Multimodal profiling of HER2/neu-specific TCR-T cells reveals clonotype-dependent activation programs underlying differential cytotoxic efficacy

T cell isolation, activation, and lentiviral transduction Peripheral blood samples were obtained from healthy HLA-A*02:01-positive donors (n = 15) after written informed consent in accordance with institutional ethical guidelines. Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation using Ficoll™ (PanEco, Russia) at 400 × g for 40 min at room temperature. CD3⁺ T lymphocytes were purified from PBMCs by negative magnetic selection using the MojoSort™ Human CD3 T Cell Isolation Kit (480131, BioLegend, USA) following the manufacturer’s protocol. For activation, T cells (1 × 10⁶ cells mL⁻¹) were seeded in 24-well plates pre-coated overnight at 4 °C with the recombinant fibronectin fragment Retronectin (10 µg mL⁻¹; Sci Store, Russia) and Human CD3/CD28 T Cell Activation Beads (422604, BioLegend, USA). Cells were cultured for 24 h in RPMI-1640 medium supplemented with recombinant human IL-7 (10 ng mL⁻¹; PeproTech, USA) and IL-15 (5 ng mL⁻¹; PeproTech, USA) at 37 °C in 5 % CO₂. Activated T cells were harvested, washed, and resuspended at 0.5–1.0 × 10⁶ cells mL⁻¹ in complete RPMI-1640 medium containing 2 % human serum albumin (HSA). Cells (2 × 10⁵ per well) were seeded into Retronectin-coated wells, and lentiviral particles encoding the HER2/neu-specific T cell receptor (TCR) were added at a multiplicity of infection (MOI) of 4 in the presence of protamine sulfate (5 µg mL⁻¹). Plates were centrifuged at 1200 × g for 90 min at 32 °C and incubated for 8 h at 37 °C. After incubation, cells were washed to remove residual virus and protamine, resuspended in fresh complete medium containing recombinant human IL-2 (300 IU mL⁻¹; Biotech LLC, Russia), and transferred to 12-well plates. Cultures were maintained by partially replacing medium every 48 h with fresh IL-2–supplemented medium. On day 7 post-transduction, cells were harvested, washed, and counted using trypan blue exclusion (PanEco, Russia) and Calcein AM (C1430, Thermo Fisher Scientific, USA) viability staining on an Attune NxT Flow Cytometer (A29001, Thermo Fisher Scientific, USA). Co-culture of TCR-engineered T cells with tumor cell lines To assess cytotoxicity and transcriptional responses, HER2/neu-specific TCR-engineered T cells were co-cultured with tumor cell lines. Two HER2/neu-positive melanoma lines (SK-MeL-5 and SK-MeL-37) and two HER2/neu-negative controls (HCT and MDA) were maintained in logarithmic growth phase and detached using a 1:3 mixture of 0.25 % trypsin (PanEco, Russia) and Versen solution (Vector, Russia). Tumor cells (4 × 10⁴ per well) were seeded into 96-well flat-bottom plates and allowed to adhere for 2–3 h at 37 °C in RPMI-1640 medium supplemented with 5 % fetal calf serum (FCS). Following adhesion, TCR-transduced (anti-HER2/neu) and non-transduced (Non-TD) T cells were added at 4 × 10⁵ cells per well, corresponding to an effector-to-target (E:T) ratio of 10:1. Co-cultures were incubated for 24 h at 37 °C in 5 % CO₂. After incubation, both anti-HER2/neu and Non-TD T cells were collected by gentle pipetting. T cell preparation and AbSeq antibody staining for single-cell multi-omics For single-cell capture, 1 × 10⁶ anti-HER2/neu Construct #2 T cells and non-transduced T cells (pre- and post-tumor co-culture) were washed twice in PBS + 0.04 % BSA, filtered through 40 µm filters, and resuspended in BD OMICS-Guard Sample Preservation Buffer (570911, BD Biosciences, USA). Pre-co-culture aliquots were stored at 4 °C during the 24 h of tumor co-culture. Before loading onto the BD Rhapsody cartridge, cells were centrifuged at 800 × g for 5 min, and the supernatant was discarded to remove OMICS-Guard buffer. For sample multiplexing and surface protein quantification, cells were incubated with 15 BD AbSeq antibody–oligonucleotide conjugates and 12 BD Sample Tags (1 tag per sample) for 30 min at room temperature in the dark, followed by two washes in staining buffer. The AbSeq panel was optimized to resolve naïve, memory, activated, and innate-like T-cell subsets and included the following specificities: CCR7, CD117 (KIT), CD122 (IL-2RB), CD127 (IL-7R), CD25 (IL-2RA), CD294 (CRTH2), CD45RA, CD45RO, CD4, CD56, CD62L, CD71, CD86, CD8, and CD95 (FAS) (BD Biosciences, USA). Plates were centrifuged at 1200 × g for 90 min at 32 °C and incubated for 8 h at 37 °C. After incubation, cells were washed to remove residual virus and protamine, resuspended in fresh complete medium containing recombinant human IL-2 (300 IU mL⁻¹; Biotech LLC, Russia), and transferred to 12-well plates. Cultures were maintained by partially replacing medium every 48 h with fresh IL-2–supplemented medium. On day 7 post-transduction, cells were harvested, washed, and counted using trypan blue exclusion (PanEco, Russia) and Calcein AM (C1430, Thermo Fisher Scientific, USA) viability staining on an Attune NxT Flow Cytometer (A29001, Thermo Fisher Scientific, USA). Single-cell library preparation and sequencing Single-cell capture, cDNA synthesis, and library preparation were performed using the BD Rhapsody™ Single-Cell Analysis System (BD Biosciences, USA) following the manufacturer’s instructions (Targeted mRNA, AbSeq, and Sample Tag Library Preparation Protocol, Doc ID 23-22025, BD Biosciences). Cells were loaded onto the BD Rhapsodycartridge at 55 cells µL⁻¹. Cell concentration was verified on an InCell Analyzer 6000 (GE Healthcare, USA) by counting Calcein AM-positive cells in five fields of view and calculating expected occupancy (175 microwells per field, 200 000 microwells total). After cell lysis, poly-A mRNA and oligonucleotide tags from AbSeq and Sample Tag antibodies were hybridized to bead-bound capture oligonucleotides. Beads were recovered and reverse-transcribed to generate cDNA for mRNA, AbSeq, and Sample Tag targets. Targeted transcriptome enrichment used the BD Rhapsody Human T Cell Expression Panel (633751, BD Biosciences, USA), covering 259 genes related to T-cell phenotype, activation, cytokine signaling, cytotoxicity, and immunoregulation. After reverse transcription, mRNA, AbSeq, and Sample Tag PCR1 products were purified with AMPure XP magnetic beads (A63880, Beckman Coulter, USA) and size-separated. Purified mRNA and Sample Tag PCR1 products underwent semi-nested PCR2 amplification to improve specificity, followed by an additional bead purification step. Library concentrations were determined using a Qubit 4 High-Sensitivity dsDNA Assay Kit (Q32853, Thermo Fisher Scientific, USA), and fragment sizes were verified by Qsep1 DNA High-Sensitivity Capillary Electrophoresis (BiOptic, Taiwan). Indexed libraries were pooled at an approximate ratio of 39 : 57 : 4 (mRNA : AbSeq : Sample Tag) to a final concentration of 10 nM, targeting sequencing depths of 10 000, 15 000, and 1 000 reads per cell for each library type, respectively. Sequencing was performed on an Illumina NovaSeq X system using a 10B flow cell (R1 = 51 bp, R2 = 51 bp), yielding ~1.5 × 10⁹ paired-end reads. Raw sequencing data processing Sequencing data were processed using the BD Rhapsody Analysis Pipeline (v2.2, BD Biosciences). After initial quality control to exclude low-quality reads, R1 reads were used to extract cell and UMI barcodes, and R2 reads were aligned to the BD Rhapsody Human T Cell Expression Panel (633751, BD Biosciences) and corresponding AbSeq references via Bowtie2. Reads sharing cell tags and UMIs were collapsed into unique molecules. Recursive substitution error correction (RSEC) and distribution-based error correction (DBEC) were applied to minimize amplification bias and sequencing errors. Valid cell-associated barcodes were identified via second-derivative inflection analysis, excluding background RNA. Sample Tag barcodes enabled sample demultiplexing and multiplet exclusion. Across all samples, 30 467 high-quality cells (n = 12; three biological replicates per condition) were detected across anti-HER2/neu TCR-transduced and non-transduced T cells before and after SK-MeL-37 co-culture. Sequencing saturation was 97.67 %, with a mean DBEC-adjusted depth of 7.09, where values ≥ 8 represent deep coverage for BD Rhapsody datasets.