Anti-HER2/neu TCR-T Cells in Action: Linking Transcriptional Signatures, Secretomics, and In Vivo Tumor Suppression

Extract and growth protocol One day before initiating the experiment, 24-well plates were coated with Retronectin (25 µg/mL, Sci Store, Russia) and anti-CD3 antibodies (5 µg/mL, BioLegend) in ACDA buffer (415 µL per well) and incubated overnight. Peripheral blood (n = 4) was drawn from HLA-A*02-positive conditionally healthy donors into EDTA-coated vacuum tubes. Peripheral blood mononuclear cells (PBMCs) were then isolated by Ficoll™ (PanEco, Russia) density gradient centrifugation at 400 × g for 40 minutes at room temperature, with the buffy coat layer subsequently collected. All the donors signed an informed consent to participate in the study. From these PBMCs, CD3+ T cells were purified using the MojoSort™ Human CD3 Negative Magnetic Selection Kit (480134, BioLegend, United States) according to the manufacturer’s instructions. The isolated CD3+ T cells (750000 cells/mL) were seeded into the pre-coated 24-well plates and activated with IL-2 (300 U/mL, Biotech LLC, Russia) for 48 hours. On the day preceding transduction, an additional set of 24-well plates was prepared by coating with Retronectin (25 µg/mL) in ACDA buffer (255 µL per well). Following the 48-hour activation period, the T cells were harvested, centrifuged at 350 g for 10 min, resuspended at a concentration of 4 × 105 cells/mL in GT-T551 (WK552S, Takara Bio, Mountain View, CA, USA) serum-free medium, and counted. The cells were then split into cells for transfection (anti-HER2/neu T cells) and non-transfected T cells (LV-neg T cells). Subsequently, 200000 cells deemed for transfection were transferred into each well of the Retronectin-coated plates in a volume of 500 µL. Lentiviral particles encoding the anti-HER2/neu TCR (200000 particles per well) at a Multiplicity Of Infection (MOI) = 1 and protamine sulfate (5–8 µg/mL) were added to to facilitate T cell transfection. The plates were centrifuged at 600 × g for 2 hours at 32°C. Post-centrifugation, 500 µL of warm GT-T551 serum-free medium containing IL-2 (final concentration 300 U/mL) was added, and the cells were incubated overnight. The next morning, the transduced T cells were transferred to 12-well plates with an equal volume of complete medium supplemented with IL-2 (300 U/mL). Cell growth was observed regularly, and every two days, partial media replacement was performed with the addition of fresh IL-2. Throughout the culture period, cell growth, conglomerate formation, and the cell culture medium state were visually monitored. Seven days post-transduction, the transduced cells were collected, centrifuged at 350 × g for 10 minutes, and the cell count and viability were assessed in a counting chamber with trypan blue staining, transduction efficiency was also evaluated using Flex-T tetramers loaded with the KIFGSLAFL peptide as previously described [https://doi.org/10.3390/cancers16234020], having positive cells stained simultaneously with two fluorochromes for their identification. Library construction protocol The scRNAseq was performed using the BD Rhapsody™ System mRNA Targeted and Sample Tag Library Preparation Protocol Data processing Sample Tag-demultiplexed immune transcriptome data was obtained via BD Rhapsody pipeline ver. 2.0 from R1 and R2 FASTQ files Reference Panel BD Rhapsody™ Onco-BC Targeted Panel (Human) (https://scomix.bd.com/hc/article_attachments/13766899704717).