Fig 2. mRNA-promoted cytokine response and adjuvant effect.

(a) PBMC were co-cultured with autologous MoDC loaded with either non-coding control mRNA (NC), spa mRNA, SpA protein or the lipofection control (LF). IFNγ was detected after overnight incubation by ELISpot. The individual results of the single donors (n = 7) were connected with lines and displayed as IFNγ spots. Analysis was carried out in technical duplicates. Following testing for normal distribution, paired Student’s t-test was used to calculate significance. (b) Cytokine production by MoDC stimulated with mRNA-encoded staphylococcal antigens or the respective proteins and controls (lipofectamine (LF), non-coding control mRNA (NC), Influenza MP1 peptides (MP1) and tetanus toxoid (TT)) was measured in supernatants after 24 hours. The results of duplicates for TNF and IFNα are shown as mean values ± SEM of n = 4 donors. p≥ 0.05 n.s. (One-way ANOVA) (c) Stimulation of MoDC/CD8⁺ T cell co-cultures with PBP2a or SpA in the presence and absence of NC mRNA after overnight IFNγ ELISPOT analysis. The ELISPOT enzymatic activity relative to the unstimulated control is shown as mean values ± SEM of n = 8 independent donors. Statistical analysis was done using the Wilcoxon matched-pairs signed rank test. Experiments were carried out in duplicates. (d) Upon transfection of MoDC with spa mRNA and co-culture with CD8⁺ T cells, blocking antibodies against IFNα and TNFα alone or in combination were added and IFNγ secretion of duplicates was quantified by ELISPOT. IFNγ enzymatic activity of n = 5 independent donors is normalized to the unstimulated isotype control and displayed as mean ± SEM. Following testing for normal distribution, paired Student’s t-test was used to calculate significance. p**<0.01, p*< 0.05.