Cell death was assessed 4 hours later. (which we termed type II mAbs) evoke a nonapoptotic mode of cell death that appears to be linked with the induction of homotypic adhesion. Here, we reveal that peripheral relocalization of actin is critical for the adhesion and cell death induced by both the type II CD20-specific mAb tositumomab and an HLA-DRCspecific mAb in both human lymphoma cell lines and main chronic lymphocytic leukemia cells. The cell death elicited was quick, nonapoptotic, nonautophagic, and dependent on the integrity Taltobulin of plasma membrane cholesterol and activation of the V-type ATPase. This cytoplasmic cell death involved lysosomes, which swelled and then dispersed their contents, including cathepsin B, into the cytoplasm and surrounding environment. The producing loss of plasma membrane integrity occurred independently of caspases and was not controlled by Bcl-2. These experiments provide what we believe to be new insights into the mechanisms by which 2 clinically relevant mAbs elicit cell death and show that this homotypic adhesionCrelated cell death occurs through a lysosome-dependent pathway. Introduction mAbs are becoming increasingly utilized in the treatment of lymphoid disorders (1, 2). In particular, mAb directed to cell-surface antigens on malignant B cells has proven the most clinically effective, with the anti-CD20 mAb, rituximab, being the first to be approved by the US FDA for the treatment of cancer. Rituximab has substantially improved end result for patients with many different types of non-Hodgkin lymphoma and has now been administered to over 1 million patients in the decade since its approval. Despite such success, treatment is not curative and there is intense preclinical and clinical investigation of many other engineered mAbs directed to both CD20 and a host of other cell-surface antigens (2). The current challenge in the development of novel anti-cancer mAbs is usually to discover cell-surface antigens that will lead to efficient tumor cell killing and to identify biological pathways that will augment the cytotoxic effects of these mAbs. Central to this task is the need to identify the crucial effector mechanisms involved in mAb therapy. Although Fc-FcR interactions are thought to explain much of the therapeutic effect of mAbs, this does not explain why certain mAb specificities are more potent than others. In addition to classical Fc-dependent effector mechanisms such as complement-dependent cytotoxicity (CDC) and Ab-dependent cellular cytotoxicity (ADCC), certain mAbs are also able to trigger intracellular signaling in the target cell and directly induce programmed cell death (PCD) (examined in refs. 3, Taltobulin 4). Whereas ADCC and CDC are dependent only upon the level of surface expression and degree of modulation of a target molecule, PCD is usually reliant upon the nature of the target molecule with its associated transmission transduction cascade. An enhanced understanding of how different mAbs evoke PCD is clearly central to providing new insights regarding not only their mode of action, but also how they might function in vivo and how mAb efficacy might be augmented. We previously defined anti-CD20 mAbs as either type I or II, based upon Taltobulin their Rabbit Polyclonal to OR5I1 ability to redistribute CD20 into plasma membrane lipid rafts and their potency in various assays measuring CDC, homotypic adhesion (HA), and PCD (5C7). These experiments indicated that type II mAbs (such as tositumomab/B1), with their greater tendency to promote PCD but not CDC, are more effective at depleting malignant B cells in xenograft models (6). We have recently confirmed that this superior efficacy also translates to syngeneic models of B cell depletion in human CD20 Tg mice (8). In both cases, this enhanced activity was independent of the need for match (6, 8) and no differences in ADCC (6) or macrophage uptake were apparent (8). Therefore, in the absence of other apparent effector mechanisms, it is possible that this enhanced induction of PCD may contribute to the greater efficacy of type II reagents. Previously, we exhibited that this cell death induced by these type II mAbs was nonapoptotic, impartial of caspase activity, and correlated with HA (5). The induction of physiological or cytotoxic responses by extracellular signals requires the spatial and temporal coordination of unique signaling pathways. Actin cytoskeleton remodeling has been established as an integrating mechanism in lymphocyte activation and antigen presentation, establishing immunological synapses and kinapses (examined in ref. 9). Disruption of the actin cytoskeleton inhibits Taltobulin both HA and cell death induced by antiCHLA-DR (10, 11) and anti-CD99 Abs (12, 13). Moreover, this form of cell death, like that induced by type II anti-CD20 mAbs, is usually impartial of caspase activation (12). HA has also been reported for B cells following the ligation of CD19, CD20, CD39, CD40 (10), and CD53 (14). Therefore, we hypothesized that cell signaling Taltobulin events leading to anti-CD20 mAbCinduced HA.