Journal: bioRxiv

Article Title: Novel Fas-TNFR chimeras that prevents Fas ligand-mediated kill and signals synergistically to enhance CAR T-cell efficacy

doi: 10.1101/2023.02.22.529492

Figure Lengend Snippet: (A) Left: Upon binding FasL, Fas trimerisation recruits FADD, initiating apoptosis. Middle: FasΔDD acts as a decoy receptor to FasL by being unable to recruit FADD. Right: Schematic of Fas-TNFR structure; the ectodomain and transmembrane domain of Fas are fused to the endodomains of TNFRs. Upon FasL binding the Fas-TNFR chimera converts the death signal into a survival/growth signal. (B) Members of the TNFR superfamily. Those highlighted in gold were included in the Fas-TNFR screen. (C) Schematic of polycistronic transgene transduced into human T cells. 19-ζ: Fmc63 binder fused to the endodomain of CD3ζ via a CD8 stalk/transmembrane domain. (D) NF-κB reporter Jurkat cells transduced to express either 19-ζ alone or co-express FasΔDD or the Fas-TNFRs were cultured with or without immobilised recombinant FasL (20 μg/ml) overnight, where NF-κB activity was measured. Experiment performed with technical triplicates, error bars are SEM. (E) 5×10 4 human T cells expressing 19-ζ and FasΔDD or the Fas-TNFRs were cultured with or without immobilised recombinant FasL (20 μg/ml) for five days, at which point cell counts were analysed by flow cytometry. Due to the large number of possible Fas-TNFR chimeras, they were tested over two separate experiments (screen 1 and 2) with the data being compiled onto one graph. Five independent donors were tested in screen 1 and four independent donors were tested in screen 2, error bars are SEM. (F) Human T cells from five independent donors were transduced to express 19-ζ or co-express FasΔDD or the stated Fas-TNFRs and then cultured with or without immobilised recombinant FasL (20 μg/ml) for three days, at which point RNA was extracted and analysed using the nCounter® NanoString® platform with the CAR-T Characterization Panel. 19-ζ cells co-expressing FasΔDD or the Fas-TNFRs were made relative to 19-ζ alone, and the number of significantly ( P < 0.05) upregulated differentially expressed genes (DEGs) were categorised by pathway involvement. (G) Significant DEGs relative to 19-ζ with greatest Log 2 fold change (FC) from experiment described in F. (H) Volcano plot from experiment described in F of Fas-CD40-19-ζ cells compared to 19-ζ alone after incubation with immobilised FasL. (I) Significant DEGs relative to FasΔDD-19-ζ with greatest Log 2 (FC) from experiment described in F. (J and K) 19-ζ cells were cultured in the presence or absence of immobilised FasL (20 μg/ml) for five days and then stained for CCR8, ICOSL and ICOS expression by flow cytometry (I), or the cell culture supernatant analysed for CCL1, CXCL10 and CXCL13 secretion (J). Six independent donors tested, error bars are SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, two-way ANOVA.

Article Snippet: Cytokine concentrations in cell culture supernatants were measured by ELISA using kits to detect IFNγ (Biolegend; 430104), IL-2 (Biolegend; 431804), CCL1 (R&D Systems; DY272), CXCL10 (R&D Systems; DY266), CXCL13 (R&D Systems; DY801), according to the manufacturer’s instructions using a MultiskanTM FC microplate photometer (Thermo Scientific).

Techniques: Binding Assay, Cell Culture, Recombinant, Activity Assay, Expressing, Flow Cytometry, Incubation, Staining