Isolation of a tumor neoantigen specific CD8+ TCR from a skin biopsy of a vaccination site
The use of adoptive T-cell therapy for cancer treatment has shown great potential. This approach involves the transplantation of tumor-infiltrating lymphocytes or genetically modified T cells with novel T-cell receptors, obtained either from the patient’s own cells or from a donor. It has yielded long-lasting responses and even complete remission in certain cases. Therefore, the effective, rapid and safe acquisition of tumor antigen-specific T cells has become a central focus in clinical research. The classical approach, known as TIL therapy, utilizes expanded T cells from the bulk tumor as a direct cell therapy administered into the patient after an immunodepleting preparative regimen. Recently, FDA approved the first TIL therapy, lifileucel, for the treatment of unresectable or metastatic melanoma. , This is the first approval of a cell therapy for the treatment of solid tumors and exemplifies how cell therapies are gaining ground beyond hematologic malignancies. However, there are still obstacles to overcome associated with the TIL therapy. One major drawback is that the tumor-specific populations are terminally differentiated and dysfunctional, resulting in less efficient expansion. To overcome this T-cell dysfunction, researchers are currently developing genetically modified peripheral blood lymphocytes (PBL) expressing transgenic T-cell receptor (tg-TCR) sequences. These TCR sequences are obtained from tumor antigen-specific T cells, with current approaches aiming to identify neoantigen-specific T cells in either TIL or the peripheral blood repertoires. However, several challenges hinder the development of TCR-transduced T-cell therapy in clinical settings. First, TIL screening requires substantial amounts of fresh tumor tissue, which may not always be safely accessible. Second, isolating neoantigen-specific cells from the blood of patients has proven to be challenging, necessitating highly sensitive isolation methods due to the low frequency of neoantigen-reactive T cells in the periphery, even in cancers with high tumor mutational burden (TMB) such as melanoma. In common epithelial cancers, the average number of mutations is lower, leading to a limited repertoire of neoantigen-reactive tumor-infiltrating lymphocytes. Recent approaches to enrich neoantigen-reactive cells from the PBL compartment of cancer patients include PD-1-positive (PD-1 + ) or CD45RO + memory enrichment of CD8 + T cells, as well as MHC multimer-based isolation. However, even within the PD-1 + /memory subset, only a small fraction of T cells appear to be tumor-specific. Consequently, these methods rely on an additional ex vivo enrichment step, involving additional purification through the isolation of T cells that express activation markers upon co-culture with tumor antigen-presenting cells. Nevertheless, these methods have proven to be inefficient for certain T-cell populations with exhausted phenotypes and dysfunctional proliferation, as well as suffer from contamination by bystander cells (and our own data). Prior studies have demonstrated that vaccination can be employed to generate matured neoantigen-specific T cells, resulting in increased neoantigen reactivity and T cell clonotype diversity. , Interestingly, vaccination has been proven necessary to develop de novo neoantigen-specific CD8 + T cells despite the presence of neoantigens, suggesting ineffective neoantigen cross-presentation as a primary mechanism for immunological ignorance. , Therefore, stimulating T-cell responses through vaccination may aid in the discovery of TCRs against neoantigens. In the present study, we explore lymphocytes from a vaccination site, referred to as vaccination site infiltrating lymphocytes (VILs), with the aim of developing a strategy to facilitate the isolation, expansion, and study of mutated antigen-specific T cells. In the tissue, we found MHCII-expressing CD11c + cells and CD8 + T cells with a PD1 + TOX + KI67 − phenotype, compatible with a tissue-resident memory like T cell (Trm-like). The dominant clone identified in the VILs was neoantigen-specific and retained the proliferative capacity to survive traditional in vitro expansion. Consequently, it holds improved potential as a rapid, easy to obtain and safe starting material for direct cell therapy products or for TCR screening methods. Of note, a validated neoantigen reactive clone was not found in the PBL compartment, supporting their tissue residency. To the best of our knowledge, this study represents the first successful isolation of private mutation-reactive T cells from patients’ vaccination sites, providing a potential foundation for designing personalized immunotherapies to treat patients with advanced cancer. 

Biomaterial Whole genome and transcriptome sequencing of tumor samples and germline as well as longitudinal sampling of liquid biopsies has been performed by the CATCH ( C omprehensive A ssessment of clinical fea T ures and biomarkers to identify patients with advanced or metastatic breast C ancer for marker driven trials in H umans) precision oncology platform, which is a non-interventional, prospective registry that uses genomics and transcriptomics to guide therapeutic decisions in the clinical management of metastatic breast cancer. Methods and workflow has been carried as previously described. The study protocol was approved by the Institutional Review Board of the University Hospital of Heidelberg (UKHD) (approval number S-164/2017). In addition, the patient gave written informed consent for compassionate use treatment of a personalized vaccine in the context of the “Pilot project for the development of personalized cellular immunotherapies targeting solid cancers” (PIKT). Additional written informed consent was obtained for the collection of and investigational use of blood and tissue samples (S-207/2005). All activities were conducted in compliance with the declaration of Helsinki. Sequence data analysis and neoepitope prediction DNA and RNA from tumor tissue and corresponding blood-derived non-tumor DNA were profiled using whole-genome and transcriptome sequencing as previously described. Non-synonymous point mutations in tumor tissues, resulting from single-nucleotide variants, were identified and compared with matched non-tumor DNA from the blood. Somatic single nucleotide variants (SNVs) were identified based on DNA sequencing data (WGS), and the quantification of gene expression based on RNA sequencing data was performed with the DKFZ Omics Data Core Facility supported analyses module “One touch platform (OTP)”. Neoepitopes were predicted with our in-house pipeline utilizing netMHCpan4.1 and netMHCstabpan1.0 for MHC I epitope prediction, according to patient-specific HLA type, which are predicted by OptiType and Kourami. Prediction results are ranked by binding affinity, binding stability and mRNA expression levels. Personalized vaccine treatment Seventeen potential neoepitopes presented in the context of the patient’s haplotypes were selected to produce a personalized peptide vaccine. Selection was based on their binding predictions to matching HLAs and the level of expression of the mutant genes. After selection of the minimal epitopes, extended peptides were used to improve the vaccine immunogenicity. Long-peptides spanning upstream and downstream amino acids from the mutation site were synthesized as 25-mers by Fmoc chemistry using a fully automated multiple synthesizer (Syro II, MultiSynTech, Germany). Synthesis was carried out on preloaded Wang-resins with 2-(1 h-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) as coupling agent. Montanide ISA51 VG (SEPPIC GmbH) was used as an adjuvant to form a water-in-oil emulsion, mixed with the peptide solution containing 60 µg per peptide each (aqueous phase, 10% DMSO) in a 50:50 ratio. The treatment schedule consisted of eight cycles of intra-dermal vaccinations and is shown in . Multiplexed ion beam imaging by time of flight (MIBI-TOF) We designed a MIBI-TOF antibody panel to analyze the vaccinated lesion. To do so, we included antibodies to quantify nuclear staining, lineage markers to discriminate immune cells, immunoregulatory proteins and cytokeratin to distinguish epithelial cells (listed in Supplementary Table S1). Briefly, primary antibodies were conjugated to isotopic metal reporters as described previously. Tumor tissue was sectioned at 5 μm on conductive microscopy slides. FFPE tissues are deparaffinized and rehydrated with sequential washes. After antigen retrieval, slides were stained over night following standard procedures and dried under vacuum prior to imaging. Imaging was performed using the MIBI-TOF instrument (IonPath) which utilizes a bright ion source and orthogonal time-of-flight mass spectrometry to image metal-tagged antibodies in previously selected 800 × 800 μm field of views (FOVs). Finally, pixel data were converted to TIFF images. Prior to downstream analysis, images were processed through a pipeline (Toffy), as described previously, in order to subtract background and isotopic impurities, as well as to compensate for channel crosstalk. Next, the images were segmented using DeepCell, a convolutional neural network (CNN) for whole cell segmentation. After segmentation, cells were clustered using FlowSOM. , In order to assign each cell a lineage, well-established combinations of lineage markers were used as described previously, followed by subclustering of immune cell types, including CD4 T cells (CD4T; CD3+, CD4+, CD8-), CD8 T cells (CD8T; CD3+, CD8+, CD4-), B cells (B; CD45+, CD20+), NK cells (NK; CD3-CD56+), macrophages (Macrophages; CD45+,CD68+), myofibroblasts (Myofibroblasts; CD45-SMA+) and dendritic cells (DC; CD45+CD11c+). UMAPs were plotted using Python package Scanpy and spatial graphs were plotted using Python package Squidpy. Establishing of VILs culture VILs cultures were established using the ‘young-TIL’ protocol with slight modifications. In brief, fresh biopsies from the vaccination site were cut into small pieces of around 2 mm and placed individually in each well of 24-well culture plates. The plates were filled with T cell medium comprising AIM-V (Thermo Fisher Scientific) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin and 5% human serum (Sigma-Aldrich) and 6000 IU/mL IL-2 (Miltenyi Biotec). After 24 h, half of the medium was replaced with fresh medium containing IL-2. The plates were visually inspected every few days, and when the cells reached confluence, they were divided into new cultures. On day 12, all the wells were pooled and VILs were immunophenotyped by FACS and assayed for specific activity by ELISpot. Remaining cells were subjected to in vitro stimulation, as described below. INF-gamma screening by ELISpot assay To obtain APCs, autologous B cells were isolated by magnetic positive selection using CD19 MicroBeads (Miltenyi Biotec) and expanded with the Human CD40-Ligand Multimer Kit (Miltenyi Biotec) under manufacturer's recommendation in B cell media with 50UI/ml Human IL-4 (Miltenyi Biotec), 10UI/ml Human IL-21 (Miltenyi Biotec) and 30 ng/ml BAFF (Biolegend), hereafter, CD40-B cells. To test VILs reactivity CD40-B cells were pulsed ON with 10 µg/ml long peptides or short peptides and co-cultured the next day with VILs in filter plates with 0.45 um pore size Mixed Cellulose Esters membrane (Millipore), plating 100,000 B cells and 20,000 VILs or T cells per well in triplicates or duplicates, in screening or validation assays, respectively. Anti-CD3 coated wells (10 µg/ml, clone OKT3, Biolegend) were used as positive controls. To test T cell reactivity in the peripheral blood, 500,000 PBMCs were plated, directly pulsed with long peptides at 10 µg/ml and incubated during 48hs and the positive control wells were stimulated with SEB at 0.1 µg/ml. Viral peptide pools (PepTivator CEF MHC Class I Plus, Miltenyi Biotec) as internal controls and negative controls (unstimulated cells and wells with only media) were included in every assay. INF-gamma detection was performed using Human IFN-γ ELISpot basic kit (ALP) (Mabtech) following manufacturer's protocol. Images were acquired and analyzed with ImmunoSpot® Analyzer (CTL). Population profiling and functional analysis by FACS Cells were washed with PBS and stained for dead exclusion markers with Zombie Aqua Fixable Viability Kits (Biolegend) for 15 min at RT protected from light exposure. After a wash with FACS buffer containing 0.5% BSA and 2 mm EDTA, cells were stained with the antibody cocktails for suitable markers. Anti-human antibodies used were purchase from Biologend: APC/Fire™ 750 anti-human CD3 (clone UCHT1), Brilliant Violet 570™ anti-human CD4 (cloneRPA-T4), Brilliant Violet 785™ anti-human CD8 (clone SK1), Brilliant Violet 510™ anti-human CD56 (clone HCD56), Brilliant Violet 421™ anti-mouse TCR β chain (clone H57–597), PE anti-mouse TCR β chain (clone H57–597), PE anti-human CD137 (clone 4B4–1), PE/Cyanine7 anti-human CD134 (OX40) (clone Ber-ACT35), and APC anti-human CD69 (clone FN50). Tetramer staining was performed based on complexed easYmers (ImmunAware) as described previously. For blocking HLA-I blockade control in functional assays, anti-HLA-ABC (clone W6/32, Biotrend) was added 30 min before the co-culture to pulsed B cells at 50 µg/ml. In vitro stimulation (IVS) of VIL cells In vitro stimulations were performed as previously described, with slight modifications. CD40L-B cells were pulsed ON with long peptides and co-cultured the next day with enriched CD3+ (Pan T cell isolation kit, Miltenyi Biotec) or CD8+ (REAlease® CD8 MicroBead Kit, Milenyi Biotec) VILs in T cell medium containing IL-21 30 ng/ml. Cells were incubated at 37°C for 72 h, after which time the cultures were fed with fresh T cell medium containing IL-21 30 ng/ml, IL-2 3000 IU/ml, IL-15 2.5 ng/ml, IL-7 2.5 ng/ml. The splits were performed after 72 h. After the second split, VIL reactivity was rechecked. The complete scheme of the cultures is shown in Suppl. Figure S1. Peptide binding stabilization of HLA-I molecules by peptide-HLA-I complex formation assay Short peptides with binding predictions to patient’s HLAs (HLA-A *02:01, HLA-A *03:01, HLA-B * 07:02, HLA-B * 15:01, HLA-C * 03:03, HLA-C * 07:02), were synthetized and tested in a cytometry-based assay of peptide-HLA-I complex formation (easYmer, ImmunAware) performed according to manufacturer’s instructions. Percentage of stabilization for each peptide was calculated by setting 0% to the fluorescence intensity of negative controls (no peptide) and 100% to the positive control peptides provided in the kit by ImmunAware for each allele. Positive controls for evaluation analysis of peptide-HLA folding were: CMV pp65 495–503 (NLVPMVATV), a known HLA-A *02:01 binder; CMV IE1 99–107 (RIKEHMLKK), a known HLA-A *03:01 binder; CMV pp65 417–426 (TPRVTGGGAM), a stable HLA-B * 07:02 binder; YFV NS3 73–18 (SVKEDLVAY), a stable HLA-B * 15:01 binder; Burkholderia mallei N-ethylmaleimide reductase 137–145 (YVTPRALEL), a stable HLA-C * 03:03 binder; E. coli DH10B conserved protein 202–210 (FRAPNTREL), a known HLA-C * 07:02 binder. TCR sequencing and analysis from VILs and PBL TCR sequencing (TCR-seq) was performed from cryopreserved VILs and PBMC at Genewiz (Azenta). Briefly, single cell TCR sequencing of VDJ regions was performed using the Chromium Single Cell Immune Profiling kit (10× Genomics), followed by libraries sequencing on a MiSeq (Illumina). Analysis of full-length V(D)J sequences for paired T-cell receptors was performed using the Loupe VDJ Browser v4.0.0 (10× Genomics). TCR cloning, lentivirus production and transduction of T cells The retrieved VDJ sequences of the alpha and beta chains of TCR-1131 and TCR-1132 were fused in silico to the sequences of the respective murine constant alpha and beta TCR domains in order to propagate an exclusive pairing of the chosen sequences to form the artificial TCR and thereby avoiding a mispairing with endogenously expressed TCRs of donor T cells, which might generate a novel unpredictable specificity of the T cell product. The full-length alpha and beta chains were then linked by a P2A cleavage sequence that allows the combination of multiple gene products into one linear vector sequence. The obtained sequences of TCR-1131 and TCR-1132 were then synthesized by GeneArt and cloned into the backbone of a self-inactivating lentiviral vector. Subsequently, lentiviral particles containing TCR-1131 and TCR-1132 were produced by standard transfection methods. Primary patient T cells were isolated from PBMCs (Pan T Cell Isolation Kit, Miltenyi Biotec) and activated using TransAct reagent and TexMACS media containing IL-7 and IL-15 (all Miltenyi Biotec). Lentiviral transduction was performed 2 d post activation. TCR repertoire analysis from RNA sequencing data TRUST4, an open-source algorithm for reconstructing immune receptor repertoires in αβ/γδ T cells from RNA sequencing (RNA-seq) data, was used to obtain an overview of the immune repertoire of tumors and to query the VDJ sequence of the validated TCR. Briefly, candidate reads from either BAM files of bulk RNA-seq data from tumors were extracted, candidate read assembly was prioritized by abundance and assembly of all candidate reads with partial overlaps against contigs was annotated. Finally, we tested the occurrence of the validated TCR sequence in the reconstructed immune repertoire. HLA-I immunohistochemistry (IHC) Immunohistochemical staining for HLA-I was performed using the mouse monoclonal antibody EMR8–5 (Cell Signaling Technology). Staining was conducted on the Ventana Benchmark Ultra automated staining platform (Ventana Medical Systems, Tucson, USA). Antigen–antibody binding was reveled using the biotin-free OptiView DAB IHC detection kit (Ventana Medical Systems). Counterstaining was performed with hemalum. Whole slide images were acquired using a VS200 slide scanner (Evident, Japan). Statistical analysis and clustering analysis Analysis and graphical representations were conducted using GraphPad Prism 10 software (GraphPad Software Inc.), R and Python. We downloaded the transcriptomic data (RNA-seq) of 113 normal samples from breast cancer patients from The Cancer Genome Atlas (TCGA) cohort via the UCSC Xena database ( https://xenabrowser.net/datapages/ ). RNA-seq expression levels, available as Fragments-Per-Millions (FPPM) were transformed to log2(FPKM +1). To test for differential gene expression across our samples and normal samples, we used the DESeq2 R package. The Clustering and visualization of gene expression profiling was done with the software Genesis. 

Whole genome and transcriptome sequencing of tumor samples and germline as well as longitudinal sampling of liquid biopsies has been performed by the CATCH ( C omprehensive A ssessment of clinical fea T ures and biomarkers to identify patients with advanced or metastatic breast C ancer for marker driven trials in H umans) precision oncology platform, which is a non-interventional, prospective registry that uses genomics and transcriptomics to guide therapeutic decisions in the clinical management of metastatic breast cancer. Methods and workflow has been carried as previously described. The study protocol was approved by the Institutional Review Board of the University Hospital of Heidelberg (UKHD) (approval number S-164/2017). In addition, the patient gave written informed consent for compassionate use treatment of a personalized vaccine in the context of the “Pilot project for the development of personalized cellular immunotherapies targeting solid cancers” (PIKT). Additional written informed consent was obtained for the collection of and investigational use of blood and tissue samples (S-207/2005). All activities were conducted in compliance with the declaration of Helsinki. 

DNA and RNA from tumor tissue and corresponding blood-derived non-tumor DNA were profiled using whole-genome and transcriptome sequencing as previously described. Non-synonymous point mutations in tumor tissues, resulting from single-nucleotide variants, were identified and compared with matched non-tumor DNA from the blood. Somatic single nucleotide variants (SNVs) were identified based on DNA sequencing data (WGS), and the quantification of gene expression based on RNA sequencing data was performed with the DKFZ Omics Data Core Facility supported analyses module “One touch platform (OTP)”. Neoepitopes were predicted with our in-house pipeline utilizing netMHCpan4.1 and netMHCstabpan1.0 for MHC I epitope prediction, according to patient-specific HLA type, which are predicted by OptiType and Kourami. Prediction results are ranked by binding affinity, binding stability and mRNA expression levels. 

Seventeen potential neoepitopes presented in the context of the patient’s haplotypes were selected to produce a personalized peptide vaccine. Selection was based on their binding predictions to matching HLAs and the level of expression of the mutant genes. After selection of the minimal epitopes, extended peptides were used to improve the vaccine immunogenicity. Long-peptides spanning upstream and downstream amino acids from the mutation site were synthesized as 25-mers by Fmoc chemistry using a fully automated multiple synthesizer (Syro II, MultiSynTech, Germany). Synthesis was carried out on preloaded Wang-resins with 2-(1 h-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) as coupling agent. Montanide ISA51 VG (SEPPIC GmbH) was used as an adjuvant to form a water-in-oil emulsion, mixed with the peptide solution containing 60 µg per peptide each (aqueous phase, 10% DMSO) in a 50:50 ratio. The treatment schedule consisted of eight cycles of intra-dermal vaccinations and is shown in . 

We designed a MIBI-TOF antibody panel to analyze the vaccinated lesion. To do so, we included antibodies to quantify nuclear staining, lineage markers to discriminate immune cells, immunoregulatory proteins and cytokeratin to distinguish epithelial cells (listed in Supplementary Table S1). Briefly, primary antibodies were conjugated to isotopic metal reporters as described previously. Tumor tissue was sectioned at 5 μm on conductive microscopy slides. FFPE tissues are deparaffinized and rehydrated with sequential washes. After antigen retrieval, slides were stained over night following standard procedures and dried under vacuum prior to imaging. Imaging was performed using the MIBI-TOF instrument (IonPath) which utilizes a bright ion source and orthogonal time-of-flight mass spectrometry to image metal-tagged antibodies in previously selected 800 × 800 μm field of views (FOVs). Finally, pixel data were converted to TIFF images. Prior to downstream analysis, images were processed through a pipeline (Toffy), as described previously, in order to subtract background and isotopic impurities, as well as to compensate for channel crosstalk. Next, the images were segmented using DeepCell, a convolutional neural network (CNN) for whole cell segmentation. After segmentation, cells were clustered using FlowSOM. , In order to assign each cell a lineage, well-established combinations of lineage markers were used as described previously, followed by subclustering of immune cell types, including CD4 T cells (CD4T; CD3+, CD4+, CD8-), CD8 T cells (CD8T; CD3+, CD8+, CD4-), B cells (B; CD45+, CD20+), NK cells (NK; CD3-CD56+), macrophages (Macrophages; CD45+,CD68+), myofibroblasts (Myofibroblasts; CD45-SMA+) and dendritic cells (DC; CD45+CD11c+). UMAPs were plotted using Python package Scanpy and spatial graphs were plotted using Python package Squidpy. 

VILs cultures were established using the ‘young-TIL’ protocol with slight modifications. In brief, fresh biopsies from the vaccination site were cut into small pieces of around 2 mm and placed individually in each well of 24-well culture plates. The plates were filled with T cell medium comprising AIM-V (Thermo Fisher Scientific) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin and 5% human serum (Sigma-Aldrich) and 6000 IU/mL IL-2 (Miltenyi Biotec). After 24 h, half of the medium was replaced with fresh medium containing IL-2. The plates were visually inspected every few days, and when the cells reached confluence, they were divided into new cultures. On day 12, all the wells were pooled and VILs were immunophenotyped by FACS and assayed for specific activity by ELISpot. Remaining cells were subjected to in vitro stimulation, as described below. INF-gamma screening by ELISpot assay To obtain APCs, autologous B cells were isolated by magnetic positive selection using CD19 MicroBeads (Miltenyi Biotec) and expanded with the Human CD40-Ligand Multimer Kit (Miltenyi Biotec) under manufacturer's recommendation in B cell media with 50UI/ml Human IL-4 (Miltenyi Biotec), 10UI/ml Human IL-21 (Miltenyi Biotec) and 30 ng/ml BAFF (Biolegend), hereafter, CD40-B cells. To test VILs reactivity CD40-B cells were pulsed ON with 10 µg/ml long peptides or short peptides and co-cultured the next day with VILs in filter plates with 0.45 um pore size Mixed Cellulose Esters membrane (Millipore), plating 100,000 B cells and 20,000 VILs or T cells per well in triplicates or duplicates, in screening or validation assays, respectively. Anti-CD3 coated wells (10 µg/ml, clone OKT3, Biolegend) were used as positive controls. To test T cell reactivity in the peripheral blood, 500,000 PBMCs were plated, directly pulsed with long peptides at 10 µg/ml and incubated during 48hs and the positive control wells were stimulated with SEB at 0.1 µg/ml. Viral peptide pools (PepTivator CEF MHC Class I Plus, Miltenyi Biotec) as internal controls and negative controls (unstimulated cells and wells with only media) were included in every assay. INF-gamma detection was performed using Human IFN-γ ELISpot basic kit (ALP) (Mabtech) following manufacturer's protocol. Images were acquired and analyzed with ImmunoSpot® Analyzer (CTL). 

To obtain APCs, autologous B cells were isolated by magnetic positive selection using CD19 MicroBeads (Miltenyi Biotec) and expanded with the Human CD40-Ligand Multimer Kit (Miltenyi Biotec) under manufacturer's recommendation in B cell media with 50UI/ml Human IL-4 (Miltenyi Biotec), 10UI/ml Human IL-21 (Miltenyi Biotec) and 30 ng/ml BAFF (Biolegend), hereafter, CD40-B cells. To test VILs reactivity CD40-B cells were pulsed ON with 10 µg/ml long peptides or short peptides and co-cultured the next day with VILs in filter plates with 0.45 um pore size Mixed Cellulose Esters membrane (Millipore), plating 100,000 B cells and 20,000 VILs or T cells per well in triplicates or duplicates, in screening or validation assays, respectively. Anti-CD3 coated wells (10 µg/ml, clone OKT3, Biolegend) were used as positive controls. To test T cell reactivity in the peripheral blood, 500,000 PBMCs were plated, directly pulsed with long peptides at 10 µg/ml and incubated during 48hs and the positive control wells were stimulated with SEB at 0.1 µg/ml. Viral peptide pools (PepTivator CEF MHC Class I Plus, Miltenyi Biotec) as internal controls and negative controls (unstimulated cells and wells with only media) were included in every assay. INF-gamma detection was performed using Human IFN-γ ELISpot basic kit (ALP) (Mabtech) following manufacturer's protocol. Images were acquired and analyzed with ImmunoSpot® Analyzer (CTL). 

Cells were washed with PBS and stained for dead exclusion markers with Zombie Aqua Fixable Viability Kits (Biolegend) for 15 min at RT protected from light exposure. After a wash with FACS buffer containing 0.5% BSA and 2 mm EDTA, cells were stained with the antibody cocktails for suitable markers. Anti-human antibodies used were purchase from Biologend: APC/Fire™ 750 anti-human CD3 (clone UCHT1), Brilliant Violet 570™ anti-human CD4 (cloneRPA-T4), Brilliant Violet 785™ anti-human CD8 (clone SK1), Brilliant Violet 510™ anti-human CD56 (clone HCD56), Brilliant Violet 421™ anti-mouse TCR β chain (clone H57–597), PE anti-mouse TCR β chain (clone H57–597), PE anti-human CD137 (clone 4B4–1), PE/Cyanine7 anti-human CD134 (OX40) (clone Ber-ACT35), and APC anti-human CD69 (clone FN50). Tetramer staining was performed based on complexed easYmers (ImmunAware) as described previously. For blocking HLA-I blockade control in functional assays, anti-HLA-ABC (clone W6/32, Biotrend) was added 30 min before the co-culture to pulsed B cells at 50 µg/ml. 

stimulation (IVS) of VIL cells In vitro stimulations were performed as previously described, with slight modifications. CD40L-B cells were pulsed ON with long peptides and co-cultured the next day with enriched CD3+ (Pan T cell isolation kit, Miltenyi Biotec) or CD8+ (REAlease® CD8 MicroBead Kit, Milenyi Biotec) VILs in T cell medium containing IL-21 30 ng/ml. Cells were incubated at 37°C for 72 h, after which time the cultures were fed with fresh T cell medium containing IL-21 30 ng/ml, IL-2 3000 IU/ml, IL-15 2.5 ng/ml, IL-7 2.5 ng/ml. The splits were performed after 72 h. After the second split, VIL reactivity was rechecked. The complete scheme of the cultures is shown in Suppl. Figure S1. 

Short peptides with binding predictions to patient’s HLAs (HLA-A *02:01, HLA-A *03:01, HLA-B * 07:02, HLA-B * 15:01, HLA-C * 03:03, HLA-C * 07:02), were synthetized and tested in a cytometry-based assay of peptide-HLA-I complex formation (easYmer, ImmunAware) performed according to manufacturer’s instructions. Percentage of stabilization for each peptide was calculated by setting 0% to the fluorescence intensity of negative controls (no peptide) and 100% to the positive control peptides provided in the kit by ImmunAware for each allele. Positive controls for evaluation analysis of peptide-HLA folding were: CMV pp65 495–503 (NLVPMVATV), a known HLA-A *02:01 binder; CMV IE1 99–107 (RIKEHMLKK), a known HLA-A *03:01 binder; CMV pp65 417–426 (TPRVTGGGAM), a stable HLA-B * 07:02 binder; YFV NS3 73–18 (SVKEDLVAY), a stable HLA-B * 15:01 binder; Burkholderia mallei N-ethylmaleimide reductase 137–145 (YVTPRALEL), a stable HLA-C * 03:03 binder; E. coli DH10B conserved protein 202–210 (FRAPNTREL), a known HLA-C * 07:02 binder. 

TCR sequencing (TCR-seq) was performed from cryopreserved VILs and PBMC at Genewiz (Azenta). Briefly, single cell TCR sequencing of VDJ regions was performed using the Chromium Single Cell Immune Profiling kit (10× Genomics), followed by libraries sequencing on a MiSeq (Illumina). Analysis of full-length V(D)J sequences for paired T-cell receptors was performed using the Loupe VDJ Browser v4.0.0 (10× Genomics). 

The retrieved VDJ sequences of the alpha and beta chains of TCR-1131 and TCR-1132 were fused in silico to the sequences of the respective murine constant alpha and beta TCR domains in order to propagate an exclusive pairing of the chosen sequences to form the artificial TCR and thereby avoiding a mispairing with endogenously expressed TCRs of donor T cells, which might generate a novel unpredictable specificity of the T cell product. The full-length alpha and beta chains were then linked by a P2A cleavage sequence that allows the combination of multiple gene products into one linear vector sequence. The obtained sequences of TCR-1131 and TCR-1132 were then synthesized by GeneArt and cloned into the backbone of a self-inactivating lentiviral vector. Subsequently, lentiviral particles containing TCR-1131 and TCR-1132 were produced by standard transfection methods. Primary patient T cells were isolated from PBMCs (Pan T Cell Isolation Kit, Miltenyi Biotec) and activated using TransAct reagent and TexMACS media containing IL-7 and IL-15 (all Miltenyi Biotec). Lentiviral transduction was performed 2 d post activation. 

TRUST4, an open-source algorithm for reconstructing immune receptor repertoires in αβ/γδ T cells from RNA sequencing (RNA-seq) data, was used to obtain an overview of the immune repertoire of tumors and to query the VDJ sequence of the validated TCR. Briefly, candidate reads from either BAM files of bulk RNA-seq data from tumors were extracted, candidate read assembly was prioritized by abundance and assembly of all candidate reads with partial overlaps against contigs was annotated. Finally, we tested the occurrence of the validated TCR sequence in the reconstructed immune repertoire. 

Immunohistochemical staining for HLA-I was performed using the mouse monoclonal antibody EMR8–5 (Cell Signaling Technology). Staining was conducted on the Ventana Benchmark Ultra automated staining platform (Ventana Medical Systems, Tucson, USA). Antigen–antibody binding was reveled using the biotin-free OptiView DAB IHC detection kit (Ventana Medical Systems). Counterstaining was performed with hemalum. Whole slide images were acquired using a VS200 slide scanner (Evident, Japan). 

Analysis and graphical representations were conducted using GraphPad Prism 10 software (GraphPad Software Inc.), R and Python. We downloaded the transcriptomic data (RNA-seq) of 113 normal samples from breast cancer patients from The Cancer Genome Atlas (TCGA) cohort via the UCSC Xena database ( https://xenabrowser.net/datapages/ ). RNA-seq expression levels, available as Fragments-Per-Millions (FPPM) were transformed to log2(FPKM +1). To test for differential gene expression across our samples and normal samples, we used the DESeq2 R package. The Clustering and visualization of gene expression profiling was done with the software Genesis. 

Case presentation and personalized treatment A 41-year-old female with a metastatic triple-negative breast cancer (TNBC) was enrolled in our institutional diagnostic and registry trial (NCT05652569) in February 2020. Longitudinal, molecular profiling of tumor- and liquid biopsies were expanded by a translational program of immunomonitoring as well as a research to identify and clone neoantigen‑specific T cells. Timeline of treatment regimen, personalized vaccination and translational activities are shown in . Of note, the personalized immunotherapy was performed in parallel to the standard of care, including PD-L1 directed immunotherapy, starting in August 2020. Neoantigen calling from whole-genome and transcriptome sequencing of the first analyzed tumor lesion (Tumor 1) by neoepitope identification using NetMHCpan4.1 revealed 254 putative binding candidates to patient’s HLA-I alleles (Supplementary Table S2) from 54 non-synonymous single-nucleotide variants (SNV) considering a filter based on expression of the altered allele (FPKM > 0) and %RANK_EL < 2 (predicted binders). We selected 17 candidates based on gene expression, allele frequency, and high affinity of the peptide-MHC, with the aim to select neoepitopes that best contribute to tumor immunogenicity. For the vaccination, extended peptide sequences flanking both sides of the predicted epitope were designed, as long peptides have the potential to cover CD4 + T helper cell (Th) epitopes and the length of the peptide used for vaccination strongly influences the magnitude of the induced CD8+ T-cell response therefore increasing the efficacy of the vaccination. The list of peptides used in the vaccination can be found in . Intradermal application of the personalized vaccine started in February 2021 and after three rounds (every 4–6 weeks), the patient presented with a strong local inflammatory reaction at the vaccination site, manifesting induration, redness and swelling. Considering inflammation-related adverse reactions as a consequence of a strong and antigen-specific immune response, the vaccinated lesion was referred to biopsy followed by an analysis of immune repertoire and antigen specificity. Deep exploration of vaccination site A fresh biopsy was taken and processed following a “young TIL” protocol. After VIL culture establishment, expanding cells were harvested and analyzed for their phenotype. Most of the cells corresponded to CD3 + T cells (64.4% of the culture); among the T cells, CD4 + , CD8 + and a double negative (DN) population were present at 14.7%, 38.6% and 10.2%, respectively (percentages refer to their frequencies in the whole culture) . To interrogate the phenotype of the cells and their spatial distribution in the original tissue we performed a multiplexed staining and multiplexed ion beam image acquisition (MIBI-TOF). Based on tissue integrity and overall topography in a hematoxylin and eosin stain (HE), we selected four fields of view (FOVs, Suppl. Figure S1). Across all FOVs, we found a number of dermal follicles, along with MHC-II-expressing CD11c + cells, presumably dendritic and Langerhans cells, migrating in response to the vaccination. Whole cell segmentation and single cell clustering revealed CD4 + and CD8 + T cells to be most abundant within the immune cell cluster . Noteworthy, CD8 + T cells expressed low to intermediate levels of PD-1, along with intermediate TOX levels in a subset of cells , compatible with a non-terminally exhausted memory phenotype. Moreover, the low proliferation index whilst maintaining cytotoxic potential (GZMB + ) and high proliferative response to IL-2 ex vivo , is suggestive of a niche of Trm-like T cells. Besides, we observed distinct FOXP3 expression within the CD4 + T cell population possibly reflecting finely regulated immunosuppression in orchestration of response to the vaccination. T cells targeting private mutations are present in the vaccination site To investigate the presence of neoantigen-specific T cells at the vaccination site, we screened the expanded VILs for antigen-specific reactivity by measuring IFNγ secretion from activated T cells using an IFNγ ELISpot assay. The results of the IFNγ ELISpot showed reactivity to four peptides (LYRM1 C63Y /NCOR1 L1475R /SLC9A5 S562A /U2SURP A656V ) in the VILs . Notably, the reactivities of T cells that infiltrated the vaccination site were distinct from those detected in the patient’s blood (directed to ANPEP T512I and HSDL1 A202V ) . To enrich the culture in neoantigen-specific T cells, we proceeded with an in vitro stimulation (IVS)-based enrichment approach to expand the reactive cells. In the IVS process, we first enriched pan T cells and then initiated two separate cultures: one for purified CD8 + T cells and another for a bulk T cell fraction, both using autologous expanded CD40-activated B cells (CD40-B cells) loaded with a pool of peptides. The four peptides that had activated cells from the vaccination site (LYRM1 p.C63Y /NCOR1 L1475R /SLC9A5 S562A /U2SURP A656V ) and two peptides that had activated cells from peripheral blood were included in the stimulation (ANPEP T512I /HSDL1 A202V ). A summary of the IVS performed is shown in Suppl. Figure S2. After IVS, we evaluated the IFN-γ-secreting cells through ELISpot. The results revealed the expansion of T cell clones with diverse reactivity profiles in the different cultures . Interestingly, in the post-IVS bulk IVS culture (confirmed to be a mix of CD4 + and CD8 + cells), reactive cells to three different peptides (LYRM1 C63Y /NCOR1 L1475R /U2SURP A656V ) were expanded , while only NCOR1 L1475R -reactive cells showed significant expansion that were clearly distinguishable from background levels in the CD8 + culture . In conclusion, VILs cells were successfully expanded and contained T cells, including both CD4 + and CD8 + T cells, reactive against vaccination peptides. Vaccine predicted epitopes are presented on HLA-I molecules Next, we sought to validate the class-I T cell epitopes from the in silico predictions that are able to bind HLA-I molecules. Based on these predictions, we selected and synthesized short peptides corresponding to the four long peptides that showed reactivity in the in vitro cultures . To evaluate the binding and stability of these short peptides to HLA molecules (p-HLA), we used recombinant HLA-I tetramers . Notably, one of the predicted short peptides from NCOR1 L1475R (GPSVRRSTL) exhibited a high capacity to form stable HLA-B * 07:02 peptide-MHC complexes . Taken together, these findings suggested the presence of a CD8-reactive T cell population targeting the mutation NCOR1 L1475R , which is presented in the context of HLA-B * 07:02. Consequently, we focused our efforts on analyzing the CD8-reactive population specific to NCOR1 L1475R (covered in the sequence of the long peptide NCOR1 L1475R used in the vaccination). TCR identification and validation Analysis of single cell TCR sequencing (scTCR-seq) data revealed the presence of three common clonotypes at the original vaccination site (>5%, clonotypes 1–3, and Suppl. Table S3), from which two exhibited significant expansion after IVS (both in the bulk and the pure CD8 cultures) with the long peptide NCOR1 L1475R (clonotypes 1 and 2, and Suppl. Table S3). We selected the TCRs from these two most prominent clonotypes (designated as TCR-1131 and TCR-1132), and we proceeded with their cloning and subsequent validation. To validate the functional potential of TCR-1131 and TCR-1132, transgenic (Tg) T cells were generated through lentiviral transduction of primary T cells from the patient. Upon confirmation of successful TCR expression by FACS ( , top panel), the Tg-T cells were exposed to autologous CD40-B cells pulsed with various peptides ( , middle and lower panels, and ). Our investigation revealed that only TCR-1131 demonstrated specific reactivity against the NCOR1 mutation L1475R , while TCR-1132 did not show reactivity to any of the tested peptides (not shown). The observed reactivity appeared to be primarily associated with CD8+ T cells, as its activation was predominantly directed toward this subset. Furthermore, HLA-I blockade partially reduced this activation in our in vitro assay . Interestingly, when the short peptides were pulsed on APCs, the TCR-1131 reacted with the GPHVRRSTL 9-mer and GPHVRRSTLH 10-mer, although the latter was not suggested by the p-HLA stabilization assay as the peptide is a weaker B * 07:02 binder. This may be due to the natural processing and presentation pathways present in APCs that can sometimes overcome suboptimal peptide length, leading to effective T cell activation. In addition, the reactivity against the long peptide confirms that the peptide is being processed and cross-presented on HLA-I by B cells . Notably, reactivity toward the wild-type (WT) peptide remained similar to the background levels displayed by other irrelevant peptides tested . To further validate the specificity of TCR-1131, tetramer staining was performed using the pHLA complex of the GPSVRRSTL peptide in HLA-B * 07:02. This analysis demonstrated specific binding of the tetramer to the TCR-1131 + Tg-T cells (co-detected with an antibody recognizing msTCRβ), with no cross-reactivity to the WT peptide . In conclusion, we successfully validated a TCR isolated from a vaccination site as a neoantigen-specific TCR capable of detecting the patient’s private mutation NCOR1 L1475R , presented in the context of HLA-B * 07:02, while exhibiting no reactivity to the WT peptide. Personalized immunotherapy induced immune cell gene expression without evidence of neoantigen specific T cells in the tumor microenvironment During the process of TCR identification and subsequent to the personalized vaccination therapy, the patient experienced further disease progression despite receiving immune-checkpoint blockade and chemotherapeutic regimens . A longitudinal biopsy, referred to as Tumor 2, was performed and analyzed . Differential analysis of RNA-seq data of Tumor 1 and Tumor 2 confirmed an increase in the expression of genes associated with the antigen-presenting machinery and immune activation in Tumor 2, suggesting an anti-tumor immune response was elicited by the treatment ( and Suppl. Tables S4 and S5). However, immunohistochemical staining of the same tumors revealed a complete loss of HLA-I protein expression in Tumor 2 . Upon analyzing the TCR repertoire from RNA-seq data of the three tumor samples, including pre-vaccination tumor (Tumor 1), post-vaccination (Tumor 2), and a later malignant pleural effusion (Tumor 3), we observed that the TCR sequences associated with the neoantigen-reactive T cells were not detected in either the pre-vaccination tumor or the post-vaccination tumors (Suppl. Tables 6–8, respectively). Of note, both post-vaccination tumors lost the NCOR1 L1475R mutation ( and Suppl. Tables S9 and S10). Interestingly, another mutation within the NCOR1 gene (NCOR1 I966V ), which was part of the vaccination but failed to induce a reactive TCR at the vaccination site , was still detected ( and Suppl. Tables s9 and s10). NCOR1 I966V was expressed at higher frequency in Tumor 1 than NCOR1 L1475R ( and Suppl. Tables s9 and s10), thus all the NCOR1 L1475R -harboring clones may have been lost, without affecting the persistence of NCOR1 I966V ones. Given that, both the negative selection of NCOR1 L1475R harboring tumor clones and HLA-I loss during the disease course, are known mechanisms of resistance to TCR T cell therapies, this therapeutic approach was not further pursued in light of additional existing standard-therapies. Despite this, the collective findings demonstrated, for the first time, the feasibility of isolating reactive T cells from vaccination site biopsies. Neoantigen specific TCR sequences are exclusively found in the vaccination-site Finally, we considered the possibility of detecting the validated NCOR1 L1475R -specific CD8 + TCR in the peripheral blood. Therefore, we conducted TCR single-cell sequencing of peripheral blood cells at three different time points: pre-vaccination, a sample taken after the sixth vaccination (closest time point after analysis of the vaccination site biopsy where we identified the reactive TCR, Blood 3), and a final sample after completing the vaccination scheme (Blood 4) . However, our analysis of all three samples showed no evidence of T cells with the NCOR1 L1475R -specific TRA and TRB CDR3 sequences ( and Suppl. Tables s11-s13), confirming the results by IFNγ ELISpot ( , left panel). In conclusion, VILs were the sole source where the NCOR1 L1475R -specific CD8 + TCR sequence was detectable. Collectively, these findings demonstrate the feasibility of isolating tumor neoantigen-specific CD8 + TCRs exclusively from vaccination sites. 

A 41-year-old female with a metastatic triple-negative breast cancer (TNBC) was enrolled in our institutional diagnostic and registry trial (NCT05652569) in February 2020. Longitudinal, molecular profiling of tumor- and liquid biopsies were expanded by a translational program of immunomonitoring as well as a research to identify and clone neoantigen‑specific T cells. Timeline of treatment regimen, personalized vaccination and translational activities are shown in . Of note, the personalized immunotherapy was performed in parallel to the standard of care, including PD-L1 directed immunotherapy, starting in August 2020. Neoantigen calling from whole-genome and transcriptome sequencing of the first analyzed tumor lesion (Tumor 1) by neoepitope identification using NetMHCpan4.1 revealed 254 putative binding candidates to patient’s HLA-I alleles (Supplementary Table S2) from 54 non-synonymous single-nucleotide variants (SNV) considering a filter based on expression of the altered allele (FPKM > 0) and %RANK_EL < 2 (predicted binders). We selected 17 candidates based on gene expression, allele frequency, and high affinity of the peptide-MHC, with the aim to select neoepitopes that best contribute to tumor immunogenicity. For the vaccination, extended peptide sequences flanking both sides of the predicted epitope were designed, as long peptides have the potential to cover CD4 + T helper cell (Th) epitopes and the length of the peptide used for vaccination strongly influences the magnitude of the induced CD8+ T-cell response therefore increasing the efficacy of the vaccination. The list of peptides used in the vaccination can be found in . Intradermal application of the personalized vaccine started in February 2021 and after three rounds (every 4–6 weeks), the patient presented with a strong local inflammatory reaction at the vaccination site, manifesting induration, redness and swelling. Considering inflammation-related adverse reactions as a consequence of a strong and antigen-specific immune response, the vaccinated lesion was referred to biopsy followed by an analysis of immune repertoire and antigen specificity. 

A fresh biopsy was taken and processed following a “young TIL” protocol. After VIL culture establishment, expanding cells were harvested and analyzed for their phenotype. Most of the cells corresponded to CD3 + T cells (64.4% of the culture); among the T cells, CD4 + , CD8 + and a double negative (DN) population were present at 14.7%, 38.6% and 10.2%, respectively (percentages refer to their frequencies in the whole culture) . To interrogate the phenotype of the cells and their spatial distribution in the original tissue we performed a multiplexed staining and multiplexed ion beam image acquisition (MIBI-TOF). Based on tissue integrity and overall topography in a hematoxylin and eosin stain (HE), we selected four fields of view (FOVs, Suppl. Figure S1). Across all FOVs, we found a number of dermal follicles, along with MHC-II-expressing CD11c + cells, presumably dendritic and Langerhans cells, migrating in response to the vaccination. Whole cell segmentation and single cell clustering revealed CD4 + and CD8 + T cells to be most abundant within the immune cell cluster . Noteworthy, CD8 + T cells expressed low to intermediate levels of PD-1, along with intermediate TOX levels in a subset of cells , compatible with a non-terminally exhausted memory phenotype. Moreover, the low proliferation index whilst maintaining cytotoxic potential (GZMB + ) and high proliferative response to IL-2 ex vivo , is suggestive of a niche of Trm-like T cells. Besides, we observed distinct FOXP3 expression within the CD4 + T cell population possibly reflecting finely regulated immunosuppression in orchestration of response to the vaccination. 

To investigate the presence of neoantigen-specific T cells at the vaccination site, we screened the expanded VILs for antigen-specific reactivity by measuring IFNγ secretion from activated T cells using an IFNγ ELISpot assay. The results of the IFNγ ELISpot showed reactivity to four peptides (LYRM1 C63Y /NCOR1 L1475R /SLC9A5 S562A /U2SURP A656V ) in the VILs . Notably, the reactivities of T cells that infiltrated the vaccination site were distinct from those detected in the patient’s blood (directed to ANPEP T512I and HSDL1 A202V ) . To enrich the culture in neoantigen-specific T cells, we proceeded with an in vitro stimulation (IVS)-based enrichment approach to expand the reactive cells. In the IVS process, we first enriched pan T cells and then initiated two separate cultures: one for purified CD8 + T cells and another for a bulk T cell fraction, both using autologous expanded CD40-activated B cells (CD40-B cells) loaded with a pool of peptides. The four peptides that had activated cells from the vaccination site (LYRM1 p.C63Y /NCOR1 L1475R /SLC9A5 S562A /U2SURP A656V ) and two peptides that had activated cells from peripheral blood were included in the stimulation (ANPEP T512I /HSDL1 A202V ). A summary of the IVS performed is shown in Suppl. Figure S2. After IVS, we evaluated the IFN-γ-secreting cells through ELISpot. The results revealed the expansion of T cell clones with diverse reactivity profiles in the different cultures . Interestingly, in the post-IVS bulk IVS culture (confirmed to be a mix of CD4 + and CD8 + cells), reactive cells to three different peptides (LYRM1 C63Y /NCOR1 L1475R /U2SURP A656V ) were expanded , while only NCOR1 L1475R -reactive cells showed significant expansion that were clearly distinguishable from background levels in the CD8 + culture . In conclusion, VILs cells were successfully expanded and contained T cells, including both CD4 + and CD8 + T cells, reactive against vaccination peptides. 

Next, we sought to validate the class-I T cell epitopes from the in silico predictions that are able to bind HLA-I molecules. Based on these predictions, we selected and synthesized short peptides corresponding to the four long peptides that showed reactivity in the in vitro cultures . To evaluate the binding and stability of these short peptides to HLA molecules (p-HLA), we used recombinant HLA-I tetramers . Notably, one of the predicted short peptides from NCOR1 L1475R (GPSVRRSTL) exhibited a high capacity to form stable HLA-B * 07:02 peptide-MHC complexes . Taken together, these findings suggested the presence of a CD8-reactive T cell population targeting the mutation NCOR1 L1475R , which is presented in the context of HLA-B * 07:02. Consequently, we focused our efforts on analyzing the CD8-reactive population specific to NCOR1 L1475R (covered in the sequence of the long peptide NCOR1 L1475R used in the vaccination). 

Analysis of single cell TCR sequencing (scTCR-seq) data revealed the presence of three common clonotypes at the original vaccination site (>5%, clonotypes 1–3, and Suppl. Table S3), from which two exhibited significant expansion after IVS (both in the bulk and the pure CD8 cultures) with the long peptide NCOR1 L1475R (clonotypes 1 and 2, and Suppl. Table S3). We selected the TCRs from these two most prominent clonotypes (designated as TCR-1131 and TCR-1132), and we proceeded with their cloning and subsequent validation. To validate the functional potential of TCR-1131 and TCR-1132, transgenic (Tg) T cells were generated through lentiviral transduction of primary T cells from the patient. Upon confirmation of successful TCR expression by FACS ( , top panel), the Tg-T cells were exposed to autologous CD40-B cells pulsed with various peptides ( , middle and lower panels, and ). Our investigation revealed that only TCR-1131 demonstrated specific reactivity against the NCOR1 mutation L1475R , while TCR-1132 did not show reactivity to any of the tested peptides (not shown). The observed reactivity appeared to be primarily associated with CD8+ T cells, as its activation was predominantly directed toward this subset. Furthermore, HLA-I blockade partially reduced this activation in our in vitro assay . Interestingly, when the short peptides were pulsed on APCs, the TCR-1131 reacted with the GPHVRRSTL 9-mer and GPHVRRSTLH 10-mer, although the latter was not suggested by the p-HLA stabilization assay as the peptide is a weaker B * 07:02 binder. This may be due to the natural processing and presentation pathways present in APCs that can sometimes overcome suboptimal peptide length, leading to effective T cell activation. In addition, the reactivity against the long peptide confirms that the peptide is being processed and cross-presented on HLA-I by B cells . Notably, reactivity toward the wild-type (WT) peptide remained similar to the background levels displayed by other irrelevant peptides tested . To further validate the specificity of TCR-1131, tetramer staining was performed using the pHLA complex of the GPSVRRSTL peptide in HLA-B * 07:02. This analysis demonstrated specific binding of the tetramer to the TCR-1131 + Tg-T cells (co-detected with an antibody recognizing msTCRβ), with no cross-reactivity to the WT peptide . In conclusion, we successfully validated a TCR isolated from a vaccination site as a neoantigen-specific TCR capable of detecting the patient’s private mutation NCOR1 L1475R , presented in the context of HLA-B * 07:02, while exhibiting no reactivity to the WT peptide. 

During the process of TCR identification and subsequent to the personalized vaccination therapy, the patient experienced further disease progression despite receiving immune-checkpoint blockade and chemotherapeutic regimens . A longitudinal biopsy, referred to as Tumor 2, was performed and analyzed . Differential analysis of RNA-seq data of Tumor 1 and Tumor 2 confirmed an increase in the expression of genes associated with the antigen-presenting machinery and immune activation in Tumor 2, suggesting an anti-tumor immune response was elicited by the treatment ( and Suppl. Tables S4 and S5). However, immunohistochemical staining of the same tumors revealed a complete loss of HLA-I protein expression in Tumor 2 . Upon analyzing the TCR repertoire from RNA-seq data of the three tumor samples, including pre-vaccination tumor (Tumor 1), post-vaccination (Tumor 2), and a later malignant pleural effusion (Tumor 3), we observed that the TCR sequences associated with the neoantigen-reactive T cells were not detected in either the pre-vaccination tumor or the post-vaccination tumors (Suppl. Tables 6–8, respectively). Of note, both post-vaccination tumors lost the NCOR1 L1475R mutation ( and Suppl. Tables S9 and S10). Interestingly, another mutation within the NCOR1 gene (NCOR1 I966V ), which was part of the vaccination but failed to induce a reactive TCR at the vaccination site , was still detected ( and Suppl. Tables s9 and s10). NCOR1 I966V was expressed at higher frequency in Tumor 1 than NCOR1 L1475R ( and Suppl. Tables s9 and s10), thus all the NCOR1 L1475R -harboring clones may have been lost, without affecting the persistence of NCOR1 I966V ones. Given that, both the negative selection of NCOR1 L1475R harboring tumor clones and HLA-I loss during the disease course, are known mechanisms of resistance to TCR T cell therapies, this therapeutic approach was not further pursued in light of additional existing standard-therapies. Despite this, the collective findings demonstrated, for the first time, the feasibility of isolating reactive T cells from vaccination site biopsies. 

Finally, we considered the possibility of detecting the validated NCOR1 L1475R -specific CD8 + TCR in the peripheral blood. Therefore, we conducted TCR single-cell sequencing of peripheral blood cells at three different time points: pre-vaccination, a sample taken after the sixth vaccination (closest time point after analysis of the vaccination site biopsy where we identified the reactive TCR, Blood 3), and a final sample after completing the vaccination scheme (Blood 4) . However, our analysis of all three samples showed no evidence of T cells with the NCOR1 L1475R -specific TRA and TRB CDR3 sequences ( and Suppl. Tables s11-s13), confirming the results by IFNγ ELISpot ( , left panel). In conclusion, VILs were the sole source where the NCOR1 L1475R -specific CD8 + TCR sequence was detectable. Collectively, these findings demonstrate the feasibility of isolating tumor neoantigen-specific CD8 + TCRs exclusively from vaccination sites. 

In our study, we administered a personalized peptide vaccine to a metastatic breast cancer patient with the aim of inducing an immune response against private neoantigens. Using tissue from a vaccination site as a source of T cells, we were able to isolate a CD8 + neoantigen reactive TCR targeting an NCOR1 L1475R mutation presented in the context of HLA-B * 07:02. The primary objective of a therapeutic cancer vaccine is to induce a robust and enduring response that culminates in the generation of memory T cells that circulate throughout the body, enabling continuous surveillance and immune monitoring. Accordingly, correlations between immunologic measures and clinical outcome have been found in several trials. However, the most striking discovery of our study was the lack of overlapping of TCR sequences between the VILs and PBL. By tracking IFN-γ + T cell responses against vaccine peptides, specific T-cell responses against two of the vaccine peptides could be detected in the peripheral blood and against four different ones in the VILs. Previous studies have found that, even when the algorithms used did specifically search for neoantigens that were predicted to induce CD8+ CTLs (i.e. HLA-I binding peptides), the neoantigens represented in the vaccine that were recognized by CD4 + T cells generated a greater immune response in the periphery. This does not exclude dual activation of both CD4+ and CD8+ T cells but may indicate that sometimes the CD4+ response is more dominant or easier to detect in the periphery. In line with our findings, previous studies support the detection of tumor-specific CD8 cytotoxic T cells at vaccination sites. , In another case-study of a melanoma patient, persistent TCR clones sequenced from a vaccination site were found to infiltrate a regional cutaneous metastasis, although antigen specificity was not confirmed. Memory CD8 + T cells are classified into different subsets based on their location and circulation within the body after activation. These subsets include central memory, effector memory, and tissue-resident memory T cells. Among them, tissue-resident memory T cells are distinct as they do not circulate in the bloodstream but instead reside in various tissues throughout the body. These cells serve a critical role in local immune responses and rapid recall reactions. Our comprehensive analysis of the vaccination site provides evidence supporting the development of local memory CD8 CTLs T cells compatible with a Trm-like phenotype. The induction of memory cells within the vaccination site could play a crucial role in effective anti-tumor immunity. However, challenges remain in ensuring the migration and efficacy of cytotoxic T lymphocytes (CTLs) from the vaccination site to distant tumors. Whether these cells served as a source of antitumor T cells with potential to re-circulate distant tissues in our analyzed patient remains unresolved. Arguing for their efficacy, re-biopsy of new tumors allowed us for the re-assessment of the tumor mutations and the TME. One notable observation was the transition from a “cold” to a “hot” TME, with an increased presence of immune cells and a more favorable immunological environment. This shift suggests that the vaccine has stimulated an immune response targeting the tumor. However, we confirmed complete loss of HLA-I at the protein level, a well-known tumor escape immune escape mechanism. Furthermore, genomic and expression analyses of the re-biopsied tumors revealed the loss of the neoantigen NCOR1 L1475R targeted by the vaccine (and the validated TCR-1131), but not of a NCOR1 mutation included in the vaccination for which we could not detect a T cell reactivity, indicative of successful immune recognition and elimination of tumor cells only presenting the specific NCOR1 L1475R neoantigen from the original HLA-I-positive tumor. Moreover, we observed alterations in other neoantigens following vaccination, but it remains uncertain whether this phenomenon was a consequence of antigen spreading or clonal drift. Despite this uncertainty, our findings underscore the potential of selecting therapeutically valuable targets from individual passenger mutations. This suggests that focusing solely on driver mutations may overlook potentially effective targets among the broader landscape of passenger mutations and highlights the existence of an effective immunogenicity despite lack of functional relevance. On the other hand, it is important to acknowledge the limitations of our study to validate the NCOR1 L1475R specific T cells efficacy in vivo . The immune repertoire analysis of all the tumor lesions did not reveal detectable neoantigen-specific TCR clones. This does not necessarily indicate the absence of an immune response or the ineffectiveness of the vaccine since several factors need to be considered when interpreting these results. For instance, it is expected that the immune edition of the NCOR1 L1475R as well as the loss of HLA-I expression will result in reduced detectability of cognate TCR clones. However, by further analyzing peripheral blood at different time-points comprising pre- and post-vaccination samples, our validated TCR sequence was also non-detectable. These considerations highlight the complexities involved in studying the immune response to vaccines and the challenges in detecting specific T cell clones. Despite the uncertainty of the role of the validated TCR clone against NCOR1 L1475R in the active anti-tumor immune response and immune-edition of this neoantigen, the finding of tumor-specific CTL in high numbers at the vaccination site holds promise for novel approaches in adoptive cell therapy. The retention of functional and proliferative capacity overcomes current hurdles for TCR screening and TIL expansion. Although we did not fully characterize them, VILs demonstrated reactivity to multiple tumor neo-epitopes. Their ability to leverage multiple tumor-reactive TCRs to target diverse tumor epitopes makes them promising candidates for future immunotherapies. This approach minimizes immune escape, aligning with recent studies highlighting the importance of single TCRs capable of multi-epitope targeting in successful TIL therapy. In conclusion, the remarkable abundance of CD8 neoantigen-specific T cells observed in our study highlights the robust immune response elicited against tumor mutations at the local site of vaccine administration. Furthermore, the accessibility and safety of obtaining tissue biopsies from a cutaneous vaccination site offer practical advantages over invasive procedures, making this approach feasible for widespread implementation. The generation and isolation of tumor mutation-specific T cells at high frequencies, coupled with the use of standard TCR screening methods, open new avenues in the development of transgenic TCR T cells or ex vivo expansion of VILs for clinical use. Further research and clinical investigations are warranted to explore the full therapeutic potential of VILs in the fight against cancer. 

Individual supp tablesTable S4.xlsx Suppl Figure 1.eps Individual supp tablesTable S8.xlsx Individual supp tablesTable S13.xlsx Individual supp tablesTable S3.xlsx Individual supp tablesTable S11.xlsx Individual supp tablesTable S12.xlsx Suppl Figure 2.eps Individual supp tablesTable S1.xlsx Individual supp tablesTable S10.xlsx Individual supp tablesTable S9.xlsx Individual supp tablesTable S2.xlsx Individual supp tablesTable S5.xlsx Individual supp tablesTable S6.xlsx Individual supp tablesTable S7.xlsx 

