Female C57BL/6 mice were obtained from Janvier (France). All mice were kept with a 12-h light/dark cycle, water ad lib. and regular chow diet (sniff, Soest, Germany), at the University of Munich, Munich, Germany. The KrasG12D p53fl/R172H Ptf1a-Cre (KPC)-derived T110299 pancreatic tumor cell line was provided by Prof. Jens Siveke, (University Hospital Essen, Germany), Ifnar1−/− mice (Ifnar1tm1Agt) were provided by Prof. Simon Rothenfußer (LMU Munich, Germany). Age- and sex-matched 6–12 weeks old wild type mice and OT-I TCR-transgenic mice (C57BL/6Tg(TcraTcrb)1100Mjb/J) were purchased from Jackson Laboratory (Stock number 003831).
Primary cells were cultured in RPMI-1640 medium (Sigma-Aldrich, Taufkirchen, Germany), supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 U/l penicillin, 0.1 mg/ml streptomycin, 100 mM non-essential amino acids (all gibco®, Thermo Fisher Scientific, Karlsruhe, Germany), 1 mM sodium pyruvate and 50 mM 2-mercaptoethanol (both Sigma Aldrich). Tumor cells were cultured in DMEM high glucose media (Sigma-Aldrich), supplemented with 10% FCS, 2 mM L-glutamine, 100 U/l penicillin and 0.1 mg/ml streptomycin. OVA expression T110299 cell were generated by transfection with the pAc-Neo-OVA plasmid  using the Novagen Genejuice® transfection reagent, according to the manufacturer’s instructions. OVA+ T110299 cells were selected with G418 (geneticin). All cells were kept in a humidified incubator at 37 °C and 5% CO2. For the assessment of MHC-I expression of explanted tumors, we made use of EpCAM-expressing T110299 tumors cells, which were generated by transducing T110299 cell with a pMXs vector harboring murine EpCAM, thus, allowing labeling with mAb for flow cytometry analysis.
Orthotopic tumor induction and poly(I:C)c treatment
Orthotopic tumors were induced by surgical implantation, as described before . Briefly, mice were anesthetized and, by surgical incision, the pancreas was carefully mobilized for injection. After the injection of 2 × 105 T110299 cells in 25 μl PBS, the pancreas was relocated and the incision was closed by surgical suture. Mice were monitored daily and distressed mice were sacrificed. For treatment, 50 μg VacciGrade™ HMW polyinosinic-polycytidylic acid (poly(I:C)) (InvivoGen, Toulouse, France) were complexed with in vivo-jetPEI® (VWR International GmbH, Darmstadt, Germany), at a N/P ratio of 6 in 5% glucose solution, according to the manufacturer’s instructions (referred to as poly(I:C)c). Mice were treated i.v. at day 18 and 20 after tumor induction with either poly(I:C)c or glucose as control. 6 h after the first treatment cytokine levels of CXCL10 and IL-6 were measured using enzyme-linked immunosorbent assays (ELISA) from R&D systems (Minneapolis, USA). 12 h after the second treatment IFNβ levels were measured using ELISA from R&D systems. On day 14 and day 21 following tumor induction, serum G-CSF levels were measured by ELISA (R&D systems, Bio-Techne GmbH, Wiesbaden-Nordenstadt, Germany). Tumor-free mice served as controls. All other serum cytokines were measured 6 h after the first treatment by multiplex analysis, using a Procarta Plex Mix&Match Panel (Invitrogen, Thermo Fisher Scientific, Karlsruhe, Germany) and a MAGPIX® system (Merck, Darmstadt, Germany), according to the manufacturer’s protocol.
Spleens were processed through a 70 μm cell strainer, followed by red blood cell lysis (BD Pharm Lyse™, BD Biosciences, Heidelberg, Germany). Tumor tissue was minced into pieces and mechanically dissociated using the mouse Tumor Dissociation Kit with the gentleMACS™ Dissociator application (both Miltenyi Biotech, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. The cell suspension was separated from tissue debris by sequentially using 100 μm and 70 μm cell strainers. For functional assays, T cells were isolated using the Pan T cell isolation Kit II and stained with 2.5 μM CFSE (Thermo Fisher Scientific, Karlsruhe, Germany) for 4 min at room temperature. For MDSC isolation, the Myeloid-Derived Suppressor Cell Kit was used. Macrophages/TAM were isolated using the anti-F4/80 MicroBeads UltraPure (all Miltenyi Biotec). Cell purity yielded > 95% for T cells, 60 - 95% for macrophages and 75 - 90% for MDSC. For RNA analyses, single cell suspensions were enriched for myeloid cells using the CD11b+ MACS kit (Miltenyi Biotec) and stained with Fixable Viability Dye (eBioscience, Frankfurt, Germany), anti-CD45 (clone: 30-F11), anti-CD11b (clone: M1/70), anti-Ly6G (clone: 1A8) and anti-Ly6-C (clone HK1.4; all BioLegend, London, UK) for 30 min on ice. Cells were washed and sorted for CD11b+Ly6CloLy6G+ PMN-MDSC or CD11b+Ly6ChiLy6G− M-MDSC on a BD FACSAria III (BD Biosciences), yielding mean purities of > 90% (tumor) and > 95% (spleen) (Additional file 1: Figure S3A).
Prior to fluorochrome staining, FcRIII/II blocking was performed using the TrueStain fcX™ antibody (Biolegend, London, UK). Cell surface staining was done with anti-CD3 (clone 145-2C11), anti-CD4 (clone GK1.5), anti-CD8 (clone 53–6.7), anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD19 (clone 6D5), anti-CD26 (clone H194–112), anti-CD45 (clone 30-F11), anti-CD69 (clone H1.2F3), anti-CD172a (clone P84), anti-CD206 (clone C068C2), anti-EpCAM (clone G8.8), anti-F4/80 (clone BM8), anti-Ly6C (clone HK1.4), anti-Ly6G (clone 1A8), anti-MHC-I (clone AF6–88.5), anti-MHC-II (clone AF6–120.01), anti-NK1.1 (clone PK136), anti-PD-1 (clone 29F.1A12), anti-PD-L1 (clone 10F.9G2), anti-CD86 (clone GL-1), anti-CD40 (clone 3/23), anti-XCR1 (clone ZET; all BioLegend, London, UK) and anti-CD204 (clone 2F8, Biorad, Munich, Germany) antibodies, and Fixable Viability Dye (Thermo Fisher Scientific, Karlsruhe, Germany) was used to exclude dead cells. The gating strategy is depicted in Additional file 1: Figure S1. Intracellular staining was done for arginase-1 (Polyclonal Sheep IgG; R&D Systems, Minneapolis, USA) using the eBioscience™ FoxP3/Transcription Factor Staining Buffer Kit (Thermo Fisher Scientific, Karlsruhe, Germany). Data were acquired on a BD LSRFortessa system (BD Bioscience, Heidelberg, Germany) and analyzed with FlowJo X software (FLOWJO LLC, Ashland, OR, USA).
RNA from MDSC and tumor tissue was isolated using the QIAzol Lysis buffer together with the RNeasy Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. RNA concentration and rRNA integrity was measured using a Pico 6000 Assay (Agilent Technologies, Ratingen, Germany). RIN values were reached > 7 (Additional file 1: Figure S3A) and total RNA yield was 6.8–350 ng. RNA sequencing library was prepared using the SMARTer® Stranded Total RNA-Seq Kit v2 - Pico-Input Mammalian (Takara, Saint-Germain-en-Laye, France). Briefly, ~ 10 ng RNA was fragmented for 4 min at 94 °C, followed by first-strand cDNA synthesis following adding of Illumina Adapters and Indexes. RNA sequencing library was isolated using AMPure beads, and ribosomal RNA was depleted using ZapRv2 and R-Probes v2. RNAseq library was amplified in 13 cycles and isolated using AMPure beads. Mean tumor cell contamination, as determined by expression of cytokeratin 8 or 18, was < 1%, for tumor-derived MDSC populations (Additional file 1: Figure S3B).
Bioinformatic data analysis
Quality of sequencing reads was assessed using fastQC (http://www.bioinformatics. babraham.ac.uk/projects/fastqc). Reads were mapped against the mouse genome (mm10) and mouse rRNA sequences with ContextMap version 2.7.9 , using BWA  as internal short read aligner and allowing at most 4 mismatches per read. Number of read fragments per gene were determined in a strand-specific manner from mapped RNA-Seq reads, using featureCounts  and Gencode (v16) annotations. Gene expression was quantified as numbers of fragments per kilobase of transcript per million mapped reads (FPKM). Principal component analysis (PCA) was performed in R for all genes with a median FPKM ≥1, for conditions compared. Differential gene expression analysis was performed on gene read counts using DEseq2 for all genes with an average of 25 reads per sample . P-values were adjusted for multiple testing using the method by Benjamini and Hochberg , and genes with an adjusted p-value < 0.001 and at least a 2-fold change in expression (fold-change ≥2 or log2 fold-change ≤1/2) were considered significantly differentially expressed. The RNA-Seq analysis workflow was implemented and run using the workflow management system Watchdog . Gene set enrichment analysis for all genes ranked by gene expression log2 fold-change was performed using GSEA  for MSigDB gene sets (FDR q-value cutoff 0.05):
GSE24102_GRANULOCYSTIC_MDSC_VS_NEUTROPHIL_DN/UP, GSE5099_CLASSICAL_M1_VS_ALTERNATIVE_M2_MACROPHAGE_DN/UP, GSE5099_MONOCYTE_VS_CLASSICAL_M1_MACROPHAGE_DN/UP.
Functional enrichment analysis for up- and downregulated genes was performed using the DAVID webserver , against the background of all genes included in the differential gene expression analysis (adj. p-value < 0.01).
Total RNA was isolated using the peqGold TriFast™ Kit (VWR International GmbH, Darmstadt, Germany) according to the manufacturer’s instructions. cDNA synthesis was done with the RevertAID™ First strand cDNA Synthesis kit (Thermo Fisher Scientific, Karlsruhe, Germany) and qRT-PCR was performed with KAPA PROBES FAST qPCR Maser Mix (2x) Kit (Sigma-Aldrich, Taufkirchen, Germany), on the LightCycler 480 II system (Roche Diagnostics, Penzberg, Germany). Primers were designed with the Universal Probes Library.
T cell suppression assay
For the assessment of suppressive capacity of MDSC or macrophages, a co-culture with T cells was done. For this, 5 × 104 CFSE-labeled T cells (per well) from tumor-naïve C57BL/6 mice were seeded into 96-well plates and co-cultured with 1.25 × 104 (0.25:1), 2.5 × 104 (0.5:1) or 5 × 104 (1:1) MDSC or macrophages. Each well was supplemented with 1 μl anti-CD3/anti-CD28 mAb-coated beads (gibco®, Thermo Fisher Scientific, Karlsruhe, Germany). After 72 h, CFSE dilution of CD4+ and CD8+ T cells was analyzed by flow cytometry. IFN-γ secretion following co-culture was measured from supernatants at an E:T ratio of 1:1, with ELISA (BD OptEIA, BD Biosciences, Heidelberg, Germany).
Antigen presentation assay
To assess antigen presentation of MDSC, 5 × 104 CFSE-labeled OT-I T cells were seeded into 96-well plates and co-cultured with 1.25 × 104 (0.25:1), 2.5 × 104 (0.5:1) or 5 × 104 (1:1) MDSC. Tumor-derived MDSC were co-cultured without further treatment. Splenic MDSC were incubated with 1 μg/ml OVA protein over night at 37 °C or loaded with SIINFEKL (100 μg/ml). Subsequently, MDSC were washed and seeded as described above. After 72 h, CFSE dilution of CD8+ T cells was analyzed by flow cytometry.
Data present means +/− standard error of the mean (SEM) of biological replicates. Significant differences between two groups were calculated using the Mann Whitney U test or if indicated using an unpaired two-sided students t test. Multiple comparisons were analyzed using Kruskal Wallis test. In case of significant results, subsequent post hoc test was calculated for selected comparisons as indicated. Spearman’s rank-order correlation was performed to analyze associations. To analyze the influence of genotype and treatment a 2-way ANOVA was performed. In case of a significant result, post hoc test between treatments was performed as indicated. Statistical analysis was performed using GraphPad Prism software (version 7.04); p-values < 0.05 were considered significant.