Figure 5.

mRNA-loaded CMRF-56⁺ BDC induce recall T-cell responses to Influenza viral antigens and primary T-cell responses response to the TAA, WT1. (A) CMRF-56⁺ BDC isolated from HLA-A*0201⁺ donors were activated with GM-CSF and either transfected with IVT-FMP mRNA or with vehicle alone (Mock). These were mixed at a 10:1 ratio of autologous CMRF-56⁻ cells: BDC. The cells were supplemented with IL-2, IL-7 and IL-15 at day 3 and again with IL-2 at day 5. On day 7, cells were re-stimulated with FMP_58–66 peptide for 6 h and Brefeldin A for the last 4 h and analyzed by flow cytometry. (B) CD4 and CD8 IFNγ responses were quantified compared to Mock controls (n = 4 independent donors). Mock or WT1 mRNA transfected, HLA-A*0201 CMRF-56⁺ BDC were used to prime autologous CMRF-56⁻ cells for 7 d. (C and D) Antigen specific WT1_126–134 peptide specific IFNγ production (n = 7, paired t-test) and (E and F) extracellular CD107a expression (n = 3, paired t-test) was measured by flow cytometry following 6 h of peptide re-stimulation. (G and H) The frequency of dextramer⁺ WT1_126–134-specific CD8⁺ T cells was increased following two additional rounds of expansion with WT1_126–134 peptide for two donors and these cells had (I) the capacity to lyse T2 target cells pulsed with WT1_126–134 but not control cells pulsed with KLK4 _11–19 peptide. Effector: target ratio of 50:1 is shown. n = 3; one-tailed t-test.