(2015) Extensive crosslinking of CD22 by epratuzumab triggers BCR signaling and caspase-dependent apoptosis in human lymphoma cells, mAbs, 7:1, 199-211, DOI: 10.4161/19420862.2014.979081 To link to this article: https://doi.org/10. 4161/19420862.2014 Abbreviations: Anti-IgM, F(ab') 2 fragment of affinity-purified goat anti-human IgM, Fc 5m fragment; BCR, B-cell antigen receptor; BSA, bovine serum albumin; CM-H 2 DCF-DA, 2 0 ,7 0 -dichlorodihydrofluorescein diacetate; Dc m , mitochondria membrane potential; DNP, 2,4-dinitrophenyl; EC, endothelial cells; ERKs, extracellular signal-regulated kinases; FBS, fetal bovine serum; FITC-DNase I, fluorescein isothiocyanate-conjugated DNase I; 488-annexin V, Alexa Fluor 488-conjugated annexin V; GAH, F(ab 0 ) 2 fragment of affinity-purified goat anti-human IgG Fcg fragment-specific; HUV-EC, human umbilical vein endothelial cells; ITIM, immunoreceptor tyrosine-based inhibition motif; JNKs, c-Jun N-terminal kinases; JP, jasplakinolide; LatB, latrunculin B; Lyn, Lck/Yes novel tyrosine kinase; MAP kinases, mitogen-activated protein kinases; mIgM, membrane IgM; MTS, (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; PLCg2, phospholipase C, isotype gamma 2; Rhodamine-anti-IgG, rhodamine-conjugated F(ab 0 ) 2 fragment of affinity-purified goat anti-human IgG, F(ab 0 ) 2 fragment-specific; ROS, reactive oxygen species; 7-AAD, 7-aminoactinomycin D, Syk, spleen tyrosine kinase; TMRE/tetramethylrhodamine/ethyl ester Epratuzumab has demonstrated therapeutic activity in patients with non-Hodgkin lymphoma, acute lymphoblastic leukemia, systemic lupus erythematosus, and Sj€ ogren's syndrome, but its mechanism of affecting normal and malignant B cells remains incompletely understood. We reported previously that epratuzumab displayed in vitro cytotoxicity to CD22-expressing Burkitt lymphoma cell lines (Daudi and Ramos) only when immobilized on plates or combined with a crosslinking antibody plus a suboptimal amount of anti-IgM (1 mg/mL). Herein, we show that, in the absence of additional anti-IgM ligation, extensive crosslinking of CD22 by plate-immobilized epratuzumab induced intracellular changes in Daudi cells similar to ligating B-cell antigen receptor with a sufficiently high amount of anti-IgM (10 mg/mL). Specifically, either treatment led to phosphorylation of CD22, CD79a and CD79b, along with their translocation to lipid rafts, both of which were essential for effecting caspase-dependent apoptosis. Moreover, such immobilization induced stabilization of F-actin, phosphorylation of Lyn, ERKs and JNKs, generation of reactive oxygen species (ROS), decrease in mitochondria membrane potential (Dc m ), upregulation of pro-apoptotic Bax, and downregulation of anti-apoptotic Bcl-xl and Mcl-1. The physiological relevance of immobilized epratuzumab was implicated by noting that several of its in vitro effects, including apoptosis, drop in Dc m , and generation of ROS, could be obse...