Journal: Journal for Immunotherapy of Cancer

Article Title: Membrane anchored IL-18 linked to constitutively active TLR4 and CD40 improves human T cell antitumor capacities for adoptive cell therapy

doi: 10.1136/jitc-2020-001544

Figure Lengend Snippet: MemIL-18 constructs: design, expression and signaling. (A) Sketches of the membrane-anchored protein products. S, short and L, long linker. (B) Immunoblot analysis for the indicated protein products in lysates of K562 cells. Twenty-four hours post-transfection, cell lysates were subjected to PAGE analysis and probed by using rabbit anti-human IL-18 Ab and HRP-conjugated goat anti-rabbit Ab. (C) Quantitative fluorescence imaging in CD8 T cells engineered by memIL-18 mRNA electroporation. Images were acquired using the WiScan Hermes High-Content Imaging System using an X60 objective and quantification was performed with the WiSoft Athena software protein expression application. Graph summarizes mean intensity ±SEM of at least a thousand cells in 137 images. Nuclei (blue). Scale bar: 12.5 µm. Indicated significance was determined by one-way ANOVA with Tukey’s multiple comparison test; (***p≤0.001). (D) Representative flow cytometry data for the surface expression of memIL-18 in TIL14, 24 hours post-mRNA transfection. The experiment was repeated twice. (E) Upregulation of T-bet in mRNA-transfected CD8 + T cells of a healthy donor. Cells were transfected with the indicated mRNAs and stained for cell surface IL-18 and for intracellular T-bet, using a PE-conjugated anti-human T-bet mAb. The experiment was repeated twice. (F) Reporter assay for activation of the NF-κB pathway in HEK293T cells. Cells were transiently transfected with the NF-κB-Luciferase reporter plasmid. Twenty-four hours later, cells were transfected with 10 µg of the indicated mRNAs and after 24 hours luciferase activity in cell lysates was monitored. caTLR4 and caCD40, constitutively active TLR4 and CD40, respectively. All experimental data show mean values±SEM from four independent experiments. Indicated significance was determined by one-way ANOVA with Tukey’s multiple comparison test; (**p≤0.01; ***p≤0.001). (G) The two memIL-18 products engage IL-18R both in cis and in trans. CD8 + T cells were electroporated with mRNA encoding the two memIL-18 products or GFP. Six hours post-transfection, coculture was performed at a 1:1 GFP/CD8 + :memIL-18/CD8 + cell ratio. Fourteen hours later, cells were stained for cell surface IL-18 and for intracellular T-bet as described above. (H) CD8 + T cells were transfected with the indicated mRNAs, with or without the anti-melanotransferrin CAR mRNA, and cocultured at a 1:1 ratio with 579 melanoma cells for 24 hours. Growth medium was then collected and analyzed for the presence of IL-18 by MAGPIX Luminex. Graph bars present the mean cytokine concentration (pg/mL)±SEM (triplicate) from three healthy donors (n=3). (I) TIL14 and TIL86 were transfected with the indicated mRNAs, cultured in the presence or absence of the 624 melanoma cells at a 1:2 E:T ratio for 24 hours and growth medium was similarly analyzed for IL-18. Some error bars obtained with MAGPIX are too small to be visible. ANOVA, analysis of variance; E:T, effector:target; mAb, monoclonal antibody; N.T., non-transfected; P.O., pulse-only.

Article Snippet: Image quantification was performed with the WiSoft Athena software protein expression Application (IDEA Bio-medical, Rehovot, Israel).

Techniques: Construct, Expressing, Membrane, Western Blot, Transfection, Fluorescence, Imaging, Electroporation, Software, Comparison, Flow Cytometry, Staining, Reporter Assay, Activation Assay, Luciferase, Plasmid Preparation, Activity Assay, Luminex, Concentration Assay, Cell Culture