The study was approved by the competent regulatory authority and independent ethics committee. UV1 consists of three synthetic long peptides derived from the active site of telomerase reverse transcriptase (hTERT 660–689 termed p719-20; hTERT 691–705 termed p725; hTERT 651–665 termed p728). A total of 300 µg of lyophilized peptides in equimolar amounts was reconstituted in water for injection and administered intradermally to the lower abdomen. Granulocyte–mac - rophage colony-stimulating factor (GM-CSF, sargra - mostim) (Leukine, Sanofi Aventis, Bridgewater, NJ, US) was used as a vaccine adjuvant at 75 µg and was injected intradermally at the same site 10–15 min before UV1. Ipilimumab (3 mg/kg) was administered according to the label for up to 4 infusions. Patients received up to nine UV1 vaccinations, initiated one week prior to the first dose of ipilimumab. Immune response assessment Peripheral blood mononuclear cells (PBMCs) were iso - lated from whole blood samples (50 ml in acid dextrose tubes) at baseline and at frequent intervals during the treatment and long-term follow-up periods, as previously described [29]. Briefly, vaccine-specific T cell immune responses were assessed using a standard prolifera - tion assay (3H-Thymidine incorporation). PBMCs were pre-stimulated in vitro for 10–12 days with a mixture of vaccine peptides at 10 μM. After pre-stimulation, the cells were re-stimulated for 48 h with or without 10 μM vaccine peptides using irradiated autologous PBMCs as antigen-presenting cells, and tested in triplicate for pro - liferation by 3H-thymidine incorporation. The stimu - lation index (SI) was calculated by dividing the mean proliferation count in vaccine peptide-stimulated wells by the mean proliferation count in unstimulated wells. A three-fold increase in proliferation towards any of the three peptides, or a mixture of these, was considered an immune response-positive sample. Staphylococcus aureus enterotoxin C3 (SEC3) was used as the positive control in the immune response assay. The reported SI values were those observed during the treatment period, defined as up to 16 weeks after the last vaccination. TCR sequencing DNA was extracted from PBMC samples of all patients at baseline and up to two time points thereafter using the GenElute Mammalian Genomic DNA Kit (Sigma Aldrich), according to the manufacturer´s instructions (Additional file 1: Table S1). In addition, DNA extrac - tion was performed on stimulated PBMC samples after a 10-day in vitro stimulation (N01, N02, N07, and N09) and biopsies from patients N02 and N03. The T cell receptor beta (TRB) locus was amplified from up to 250 ng of genomic DNA, as described pre - viously [36, 37]. Briefly, a TRB repertoire library was generated using two consecutive polymerase chain reac - tions. First, the rearranged TRB locus was amplified and sample-specific barcodes were added to the amplicons. Library concentrations and sizes were determined using a Qubit (Thermo Fisher) and Bioanalyzer (Agilent), respectively. The final library was sequenced on an Illu - mina MiSeq with the MiSeq Reagent Kit v3 (600-cycles) chemistry. TCR profiling and processing Sequencing quality was assessed before and after reper - toire sequencing data processing with IGX Inspect (IGX Platform 3.0.6 August 2021), a quality control application designed for immune receptor sequencing data. Checks included standard fastq quality metrics, such as average read quality, Q30 scores, as well as V and J gene align - ment distributions and read fate, and a percentage break - down of the receptor extraction status of raw reads. All the samples had good sequence quality and a high per - centage of reads with successfully extracted receptors (all > 95%, except for one sample with 90% and one with 70%). Raw fastq files were processed using the IGX Pro - file (IGX Platform 3.0.6 August 2021), a tool that parses immune receptor structural components by aligning germline genes and adaptively correcting errors based on the overall sample quality. The IGX Profile provided Page 4 of 13 Ellingsen et al. Journal of Translational Medicine (2022) 20:419 receptor annotation with complementarity-determining region 3 (CDR3) sequences, V and J gene assignments, functionality, alignment scores, and quality information. All receptors with the same CDR3 amino acid sequence were considered instances of the same clone and all anal - yses were performed at the clone level. Count normalization of TCR repertoires was per - formed by downsampling. Identification of significantly expanded clones was performed using EdgeR [38]. The expanded clones already present in the baseline sample were filtered out. A more detailed description of these methods is provided in the Additional file 1. Multiplex immunofluorescence staining Biopsies were harvested at baseline from nine patients and at week 12–15 from five patients (Additional file 1: Table S2). One part of the biopsy was snap-frozen in liq - uid nitrogen and stored at − 80 °C, whereas the other was formalin-fixed and paraffin-embedded (FFPE). FFPE biopsies were used for multiplex immunofluorescence staining. Biopsy Sects. (4 µm thick) were stained using a custom-based 5-color IHC kit (Akoya Biosciences, Marl - borough, MA, USA) and the fully automated Leica Bond RXm (Leica Biosystems, Buffalo Grove, IL, USA). The slides were deparaffinized, rehydrated, and rinsed with distilled H2O. Antigen retrieval and removal of antibod - ies from the previous cycles were performed by boiling at 95 °C at pH 9 (first cycle) or pH 6 (all remaining cycles). For multiplex immunofluorescence staining, a panel of immune markers was developed using antibod - ies against CD4 (rabbit/ERP6855, Abcam, 1:80), CD8α (mouse/144B, Invitrogen/MA5-13,473, 1:100), PD-L1 (rabbit/E1L3N, Akoya, ready to use), and TERT (rabbit/ ab230527, Abcam, 1:400). A cocktail of two antibod - ies was used to identify the melanoma cells: anti-Sox10 (rabbit/EP268-1, Akoya, ready to use) and anti-S100 (mouse/4C4.9, Akoya, ready to use). Staining was devel - oped using amplification HRP-polymer systems and Opal fluorophore dyes (see Additional file 1: Table S3). To visualize the cell nuclei, the tissue was stained with 4′,6-diamidino-2-phenylindole (Spectral DAPI, Akoya). The slides were mounted with Prolong Diamond Anti - fade Mountant (Thermo Fisher, Waltham, MA, USA) and imaged at × 20 magnification using the Vectra® Pola - ris™ Automated Quantitative Pathology Imaging Sys - tem (Akoya Biosciences, Marlborough, MA, USA). Each image was manually reviewed and curated by a patholo - gist to exclude artifacts and staining defects. Whole‑exome and RNA sequencing and downstream analyses Snap-frozen biopsies were disrupted on a TissueLyser LT, followed by DNA extraction using the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen, Hilden, Ger - many). RNA extraction was performed using a GenEl - ute™ Total RNA Purification Kit (Merck). Biopsy DNA and RNA extracts were obtained from nine patients at baseline and five at week 12–15. Whole-exome sequencing (WES) was performed as previously described in Aamdal et al. [33]. Briefly, 1 µg of DNA was used as the starting material for exome library preparation using the Agilent AllExome V5 kit, according to the manufacturer’s protocol. Sequencing was performed pair-ended, generating approximately 90 M PE reads per tumor and 40 M PE reads per nor - mal, using sequencing by synthesis chemistry on a HiSeq4000 system. Variant calling was performed as previously described [33]. Tumor mutational burden (TMB) was defined as the number of non-synonymous variants with an allelic frequency of > 5% per megabase. RNA samples were processed using an Illumina TruSeq stranded mRNA kit with 100 ng as the start - ing material. RNA sequencing was performed on the NextSeq500 using two HighOutput flow cells with 75 bp single-read sequencing. Hierarchical clustering of the genes included in the IFN-γ signature [8 ] was per - formed using Euclidean distance with the Morpheus tool (https:// softw are. broad insti tute.