Figure 3: Luciferase IVT RNA electroporation permits assessment of antigen-specific CTL activity comparable to the 51Cr release and superior in the ability to monitor killing kinetics. (a) Cytolytic activity of primary CMV-pp65-specific T cells. CMV+ donor-derived CD8+ T cells were expanded for one week and used to assess the killing of autologous mDCs transfected with 50 μg luc2 RNA and loaded with overlapping peptide pools representing either CMV-pp65 or HIV-gag as control. Specific lysis was determined after 4 h incubation of peptide-loaded target cells with CD8+ effector cells using different E : T ratios. (b) Kinetics of killing mediated by TCR-transfected CD8+ T cells. OKT3-stimulated CD8+ T cells of a CMV-   donor were transfected with 20 μg TCR-8-CMV#14 alpha and beta chain IVT RNAs. Autologous iDCs transfected with 20 μg luc2 RNA were loaded with either peptide pp65495–503 or tyr368–376 as control. iDCs and CD8+ T cells were cocultured at different E : T ratios and specific killing was assessed at different time points. (c) Dose-dependent killing of target cells using different antigen formats. OKT3-stimulated CD8+ T cells from a CMV-   donor were electroporated with 20 μg TCR-8-CMV-#14 IVT RNA. Autologous iDCs were cotransfected with 20 μg luc2 IVT RNA and decreasing amounts of a CMV-pp65 antigen-encoding IVT RNA or were luc2 transfected and subsequently pulsed with titrated amounts of peptide pp65495–503. iDC transfected with luc2 IVT RNA and pulsed with 1000 nM SSX241–49 peptide served as a control. Effector and target cells were incubated at an E : T ratio of 19 : 1. ((d) and (e)) Comparability of the luc2 IVT RNA assay with the 51Cr assay. Cytotoxicity of CMV-pp65-specific CD8+ T cells of a CMV+ donor against (d) K562-A2 cells or (e) autologous mDCs was assessed after one week antigen-specific expansion using the luc2 IVT RNA assay in comparison to the 51Cr assay. 20 h after luc2 RNA electroporation, target cells were loaded with pp65495–503 peptide either alone or together with 100 μCi of 51Cr. 1 × 104 peptide-loaded targets were incubated at different E : T ratios with CD8+ effector cells for 4 h. Cytotoxicity was determined via measurement of luminescence after addition of D-luciferin substrate or via measurement of released 51Cr after harvesting of supernatant. All graphs represent the mean ± SD lysis ().