Figure 2.

TETARs can be activated more efficiently than a mixture of 2 pools of CD8⁺ T cells each reprogrammed with only one specificity. CD8⁺ T cells were RNA-transfected with the single TCRs (plain-colored columns): the gp100 TCR (white columns), the human CCT6A^m (hCCT6A^m) TCR (light gray column), or the murinised CCT6A^m (mCCT6A^m) TCR (dark gray columns). TETARs (hatched columns) were generated by transfecting CD8⁺ T cells with the gp100 TCR in combination with the human CCT6A^m TCR (hTETAR; white and light gray), or the murinised CCT6A^m TCR (mTETAR; white and dark gray). As a control, T cells were mock electroporated. Additionally, T cells transfected with gp100 TCR were mixed in a 1:1 ratio with T cells transfected with either the hCCT6A^m TCR (white and light gray chequered columns) or the mCCT6A^m TCR (white and dark gray chequered columns). Cells were stimulated with gp100 peptide (A) - or CCT6A^m peptide-loaded (B) D05-Mel#6 tumor cells overnight, and CD25-expression was measured by flow cytometry. The specific increase in the percentage of CD25-positive T cells was determined by subtraction of non-peptide-stimulated T cells. Cytotoxicity assays were performed with gp100 peptide-loaded (C), or CCT6A^m peptide-loaded (D) D05-EBV cells as target cells with a target-to-effector cell ratio of 1:60. Average values of 3 (A and B) or 5 (C and D) independent experiments ± SEM are shown. p-values were calculated with the Student's t-test (*p ≤ 0.05, **p ≤ 0.01, ns = not significant).