We examined complement-dependent cytotoxicity (CDC) by hexamer formation-enhanced CD20 mAb Hx-7D8 of patient-derived chronic lymphocytic leukemia (CLL) cells that are relatively resistant to CDC. CDC was analyzed in normal human serum (NHS) and serum from an individual genetically deficient for C9. Hx-7D8 was able to kill up to 80% of CLL cells in complete absence of C9. We conclude that the narrow C5b-8 pores formed without C9 are sufficient for CDC due to efficient antibody-mediated hexamer formation. In the absence of C9, we observed transient intracellular increases of Ca 2 + during CDC (as assessed with FLUO-4) that were extended in time. This suggests that small C5b-8 pores allow Ca 2 + to enter the cell, while dissipation of the fluorescent signal accompanying cell disintegration is delayed. The Ca 2 + signal is retained concomitantly with TOPRO-3 (viability dye) staining, thereby confirming that Ca 2 + influx represents the most proximate mediator of cell death by CDC. • Hexamer-formation enhanced CD20 mAbs kill CLL cells by CDC in serum genetically deficient in C9 • Complement activated to the C5b-8 stage is adequate to allow influx of lethal amounts of Ca 2 + in CLL cells. • The transition state intermediate (high FLUO-4 signal intensity) has a longer lifetime in cells killed by C5b-8.
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Taylor et al. (2017) studied this question.
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