Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity

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 pp65_495–503 or tyr_368–376 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 pp65_495–503. iDC transfected with luc2 IVT RNA and pulsed with 1000 nM SSX2_41–49 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 pp65_495–503 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 ().

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