Epitope selection and synthetic peptides

The thirty-four peptides derived from REα were predicted to bind multiples HLA-A/B and HLA-DR molecules using SYFPETHI and/or NetMHCpan 3.0 software (access via www.syfpeithi.de/, www.cbs.dtu.dk/services/NetMHCpan/) according to the mutations in positions 537 and 538 (Y537S, Y537C, Y537N, and D538G). Twenty-two peptides were retained based on their predictive score to bind to HLA-A/B molecules. In addition twelve peptides were selected based on their predictive score to bind to HLA-DR molecules. All synthetic peptides (> 80% purity) were purchased from JPT (Berlin, Germany). To assess antiviral T cell immunity, a pool of 23 peptides containing epitopes from influenza virus, Epstein–Barr virus, and cytomegalovirus was used (Cellular Technology Limited, Shaker heights, OH, USA). Lyophilized peptides were dissolved in PBS containing 5% DMSO at a concentration of 4 mg/mL and stored at − 80 °C.

Cell culture

Authenticated Tap-deficient T2 (174x CEM.T2) cell line and MCF7 (HTB-22) was purchased from the ATCC and (upon thawing) never passed for more than 3 weeks before use in experimental determinations. All cell lines were negative for known human’s pathogens, including mycoplasma. T2 and MCF7 cells were cultured respectively in RPMI and DMEM (Fisher, France) supplemented with 10% FBS (Fetal Bovine Serum, Eurobio, France), 1% penicillin and 1% streptomycin (Eurobio, Courtaboeuf, France). Cells were pulsed with or without peptides and stained with a FITC-conjugated anti-HLA-A2 mAb (BB7.2, BD Biosciences). Flow cytometry was performed using a DxFlex (Beckman Coulter, Brea, CA, USA) and the data were analyzed using FlowJo software 10.6.2 (Treestar Inc., Ashland, OR, USA).

Peptide binding assay

T2 cells are a hybrid human cell line which lack most of the MHC class II region including the known TAP (transporter proteins for antigenic peptide) and proteasome genes. They contain the gene HLA-A*0201, but express very low levels of cell-surface HLA-A2.1 and are unable to present endogenous antigens [19]. T2 binding assays were performed to test peptide binding affinity to HLA-A*02:01 molecules. 500 000 T2 cells were incubated with 50 µg/mL of mutant, wild-type, or control peptides (V8L for negative control and L10L for positive control) for 12 h at RT in RPMI1640 media supplemented with 10% FBS. Then, T2 cells were incubated with 10 µg/mL of peptide for 30 min at RT. Surface expression of HLA-A*02:01 in T2 cells was determined by staining with an anti-HLA-A2 mAb (BB7.2, BD Biosciences). The median fluorescence intensity (MFI) was measured using a FACSCanto II (BD Biosciences).

Mice model

HLA-DRB1*0101/HLA-A*02:01 transgenic mice C57BL/6J (A2/DR1 mice) described in [20] were used to demonstrate in vivo the immunogenicity of ERα-derived peptides. Female mice aged of 8 to 10 weeks were used in the experiments. Hockpad vaccination [21] was performed on D0 with 100 µg of ERα-derived peptides and 2 µM of CpG (Invivogen, USA). Three other peptide injections were performed on D7, D14 and D121 with 50 µg of peptides and 1 µM of CpG. The mice were sacrificed on D23 by euthanasia, in order to remove the splenocyte and popliteal lymph nodes. The presence of T lymphocytes specific for mutated REα in these nodes was assessed ex vivo by 24 h IFN-γ ELISpot assay.

All experiments were carried out according to the good laboratory practices defined by the animal experimentation ethic rules in France.

Patients and healthy donors

Breast cancer patients (n = 25) were recruited from the department of Oncology at the Franche-Comté Hospital in the CRC01 cohort D. Patients’ characteristics are detailed in additional Table S3. This trial was registered with ClinicalTrials.gov (https://clinicaltrials.gov/) numbers (NCT02838381). All patients were enrolled after the signature of informed consent, in accordance with the French regulation and after approval by the local and national ethics committee. Blood was collected after cancer specific treatment.

Blood cells from anonymous healthy donors (HD) were collected at the Etablissement Français du Sang (EFS, Besançon, France) from apheresis kit preparations after the signature of informed consent and following EFS guidelines. The major exclusion criteria for blood collection in HD were minors (< 18 years), age over 65 years, dehydration, fatigue, low levels of hemoglobin (> 120 g/L for women and 130 g/L for men), flu symptoms, HIV, HTLV or hepatitis B/C positive status, autoimmune diseases, surgical procedures in the last 4 months and vaccine less than 4 weeks old. Blood collection allows the storage and use of this sample in anonymous fashion precluding clinical characterization of these donors.

PBMC (peripheral blood mononuclear cells) from HD and cancer patients were isolated by density centrifugation on Ficoll-Hyperpaque gradients (Eurobio, France).

Isolated PBMC were cryopreserved at a cell density of 8–15 × 106 cells per vial in CryoStor (CS10 and CS5) cell preservation media (Sigma-Aldrich, St. Louis, MO, USA) and were conserved at − 196 °C for IFN-γ ELISpot analysis.

IFN-γ ELISpot assay

T cell responses were assessed by IFN-γ ELISPOT after a short in vitro stimulation of fresh or thawed PBMC (viability > 80%) with ESR1-derived peptides during 7 days. For in vitro stimulation, at day 0, PBMC were plated at 1.106 cells/well for 7 days in 48-well plates with the pool of peptides derived from ESR1 (5 µg/ml). Recombinant interleukin IL-7 (5 ng/mL; PeproTech, Neuilly-sur-seine, France) was added at day 1. Recombinant IL-2 (20 UI/mL; PROLEUKIN aldesleukin, Novartis Pharma, CH, EU) was added at day 3. At day 7, the specificity of T cells was investigated by IFN-γ ELISpot, according to the manufacturer’s instructions (Diaclone, Besançon, France). Briefly, T cells (105 per well) were cultured in anti-human IFN-γ monoclonal antibody pre-coated IFN-γ ELISpot plates with ESR1-derived peptides (5 mg/mL) in X-vivo 15 medium (Lonza, Bâle, Switzerland) for 18 h at 37 °C. Cells cultured with medium alone or phorbol myristate acetate (PMA; 1 ng/ml)/ionomycin (500 ng/ml) Sigma -Aldrich, St. Louis, MO, USA) were used as negative and positive controls, respectively. The IFN-γ spots were revealed following the manufacturer’s instructions (Diaclone, Besançon, France). The number of specific T cells expressed as spot forming cells/ 105 cells was calculated after subtracting negative control values (background). Spot-forming cells were counted using the C.T.L. Immunospot system (Cellular Technology Limited, Shaker heights, OH, USA). Responses were considered positive when IFN-γ spots numbers were more than 10 and more than two times the background. All the experiments were conducted in duplicates.

Production of lentiviral vector

For production of ESR1 D538G lentiviral vectors, 2.6 × 106 293FT cells were plated into a 100-mm dish (131050 C Culture TC ClearLine 95.6 × 22 mm, sterile). Next day cells were transfected with 30 µg of lentiviral “ESR1 D538G” (VectorBulder, Neu-Isenburg, Germany), 2.7 µg of PMD2G (intern production), 8.6 µg of pSPAX2(intern production), using calcium phosphate transfection kit (Sigma-Aldrich, St. Louis, MO, USA) in the presence of 25 µM chloroquine (Sigma-Aldrich St. Louis, MO, USA). Eleven hours after transfection, the medium was changed with Optimem medium (Fisher, Courtaboeuf, France), thereafter virus containing supernatant was collected 2 days after transfection (Amicon Ultra-15 Centrifugal Filters, Sigma-Aldrich, St. Louis, MO, USA), and stored at − 80 °C until further use. A small aliquot from each production was used to determine viral titers by transduction of 293 cells with serially diluted amounts of virus supernatant. The virus supernatant was used for MOI (Multiplicity of Infection) at 15.

Lentiviral transduction

For each lentiviral transduction, 5 × 105 tumor cells per well were seeded in a 24-well plate and mixed with an appropriate amount of virus supernatant in the presence of 8 µg/ml of hexadimethrine bromide (Polybrene®) (2 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA) in a final volume of no more than 500ul. Plates were centrifuged at 3000×RPM for 15 min at room temperature. After centrifugation, without removing viral supernatants, the plates were incubated at 37 °C, 5% CO2 for 12 h. At the end of the incubation, the supernatants were removed from the wells and 1 ml of fresh tumor cell growth medium (RPMI supplemented with 10% fetal serum and 1% PS, Gibco, Illkirch, France) per well was added. The cells were maintained in this medium for at least 2 days before acquisition of enhanced green fluorescent protein (eGFP) expression was carried out. If the number of translated cells is suitable, the cells are sorted (SONY SH800S, Puteaux, France) then amplified and cloned at 1 cell/well in a 96-well plate.

Generate a T cell clone

T cell clones were generated either from HLA-A*02:01 HD PBMC. PBMC (2 × 107) were cultured in 24-well plates (4 × 106 cells per well), in RPMI medium containing 10% of human serum, 1% PS enriched with R-848 (resquimod, 3 µg/ml; Invivogen), high–molecular weight poly-IC (polyinosine-polycytidylic acid) (50 µg/ml; Sigma-Aldrich, St. Louis, MO, USA), interleukin-2 (20 IU/ml; IL-2; PROLEUKIN aldesleukin, Novartis Pharma, CH, EU), at day 0. At day 1, the peptide of interest (10 µg/mL) was added. After 2, 6, and 12 days, 100 µl of medium were replaced by enriched fresh medium (IL-7 (50 ng/mL; PreproTech 200-07) only at day 2 and IL-7 (5 ng/mL) and IL-2 (50 UI/mL) at day 6, 12) and splited if necessary. On day 14, cells were collected and functional analyses of T-cell were done by using intracytoplasmic IFN-g staining. ESR1-reactive T cells were isolated by IFN-γ T cell sorting (Miltenyi Biotec, 130-054-201), according to manufacturer’s instructions. ESR1-specific HDB.5 T cell clone was generated by limiting dilution in terasaki plates (T/C Microtitre 60 well plate, Sarstedt, Germany) and amplified after stimulation by PhytoHémaglutinnine (PHA) (Sigma-Aldrich, St. Louis, MO, USA, 1 µg/mL) and 150 UI/mL of IL-2 in presence of irradiated allogenic PBMC (25 Gy), B-EBV cell line (50 Gy).

Flow cytometry

To discriminate living from dead cells, PBMC were first washed in 1× phosphate-buffered saline (PBS) (Gibco, Grand Island, NY, USA) and stained with eFluor 780 viability dye according to the manufacturer’s instructions (eBioscience, Villebon-sur-Yvette, France ). For T cell clones analysis, samples were surface-stained in the dark for 30 min at 4 °C with the following antibodies: BV605-CD3 (BD Biosciences clone HIT3a), BV421-CD4 (SBD Biosciences clone RPA-T4), BV786-CD8 (BD Biosciences clone RPA-T8). For intracytoplasmic cytokine staining, the cells were fixed and permeabilized using cytofix/CytoPerm Plus KIT (BD Bioscience) before staining with APC-IFN-γ (BD Pharmingen, clone B27), FITC-TNFα (BD Pharmingen, clone Mab11) and PE-IL2 (Sony clone MQ1-17H12) and washed. The samples were directly acquired on DxFlex (Beckman Coulter, Brea, CA, USA) and data analyzed with FlowJo software 10.6.2 (Treestar Inc., Ashland, OR, USA).

For ESR1-tetramer staining: ESR1D538G-HLA-A2 tetramer was provided by the peptide and Tetramer Core Facility, UNIL-CHUV, Epalinges, Switzerland. One million cells were incubated with 2 µL phycoerythrin (PE)-labeled tetramers loaded with HLA-A2-derived peptide in 100 µL FACS buffer for 20 min at RT (Room Temperature). Thereafter, cells were stained with anti-CD3-BV605, anti-CD8-BV786 for 20 min at 4 °C. Cells were analyzed on a DxFlex flow cytometer and FlowJo software.

Peptide-MHC tetramer

ESR1D538G-HLA-A2 tetramer was designed using the p1 peptide (ESR1D538G).

Bulk RNA-sequencing analysis

Bulk RNA-Seq reads were aligned using STAR [22] v2.7.9a to GENCODE Human Release 39 (GRCh38.p13) and read counts matrix produced with FeatureCounts [22] v2.0.1, both with default parameters. The Bioconductor RNA-seq workflow was followed to detect differential expression genes with DESEQ2 [23] v1.34.0. A total of 19,258 coding genes filtered with biomaRt 2.5.3 were analyzed for transcript abundance and poorly expressed genes were eliminated based on the criteria of a maximum read count > 10 for all samples. Data visualization was achieved in R v4.1.1 with ComplexHeatmap package v2.11.1 and EnhancedVolcanoPlot [24] v1.12 using DESeq 2’s regularized-logarithm transformation (rlog) of the count data. HLA Typing has been performed by ArcasHLA [25] v0.5.0 and been use with default parameters. TCR Analysis has been executed by the tool MIXCR [26] v3.0.13 with the default parameter “analyze shotgun”. Gene set enrichment analysis was performed with FGSEA R package v1.20.0 on a gene set subset from msigdb including “ANTIGEN_PROCESSING” in pathways from c2 collection CP: KEGG subcollection. Figure showing differential expression of pathway genes using pathview R package v1.34.0. Computations have been performed on the supercomputer facilities of the Mésocentre de calcul de Franche-Comté.

Availability of RNA-seq data and materials

The dataset used in this study is the property of the Gustave Roussy Institute and is available upon reasonable request.

METAPRISM (PMID: 36862804) EGAD00001009684

Statistics

Statistical analyses were carried out with Prism 8 software (San Diego, CA, USA). Continuous parameters were summarized with median and interquartile range (IQR) and compared between subgroups of interest using the Wilcoxon-Mann–Whitney test. Proportions were compared using the Chi2 test (or Fisher exact test, if appropriate). The level of significance was set at p < 0.05 for all tests (∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ 0.0001).