Journal: Journal for Immunotherapy of Cancer

Article Title: A new synthetic toll-like receptor 1/2 ligand is an efficient adjuvant for peptide vaccination in a human volunteer

doi: 10.1186/s40425-019-0796-5

Figure Lengend Snippet: Pam 3 Cys-GDPKHPKSF (XS15) is a TLR1/2 ligand activating immune cells and stimulating DCs and cytokine release . ( a ) Structure of Pam 3 Cys-GDPKHPKSF: Skeletal structural formula of the molecular structure of the lipopeptide Pam 3 Cys-GDPKHPKSF termed XS15. ( b ) Dual-luciferase assay on HEK293T cells transfected with TLR2: HEK293T cells were transiently transfected with a human TLR2 plasmid and a NF-κB luciferase reporter plasmid or left untreated (− ctrl.). Culture medium was replaced after 30 h and stimuli added at the stated concentrations. The cells were incubated for 18 h and lysates were prepared and analysed by dual-luciferase assay. Pam 3 CysSK 4 (P3CSK4) and two different lots of XS15 (XS15#1/ XS15#2) were used. ( c ) HEK-Dual hTLR2 cells, stably expressing a NF-κB/AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter, were incubated for 1 h with TLR1, TLR2 and TLR6 blocking antibodies, isotype control or negative controls (no Abs) (4 μg/ml). Then, cells were stimulated for 24 h with the established TLR2/6 agonist FSL-1 (1 ng/ml), XS15 (10 ng/ml) or left unstimulated (− ctrl.). Supernatants were collected and SEAP levels determined using QUANTI-Blue detection assay. Error bars represent SD. The graph shows the mean + SEM of n = 2 experiments, significance was assessed by two-way ANOVA. ( d ) Immune cell activation by XS15: Fresh PBMCs were cultured for 40 h in the presence of Phytohemagglutinin-L (PHA) + Pokeweed (PWM) (P + P), Pam 3 CysSK 4 (P3CSK4), XS15 or left untreated (− ctrl.). Activated NK ( left panel ) and B cells ( right panel ) were assessed with the marker CD69 following the gating strategy: time gate, single cells (FSC-H/ FSC-A), living cells (Zombie-Aqua/ FSC-A), lymphocytes (FSC-A/ SSC-A); B-cells were defined as CD14 neg CD3 neg CD19 + cells and NK cells as CD14 neg CD3 neg CD19 neg CD56 + cells. Healthy donors ( n = 6), means are shown, significance was assessed by one-way ANOVA. ( e ) Dendritic cell (DC) stimulation by XS15: DCs were differentiated from blood monocytes and then matured as described in the material and methods section. Gating strategy was: time gate, single cells (FSC-H/ FSC-A), living cells (Zombie Aqua/ FSC-A). Upper panel : scatter plots for healthy donors ( n = 6), means are shown significance was assessed by one-way ANOVA. Lower panel : modal histograms and median fluorescences for one representative donor. Medium control without maturation cocktail = − ctrl. Standard maturation cocktail = Mat. ( f ) Induction of cytokine release by XS15: Anticoagulated whole blood was incubated with XS15 (10 μg/ml) as well as LPS (100 ng/ml) and PHA (2 μg/ml)/ PWM (1 μg/ml) as positive (+ ctrl.) and medium only as negative controls (− ctrl.) and supernatants harvested after 20 h. Multiplexed bead-based sandwich immunoassays were performed using a LUMINEX device with a 42-analyte panel. Exemplary findings obtained in three healthy donors (HD) for IL-8 ( left ), MCP1 ( middle ) and MIP-1β ( right ) are shown with means. HD1 (blue square) designates the vaccinated volunteer characterized in more detail subsequently. Additional results are provided in Additional file : Table S1. In case of saturation, the upper limit of quantification (ULOQ) was assigned. p ≤ 0.05*; ** p ≤ 0.01; *** p ≤ 0.001

Article Snippet: The kit components and software for data analysis of the multiplexed immunoassay were kindly provided by Myriad RBM, Austin, TX ( http://rbm.myriad.com ) and used as specified.

Techniques: Luciferase, Transfection, Plasmid Preparation, Incubation, Stable Transfection, Expressing, Blocking Assay, Control, Detection Assay, Activation Assay, Cell Culture, Marker, Luminex