Extended Data Fig. 4: Higher levels of inducible gene-cargo are produced by TCR-T cells transduced with the dual antisense versus sense lentiviral vector.

(a) Schematic of sense and antisense constructs encoding an HLA-A2 restricted NY-ESO-1_157-165 specific TCR (Gene A) 1under the control of the PGK promoter and mCherry or h-IL-2 (Gene B) under the 6xNFAT promoter. (b) Top and bottom left; percentage TCR expression as measured by tetramer staining of primary human CD4⁺ and CD8⁺ T cells transduced with sense and antisense lentivirus vector supernatant produced in the presence of TNFα and NovB2. Top and bottom right; TCR expression levels (MFI values) for primary human CD4⁺ and CD8⁺ T cells transduced with sense and antisense lentivirus vector supernatant produced in the presence of TNFα and NovB2. Bar graph shows the mean + /- S.E.M. for n = 6 human donors for two independent experiments (n = 3 per experiment); ***P < 0,001 upper panel TCR sense versus TCR IL2 antisense; ****P < 0,001 bottom panel TCR IL2 sense versus TCR IL2 antisense) (c) Killing assay for TCR-T cells and UTD T cells against A2⁺/NY⁺ Saos-2 tumour cells labelled with nuclei green at ratio of 2:1 as measured by the IncuCyte instrument over time. Loss of total green area/μm² is proportional to killing activity. Shown are mean values + /- S.E.M. for T cells from n = 3 human donors. (d) IFNγ quantification by ELISA assay of TCR- and UTD-T cells co-cultured with A2⁺/NY⁺ Saos-2 tumour cells at ratio of 2:1. Shown are mean values + /- S.E.M. for T cells from n = 6 human donors for two independent experiments (left panel ns=0,9392 TCR IL2 sense versus TCR IL2 antisense, ns>0,9999 TCR mC sense versus TCR mC antisense; right panel ns=0,9959 TCR IL2 sense versus TCR IL2 antisense, ns=0,3562 TCR mC sense versus TCR mC antisense). (e) Human (h)IL-2 quantification by ELISA assay of TCR- and UTD-T cells co-cultured with A2⁺/NY⁺ Saos-2 tumour cells at a ratio of 2:1. Shown are mean values + /- S.E.M. for T cells from n = 6 human donors for two independent experiments (panel left ****P < 0,0001 TCR IL2 sense versus TCR IL2 antisense; panel right **P < 0,0023 TCR IL2 sense versus TCR IL2 antisense;). (f) hIL-2 quantification by ELISA assay of TCR- and UTD-T cells cultured overnight in the presence of PMA-Ionomycin (left panel ns=0,953 TCR IL2 sense versus TCR IL2 antisense, ns>0,9999 TCR mC sense versus TCR mC antisense). (g) Induced mCherry expression by TCR- and UTD-T cells against A2⁺/NY⁺ Saos-2 tumour cells at a ratio of 2:1 as measured by the IncuCyte instrument (total red area/μm²) over time. Shown are mean values + /- S.E.M. for T cells from n = 6 human donors (**P = 0,0031 TCR mC sense versus TCR mC antisense at endpoint). (h) Flow cytometric analysis of mCherry expression for TCR- and UTD-T cells cells co-cultured with A2⁺/NY⁺ Saos-2 tumour cells at a ratio of 2:1. Shown are mean values + /- S.E.M. for T cells from n = 3 human donors. Left; percentage of mCherry⁺ cells (*P = 0,0461 TCR mC sense versus TCR mC antisense). Right; mCherry expression levels (MFI) (**P = 0,0092 TCR mC sense versus TCR mC antisense). (i) Flow cytometric analysis of mCherry expression for TCR- and UTD-T cells after overnight stimulation with PMA-Ionomycin. Shown are the mean values + /- S.E.M. for T cells from n = 3 human donors Left; percentage of mCherry⁺ cells (ns=0,8478 TCR mC sense versus TCR mC antisense). Right; mCherry expression levels (relative MFI) (****P < 0,0001 TCR mC sense versus TCR mC antisense). Two-way (panel c and g) and One-way Anova (panels b,d,e,f,h and i) tests were used to determine statistical significance.

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