CD20 antibodies induce production and release of reactive oxygen species by neutrophils

To the editor:

In a recent study, Golay and coworkers address the commonly overlooked role of neutrophils in therapy of chronic lymphocytic leukemia (CLL) with monoclonal antibodies (mAbs).¹  Convincingly, they demonstrate that both rituximab (RTX) and, to a greater extent, glycoengineered obinituzumab, trigger neutrophil effector functions via the Fc receptors CD16b and CD32. Moreover, utilizing a 2′,7′-dichlorofluorescein diacetate (H2DCFDA)–based assay, the authors claim that neutrophil activation occurs without concomitant production of reactive oxygen species (ROS; oxygen radicals). However, the experimental approach used to assess ROS production has serious limitations: first, H2DCFDA assays are relatively insensitive and unspecific²  and second, in the study, neutrophils were stained with a fluorescein-conjugated antibody preventing the ability to correctly assess the ROS formation in response to CD20 antibodies with a fluorescein-based assay. Thus, in a series of experiments, we used a sensitive isoluminol-enhanced chemiluminescence method to monitor ROS responses in neutrophils exposed to CD20 mAbs or αCD20-opsonized leukemic cells. These experiments showed that malignant CLL cells in the presence of either RTX or the second-generation agent ofatumumab (OFA), triggered a robust extracellular release of oxygen radicals from neutrophils. ROS production was readily blocked by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenyleneiodonium (DPI). Similar results were obtained in experiments where we exposed CLL patient-derived neutrophils to immobilized CD20 mAbs (Figure 1A-D).

A significant part of the benefit of CD20 mAbs in therapy of CLL is attributed to antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells.^3,4  However, cytotoxic NK cells are also highly sensitive to oxygen radical-mediated inactivation.^5,6  Thus, we investigated whether αCD20-induced neutrophil ROS production had an impact on NK-cell viability.⁵  Indeed, we found that NK cells displayed significant cell death after exposure to neutrophils in the presence of either RTX or OFA, but not to either agent alone. The addition of DPI rescued NK cells, strongly suggesting NADPH oxidase– and ROS-dependent NK cell death (Figure 1E). During the course of these experiments we did not have access to the glycoengineered antibody obinituzumab, but given its profound capacity to stimulate neutrophils, it is likely to share the ROS-triggering characteristics of RTX and OFA.

Collectively, our findings raise the question of whether oxygen radical release from αCD20-exposed neutrophils may inactivate NK cells also in vivo and thus limit the efficacy of therapeutic mAbs in CLL. More studies are warranted to investigate whether neutrophils or neutrophil-derived ROS are important effector arms in antibody treatment of CLL, and whether it may be beneficial to supplement αCD20 therapy with antioxidative strategies to unravel the full effector function of NK cells in CLL.

Approval was obtained from the Ethical Review Board of Gothenburg for these experiments. Informed consent was provided according to the Declaration of Helsinki.