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Short peptide vaccine induces CD4+ T helper cells in patients with different solid cancers – desription of methods according to MIATA guidelines (MIATA checklist) Information on…. ….the sample patients techniques ….the assay detailed MFTC assay description HLA types: HLA-A1, HLA-A2, HLA-A3, HLA-A24, HLA-B7 Disease: patients had a variety of different solid survivin expressing cancers Treatment: vaccination with EMD640744 in Montanide ISA 51 VG Co-medication: none see detailed description of the trial in: “Lennerz V, Gross S, Gallerani E, Sessa C, Mach N, Boehm S, et al. Immunologic response to the survivin-derived multi-epitope vaccine EMD640744 in patients with advanced solid tumors. Cancer Immunol Immunother. 2014.” Whole blood collection and PBMC processing was done in the five Swiss study centers by trained personnel; 95 ml whole blood was obtained by venipuncture using LiHeparin blood collection tubes at different time points pre and post vaccination; Maximum time between collection and processing was two hours. PBMC were isolated using Ficoll densitiy gradient centrifugation with Leukosep separation tubes (Greiner Bio-one, #227290); whole blood was not diluted; leukaphereses were diluted 1:2 with PBS/EDTA; PBMC were frozen in FCS/10% DMSO at 1x107/vial using 2propanol-filled cryobox freezing containers (Nalgene, #51000001) at -80°C overnight and afterwards stored in liquid nitrogen until transport to the experimental laboratories in Mainz and Erlangen PBMC were transported from study centers to experimental lab on dry ice within 24 hours Upon arrival, all shipped vials were controlled for completeness and proper condition, and the vials were rapidly transferred to liquid nitrogen for storage until use; (every step of the shipment was monitored, respective data loggers were signed and sent to the study sponsor and a monitoring agency) Samples for direct comparison (i.e. samples from different timepoints of the same patient) were always thawed, stimulated and assayed on the same day to ensure same assay conditions. Vials with 10x106 cells each were thawed, washed and counted (median recovery: 7.4x106 per vial) using a CasyCellCounter (Schärfe Systems). No dead cell determination was performed at this time point. Cells were then seeded in a 12-Well-Plate (2–4 million cells per controls reagents ml) in MLPC-Medium. Cells were then prestimulated with the corresponding peptide or the EMD640744 peptide mix (5-10 g/ ml). The next day, IL-2 and IL-7 were added. Half of the medium was replaced every 3–4 days with fresh MLPC-medium containing IL-2. No IL-2 was given to the in vitro stimulated PBMCs in the last two days before the assay. On day 12 – 15 cells were washed with MLPC medium and stimulated overnight (37°C, 5% CO2) in sterile 5ml FACS tubes (BD) in MLPC medium with or without the corresponding peptide and in some cases blocking antibodies (as indicated in Fig.2) in the presence of BrefeldinA and Monensin and CD107a- and CD154- antibodies. In some experiments peptides were loaded onto HLA-matched or mismatched EBV-transformed lymphoblastic cell lines (EBV-LCLs). Loading was for one hour at room temperature with 5 μg/ml peptide. Cells were then washed and used at a ration of 1:1 in the assay. The next day, cells were washed with PBS and stained with deadcell stain Live/Dead aqua (Invitrogen) according to the manufacturer’s instructions. The cells were then spun down, the supernatant was discarded and the cells were resuspended in PBS containing surface-staining antibodies (CD8, CD4, CD14). After 20 minutes at 4˚C cells were washed with PBS and fixed and permeabilized with fix/perm solution and perm/wash (both eBioscience) according to the manufacturer’s instructions. Intracellular staining was performed in perm/wash with IL2-, TNFalpha- and IFNgamma- antibodies, for 30 minutes. Cells were then washed and resuspended in PBS. For each sample all cells within a tube were acquired. For MFTC assays total events ranged from 1.0 x105 to 4.1 x106 (mean: 1.8 x106). For each sample a negative buffer control (prepared under the same assay conditions but without peptide) was assessed. MLPC-Medium (RPMI1640 with 10% pretested human pooled serum (Lonza), gentamycin, pyruvate and nonessential amino acids) Survivin-peptides (Bachem) had a purity of >95% HLA-A1-binding FTELTLGEF, Sur93-101/T2 HLA-A2-binding LMLGEFLKL, Sur96-104/M2 HLA-A3-binding RISTFKNWPK, Sur18-27/K10 HLA-A24-binding STFKNWPFL, Sur20-28 HLA-B7-binding LPPAWQPFL, Sur6-14 IL-2 (5 U/ml, Roche) and IL-7 (10 ng/ml, TEBU) BrefeldinA (5ng/ml) and Monensin (750 pg/ ml), both from Sigma Antibodies (vendor and concentration or dilution): anti-HLA-ABC (BD, DX17, 10 μg/ ml), anti-HLA-DR/DP/DQ (BD, Tu39, 10 μg/ ml), anti-HLA-DR (Biolegend, L243, 10 μg/ ml), anti-HLA-DQ (Beckman Coulter, SPVL3, 10 μg/ ml), anti-HLA-DP (abcam, B7/21, 10 μg/ ….data acquisition Materials/ Equipment Machines/ Software Calibration ….results Gating Response criteria Auditing Correlation between data sets ….the lab environment SOPs Qualification/ Training of personnel ml), anti-CD107a-FITC (BD, 1:50), anti-CD154-PE (BD, 1:50), antiCD8-PerCP (BD, 1: 10) and anti-CD4-V450 (BD, 1:20), anti-CD14PacificOrange (Invitrogen, 1:30), anti-IL2-APC (BD, 1:25), antiTNFalpha-PE-Cy7 (BD, 1:20) and anti-IFNgamma-AlexaFluor700 (BD, 1:30) cell incubators, centrifuges and laminar flow cabinets were state-of-the-art devices for the sterile handling of human cell material The Flow Cytometry assays were aquired on a FACS Canto II Flow Cytometer (BD Biosciences) equipped with FACS Diva (6.1) software; for Flow Cytometry analyses, FlowJo 9.7.5 was used; the FACS Canto II Flow Cytometer was calibrated according to the manufacturers recommendations on a regular basis (CS&T Performance Check on every day of usage); The gating strategy is shown in Supplemental Figure 1 The values obtained from negative controls were subtracted from the corresponding test samples. Criteria for a positive response were: results of individual cytokine-producing subsets at least two times higher than the corresponding subsets in the negative control (background), and the sum of all cytokine-producing subsets greater than 0.03%. Representative example data for an MFTC assay, including gating strategy, can be seen in Supplemental Figure 1. All plots were audited and cells within the cytokine gates were regularly checked by backgating A correlation of the immune response data with disease stage of the patients and treatment was not analyzed since objective clinical response rates are generally low after cancer vaccination, clinical efficacy can only be reasonably assessed in an appropriately designed trial with a time-toevent endpoint. Standardized and validated working practices were in place for cell thawing, counting, stimulation, the MFTC assay (staining and data aquistion including quality-control for Canto II) and analysis Only well trained personnel with more than five years’ experience with all methods and technologies performed the experiments and data analyses as well as interpreted and reported results;