Vegf c

ILC migratory effect was assessed ex vivo using CytoSelect 96-well Cell Migration Assay, in 5 μm pore size Transwell plates (Cell Biolabs). Uncoated Transwell inserts were placed in Yssel’s medium (1% human serum) containing recombinant protein VEGF-165 (25 ng/mL; BioLegend) or VEGF-C (50 ng/mL) or CXCL13 (200 ng/mL; BioLegend). A solution of ILC (20,000 in 0.1 mL of serum-free Yssel’s medium) was added to the upper well of the Transwell, followed by 12 hr incubation at 37°C. Following CytoSelect Cell Migration manufacturer protocol, migratory cells were lysed and stained with fluorescence dye, followed by quantification of chemotaxis activity using fluorescence plate reader (BMG Polarstar; MTX Lab Systems).

CAR-T cells were incubated with 10 µg/mL recombinant mVEGFR-2-hIgG-Fc fusion protein (R&D Systems) in the presence of control protein or soluble murine VEGF-A (VEGF-164, BioLegend), VEGF-C (BioLegend), or VEGF-D (R&D Systems), at a VEGF/VEGFR-2 molar ratio of 3:1 in a total volume of 10 μL. Alternatively, VEGF-A was incubated with anti-VEGF-A antibody (Clone B20‐4.1.1; a kind gift from Genentech, San Francisco, California, USA) at a VEGF-A:anti-VEGF-A antibody molar ratio of 1:3 for 30 min at 37°C prior its addition to CAR-T cells. The cells were then washed and stained to evaluate mVEGFR-2-hIgG-Fc binding levels on the CAR-T cells as described above.