High-resolution quantification of discrete phagocytic events by live cell time-lapse high-content microscopy imaging.

Phagocytosis is a dynamic process central to immunity and tissue homeostasis. Current methods for quantification of phagocytosis largely rely on indirect or static measurements, such as target clearance or dye uptake, and thus provide limited information about engulfment rates or target processing. Improved kinetic measurements of phagocytosis could provide useful, basic insights in many areas. We present a live-cell, time-lapse and high-content microscopy imaging method based on the detection and quantification of fluorescent dye 'voids' within phagocytes that result from target internalization to quantify phagocytic events with high temporal resolution. Using this method, we measure target cell densities and antibody concentrations needed for optimal antibody-dependent cellular phagocytosis. We compare void formation and dye uptake methods for phagocytosis detection, and examine the connection between target cell engulfment and phagolysosomal processing. We demonstrate how this approach can be used to measure distinct forms of phagocytosis, and changes in macrophage morphology during phagocytosis related to both engulfment and target degradation. Our results provide a high-resolution method for quantifying phagocytosis that provides opportunities to better understand the cellular and molecular regulation of this fundamental biological process.

Macrophage hypophagia as a mechanism of innate immune exhaustion in mAb-induced cell clearance.

Macrophage antibody (Ab)-dependent cellular phagocytosis (ADCP) is a major cytotoxic mechanism for both therapeutic unconjugated monoclonal Abs (mAbs) such as rituximab and Ab-induced hemolytic anemia and immune thrombocytopenia. Here, we studied the mechanisms controlling the rate and capacity of macrophages to carry out ADCP in settings of high target/effector cell ratios, such as those seen in patients with circulating tumor burden in leukemic phase disease. Using quantitative live-cell imaging of primary human and mouse macrophages, we found that, upon initial challenge with mAb-opsonized lymphocytes, macrophages underwent a brief burst (<1 hour) of rapid phagocytosis, which was then invariably followed by a sharp reduction in phagocytic activity that could persist for days. This previously unknown refractory period of ADCP, or hypophagia, was observed in all macrophage, mAb, and target cell conditions tested in vitro and was also seen in vivo in Kupffer cells from mice induced to undergo successive rounds of αCD20 mAb-dependent clearance of circulating B cells. Importantly, hypophagia had no effect on Ab-independent phagocytosis and did not alter macrophage viability. In mechanistic studies, we found that the rapid loss of activating Fc receptors from the surface and their subsequent proteolytic degradation were the primary mechanisms responsible for the loss of ADCP activity in hypophagia. These data suggest hypophagia is a critical limiting step in macrophage-mediated clearance of cells via ADCP, and understanding such limitations to innate immune system cytotoxic capacity will aid in the development of mAb regimens that could optimize ADCP and improve patient outcome.