Figure 4.

TCR expression and cytotoxicity of transduced primary CD8⁺ T cells against malignant cell lines

(A) The TCR expression of selected TCRs (6G4 (MAGE-A1 LTQ/A1), 4F7 (MAGE-A1 KVL/A2), 3H4 (MAGE-A1 SLF/A3), 3G2 (MAGE-A1 RVR/B7), 10C1 (MAGE-A1 VRF/C7), 2H9 (MAGE-A3/A6 EVD/B35), and 2D8 (MAGE-A9 YVG/A1) was determined after transduction in primary CD8⁺ T cells. TCR-T cells were stained with mTCRβ APC (left) and pHLA tetramer PE (right). In the graph, the delta mean fluorescent intensity (MFI) (sample MFI – control MFI) of the tetramer and mTCRβ stain are depicted. Untransduced or CMV TCR-T cells were included as negative control (depicted in gray). (B) Cytotoxicity of the TCR-T cells against multiple tumor cell lines was determined by 6-h ⁵¹Cr-release assay with E:T ratio of 9:1 or 1:1. T cell clones were stimulated with tumor cell lines of different origin, including multiple myeloma (L363, U266, RPMI8226, UM9), melanoma (518A2, SK2.3), osteosarcoma cell line Saos-2, mammary carcinoma cell line CAMA-1, and prostate carcinoma cell line PC-3M-PRO4. Target cells naturally express target HLA or were transduced with target HLA alleles (+HLA). The MAGE-gene expression levels, measured by qPCR, are depicted between brackets as percentage relative to HKGs. CMV TCR-T cells were included as negative control and allo-HLA reactive T cell clones as positive control for HLA expression and killing capacity. Values and error bars represent means and standard deviations of technical triplicates, and experiments are representative of at least two independent experiments.