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Supplemental Information Supplemental information Supplemental Material and Methods Isolation of mononuclear cells from the frest ovarian tumor specimens Fresh ovarian tumor specimens were minced with scissors, digested in PBS containing 1 mg/mL of Collagenase D (Roche) and 100μg/mL DNase I at 37°C for 30 min, mechanically dissociated using the gentleMACS dissociator (Miltenyi Biotec) and passed through a 100µm nylon cell strainer (BD Biosciences). Degranulation and IFNγ production after in vitro stimulation Mononuclear cells isolated from fresh tumor specimens were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) + 1 μg/mL ionomycin in the presence of anti-CD107a FITC monoclonal antibody (BioLegend) for 1h followed by 3h incubation with brefeldin A (BioLegend). Unstimulated cells were used as control. Cells were then washed in PBS, stained with anti-CD45 PerCP (EXBIO), anti-CD3 Alexa Fluor 700 (EXBIO), anti-CD4 ECD (Beckman Coulter) and anti-CD8 HV500 (BD Biosciences) monoclonal antibodies, fixed in fixation/permeabilization buffer (eBioscience), further permeabilized with permeabilization buffer (eBioscience) and stained with anti-IFNγ PE-Cy7 (eBioscience), anti-GZMB Brilliant Violet 421 (BD Biosciences) and anti-PRF1 APC (BioLegend) monoclonal antibodies. The percentage of CD3+CD8+ T cells producing IFNγ and degranulating upon PMA/ionomycin stimulation were determined by flow cytometry. The data were analyzed with the FlowJo software package (Tree Star, Inc.). “Expression profile of co-inhibitory receptors on CD8+ T cells upon exposure to rIFNγ in vitro” Mononuclear and malignant cells were isolated from fresh HGSC specimens and cultured with rIFNγ under different conditions (Supplementary Fig. 6A). (1) Malignant cells and leukocytes were co-cultured in presence or absence of 20ng/mL rIFNγ for 24 hours. (2) Malignant cells were isolated from freshly resected HGSC samples by positive selection on EpCAM+ cells using Human EpCAM kit (EasySepTM). Population of isolated leukocytes were separately cultured with or without rIFNγ for 24 hours. (3) Malignant cells and leukocytes separated using positive selection on EpCAM+ cells by Human EpCAM kit (EasySepTM) were co-cultured in conditions of transwell separation (no physical contact) with or without rIFNγ for 24 hours. Furthermore, cells were washed in PBS, stained with anti-CD45 PE-Texas Red (Life Technologies), anti-CD3 A700 (EXBIO), anti-CD4 PE-Cy7 (eBioscience), anti-CD8 HV500 (BD Biosciences), anti-PD-L1 BV421 (BioLegend), anti-PD-1 FITC (BioLegend), anti-CTLA4 APC (BioLegend) monoclonal antibodies. The percentage of CD45+CD3+CD8+PD-L1+, CD45+ CD3+CD8+PD-1+ and CD45+CD3+CD8+CTLA-4+ were determined by flow cytometry. The data were analyzed with the FlowJo software package (Tree Star, Inc.). Library preparation and sequencing Twenty FFPE samples suitable for library preparation according to quality/quantity evaluations were processed following the manufacturer’s specifications with minor modifications by using the Illumina TruSeq® RNA Access Library Prep (Illumina) along with purification steps employing SPRI beads. DV200 values for all samples exceeded 40%, and 100 ng of total RNA was used for the cDNA synthesis. Each library was quantified with the fluorimeter Qubit 2.0 (dsDNA HS kit; Thermo Fisher), and the size distribution was determined using a DNA 1000 kit on a 2100 Bioanalyzer instrument prior to pooling. All libraries had a similar size distribution of approximately 260 bp. A 4-plex pool of libraries was made by combining 200 ng of each DNA library. The libraries were sequenced on an Illumina NextSeq 500. At least 180 M pair-end 2x75 bp reads were generated per library. The libraries were prepared and sequenced at EMBL Genomics Core Facility (Heidelberg, Germany). NGS data processing The raw FASTQ sequencing files were aligned to human reference genome (build h19) with bowtie2 (version 2.3.2) and tophat2 (version 2.1). The levels of expression as raw “counts” were calculated from aligned reads with mapping quality at least 10 using htseq-count (version 0.6.0). Count files were transferred to the GALAXY environment (Galaxy version 18.01), and differentially expressed genes (DEGs) between groups were determined using DEseq2 (Galaxy Version 2.11.40.1) with default settings (Love et al, Genome Biology, 2014). Supplemental figures Supplemental Figure 1. Experimental design of the study. Supplemental Figure 2. Tumor infiltration by CD8+ T cells, CD20+ B cells and DC-LAMP+ cells correlate with prolonged survival in patients with HGSC. (A) Representative images of CD8, CD20 and DC-LAMP immunostaining. Scale bar = 50 µm. RFS (B) and OS (C) of 80 patients with HGSC who did not receive neoadjuvant chemotherapy, upon stratifying patients based on median density of CD8+, CD20+ and DC-LAMP+ cells in the tumor microenvironment. Survival curves were estimated by the Kaplan-Meier method, and differences between groups were evaluated using log-rank test. Number of patients at risk are reported. RFS (D) and OS (E) of HGSC patients who did not receive neoadjuvant chemotherapy, upon stratification based on median density of CD8+ cells plus CD20+ cells and CD8+ cells plus DC-LAMP+ cells, respectively. Supplemental Figure 3. Representative images of PD-L1 (scale bar = 100 μm) and PD-1 (Scale bar = 50 μm ) immunostaining. Supplemental Figure 4. Representative images of CTLA4 (scale bar = 50 μm) and LAG- 3 (Scale bar = 50 μm ) immunostaining. Supplemental Figure 5. PD-L1 levels and infiltration by PD-1+, CTLA4+ and LAG-3+ cells were heterogeneous across samples but did not differ based on site of assessment. PD-L1 levels and density of PD-1+, CTLA-4+ and LAG-3+ in the tumor stroma and tumor nest (A) and among different pathological disease stages (B) of patients with HGSCs (n=80). Supplemental Figure 6. PD-L1, PD-1 and CTLA4 expression on freshly isolated CD8+ T cells after exposure to recombinant IFNγ. (A) Experimental design of study. (B) Gating strategy for CD8+ T cells. The percentage of cells in each gate is reported. (C) Percentage of PD-L1+, PD-1+ and CTLA4+ CD8+ T cells from 9 HGSC patients before and after rIFNγ treatment as determined by flow cytometry. Supplemental Figure 7. Representative images of 3 different samples of PD-1Lo/CD8Lo, PD- 1Hi/CD8Hi, PD-1Lo/CD20/DC-LAMPLo and PD-1Hi/CD20/DC-LAMPHi immunostaining. Scale bar = 100 μm. Supplemental Figure 8: The combined prognostic impact of CD8+ T cells and PD-1+, LAG- 3+ and CTLA4+ cells in HGSC. RFS and OS of HGSC patients who did not receive neoadjuvant chemotherapy, upon stratification based on median density of CD8+ T cells and PD-1+ cells (A), CD8+ T cells and LAG-3+ cells (B) and CD8+ T cells and CTLA4+ cells (C). Survival curves were estimated by the Kaplan-Meier method, and differences between groups were evaluated using log-rank test. Number of patients at risk are reported. (D) Fold change of CD8+ IFNγ+ T cells from 4 TIM-3Lo versus 4 TIM-3Hi patients after incubation with anti-PD-1, anti-TIM-3, anti-CTLA4 antibodies, alone or in combination. Study group 2 Variable (n=20) Age: Mean age (y) ± SEM 60.9 ± 2 Range 44-78 pTNM stage: Stage I 0 (0%) Stage II 6 (30%) Stage III 14 (70%) Debulking R0 8 (40%) R1 1 (5%) R2 11 (55%) Supplemental table 1. Main clinical and biological characteristics of 20 HGSC patients in which the freshly resected tumors were analyzed using flow cytometry (Study group 2). Target Parameter Source Producer Clone Retrieval Detection system Revelation Dilution Incubation solution time (min) CD8 rabbit Spring Bioscience SP16 pH8 EnVision™+/HRP, Rabbit DAB+ substrate Chromogen system 1:80 30 ImmPRESS-AP anti-mouse ImmPACT Vector red Alkaline CD20 mouse Dako L26 pH8 1:250 60 IgG (alkaline phosphatase) Phosphatase substrate kit Santa Cruz CTLA-4 mouse F-8 pH9 EnVision™+/HRP, Mouse DAB+ substrate Chromogen system 1:200 120 Biotechnology, Inc 1010E1.0 donkey anti-rat IgG-biot DC-LAMP rat Dendritics pH8 DAB+ substrate Chromogen system 1:80 60 1 (Jackson ImmunoResearch) Impress HRP anti-mouse IgG LAG-3 mouse Novus Biologicals 17B4 pH9 DAB+ substrate Chromogen system 1:150 90 (Peroxidase) Polymer Impress HRP anti-mouse IgG PD-1 mouse Abcam NAT105 pH9 DAB+ substrate Chromogen system 1:50 60 (Peroxidase) Polymer Impress HRP anti-rabbit IgG PD-L1 rabbit Ventana SP142 pH6 DAB+ substrate Chromogen system 1:50 60 (Peroxidase) Polymer Supplemental Table 2. The list of antibodies used for IHC staining. Parameter Source Producer Clone Fluorochrome Dilution CD107a mouse BioLegend H4A3 FITC 8:100 CD3 mouse EXBIO MEM-57 Alexa 700 5:100 CD4 mouse eBioscience RPA-T4 PE-Cy7 5:100 Beckman CD4 mouse SFCI12T4D11 ECD 5:100 Coulter Life CD45 mouse HI30 PE-Texas Red 6:100 technologies CD45 mouse EXBIO MEM-28 PerCP 6:100 CD8 mouse BD Biosciences RPA-T8 HV500 5:100 CTLA4 mouse BioLegend L3D10 APC 6:100 Granzyme mouse BD Biosciences GB11 BV421 4:100 IFNg murine eBioscience 4S.B3 PE-Cy7 1:100 PD-1 mouse BioLegend EH12.2H7 APC 6:100 PD-1 mouse BioLegend EH12.2H7 FITC 6:100 PD-1 mouse BioLegend EH12.2H7 PB 6:100 PD-L1 mouse BioLegend 29E.2A3 BV421 4:100 Perforin mouse BioLegend dG9 APC 4:100 TIM-3 mouse BioLegend F38-2E2 PE 3:100 Supplemental table 3. The list of antibodies used for flow cytometry. Myeloid CD8 Cytotoxic Monocytic T cells B lineage NK cells dendritic Neutrophils Endothelial cells Fibroblasts T cells lymphocytes lineage cells CD28 CD8B CD8A BANK1 CD160 ADAP2 CD1A CA4 ACVRL1 KDR COL1A1 CD3D EOMES CD19 KIR2DL1 CSF1R CD1B CEACAM3 APLN MMRN1 COL3A1 CD3G FGFBP2 CD22 KIR2DL3 FPR3 CD1E CXCR1 BCL6B MMRN2 COL6A1 CD5 GNLY CD79A KIR2DL4 KYNU CLEC10A CXCR2 BMP6 MYCT1 COL6A2 CD6 KLRC3 CR2 KIR3DL1 PLA2G7 CLIC2 CYP4F3 BMX PALMD DCN CHRM3-A5 KLRC4 FCRL2 KIR3DS1 RASSF4 WFDC21P FCGR3B CDH5 PEAR1 GREM1 CTLA-4 KLRD1 IGKC NCR1 TFEC HAL CLEC14A PGF PAMR1 FLT3LG MS4A1 PTGDR KCNJ15 CXorf36 PLXNA2 TAGLN ICOS PAX5 SH2D1B MEGF9 EDN1 PTPRB MAL SLC25A37 ELTD1 ROBO4 MGC40069 STEAP4 EMCN SDPR PBX4 TECPR2 ESAM SHANK3 SIRPG TLE3 ESM1 SHE THEMIS TNFRSF10C FAM124B TEK TNFRSF25 VNN3 HECW2 TIE1 TRAT1 HHIP VEPH1 VWF Supplemental Table 4.
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