Confocal microscopy images showing filler cells inhibition of T–T contacts. Treg cells were prestained with VF405 dye (rendered as green) and mixed with 30-fold excess unstained filler cells (shown as gray) before centrifugation onto a flat-bottom plate to form monolayer followed by incubation at 37°C. Shown are representative confocal images of the indicated channels of a randomly selected field taken at indicated time points. Bar graph summarizes the percentage of isolated Treg cells from five different image fields, calculated as (the number of Treg cells not in physical contact with another Treg cell)/(the total number of Treg cells) × 100. Each data point in the bar graph corresponds to the percentage of isolated Treg cells in a randomly selected image field.