Figure 2
From: A stable, engineered TL1A ligand co-stimulates T cells via specific binding to DR3
Fc-scTL1A Architecture Displayed High Monodispersity and Functional Activity. (A) Preparative gel-filtration analysis of TL1A constructs (indicated in legend). Recombinant TNFa was used as a control to show the population of trimer vs. oligomers. Main peaks in each chromatogram represent the target oligomeric species indicated in the main text. Off-target high molecular weight (HMW) oligomers and off-target low molecular weight (LMW) species are indicated in the chromatograms. (B) Analytical SEC analysis of TL1A constructs after preparative gel-filtration purification to isolate desired oligomeric species. The purified species were used in functional assays. (C, D) ELISA analysis of the ability of TL1A constructs to bind DR3 (left) or DcR3 (right). Molar concentrations of TL1A for each construct are normalized to concentration of TL1A trimers in each molecule. (E, F) Pan T cells from healthy donors were incubated with plate-bound anti-CD3 Ab (0.01 mg/mL). TL1A ligands at 0, 0.03, 0.1, 0.3, 1, 3, 10, 30 or 100 nM (trimer molarity) were added to aCD3-activated T cells. Levels of IFNg (left) and TNFa (right) produced by the activated T cells are shown. Stastical significance was determined using an unpaired T-test of unstimulated vs stimulated cells for each construct. Results represent averages from three independent experiments. Graphs were generated using Graphpad Prism (Version 9): https://www.graphpad.com/scientific-software/prism/.
