From www.bloodjournal.org by guest on October 28, 2014. For personal use only. 2014 123: 4001-4002 doi:10.1182/blood-2014-05-574285 CD20 antibodies induce production and release of reactive oxygen species by neutrophils Olle Werlenius, Rebecca E. Riise, Maria Simpanen, Johan Aurelius and Fredrik B. 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BLOOD, 19 JUNE 2014 x VOLUME 123, NUMBER 25 CORRESPONDENCE 4001 To the editor: CD20 antibodies induce production and release of reactive oxygen species by neutrophils 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 29,79-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 Figure 1. Induction of neutrophil ROS production by aCD20 mAbs. PMNs were isolated by dextran sedimentation followed by standard density gradient centrifugation. (A) PMNs from healthy donors were assessed for ROS production by isoluminol-enhanced chemiluminescence in the presence of purified CLL cells (CLL:PMN ratio, 1:2) and the presence or absence of soluble RTX (10 mg/mL; Roche) or OFA (10 mg/mL; GlaxoSmithKline) and DPI (3mM; SigmaAldrich; n 5 5). (B) Individual kinetic graph from 1 experiment shown in panel A. (C) PMNs derived from patients with CLL in the presence or absence of platebound RTX or OFA (10 mg/mL; n 5 4). (D) Individual kinetic graph from 1 experiment shown in panel C. (E) NK cell death in coculture experiments. PMNs and NK cells were added to 96-well plates, previously coated with RTX or OFA, at a ratio of 1:1. After 16 hours at 37°C, cells were washed and stained with the live/dead fixable dead cell stain kit (Invitrogen) and NK cell viability was assessed by flow cytometry (n 5 4-9). Error bars represent SEM. Data in panels A and C display total ROS production (area under curve). Statistical analyses were performed using 1-way ANOVAs with Bonferroni post hoc test for panels A and C and the Mann-Whitney U test for panel E. *P # .05, **P # .01, ***P # .001. ANOVA, analysis of variance; PMN, polymorphonuclear cell; RLU, relative light units. chemiluminescence method to monitor ROS responses in neutrophils exposed to CD20 mAbs or aCD20-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 aCD20-induced neutrophil ROS production had an impact on NK-cell viability.5 Indeed, we found that NK cells From www.bloodjournal.org by guest on October 28, 2014. For personal use only. 4002 BLOOD, 19 JUNE 2014 x VOLUME 123, NUMBER 25 CORRESPONDENCE 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 ROStriggering characteristics of RTX and OFA. Collectively, our ﬁndings raise the question of whether oxygen radical release from aCD20-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 aCD20 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. Olle Werlenius Sahlgrenska Cancer Center and Department of Hematology, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Rebecca E. Riise Sahlgrenska Cancer Center and Department of Infectious Diseases, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Maria Simpanen Sahlgrenska Cancer Center and Department of Hematology, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Johan Aurelius Sahlgrenska Cancer Center and Department of Infectious Diseases, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Fredrik B. Thoren ´ Sahlgrenska Cancer Center and Department of Infectious Diseases, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Acknowledgments: This work was supported by the Goteborg ¨ Medical Society, the Wilhelm and Martina Lundgren Foundation, the Assar Gabrielsson Foundation, and the Swedish Cancer Society. Contribution: O.W., R.E.R., and M.S. performed experiments; O.W. analyzed results and made the figure; and O.W., J.A., and F.B.T. designed the research and wrote the letter. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Olle Werlenius, Sahlgrenska Cancer Center, University of Gothenburg, Box 425, 405 30 Gothenburg, Sweden; e-mail: olle.werlenius@ gu.se. References 1. Golay J, Da Roit F, Bologna L, et al. Glycoengineered CD20 antibody obinutuzumab activates neutrophils and mediates phagocytosis through CD16B more efficiently than rituximab. Blood. 2013;122(20):3482-3491. 2. Bylund J, Bjornsdottir ¨ H, Sundqvist M, Karlsson A, Dahlgren C. Measurement of respiratory burst products, released or retained, during activation of professional phagocytes. Methods Mol Biol. 2014;1124:321-338. 3. Dall’Ozzo S, Tartas S, Paintaud G, et al. Rituximab-dependent cytotoxicity by natural killer cells: influence of FCGR3A polymorphism on the concentrationeffect relationship. Cancer Res. 2004;64(13):4664-4669. 4. Golay J, Manganini M, Facchinetti V, et al. Rituximab-mediated antibodydependent cellular cytotoxicity against neoplastic B cells is stimulated strongly by interleukin-2. Haematologica. 2003;88(9):1002-1012. 5. Hellstrand K, Asea A, Dahlgren C, Hermodsson S. Histaminergic regulation of NK cells. Role of monocyte-derived reactive oxygen metabolites. J Immunol. 1994;153(11):4940-4947. 6. Thoren ´ FB, Romero AI, Hermodsson S, Hellstrand K. The CD16-/CD56bright subset of NK cells is resistant to oxidant-induced cell death. J Immunol. 2007;179(2):781-785. © 2014 by The American Society of Hematology To the editor: Novel severe hemophilia A and moyamoya (SHAM) syndrome caused by Xq28 deletions encompassing F8 and BRCC3 genes A 10-year-old boy with severe hemophilia A and no other obvious morbidity arrived at the hospital with focal neurological signs and a suspected intracranial hemorrhage. Surprisingly, radiological studies demonstrated an ischemic stroke. Neither active thromboembolism nor genetic predisposition to thrombosis was found. Neuroimaging demonstrated severe narrowing of internal carotid arteries and their branches and development of a collateral vascular network, diagnostic of moyamoya syndrome (Figure 1). Further clinical workup revealed mild facial dysmorphia, hypertension, osteopenia, and duplication of the right renal artery, a phenotype likely caused by a genetic aberration. Next-generation sequencing followed by long-range polymerase chain reaction (Figure 1 and supplemental Materials) demonstrated a large Xq28 deletion of ;150 kbp encompassing exons 1 to 6 of F8, as well as the FUNDC2, MTCP1NB, MTCP1, and BRCC3 genes. BRCC3 was recently identiﬁed as a familial moyamoya gene.1 We demonstrate that both centromeric and telomeric breakage sites of the deletion are located in nearly identical repetitive Alu sequences that could be mutational hotspots. The patient’s sister and mother are heterozygous for the same deletion. At the age of 18, the sister presented a mild phenotype including low levels of factor VIII (22%), aortic coarctation, and hypertension, but she has no signs of moyamoya angiopathy. A review of the literature yields 3 more likely individuals/families with this novel severe hemophilia and moyamoya (SHAM) syndrome: 1 clinical description in a Japanese patient2 and 2 descriptions of Xq28 rearrangements in hemophilia A patients that disrupt BRCC3 and bear striking clinical similarity to Xq28-linked familial moyamoya, although no neuroimaging data are available to conﬁrm the diagnosis.3,4 There is also a genetic report of BRCC3 deletion in a hemophilia patient without phenotype data.5 The ratio of BRCC3 inactivation in hemophilia A is unknown because the regions telomeric to F8 are rarely subjected to genetic diagnostics. We accessed the Centers for Disease Control Hemophilia A mutation project database that contains .2000 pathological F8 mutations