dilution was performed for asparagine, serine, threonine, glutamic acid, lysine, and histidine quantification. A defined concentration∗ of a labeled amino acids mixture 98 atom % 13C, 98 atom % 15N (Sigma-Aldrich, #608254) was added to each sample in order to normalize the signals and estimate endogenous amino acid concentrations. ∗13C 415N -Asn , 0.21 μg/mL; ∗13C3, 15N-Ser, 0.27 μg/mL; ∗13C4,15N-Asp, 0.64 μg/mL; ∗13C5,15N2-Gln, 0.24 μg/mL; ∗13C2,15N-Gly, 0.34 μg/mL; ∗13C4,15N-Thr, 0.32 μg/mL; ∗13C5,15N-Glu, 0.58 μg/mL; ∗13C3,15N-Ala, 0.54 μg/mL; ∗13C6,15N2-Lys, 0.26 μg/mL; ∗13C6,15N3-His, 0.06 μg/mL; ∗13C6,15N4-Arg, 0.35 μg/mL; ∗13C5,15N-Pro, 0.2 μg/mL; ∗13C5,15N-Val, 0.31 μg/mL; ∗13C5,15N-Met, 0.12 μg/mL; ∗13C9,15N-Tyr, 0.23 μg/mL; ∗13C6,15N-Ile, 0.26 μg/mL; ∗13C6,15N-Leu, 0.56 μg/mL; ∗13C9,15N-Phe, 0.26 μg/mL; ∗13C11,15N2-Trp, 0.34 μg/mL.

Analysis of Amino Acid Residues by Liquid Chromatography Coupled to High Resolution Mass Spectrometry (LC-MS) LC-MS experiments were performed using a Dionex Ultimate chromatographic system (Thermo Fisher Scientific) coupled to an Exactive (Orbitrap) mass spectrometer (Thermo Fisher Scientific) fitted with an electrospray ion source. The mass spectrometer was externally calibrated before each analysis using the manufacturer’s predefined methods and provided recommended calibration mixture. Chromatographic separation was performed on a Discovery HS F5 PFPP 5 μm, 2.1 × 250 mm column (Sigma-Aldrich) at 30°C. The chromatographic system was equipped with an on-line prefilter (Thermo Fisher Scientifics). Mobile phases were 100% water (A) and 100% aceonitrile (B), both of which containing 0.1% formic acid. Chromatographic elution was achieved with a flow rate of 250 μL/min. After sample injection (20 μL), elution started with an isocratic step of 2 min at 5% phase B, followed by a linear gradient from 5 to 100% of phase B in 18 min. These proportions were kept constant for 4 min before returning to 5% of phase B and letting the system equilibrate for 6 min. The column effluent was directly introduced into the electrospray source of the mass spectrometer, and analyses were performed in the positive ion mode. Source parameters were as follows: capillary voltage set at 5 kV, capillary temperature at 300°C; sheath and auxiliary gas (nitrogen) flow rates at 50 and 25 arbitrary units, respectively; mass resolution power of the analyzer set at 50,000 at m/z 200 (full width at half maximum, FWHM) for singly charged ions. The acquisition was achieved from m/z 50 to 250 in the positive ionization mode during the first 12 min of the run. Under these conditions, we achieved a good chromatographic separation and detection (with an average mass accuracy better than 3ppm) of the 19 targeted amino acids (under their [M+H]+ form). These species were readily identified and quantified by the isotope dilution method using 13C, 15N-labeled homologues (see above). Corresponding extracted ion chromatograms were generated and resulting peaks integrated using the Xcalibur software (version 2.1, Thermo Fisher Scientific) for alanine ([M+H]+ at theoretical m/z 90.05496, retention time 2.98 min), arginine (m/z 175.11895, 3.19 min), asparagine (m/z 133.06077, 2.81 min), aspartate (m/z 134.04478, 2.84 min), glutamate (m/z 148.06043, 2.95 min), glutamine (m/z 147.07642, 2.84 min), glycine (m/z 76.03931, 2.84 min), histidine (m/z 156.07675, 3.03 min), isoleucine (m/z 132.10191, 5.73 min), leucine (m/z 132.10191, 6.37 min), lysine (m/z 147.11280, 3.00 min), methionine (m/z 150.05833, 4.17 min), proline (m/z 116.07061, 3.22 min), phenylalanine (m/z 166.08626, 8.57 min), serine (m/z 106.04987, 2.81 min), threonine (m/z 120.06552, 2.88 min), tryptophan (m/z 205.09715, 10.58 min), tyrosine (m/z 182.08117, 5.51 min), and valine (m/z 118.08626, 3.79 min). P-values were calculated by applying a Mann Whitney test using the GraphPad Prism Software.

Bioinformatic Analysis Lhomond et al., 2018 Lhomond S.

Avril T.

Dejeans N.

Voutetakis K.

Doultsinos D.

McMahon M.

Pineau R.

Obacz J.

Papadodima O.

Jouan F.

et al. Dual IRE1 RNase functions dictate glioblastoma development. ∗.CEL files) of the Pluquet et al., 2013 Pluquet O.

Dejeans N.

Bouchecareilh M.

Lhomond S.

Pineau R.

Higa A.

Delugin M.

Combe C.

Loriot S.

Cubel G.

et al. Posttranscriptional regulation of PER1 underlies the oncogenic function of IREα. Ritchie et al., 2015 Ritchie M.E.

Phipson B.

Wu D.

Hu Y.

Law C.W.

Shi W.

Smyth G.K. Limma powers differential expression analyses for RNA-sequencing and microarray studies. Pluquet et al., 2013 Pluquet O.

Dejeans N.

Bouchecareilh M.

Lhomond S.

Pineau R.

Higa A.

Delugin M.

Combe C.

Loriot S.

Cubel G.

et al. Posttranscriptional regulation of PER1 underlies the oncogenic function of IREα. Patients were clustered according to IRE1α activity based on the normalized z-score of gene expression for the BioInfoMiner signature of 38 genes (). The z-score was calculated by the equation (X - m)/s, X stands for normalized log2 expression data of each gene in each sample; m stands for mean of expression of each gene among all samples; and s stands for standard deviation. Raw data (.CEL files) of the GSE27306 dataset ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE27306 ) from () were processed into R/Bioconductor by using the RMA normalization and Limma package (). The deferentially expressed genes (DEGs) between DN and WT U87 cells, were selected by using a corrected p value threshold of 0.05 and fold change threshold of |log2(fc)| ≥1.5. 1051 deferentially expressed (D.E.) genes were then introduced into the BioInfoMiner tool and gene prioritization was executed based on the biomedical ontologies of the four-different functional and phenotype databases (GO), Reactome, MGI and HPO, separately. For the annotation process was used the “complete” version (this version amplifies the annotation of each gene with the ancestors of every direct correlated ontological term, exploiting the structure of ontological tree) and the hypergeometric pvalue threshold was set to 0.05. 227 highly prioritized genes including their proximal interactors was the union of the BioInfoMiner output from the four databases and 38 hub-genes were highlighted as the intersection with the IRE1α signature of 97 genes of (). The BioInfoMiner signature was composed of 19 genes; highly up-regulated in WT versus DN U87 cells (ASS1, C3, CCL20, COL4A6, CXCL2, CXCL5, CXCL8, IFI44L, IL1B, IL6, KCNN2, MMP1, MMP12, MMP3, PLA2G4A, PPP4R4, SERPINB2, TFPI2, ZNF804A), and 19 genes; highly down-regulated in WT versus DN U87 cells (ANGPT1, CFH, CFI, CLEC3B, COL3A1, COL8A1, DACH1, DCN, FHL1, GAS1, LUM, OXTR, PLAC8, RGS4, TAGLN, TGFB2, THBS1, TIMP3, TMEM255A). Lhomond et al., 2018 Lhomond S.

Avril T.

Dejeans N.

Voutetakis K.

Doultsinos D.

McMahon M.

Pineau R.

Obacz J.

Papadodima O.

Jouan F.

et al. Dual IRE1 RNase functions dictate glioblastoma development. This 38-genes signature was used to stratify 3 different types of tumors including glioblastoma multiform (GBM; TCGA and GBMmark,), melanoma (TCGA) and colorectal cancer (TCGA) into IRE1α high and IRE1α low activity tumors. Then based on these 2 tumor groups, the expression of the following T-cell markers was evaluated in the two groups using the transcriptome data: IFNG, IL12, TBX21, IRF1, STAT1, GZMA, GZMB, GZMH, PRF1, GNLY, NKG7, CXCL9, CXCL10, CCL5, CX3CL1, CXCR3, CCL2, CCL4, CXCL11, MADCAM1, ICAM1, VCAM1, CD3D, CD8A, GBP1, and all the available HLAs.