The suppressive activity of neutrophils depends on CD11b-dependent physical contact. (A) Purified T cells were cultured with anti-CD3 and anti-CD28 antibodies (red bars) and cultured either together with neutrophils in a well (blue bars) or physically separated from neutrophils by culturing the T cells on a Transwell filter insert. Neutrophils were incubated without (setup A, green bar) or with (setup B, purple bar) purified T cells in the lower compartment. T cells on top of the Transwell filter were harvested after 5 days and analyzed by flow cytometry for CFSE dilution among CD4⁺ T cells (n = 4). (B) Purified T cells (red bars) and neutrophils (blue bars) were cultured together with the indicated stimuli. Where indicated, neutrophils were pre-incubated with a CD11b-blocking antibody before they were added to the assay (n = 3-5). The isotype control had no effect on the MDSC activity (not included in graph). (C) Purified T cells from healthy donors (n = 2) were incubated with either control neutrophils or neutrophils from LAD-1 patients (n = 2). (D) Live cell imaging of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate salt (DiD)–labeled T cells (red) and neutrophils in the presence of anti-CD3 antibodies, anti-CD28 antibodies, TNF-α, and dihydrorhodamine-1,2,3 (turns green after reaction with ROS). The coculture was imaged for 4 hours. Two fields of view of 1 representative experiment of 3 is shown. The white arrows indicate the uptake of the T-cell membrane by the neutrophil. (E) DiD-labeled purified T cells were cocultured for 3 hours with neutrophils and indicated stimuli with or without a CD11b-blocking antibody, after which cells were harvested for flow cytometric analysis in which the amount of uptake of DiD-labeled T-cell membrane by neutrophils was determined (n = 4). Error bars indicate SEM. ****P < .0001; ***P < .001; **P < .01. MFI, mean fluorescent intensity.