Finally, it’ll be important to test the prediction that the size or abundance of lipid rafts in B-cell membranes is a contributing factor to the susceptibility or resistance of patients to CD20-mediated B-cell depletion. Acknowledgments The work of the authors described in this article was supported by the Canadian Institutes of Health Research (CIHR, formerly MRC). diseases such as rheumatoid arthritis, autoimmune haemolytic anaemia and idiopathic thrombocytopenic purpura, in which pathogenic autoantibodies are clearly involved. 5C8 The mechanism of rituximab action is not entirely clear, but is thought to involve complement-mediated lysis or phagocytosis, antibody-dependent cellular cytoxicity (ADCC), and direct effects of CD20-mediated signalling on cell growth and viability.9 EPHB4 Supporting a prominent role for ADCC are the observations that FcRIIIa is required for effective killing by rituximab in a human tumour xenograft mouse model,10 and that resistance to rituximab in some patients may be linked to a polymorphism in FcRIIIa affecting isotype preference.11 Although complement and ADCC are likely to be the major effectors of B-cell depletion expression and activate resting B cells.28C31 Other CD20 mAbs have inhibitory effects on proliferation and/or mitogen-driven antibody secretion.29,32C35 B1 C and to a lesser extent, rituximab C induces homotypic aggregation in B-cell lines, whereas 1F5, 2H7, and most other CD20 mAbs, do not.27,36 A variety of other biological responses to CD20 mAbs has been reported, including down-regulation of the B-cell receptor,37 shedding of CD23,38,39 increased expression of major histocompatibility complex class II and adhesion molecules,34,40 blocking of lymphocyte function-associated antigen-1-independent homotypic adhesion,41 Vincristine sulfate rescue from apoptosis,42,43 and induction of apoptosis (see below). Little is known of the signalling pathways responsible for these effects or their relevance to the normal functioning of the CD20 complex. CD20 signalling and apoptosis One of the earliest indications that CD20 might be coupled to a tyrosine kinase-dependent signalling pathway was the observation that the induction of homotypic aggregation by the B1 mAb was prevented by tyrosine kinase inhibitors.36 Subsequently, up-regulation of c-expression by the activating mAb 1F5 was also shown to be tyrosine kinase-dependent.44 The 2H7 mAb, which exhibits neither of these biological activities, nevertheless induces tyrosine kinase activation leading to phosphorylation of multiple tyrosine kinase substrates including phospholipase C-2.44Hyper-cross-linking, i.e. cross-linking of antigen-bound CD20 antibody with a secondary antibody, caused release of calcium from intracellular stores.44 These findings were confirmed by two Vincristine sulfate groups who went on to demonstrate that CD20 hyper-cross-linking up-regulated Fas expression, increased caspase activity, and induced apoptosis in B-cell lines.12,16,45 Induction of apoptosis by CD20 mAbs has now been demonstrated in various B-cell lines by several investigators. Although not a universal finding, there is general agreement that hyper-cross-linking enhances, Vincristine sulfate and is often essential for, the detection of apoptotic effects of CD20 mAbs (Table 2). family of related genes.65,66 Additionally, there are amino acid differences between murine and human CD20 in the sequence known to be critical for CD20 raft association (residues 219C225), raising the possibility that CD20 may localize and function differently in murine and human B cells. As described earlier, it is now clear that CD20 is constitutively associated with lipid rafts. Whatever the function of CD20, it is evidently performed in the environment of lipid raft microdomains. What, then, is the relevance, if any, of antibody-induced translocation of CD20 from the soluble to the insoluble fractions of 1% Triton X-100 cell lysates? Highly purified Fab fragments can also induce this change, and cross-linking Fab with secondary (anti-light-chain) antibodies has no additional effect (H. Li, unpublished data). From this it can be concluded that cross-linking is not required. All CD20 mAbs induce Vincristine sulfate CD20 translocation, but they do so to varying degrees (refs 27,48 and see Table 1). This variability appears to be independent of the isotype and affinity of Vincristine sulfate the mAb, but rather is an epitope-dependent characteristic.27 These observations indicate the sensitivity of a specific region of the extracellular loop to interactions that could be mediated either by a ligand or by lateral engagement with a cell surface receptor. Translocation of CD20 from rafts that are insoluble only in a low concentration of Triton X-100 (or other non-ionic detergents at 1%), to rafts that are insoluble in 1% Triton X-100, may reflect an increase in the affinity of CD20 for lipid rafts and/or stabilization of the rafts. The consequence of this is presently unknown, but co-localization of the B-cell receptor with CD20 (R. Petrie, in.