Complement (C) Activation Followed by Penetration of the Membrane Attack Complex (MAC) on B Cells Opsonized with CD20 Mabs Allows for Calcium Influx Which Induces Streamers. Generalization of Streaming to Include other Mabs and Target Cells.

We recently reported (J. Immunology, 2008) that C activation by ofatumumab- or rituximab-opsonized Daudi B cells and by ofatumumab-opsonized CLL cells induces rapid (less than 2 min) generation of long, thin structures (³ 3 cell diameters) protruding from cell surfaces, which we call streamers. Of note, ofatumumab induced more rapid killing and streaming on Daudi cells than rituximab. In contrast to rituximab, ofatumumab also promoted streamer formation and killing of ARH77 cells and primary B cells from patients with chronic lymphocytic leukemia. Streamers, whose constituents include membrane lipids, actin, and deposited C3b fragments, appear on cells before they are killed, and can be visualized by fluorescence and light microscopy, as well as by spinning disk confocal microscopy. We have since confirmed that C-induced streamers are also generated for several mAb/substrate cell systems including: alemtuzumab/Wien 133 cells; alemtuzumab/CLL cells; HuMax-CD38/Daudi cells; and anti-HLA DR/DP/DQ/Daudi cells. However, several mAb/cell substrate systems, such as cetuximab/SCC-25 cells and trastuzumab/BT-474 cells, which do not trigger substantial C activation or C-mediated killing, failed to produce streamers. Furthermore, robust C activation (C3b deposition) followed by insertion of the MAC, a lytic pore-forming agent, are essential for generation of streamers, and streamers are not observed in C9-deficient serum. Results also demonstrated that reaction of Daudi cells in media with either mellitin, another pore-forming agent, or with calcium ionophore A23187 induces substantial streaming, but streaming mediated by both reagents is almost completely abrogated in the presence of EDTA. We therefore hypothesized that initial penetration of cells by the MAC before the cell is killed allows rapid influx of calcium, and it is calcium influx that represents the most proximate cause of streaming. Influx of calcium promoted by other stimuli has previously been reported to generate nanotubules (Simon and Watkins, Immunity, 2005), which have structures similar to streamers and which may play a role in intercellular communication. Therefore, to test our hypothesis for the generation of streamers, ofatumumab-opsonized Daudi cells were reacted at 37°C with C5-deficient normal human serum (NHS) for periods varying between 30 seconds and 1 hour. Flow cytometry confirmed that this first step promotes deposition of C3b on the cells, but not killing. This paradigm allows for activation of the remainder of the C pathway in a subsequent step, which does not require metal ions. Therefore, in a second step, cells were washed and reacted with NHS-EDTA for 15 minutes at 37°C to provide the remaining C components. Brief initial incubation with C5-deficient NHS was shown to allow for maximum deposition of activated C3b and cell lysis in step two, whereas cells opsonized for 1 hour in the first step were not killed upon reaction with NHS-EDTA indicating that, at longer times, deposited C3b degrades to inactive fragments iC3b and C3dg. The majority (60%) of cells reacted for just 2 minutes in the first opsonization step with C5-depleted serum were killed upon addition of NHS-EDTA; however, streaming was observed to be substantially inhibited (³10-fold). These results provide strong support for the hypothesis that calcium influx induces formation and release of streamers observed with C activation or C-mediated killing in mAb/substrate cell systems.