A novel benzodiazepine increases the sensitivity of B cells to receptor stimulation with synergistic effects on calcium signaling and apoptosis.

Bz-423 is a 1,4-benzodiazepine with selective lymphotoxic properties and potent therapeutic activity against lupus-like disease in autoimmune mice. In NZB/W lupus-prone mice, Bz-423 specifically kills germinal center B cells, which are the cells that drive disease both in this model and in human systemic lupus erythematosus. In this report, the mechanistic basis for the selective action of Bz-423 is investigated. We show that Bz-423-induces superoxide as an immediate early response and that this reactive oxygen species is more effective as a second messenger death signal in B cells activated by B cell receptor stimulation compared with resting cells. As a result, low [Bz-423] that are not cytotoxic to non-stimulated cells kill stimulated cells in synergy with anti-immunoglobulin M antibodies. Subsequent experiments demonstrated that Bz-423 extends the rise in intracellular calcium that accompanies anti-immunoglobulin M stimulation, and this effect mediates the synergistic death response. Because B cell hyperactivation and altered calcium signaling is a distinguishing feature of autoreactive lymphocytes in lupus, the mechanism by which Bz-423 induces apoptosis preferentially targets disease-causing cells on the basis of their activation state. Thus, molecules like Bz-423 could form the basis for new and selective anti-lupus agents.

Systemic lupus erythematosus (SLE) 1 is characterized by a spectrum of auto-antibodies that are the products of B cells that escape peripheral tolerance (1). Although immunosuppressive, lymphotoxic drugs are effective for many patients, these drugs cause life-threatening side effects that account for a notable portion of lupus-related deaths (2). Therefore, agents with greater selectivity against disease-causing lymphocytes could significantly advance the treatment of SLE and related disorders.
Because lymphocyte toxicity is an established treatment modality, it offers a starting point to develop new classes of therapeutic molecules. Toward this end, a library of 1,4-benzodiazepines was previously screened for cytotoxic members against Ramos B cells, a neoplastic B cell line with a germinal center (GC) phenotype (3). These studies led to the identification of Bz-423 (Fig. 1), a pro-apoptotic molecule whose mechanism of action depends upon an increase in intracellular superoxide (O . 2 ), produced as a result of the interaction of Bz-423 with a mitochondrial molecular target (3). Comparison of Bz-423 with benzodiazepines used clinically and ligands of the peripheral benzodiazepine receptor reveals the unique cytotoxicity of this compound against B cells in vitro (3).
Based upon its lymphotoxic properties in vitro, we explored the cytotoxic properties of Bz-423 in two animal models of SLE, the MRL-lpr and the (NZB x NZW)F 1 (NZB/W) mouse strains. Lupus-like disease in MRL-lpr mice is T-cell-dominated and is linked to defective Fas signaling. These defects allow a population of autoreactive CD4 ϩ T cells to expand instead of undergoing apoptosis in response to physiologic cues (4,5). In these animals, treatment with Bz-423 specifically reduced activated CD4ϩ cells (6).
In contrast, an expanded population of activated B cells within GCs mediates disease in NZB/W mice. These activated B cells drive autoantibody production and pathogenicity that ultimately results in lupus nephritis (7,8). Administering Bz-423 to NZB/W mice selectively targeted activated GC B cells and reduced the number and size of GCs. These mice also had reduced autoantibody levels and improved glomerulonephritis. Although a complete molecular explanation for the abnormal GC persistence in NZB/W mice is not yet known, current evidence implicates defects in normal tolerance mechanisms, including defective B cell receptor (BCR)-mediated activationinduced cell death (AICD) (9). In normal immune BALB/c mice, Bz-423 neither decreased viability nor increased apoptosis of splenic lymphocytes nor affected physiologic GC responses. Thus, at therapeutic doses, Bz-423 selectively kills diseasecausing cells.
Because GC-derived cells have also been shown to mediate disease pathogenesis in human SLE, we were particularly interested in understanding the mechanistic basis for the selectivity of Bz-423 in these cells. B cells acquire and maintain a GC phenotype through a process that depends upon stimulation of BCR. GC homeostasis also depends upon BCR-coupled apoptosis, which is defective in SLE and NZB/W mice (10). These observations suggested that BCR stimulation might be involved in enhancing sensitivity to Bz-423, resulting in the selectivity observed in the NZB/W studies. Therefore, we embarked on a line of investigation testing the hypothesis that BCR stimulation facilitates killing by Bz-423.
Animals, Primary B Cells, Cell Lines, and Culture-6-week-old female Balb/C mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were euthanized and the spleens were removed for analysis. Splenocytes were obtained by mechanical disruption with isotonic lysis of red blood cells. B cell-rich fractions were prepared by negative selection using magnetic cell sorting with CD4, CD8a, and CD11b-coated microbeads (Miltenyi Biotec, Auburn, CA). Ramos cells were purchased from ATCC (Manassas, VA). Cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 g/ml) and L-glutamine (290 g/ml). Media for primary cells also contained 2-mercaptoethanol (50 M). In vitro studies were performed in media containing 2% FBS and 0.5% Me 2 SO.
B Cell Stimulation-Cell lines were stimulated with soluble goat Fab 2 anti-human IgM (1 g/ml) and/or purified anti-human CD40 (2.5 g/ml). Primary mouse B cells were stimulated with affinity-purified goat anti-mouse IgM (20 g/ml, ICN) immobilized in culture wells and/or soluble purified anti-mouse CD40 (clone HM40 -3, 2.5 g/ml, PharMingen). Bz-423 was added 10 min before stimuli were applied. Inhibitors were added 30 min prior to Bz-423.
Determination of Synergy-Synergistic effects upon cytotoxicity between Bz-423 and anti-IgM were evaluated using isobologram analysis as described previously (12,13). For this analysis, two series of dosage curves were obtained. First, using fixed concentrations of Bz-423, the EC 50 values (i.e. the concentration at which 50% of the cells are dead) of anti-IgM were measured. Then, at fixed concentrations of anti-IgM, the EC 50 values of Bz-423 were obtained. These EC 50 values were used to construct isobolograms.
Detection of Cell Death and Intracellular ROS-Cell viability, hypodiploid DNA, and O . 2 were measured by flow cytometry staining with propidium iodide (PI) and DHE, as described previously (3).
Measurement of Intracellular Calcium Concentration [Ca 2ϩ ] i Using Fura 2-The cell-permeable acetoxymethyl ester form of Fura 2 (2.5 M, Fura 2-AM) was added to Ramos cells (10 7 /ml) in loading buffer (1 mM CaCl 2 , 1 mM MgCl 2 , 1% FBS). Fura 2-AM is retained in the cell after being de-esterified by esterases in the cytoplasm; when excited at two alternate wavelengths (340 and 380 nm), the ratio of fluorescence emission at 510 nm is related to [Ca 2ϩ ] i independently of intracellular dye concentration. After incubating 30 min at 37°C to load with Fura 2, cells were washed and then resuspended in loading buffer at 10 6 /ml; 200 l (2 ϫ 10 5 cells) were added per well to a 96-well plate. As indicated, cells were pre-incubated with Bz-423 or solvent control for 2 min, warmed to 37°C, and anti-IgM (1 g/ml) was added, immediately after which fluorescence (510 nm) was monitored every 15 s over 8 min using a microplate spectrofluorometer (Molecular Devices). Fluorescence specific to Fura 2 was calculated as the difference between the fluorescence intensities from Fura 2-loaded cells and unloaded cells. The maximal fluorescence at 380 nm determined by the addition of ionophore Br-A23187 (2 g/ml) and minimum fluorescence at 340 nm determined by the addition of EGTA (35 mM) to Br-A23187-treated cells were used to calculate [Ca 2ϩ ] i as described (14).
Detection of [Ca 2ϩ ] i within Single Cells using Flow Cytometry-Ramos cells (10 7 /ml) were incubated (30 min, 37°C) with the cellpermeable fluorescent dye Fluo-3 AM (4 g/ml) in loading buffer (1 mM CaCl 2 , 1 mM MgCl 2 , 1% FBS) containing Pluronic (0.02%) and Probenecid (4 M). The cells were washed with phosphate-buffered saline and resuspended in loading buffer at 10 6 /ml. Cell suspensions were incubated with Bz-423 for 10 min at room temperature, warmed to 37°C, then analyzed on the flow cytometer continuously for 10 min with the temperature maintained at 37°C. [Ca 2ϩ ] i was calculated as described previously based upon maximum Fluo-3 fluorescence (determined by the addition of 2 g/ml Br-A23187) and minimum fluorescence (determined by addition of 35 mM EGTA to Br-A23187-treated cells).
Statistical Analysis-Statistical analysis was conducted by using the SPSS software package. All data are presented as mean Ϯ S.D.

BCR-stimulation Sensitizes Cells to Bz-423-B cells acquire
and maintain a GC phenotype through a process that depends upon stimulation of BCRs (10). To account for the selective GC reduction in the treated NZB/W mice, we postulated that BCR stimulation facilitates killing by Bz-423. To test this hypothesis, primary B cells were isolated by negative selection from splenocytes harvested from Balb/C mice. B cell-enriched isolates (Ͼ95% B220 ϩ /Thy1.2 Ϫ ) were incubated with immobilized polyclonal anti-IgM to extensively cross-link BCRs. This strong stimulus expectedly provoked AICD in ϳ40% of cells (Fig. 2, white bars). When added alone, Bz-423 (4 M) killed 20% of the cells (black bars). When Bz-423 (4 M) was combined with anti-IgM, killing was greater than with either agent alone (gray bars). Cells were also co-stimulated during these treatments with antibody specific for CD40, because CD40 stimulation is a GC cell-survival signal linked with lupus pathogenesis (15,16). In the presence of anti-CD40, killing by anti-IgM alone was completely abrogated (40% killing decreased to 5%; see Fig. 2) and the response to Bz-423 was also decreased (20 to 9%.) Interestingly, however, the response to Bz-423 plus anti-IgM was not reduced to the same extent by CD40 (anti-IgM alone, 5%; Bz-423 alone, 9%; anti-IgM plus Bz-423, 38% kill- ing). These data indicate that in primary B cells given a survival stimulus through CD40, BCR and Bz-423 cooperate to increase cytotoxic effects.
Because primary B cells can not be maintained in culture over long periods of time, we chose to further investigate synergy in the immortalized, follicular B cell lymphoma Ramos cell line. Ramos cells are an Epstein-Barr virus-negative B cell lymphoma line and express surface markers and Bcl-6 consistently with a GC phenotype (17,18). More importantly, because Ramos cells mount an apoptotic response to BCR ligation like GC cells (19,20), this line was suitable to examine the synergy between Bz-423 and BCR stimulation on cell death.
Soluble anti-IgM Fab 2 dose-dependently killed Ramos cells (Fig. 3A). When used at limiting concentrations (Ͻ1 g/ml), anti-IgM Fab 2 had very little effect upon their survival. When anti-IgM Fab 2 (1 g/ml) was applied together with increasing concentrations of Bz-423, the death response of Ramos cells to Bz-423 increased. Compared with cells treated with Bz-423 alone or Bz-423 plus a control antibody, the dose-response of Bz-423 in the presence of anti-IgM Fab 2 shifted to lower concentrations (Fig. 3B). Anti-CD40 did not change the response of these cells to either Bz-423 alone or the Bz-423-anti-IgM Fab 2 combination (data not shown).
Isobolograms were used to confirm that Bz-423 in combination with anti-IgM Fab 2 produce an effect whose magnitude is significantly increased relative to each individual agent. The concentrations of Bz-423 and anti-IgM Fab 2 that each result in 50% death were plotted as axial points, and the straight line connecting these points was drawn to represent the dose pairs that would produce this effect additively (Fig. 3C). When actual combinations of Bz-423 and anti-IgM Fab 2 producing 50 Ϯ 5% killing were plotted, the points fell below this line, confirming that BCR stimulation plus Bz-423 have a super-additive or synergistic effect upon cell death in Ramos cells. Together, these results indicate that Bz-423 significantly increased activity in activated B cells, which may account for the therapeutic response observed in lupus mice.
Bz-423 Augments Fab 2 -induced Apoptotic Signaling-To understand the basis for the synergistic death response, we probed the mechanism of cell death engaged in Ramos cells by a synergistic combination of Bz-423 (5 M) and anti-IgM Fab 2 (1 g/ml). To confirm that apoptosis was being induced, we treated Ramos cells with doses of reagents equipotent at inducing cell death (i.e. 10 M Bz-423 alone, 10 g/ml anti-IgM Fab 2 alone, or 5 M Bz-423 plus 1 g/ml anti-IgM Fab 2 ) and examined the cellular morphology. Each treatment resulted in cytoplasmic vacuolization, nuclear condensation, and plasma membrane blebbing, which is consistent with apoptosis (Fig. 4A, and data not shown). Next, we examined other signaling events that have been shown to be involved in cell death induced by Bz-423 or anti-IgM. O . 2 is rapidly induced by Bz-423 and is necessary for apoptosis and cell killing by this agent. However, inducible gene and protein expression, and specific elements of the apoptotic machinery, including caspase activity and the mitochondria permeability transition (MPT) are not required for Bz-423 to kill cells (3). In contrast, BCR-coupled AICD requires caspase activity, the MPT, and inducible protein synthesis, and is critically mediated by an early rise in intracellular calcium (21). Based on these differences, we treated cells with a range of specific inhibitors to distinguish which signaling events were necessary to induce the synergistic death response.
We first tested whether ROS are required for synergistic killing and whether co-treatment with anti-IgM increases the Bz-423-induced O . 2 response. The antioxidants vitamin E and MnTBAP each protected against killing by Bz-423 and the Bz-423-anti-IgM Fab 2 combination, without significantly re-ducing death caused by a high concentration of anti-IgM Fab 2 alone (Fig 4B). Because these results demonstrated that Bz-423 induced O . 2 is necessary for synergistic killing, we measured O . 2 during combined treatment to determine whether a greater ROS response correlated with the increased killing observed under synergistic conditions. Surprisingly, staining for O . 2 increased equivalently in response to Bz-423 alone or together with anti-IgM Fab 2 (Fig. 4C). Hence, the increase in ROS was strictly proportional to the concentration of Bz-423, and BCR stimulation did not affect the magnitude or duration of this response (Fig. 4C, and data not shown). Thus, although Bz-423-induced ROS is necessary for synergy, a further increase of O . 2 did not seem to account for the supra-additive death response.
Cycloheximide, cyclosporin A, Z-VAD-fmk, and 1,2-bis(2aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid (BAPTA) were used in similar experiments to determine whether protein synthesis, the MPT, caspases, and the influx of extracellular calcium, respectively, were necessary for synergistic killing. As expected, each of these agents protected against killing by anti-IgM Fab 2 and none protected against killing by Bz-423 (see Fig. 4B). Importantly, each agent abolished the synergistic effects of the Bz-423 and Fab 2 combination. These data implicate calcium influx, the MPT, and inducible protein synthesis in synergistic killing. Because each of these is a component of the apoptotic AICD pathway, these results suggest that Bz-423-induced O . 2 modulates the death mechanism normally coupled to BCR-stimulation in such a way as to produce a supraadditive death response.

Bz-423-induced ROS Affects BCR-coupled Calcium Signaling-Because increased [Ca 2ϩ
] i is an immediate and integral part of BCR-coupled signaling and ROS can modulate calcium release and re-uptake mechanisms, we hypothesized that Bz-423 amplifies BCR-coupled calcium signaling by means of a ROS-dependent mechanism. [Ca 2ϩ ] i increases rapidly in response to BCR stimulation, and the magnitude and duration of this increase determine the cellular response to stimulation (22). In some instances, more exaggerated [Ca 2ϩ ] i responses are associated with B cell developmental arrest and deletion (23). To measure the [Ca 2ϩ ] i in anti-IgM-stimulated cells in the presence of Bz-423, Ramos cells were pre-loaded with Fura-2, treated with Bz-423 or solvent control, and then stimulated with anti-IgM. Fura-2 is a calcium indicator that undergoes a shift in maximal absorption upon binding Ca 2ϩ , allowing ratiometric determination of [Ca 2ϩ ] i . Fluorescence emission at 510 nm was monitored in response excitation at 340 and 380 nm as a function of time on a microtiter plate reader. The ratio of fluorescence (340 nm/380 nm) was used to calculate the [Ca 2ϩ ] i . Brief exposure to Bz-423 (5 M) prior to treatment with anti-IgM Fab 2 resulted in a higher peak [Ca 2ϩ ] i that reached a maximum of 200 nM (as compared with 120 nM with anti-Fab 2 alone), and the normally transient response was prolonged (Fig. 5). Even after 10 min following BCR stimulation, [Ca 2ϩ ] i was ϳ180 nM in the Bz-423-treated cells, whereas in control cells, the level returned to baseline (Ϸ50 nM) within 2 min after the addition of anti-IgM Fab 2 . These results suggest that Bz-423 is indeed able to modulate calcium signaling induced by BCR activation.
Next, we sought to evaluate whether Bz-423-induced ROS was involved in modulating [Ca 2ϩ ] i . In these experiments, [Ca 2ϩ ] i was measured at the single cell level, which allowed us to determine both the fraction of cells responding and the magnitude of the response within individual cells. Fluo-3 was

Bz-423 Modulates B Cell Receptor Signaling
used to make these [Ca 2ϩ ] i measurements because it is a calcium-sensitive fluorophore with properties suitable for flow cytometry. To confirm that the findings obtained with Fura-2 on the plate reader could be repeated with Fluo-3, Ramos cells were loaded with Fluo-3-AM and incubated with Bz-423 or solvent control for 5 min. Fluo-3 fluorescence was measured as a function of time after the addition of anti-IgM, during which samples were maintained at 37°C. Consistent with the results above, Bz-423 caused the magnitude and duration of the calcium response to Fab 2 to increase in the majority of cells. 50% of cells reach a peak calcium level of Ͼ 8-fold over baseline, compared with 10% of control cells, and the elevation is prolonged. After 10 min, 64% of cells continued to have a fluorescent intensity above the baseline (Fig. 6). In the control cells, a rapid increase in calcium was induced by Fab 2 and resolved within 3 min, after which time, Ͼ 90% of cells had fluorescence at or below the median fluorescence intensity of unstimulated cells. Thus, by contrast, the calcium response in cells treated with Bz-423 is both amplified and prolonged. BAPTA, which chelates extracellular calcium and blocks synergistic killing, was used in these experiments to determine whether the increased [Ca 2ϩ ] i resulting from Bz-423 depends upon extracellular Ca 2ϩ . Fluo-3-loaded cells were pre-incubated (30 min) with BAPTA prior to Bz-423 or vehicle control. When cells pre-treated with either BAPTA were treated with Bz-423 and then anti-IgM Fab 2 , the increase in [Ca 2ϩ ] i associated with Bz-423 was significantly blunted (Fig. 6). As well, BAPTA reduced the calcium response to anti-IgM Fab 2 in the absence of Bz-423. Because vitamin E reduces Bz-423-induced O . 2 , it was used to determine the functional importance of ROS in modulating [Ca 2ϩ ] i . Vitamin E also significantly reversed the effects that Bz-423 had upon anti-IgM-induced [Ca 2ϩ ] i (Fig.  6). In contrast, vitamin E had no effect upon the calcium response to anti-IgM in the absence of Bz-423.
Taken together, our data show that Bz-423 dramatically increases BCR-coupled calcium signaling and that Bz-423-induced ROS is critical for affecting the increase. Thus, Bz-423 plus anti-IgM produce a synergistic death response because their individual signaling pathways intersect to coordinately affect [Ca 2ϩ ] i .

DISCUSSION
The selectivity of Bz-423 against GC B cells in autoimmune NZB/W mice prompted these studies, which have identified synergy between the cytotoxic effects of receptor stimulation and Bz-423 in B cells. Functionally, this synergy means that B cells undergo significant apoptosis in response to amounts of Bz-423 and anti-IgM that, as single agents, are not cytotoxic. Although ROS are not necessary for BCR-coupled AICD, the O .
2 generated by Bz-423 is critical for the synergistic response. Given that O . 2 does not diffuse across membranes (24) and only a small fraction of superoxide is vectorially released into the intermembrane space and enters the cytosol through voltage-dependent anion channels in the outer membrane (25,26), how does O . 2  Our findings show that the synergistic response depends upon extracellular calcium. However, because calcium influx across the plasma membrane is coupled to depletion of intracellular calcium stores in B cells (28), the present data do not necessarily indicate that Bz-423-induced ROS directly affects calcium channels or pumps in the plasma membrane. Although this is a possibility, extracellular calcium sources may be engaged more indirectly, through capacitative regulation.
There are multiple mechanisms by which Bz-423-induced ROS might modulate Ca 2ϩ signaling. One possibility is that Bz-423-induced ROS ultimately increases signals in the BCRinduced cell death pathway that result in sustained Ca 2ϩ levels. Bruton's tyrosine kinase and Syk (two of several protein tyrosine kinases activated by the BCR; Ref. 29) are candidates for points of intersection, because H 2 O 2 induces their activation (30,31) and each has a critical role in determining calcium signaling by phosphorylating phospholipase C␥ (32). The BCR initially phosphorylates and activates Syk as well as phosphatidylinositol 3-kinase. Phosphatidylinositol 3-kinase generates the second messenger phosphatidylinositol 3,4,5-trisphosphate that mediates membrane recruitment of Bruton's tyrosine kinase which, along with Syk, phosphorylates and activates phospholipase C␥ (29). Inositol-1,4,5-trisphosphate (InsP3) generated by phosphorylating phospholipase C␥ raises [Ca 2ϩ ] i by binding to InsP3 receptors in the endoplasmic reticulum (ER) membrane, depleting stores within the ER and triggering influx through the plasma membrane (33). Bruton's tyrosine kinase is a particularly appealing candidate, because when Bruton's tyrosine kinase is persistently activated, for example, by point mutation in its pleckstrinhomology domain, BCR cross-linking generates a sustained increase in [Ca 2ϩ ] i identical to the response observed in our experiments (34).
Alternatively, because the ER lies close to the mitochondria, ROS emanating from the mitochondria might directly regulate Ca 2ϩ release channels in the ER (ryanodine receptors and InsP3 receptors) or alter mechanisms that normally lower [Ca 2ϩ ] i such as re-uptake by the Ca 2ϩ -ATPase in the ER (35). Calcium-specific channels and pumps are sensitive to modulation by ROS either through direct oxidation of sulfhydryl groups located on the ion transport proteins (36, 37), peroxida-  (38), or inhibition by lower ATP levels (39).
Bz-423-induced ROS could also modulate ER channels in a more indirect manner, using cytochrome c as a messenger to the ER. A recent description (40) of the interplay between calcium and cytochrome c coordinating mitochondrial-ER interactions and driving apoptosis provides another explanation for the exaggerated calcium response and insight into how increased calcium may trigger apoptosis. Boehning et al. (40) identified small-scale release of cytochrome c from mitochondria in response to agents including staurosporine and ceramide. Because Bz-423 acts directly on mitochondria, it is conceivable that it also causes a similar cytochrome c release. Boehning et al. then showed that the small amount of cytochrome c released is sufficient to bind to and promote calcium conductance through InsP3 receptors in the ER. The released calcium feeds back to the mitochondria, triggering a coordinate permeability transition and massive cytochrome c release from all mitochondria in the cell, ultimately engaging the caspasedependent death machinery. It is possible that in an analogous fashion, the direct actions of Bz-423 on the mitochondria lead to a similar early, small-scale cytochrome c response that contributes to the increased BCR-induced calcium release by sensitizing InsP3 receptors.
Because [Ca 2ϩ ] i is fundamental for B cell activation, proliferation, and apoptosis, it is not surprising that the changes in calcium signaling effected by Bz-423 are functionally important. Among the parameters that influence BCR-driven apoptosis, the magnitude of the rise in [Ca 2ϩ ] i induced by receptor ligands is directly correlated with the apoptotic outcome induced by BCR stimulation (41). Furthermore, the magnitude and duration of the intracellular calcium response to BCR ligation precisely determines the differential triggering of the transcriptional regulators nuclear factor-B, c-Jun NH 2 -terminal kinase, nuclear factor of activated T cells, and extracellular signal-regulated kinase that ultimately dictate the characteristics of a cellular response (42). Thus, in addition to influencing BCR-coupled calcium signals in AICD, Bz-423 may modulate other B cell responses to antigen receptor stimulation.
In summary, we have demonstrated that the cytotoxic, immunomodulatory agent Bz-423 synergizes with BCR stimulation, and it is likely that this effect accounts (at least in part) for the selectivity observed in vivo. Considering that other receptors on B cells, which are effectively targeted by therapeutic antibodies presently in clinical use, are also coupled to calcium signals (for example the CD20 B cell-specific surface molecule targeted by Rituxan; Ref. 43), it may be possible to exploit Bz-423 for cancer chemotherapy. Our findings suggest how the properties of Bz-423 could provide therapeutic utility for autoimmunity, and current efforts are aimed at evaluating these possibilities.