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

1. TCR T cell Cytotoxicity In Vitro:

Measured the ability of engineered TCR-T cells to kill tumor cells using an LDH release assay after an 18-hour co-culture.

2. Single-cell Analysis:

Used single-cell RNA sequencing (BD Rhapsody™ platform) to profile the gene expression of TCR-T cells after they interacted with tumor cells (18-hour co-culture).

2.1. Sample Staining:

labeled each sample with a unique barcode, and pooled them for sequencing.

2.2. Library Prep:

Created sequencing libraries from the pooled cells using a targeted panel for oncology/immune genes (BD Rhapsody™ Onco-BC Panel HS).

2.3. Data Processing:

Processed raw sequencing data to generate a gene expression matrix for each cell, correcting errors and demultiplexing samples.

2.4. Data Analysis:

Analyzed the gene expression data to identify T cell clusters and find differentially expressed genes.

3. Cytokine Quantification:

Prepared co-cultures of T cells and tumor cells and collected the conditioned media to measure the levels of multiple cytokines in the conditioned media using LegendPlex assay.

4. TCR T cell Cytotoxicity In Vivo:

Tested the ability of engineered TCR-T cells to control tumor growth in immunodeficient mice with human tumor xenografts over a 45-day period.