Currently, no approved monoclonal antibody (mAb) therapies exist for human multiple myeloma (MM). Here we characterized cell surface CS1 as a novel MM antigen and further investigated the potential therapeutic utility of HuLuc63, a humanized anti-CS1 mAb, for treating human MM. CS1 mRNA and protein was highly expressed in CD138-purified primary tumor cells from the majority of MM patients (more than 97%) with low levels of circulating CS1 detectable in MM patient sera, but not in healthy donors. CS1

Integrins not only bind adhesive ligands, they also act as signalling receptors. Both functions allow the integrin alphaIIbbeta3 to mediate platelet aggregation. Platelet agonists activate alphaIIbbeta3 (inside-out signalling) to allow the binding of soluble fibrinogen. Subsequent platelet aggregation leads to outside-in alphaIIbbeta3 signalling, which results in calcium mobilization, tyrosine phosphorylation of numerous proteins including beta3 itself, increased cytoskeletal reorganisation and further activation of alphaIIbbeta3. Thus, outside-in signals enhance aggregation, although the mechanisms and functional consequences of specific signalling events remain unclear. Here we describe a mouse that expresses an alphaIIbbeta3 in which the tyrosines in the integrin cytoplasmic tyrosine motif have been mutated to phenylalanines. These mice are selectively impaired in outside-in alphaIIbbeta3 signalling, with defective aggregation and clot-retraction responses in vitro, and an in vivo bleeding defect which is characterized by a pronounced tendency to rebleed. These data provide evidence for an important role of outside-in signalling in platelet physiology. Furthermore, they identify the integrin cytoplasmic tyrosine motif as a key mediator of beta-integrin signals and a potential target for new therapeutic agents.

Signalling by membrane immunoglobulin, the B-lymphocyte antigen receptor, regulates B-cell maturation and activation. Crosslinking of membrane immunoglobulin by antigen or by anti-immunoglobulin antibodies inactivates immature B cells, eliminating many of the B cells capable of producing auto-antibodies. By contrast, crosslinking of membrane immunoglobulin promotes activation of mature B cells for clonal expansion and antibody production against foreign antigens. Crosslinking membrane IgM on the immature B-cell line WEHI-231 induces growth arrest. This response may be analogous to the deletion or inactivation of immature B cells that is induced by antigen or anti-IgM antibodies. Membrane immunoglobulin crosslinking stimulates phosphoinositide hydrolysis, which leads to increases in intracellular calcium and activation of protein kinase C. The induced phosphoinositide breakdown is important for inhibiting WEHI-231 growth (ref. 7 and D. Page, M.R.G., K. Fahey, L. Matsuuchi and A.L.D., manuscript submitted for publication), but may not be sufficient, as agents that elevate calcium and activate protein kinase C cause only partial growth arrest. We now show that in both mature splenic B cells and the immature B-cell line WEHI-231 crosslinking membrane immunoglobulin also stimulates phosphorylation of protein tyrosine, a reaction that has been implicated as a key regulator of cell growth. Most of these phosphorylations were not a consequence of the phosphoinositide pathway. Thus, tyrosine phosphorylation is a second mode of transmembrane signalling by membrane immunoglobulin.

Tyrosine phosphorylation of the ␤ 3 subunit of the major platelet integrin ␣ IIb ␤ 3 has been shown to occur during thrombin-induced platelet aggregation (1). We now show that a wide variety of platelet stimuli induced ␤ 3 tyrosine phosphorylation, but that this phosphorylation occurred only following platelet aggregation. Several lines of evidence suggest that the ␤ 3 cytoplasmic domain tyrosine residues and/or their phosphorylation function to mediate interactions between ␤ 3 integrins and cytoskeletal proteins. First, phospho-␤ 3 was retained preferentially in a Triton X-100 insoluble cytoskeletal fraction of thrombin-aggregated platelets. Second, in vitro experiments show that the cytoskeletal protein, myosin, associated in a phosphotyrosine-dependent manner with a diphosphorylated peptide corresponding to residues 740 -762 of ␤ 3 . Third, mutation of both tyrosines in the ␤ 3 cytoplasmic domain to phenylalanines markedly reduced ␤ 3 -dependent fibrin clot retraction. Thus, our data indicate that platelet aggregation is both necessary and sufficient for ␤ 3 tyrosine phosphorylation, and this phosphorylation results in the physical linkage of ␣ IIb ␤ 3 to the cytoskeleton. We hypothesize that this linkage may involve direct binding of the phosphorylated integrin to the contractile protein myosin in order to mediate transmission of force to the fibrin clot during the process of clot retraction.