Spheroid coculture and imaging
For coculture with PBMCs, PBMCs (HLA-matched if possible) pre-stimulated with IL-2 were added 3 days after spheroid formation at the ratio of 10:1. On the day of coculture, PBMCs were stained with MERCK Red Stain PKH26 following manufacturer instructions, and spheroids were stained with 0.1 uM Invitrogen Cell Event, followed by drug administration. Image acquisition was performed once a day for 3 days. For the blocking assay, PKH26-stained and CellEvent-stained spheroids were cocultured in the presence or absence of anti-CXCL10 neutralizing antibody (10 µg/mL; RandD systems; catalog MAB266). Z-stack analysis was done by transferring the individual spheroid to a flat-bottom clear-bottom 96-well plate which helped facilitate imaging. Images were obtained at 10 µm increments on the GE InCell6000 instrument using a 10x objective with an aperture of 0.6-0.8 AU. Then using free software provided by ImageJ (made available by the National Institutes of Health [NIH]), stacking of the 10 images was performed, followed by application of maximum intensity projection along the Z axis by the Fiji plug-in “Particle Analyzer”. 3D pictures were generated using “Clear Volume”.