Volume 16, Issue 6, Pages 1170-1180 (June 2008) Enhancing the T-cell Stimulatory Capacity of Human Dendritic Cells by Co- electroporation With CD40L, CD70 and Constitutively Active TLR4 Encoding mRNA  Aude Bonehill, Sandra Tuyaerts, An MT Van Nuffel, Carlo Heirman, Tomas J Bos, Karel Fostier, Bart Neyns, Kris Thielemans  Molecular Therapy  Volume 16, Issue 6, Pages 1170-1180 (June 2008) DOI: 10.1038/mt.2008.77 Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 1 Transgene expression after mRNA electroporation. (a) Dendritic cells (DCs) were electroporated with CD40L alone or in combination with CD70 and/or caTLR4. Immediately after electroporation, protein transport was blocked with GolgiPlug and, after 4 hours, cells were stained intracellularly for CD40L. Immature DCs electroporated with irrelevant mRNA were used as a negative control (Imm NGFR). The results are representative of data from three independent experiments. (b) DCs were electroporated with CD70 alone, or in combination with CD40L, or in combination with CD40L and caTLR4. At several time-points after the electroporation, the DCs were stained for determining CD70 expression. Immature DCs electroporated with irrelevant mRNA were used as a negative control. The results are representative of data from three independent experiments. NGFR, nerve growth factor receptor; caTLR4, constitutively active toll-like receptor 4. Molecular Therapy 2008 16, 1170-1180DOI: (10.1038/mt.2008.77) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 2 Nuclear factor-κB (NF-κB) activation assay. 293T cells were transfected with the pNFconluc reporter gene plasmid (encoding the firefly luciferase gene driven by a minimal NF-κB-responsive promoter) and the pHR-GLuc-YFP plasmid [encoding the humanized secreted Gaussia luciferase fused to yellow fluorescent protein (YFP)]. When indicated, the cells were co-transfected with the pcDNA3-caTLR4 or pcDNA3-CD27 expression plasmid. It is important to note that 293T cells endogenously express CD40. Transfections were performed in triplicate, and the total amount of plasmid was kept constant by adding empty pcDNA3 plasmid. After transfection, 1 × 105 dendritic cells (DCs) electroporated with CD40L or CD70 mRNA were added when indicated. These DCs were added to the 293T cells 2 hours after their electroporation. After 24 hours, luciferase activities were determined and normalized on the basis of secreted Gaussia luciferase activity. The results are shown as mean values ± SD, and are representative of data from three independent experiments. caTLR4, constitutively active toll-like receptor 4; NGFR, nerve growth factor receptor. Molecular Therapy 2008 16, 1170-1180DOI: (10.1038/mt.2008.77) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 3 Electroporating immature dendritic cells (DCs) with CD40L and/or constitutively active toll-like receptor 4 (caTLR4) mRNA induces phenotypic maturation and enhanced interleukin-12 (IL-12) secretion, and stimulates naive CD4+ T cells to differentiate into interferon-γ (IFN-γ)-secreting cells. (a) DCs electroporated with different combinations of CD40L, CD70, and caTLR4 mRNA were stained after 24 hours for costimulatory molecules CD40, CD80, CD83, and CD86 for human leukocyte antigen class I molecules, and for the migration marker CCR7. The percentage of positive cells and mean fluorescence intensity are indicated. The results are representative of data from at least eight independent experiments. (b) IL-12p70 produced within 24 hours after electroporation was dosed in the supernatant. Each dot represents an individual experiment, and the mean is indicated by a horizontal line. Statistically significant differences between DCs co-electroporated with CD40L/CD70/caTLR4 mRNA and immature (Imm)/cytokine cocktail–matured (Mat) DCs electroporated with irrelevant nerve growth factor receptor (NGFR) mRNA are indicated by “asterisks”. (c) Electroporated DCs were used for stimulating allogeneic naive CD45RA+CD4+ T cells. Six days later, CD4+ T cells were restimulated with CD3/CD28 T-cell expander beads. After 24 hours, IFN-γ secretion was assessed in the supernatant using enzyme-linked immunosorbent assay. Each dot represents an individual experiment, and the mean is indicated by a horizontal line. Statistically significant differences between DCs co-electroporated with CD40L/CD70/caTLR4 mRNA and Imm/Mat DCs electroporated with irrelevant NGFR mRNA are indicated by asterisk. Molecular Therapy 2008 16, 1170-1180DOI: (10.1038/mt.2008.77) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 4 Induction of HLA-A2-restricted MelanA-specific CD8+ T cells, cytolytic CD8+ T cells, and interferon-γ (IFN-γ)/tumor necrosis factor-α (TNF-α)-secreting CD8+ T cells by dendritic cells (DCs) electroporated with different combinations of CD40L, CD70, and constitutively active toll-like receptor 4 (caTLR4) mRNA. (a) Naive CD8+ T cells were stimulated three times with electroporated, MelanA-A2 peptide–pulsed DCs. Next, T cells were counted and stained for CD8 and MelanA specificity. The fold increase over immature (Imm) DCs electroporated with irrelevant mRNA is shown. Each dot represents an individual experiment, and the mean is indicated by a horizontal line. Statistically significant differences between DCs co-electroporated with CD40L/CD70/caTLR4 mRNA and Imm/cytokine cocktail–matured (Mat) DCs electroporated with irrelevant nerve growth factor receptor (NGFR) mRNA are indicated by asterisk. (b) Cytolytic activity of MelanA-specific T cells was determined using a CD107a mobilization assay. Primed T cells were restimulated with T2 cells pulsed with gag or MelanA peptide in the presence of anti-CD107-PE-Cy5 monoclonal antibodies (mAbs) and GolgiStop. After overnight culture, the cells were harvested, stained with anti-CD8-FITC, and analyzed using flow cytometry. T cells were gated on forward scatter (FSC)/side scatter (SSC) characteristics and CD8 positivity. The percentage of CD107a-positive cells is shown, after subtraction of background response induced by T2 pulsed with gag peptide. (c) Intracellular IFN-γ/TNF-α production by MelanA-primed CD8+ T cells was measured using flow cytometry. Primed T cells were restimulated with T2 cells pulsed with gag or MelanA peptide in the presence of GolgiPlug. After overnight culture, T cells were stained for CD8, IFN-γ, and TNF-α positivity. The T cells were gated on FSC/SSC characteristics and CD8 positivity. The percentage of IFN-γ and/or TNF-α secreting cells is shown, after subtraction of background response induced by T2 pulsed with gag peptide. The results in b and c are for experiment 2. The percentage of MelanA-A2 tetramer-positive cells is indicated. For all other experiments, CD107a positivity and IFN-γ/TNF-α secretion correlated with the percentage of MelanA-specific T cells present in the culture. (d) Phenotype of MelanA-specific CD8+ T cells. T cells were stained for CD8 and MelanA-A2 tetramer positivity in combination with the T-cell markers CD45RA, CD45RO, CD27, CD28, CCR7, and CD62L. The results are shown for the MelanA-specific CD8+ T cells induced by DCs electroporated with CD40L, CD70, and caTLR4 mRNA, and are representative for all MelanA-specific CD8+ T cells, irrespective of which DCs that were used for stimulation. (e) Naive CD8+ T cells were stimulated three times with DCs electroporated with CD40L, CD70, or caTLR4 that were either pulsed with MelanA-A2 peptide or co-electroporated with sig-MelanA-DCLamp mRNA. Next, the T cells were counted and stained for CD8 and MelanA specificity. The number of MelanA-A2 tetramer+ CD8+ T cells is shown. Data from one of the three experiments carried out are shown. HLA, human leukocyte antigen; FITC, fluorescein isothiocyanate; PE, phycoerythrin. Molecular Therapy 2008 16, 1170-1180DOI: (10.1038/mt.2008.77) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions