AbstractThe prevailing view is that therapeutic antibodies deplete cells through opsonization and subsequent phagocytosis, complement-dependent lysis or antibody-dependent cellular-cytotoxicity. We used high resolution in vivo imaging to identify a new antibody-dependent cell death pathway where Kupffer cells ripped large fragments off crawling antibody-coated iNKT cells. This antibody-dependent fragmentation process resulted in lethality and depletion of crawling iNKT cells in the liver sinusoids and lung capillaries. iNKT cell depletion was Fcy-receptor dependent and required iNKT cell crawling. Blood, spleen or joint iNKT cells that did not crawl were not depleted. The antibody required high glycosylation for sufficiently strong binding of the iNKT cells to the Fc Receptors on Kupffer cells. Using an acetaminophen overdose model, this approach functionally depleted hepatic iNKT cells and affected the severity of liver injury. This study reveals a new mechanism of antibody-dependent killing in vivo and raises implications for the design of new antibodies for cancer and auto-reactive immune cells.