In Chronic Lymphocytic Leukemia B Cells B Activity and Apoptosis Κ

Modulation of NF-κB Activity and Apoptosis in Chronic Lymphocytic Leukemia B Cells Richard R. Furman, Zahra Asgary, John O. Mascarenhas, Hsiou-Chi Liou and Elaine J. Schattner This information is current as of September 27, 2021. J Immunol 2000; 164:2200-2206; ; doi: 10.4049/jimmunol.164.4.2200 http://www.jimmunol.org/content/164/4/2200 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Modulation of NF-␬B Activity and Apoptosis in Chronic Lymphocytic Leukemia B Cells1

Richard R. Furman,* Zahra Asgary,* John O. Mascarenhas,* Hsiou-Chi Liou,2†‡ and Elaine J. Schattner2,3*‡

Chronic lymphocytic leukemia (CLL) is an indolent malignancy of CD5؉ B lymphocytes. CLL cells express CD40, a key regulator of B cell proliferation, differentiation, and survival. In nonmalignant B cells, CD40 ligation results in nuclear translocation and activation of NF-␬B proteins. Based on observations that in some CLL cases, the tumor cells express both CD40 and its ligand, CD154 (CD40 ligand), we proposed a model for CLL pathogenesis due to CD40 ligation within the tumor. To evaluate this issue, we used freshly isolated CLL B cells to examine constitutive and inducible NF-␬B activity by electrophoretic mobility shift assay. We consistently observed high levels of nuclear NF-␬B-binding activity in unstimulated CLL B cells relative to that detected in

nonmalignant human B cells. In each case examined, CD40 ligation further augmented NF-␬B activity and prolonged CLL cell Downloaded from survival in vitro. The principle NF-␬B proteins in stimulated CLL cells appear to be quite similar to those in nonmalignant human B cells and include p50, p65, and c-Rel. In a CD154-positive case, blocking CD154 engagement by mAb to CD154 resulted in inhibition of NF-␬B activity in the CLL cells. The addition of anti-CD154 mAb resulted in accelerated CLL cell death to a similar degree as was observed in cells exposed to dexamethasone. These data indicate that CD40 engagement has a profound inﬂuence on NF-␬B activity and survival in CLL B cells, and are consistent with a role for CD154-expressing T and B cells in CLL

pathogenesis. The data support the development of novel therapies based on blocking the CD154-CD40 interaction in CLL. The http://www.jimmunol.org/ Journal of Immunology, 2000, 164: 2200–2206.

hronic lymphocytic leukemia (CLL)4 is considered a ma- The NF-␬B proteins are key regulators of differentiation and lignancy of B-1 type B cells (1, 2). The disease is char- survival in B cells (10, 20). In mammals this protein family in- C acterized by coexpression of CD5, CD19, and CD23 in cludes p50, p52, p65 (RelA), c-Rel, and RelB (21–23). In the in- cells of a slowly expanding tumor (3). Like most B cells, CLL cells active state, NF-␬B proteins occur as homodimeric or het- express CD40 (4, 5), a TNF receptor-type molecule (6). In non- erodimeric complexes in the cytoplasm bound to I␬B proteins. transformed B cells, CD40 ligation results in NF-␬B activation (7, After appropriate stimulation, I␬B is phosphorylated, ubiquinated, by guest on September 27, 2021 8), proliferation (9), differentiation (10, 11), and survival (12, 13). and degraded, which allows translocation of NF-␬B to the nucleus The physiologic ligand for CD40 is CD154 (CD40 ligand, T cell- and transcription of NF-␬B target genes (24–26). Induction of B-activating molecule), a TNF-related molecule that is transiently NF-␬B activity protects cells from apoptosis induced by a variety expressed at the surface of activated CD4ϩ T cells (14, 15). We of stimuli including exposure to TNF-␣, chemotherapy, and ion- and others have reported that in some cases of CLL, the tumor B izing radiation (27–29). Recent studies have elucidated some cells express CD154 (16–18). Based on the fact that CLL B cells mechanisms by which NF-␬B inhibits apoptosis, such as induction express CD40 and under some circumstances express CD154, we of c-IAP1 and c-IAP2 (30), bcl-xL, and bfl-1 (31). Our model predicts that CD40 ligation, delivered either by proposed a model for CLL tumor growth and pathogenesis due to ϩ an autocrine or paracrine stimulatory effect within the tumor, CD154 T cells in the host, or by tumor-expressing B cells in a ␬ which might depend on NF-␬B (17, 19). subset of cases, would result in augmentation of NF- B activity and CLL cell survival (17). To test this hypothesis, we examined constitutive and inducible NF-␬B activity in freshly purified CLL B cells. We evaluated the impact of CD40 ligation on nuclear ␬ Divisions of *Hematology-Oncology and †Allergy and Immunology, Department of NF- B and survival in CLL cells using electrophoretic mobility Medicine, Weill Medical College and ‡Program in Immunology, Weill Graduate shift assays (EMSAs) and fluorescence flow cytometry. Through School of Medical Sciences of Cornell University, New York, NY 10021 these investigations, we determined that unstimulated CLL cells Received for publication July 20, 1999. Accepted for publication December 3, 1999. have high constitutive levels of NF-␬B activity, and that CD40 The costs of publication of this article were defrayed in part by the payment of page engagement enhances this activity and promotes survival in vitro. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. These results indicate that CD40-CD154 interactions may be cen- tral in CLL pathogenesis and offer new targets for therapy in this 1 This work was supported with funds from the William H. Kearns Foundation and National Institutes of Health Grants CA-71589 and CA-68155. R.F. was supported by disease. the Sass Foundation for Medical Research and the Florence A. Carter Fellowship Program in Leukemia Research of the American Medical Association Educational Research Foundation. Materials and Methods 2 H.-C.L. and E.J.S. contributed equally to the work. Sample isolation 3 Address correspondence and reprint requests to Dr. Elaine Schattner, Division of Peripheral blood CLL samples were obtained from untreated CLL patients Hematology-Oncology, Weill Medical College of Cornell University, 1300 York Av- whose diagnosis was confirmed at The New York Hospital-Cornell Med- enue, New York, NY 10021. E-mail address: [email protected] ical Center based on standard criteria (3). For studies in nonmalignant 4 Abbreviations used in this paper: CLL, chronic lymphocytic leukemia; I␬B, inhib- human B cells, we used buffy coat preparations of human peripheral blood itor of NF-␬B; EMSA, electrophoretic mobility shift assay; dex, dexamethasone. leukocytes from The New York Blood Center (four samples) or tonsillar B

FIGURE 1. Unstimulated CLL B cells demonstrate high levels of NF-␬B that is augmented by CD40 ligation. A, Nuclear extracts were prepared from normal-donor peripheral blood or CLL B cells immediately after puriﬁcation (no culture), or after culture for 20 h with media alone, IL-4 (10 ng/ml), anti-CD40 mAb (clone M3, 2 ␮g/ml), the combination of IL-4 and anti-CD40 mAb, or dexamethasone (10Ϫ7 M). A total of 5 ␮g of protein from each nuclear extract were analyzed for NF-␬B activity by EMSA using a probe containing the NF-␬B binding site from the Ig ␬ light chain promoter region. Arrows indicate speciﬁc NF-␬B bands. B, A total of 5 ␮g of nuclear protein from uncultured peripheral blood B cells from healthy donors (n ϭ 4) or CLL patients (n ϭ 4) was evaluated by EMSA within a single autoradiograph, and the signals measured by densitometry. Each bar represents the mean Downloaded from densitometry result (arbitrary units). The error bars indicate the SEM.

cells obtained from waste surgical specimens. For all samples, the mono- at room temperature before electrophoresis in a 6% native polyacrylamide nuclear cells were isolated by Ficoll-Hypaque density centrifugation and gel. The gels were dried and exposed to ﬁlm at Ϫ70°C, and the autora-

the T cells depleted by rosetting with sheep erythrocytes (Colorado Serum, diographs were analyzed by densitometry (SigmaGel; Jandel, San Rafael, http://www.jimmunol.org/ Denver, CO) according to standard techniques. For evaluation of NF-␬B CA). For supershift EMSAs, 10 ␮g of nuclear protein were incubated with activity in unstimulated cells, nuclear and cytosolic lysates were prepared 6 ␮g of polyclonal rabbit Ab to p50, p52, p65, c-Rel, or RelB (Santa Cruz from B cells immediately following purification, without culture. Biotechnology, Santa Cruz, CA) for 30 min at 0°C before addition of labeled probe. Cell culture reagents Flow cytometry and immunofluorescence analyses Purified B cells were cultured at 37°C with 5% CO2 in RPMI 1640 media supplemented with 10% FCS (Gemini Biological Products, Calabasas, Cells were washed in PBS and incubated with Abs according to standard CA), penicillin, streptomycin, and L-glutamine, and the following reagents, techniques. Fluorescence was measured using a Becton Dickinson FACS- as indicated: IL-4 (10 ng/ml; R&D Systems, Minneapolis, MN), anti-CD40 can (Becton Dickinson, San Jose, CA) and analyzed using the CellQuest

mAb (2 ␮g/ml, clone M3, murine IgG1␬, R&D Systems), anti-CD154 program (Becton Dickinson). Abs used for two-color analyses included by guest on September 27, 2021 mAb (3 ␮g/ml, TRAP1 clone, murine IgG1␬; PharMingen, San Diego, anti-CD3-PE (Immunotech, Westbrook, ME), anti-CD5-PE (Coulter Phar- CA), anti-CD23 (3 ␮g/ml, EBVCS; American Type Culture Collection, maceutical, Hialeah, FL), anti-CD19-PE (BioSource International, Cama- Ϫ7 Manassas, VA), and dexamethasone (10 M; Sigma, St. Louis, MO). In rillo, CA), anti-CD154-PE (clone 89-76; Becton Dickinson), anti-CD40- the time course experiment, anti-CD30 mAb (2 ␮g/ml, murine IgG1␬; FITC (R&D Systems), and anti-CD19-FITC (Immunotech). For PharMingen) was used as an isotype-matched control Ab. For some ex- measurement of apoptosis, cells were washed in annexin-binding buffer periments, we used two Jurkat mutant cell lines in which the cells consti- (Chemicon International, Temecula, CA) and exposed to annexin V-FITC tutively express (clone D1.1) or cannot express (clone B2.7) CD154, in (Chemicon) before analysis by flow cytometry. coculture with target B cells after irradiation (2000 rad) of the T cells, using a T:B cell ratio of 1:4. The Jurkat lines were the kind gift of Dr. Seth Lederman (Columbia University College of Physicians and Surgeons, New Results York, NY) (32). CLL cells have high constitutive levels of NF-␬B activity that is augmented by CD40 ligation Preparation of cytosolic and nuclear lysates We purified B cells from peripheral blood samples of healthy do- Nuclear and cytosolic fractions were prepared as described previously (10). For each circumstance, a minimum of 2 ϫ 107 cells were washed in cold nors and CLL patients, and exposed the cells in vitro to IL-4, PBS and resuspended in buffer A (10 mM HEPES (pH 7.9), 1.5 mM CD40 ligation, or dexamethasone before evaluation of nuclear NF- ␬ MgCl2, 10 mM KCl, and 0.5 mM DTT) with 0.1% Nonidet P-40 and B-binding activity by EMSA (Fig. 1). In this set of experiments, protease inhibitors (leupeptin, aprotinin, trypsin-chymotrypsin inhibitor, CD40 ligation was achieved using an agonistic, soluble anti-CD40 pepstatin A, and PMSF) at 0°C. For each sample, lysis of the plasma mAb. We chose also to examine the effects of IL-4, because this membrane was confirmed by trypan blue uptake before centrifugation, re- moval of the supernatant (cytosolic extract), and resuspension of the nu- cytokine is an established growth factor for CLL, and in some clear pellet in buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 0.42 M circumstances acts in concert with CD40 ligation to favor B cell NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 0.5 mM DTT) with protease survival (13, 33). Dexamethasone exposure was used for purposes inhibitors at 0°C. The nuclear material was sonicated briefly before rotation of comparison, as it is known to inhibit NF-␬B activity and pro- at 4°C for 30 min. To remove insoluble debris, cytosolic and nuclear lysates were centrifuged before storage at Ϫ70°C. The protein content of mote CLL cell apoptosis in vitro (34). each lysate was determined according to the Bradford assay (Bio-Rad, As shown in the representative analyses of Fig. 1A, CD40 liga- Hercules, CA). tion increased NF-␬B activity in both normal and CLL B cells, and high levels of NF-␬B activity were readily detected in nuclear EMSA and Ab inhibition (supershift) assay extracts prepared from CLL cells as compared with those of non- EMSAs were performed as described previously (10) using a T4 kinase malignant human B cells isolated and analyzed in the same man- end-labeled probe with the NF-␬B-binding sequence from the Ig ␬ light ner. To demonstrate that the high levels of NF-␬B activity we chain promoter region (5Ј-GGGACTTTCC-3Ј). For each EMSA, the nuclear lysates were incubated in DNA-binding buffer (10 mM Tris, 50 mM observed in unstimulated CLL cells were not a function of culture KCl, 1 mM DTT, 1 mM EDTA, and 5% glycerol), poly(dI-dC) (100 ng/ in media, we examined NF-␬B activity in four normal peripheral ␮l), Nonidet P-40 (0.25%) and 20,000 cpm of 32P-labeled probe for 15 min blood and four CLL B cell samples immediately after purification 2202 CONSTITUTIVE AND INDUCIBLE NF-␬B ACTIVITY IN CLL

FIGURE 3. Kinetics of CD40-mediated NF-␬B induction in CLL B cells. Nuclear extracts were prepared from CLL B cells immediately after puriﬁcation (no culture) or after exposure to media alone, anti-CD40 mAb, or an isotype-matched control Ab for time periods as indicated and analyzed by EMSA, as in Fig. 1. Downloaded from

ples analyzed for this effect, there were four cases in which CD40 ligation and IL-4 together appeared to induce NF-␬B activity to a greater extent than either agent alone. As anticipated, and consis- FIGURE 2. Composite results for NF-␬B modulation in a series of tent with the known capacity of glucocorticoids to induce apopto- cases. Freshly puriﬁed CLL B cells were exposed in vitro for6htomedia

alone (n ϭ 13), IL-4 (n ϭ 10), anti-CD40 mAb (n ϭ 12), IL-4 and anti- sis of CLL cells via a proteosome-dependent mechanism (34), ex- http://www.jimmunol.org/ ␬ CD40 mAb (n ϭ 8), or dexamethasone (n ϭ 11), after which nuclear posure to dexamethasone reduced NF- B activity in all but 1 of 11 extracts were prepared and NF-␬B activity was measured by EMSA. For cases. each case, the densitometry result for the NF-␬B signal in each experi- ␬ mental circumstance was normalized to the result for the same cells ex- CD40 ligation is a potent inducer of NF- B activity in CLL posed to media alone, evaluated in the same autoradiograph, and is repre- cells sented as a percent relative to the result for media alone. Each symbol To investigate the kinetics of CD40-mediated NF-␬B induction in represents the results for a single case, and the horizontal bars in each column CLL B cells, we analyzed nuclear NF-␬B immediately after puri- indicate the mean relative signal intensity among all of the cases. Note that fication or after exposure to media only, anti-CD40 mAb, or an the p values were determined according to Student’s t test (signal intensity by guest on September 27, 2021 for media vs each experimental circumstance). isotype-matched control Ab for various time intervals (Fig. 3). As shown, NF-␬B was activated within3hofCD40 ligation, peaked within 9 h, and was sustained at 21 h. Based on this and similar (Fig. 1B). As shown, the difference in signal intensity for NF-␬B experiments, we conclude that NF-␬B activity peaked between 6 activity in unstimulated CLL cells was significantly greater ( p ϭ and 9 h after CD40 ligation and was maintained for as long as 72 h 0.004) and ϳ2.5-fold increased, relative to that for normal B cells after stimulation (data not shown). This observation is consistent purified by the same method and analyzed in the same autoradio- with reports of sustained NF-␬B activity induced by CD40 ligation graph. These data indicate that CLL B cells have high levels of in nonmalignant human B cells (8). It is noteworthy that we ob- constitutive NF-␬B-binding activity compared with normal human served clear and reproducible NF-␬B induction in these experi- peripheral blood B lymphocytes. ments in which the CLL cells were stimulated only by soluble Ab Fig. 2 summarizes the composite results for all of the EMSAs in to CD40, a relatively weak effector of CD40 ligation. These ob- which NF-␬B activity was measured in CLL cells after6hof servations indicate that CLL B cells are extremely sensitive to stimulation. To circumvent some of the inherent difficulty in as- CD40-mediated transcriptional stimulation. sessing the effects of stimulation on NF-␬B activity among a het- ␬ erogeneous group of clinical samples, we used densitometry to Characterization of specific NF- B components in normal measure the EMSA results for each experimental circumstance. As human and CLL B cells shown, the results represent percent values relative to the signals Cross-linking of CD40 by anti-CD40 mAb in CLL B cells resulted observed for cells of the same case exposed to media. The 6-h time in an approximate doubling of NF-␬B activity in the cells (Fig. 2). point was chosen for these studies based on experiments in which In similar assays performed using four preparations of nonmalig- we observed maximal changes in NF-␬B activity between 6 and nant human peripheral blood B cells, we observed ϳ3-fold induc- 9 h after initiation of treatment (Fig. 3, below). In 11of 12 cases tion of NF-␬B activity (data not shown). To characterize which are examined at the 6-h time point, NF-␬B activity was augmented in the predominant NF-␬B components in CLL B cells, we performed CLL cells subjected to CD40 ligation by anti-CD40 mAb. supershift EMSAs using Abs to specific NF-␬B proteins with nu- Whereas CD40 ligation was fairly uniform in its effects on the clear extracts prepared from untreated or CD40-stimulated CLL B CLL cells, the consequences of IL-4 exposure were diverse among cells. The data in Fig. 4 demonstrate that the dominant NF-␬B the clinical samples analyzed for this effect. In some cases, the components in stimulated CLL cells include p50, p65, and c-Rel. addition of IL-4 alone to the CLL cells enhanced NF-␬B activity, These results are quite similar to those observed using nuclear but this was not the case for the majority of the samples. In four of extracts prepared from human tonsillar B cells subjected to CD40 eight cases analyzed after exposure to IL-4, there was an increase ligation by anti-CD40 mAb in vitro, and suggest that the regulation in NF-␬B activity in the CLL cells. Although the effect was not of NF-␬B activity in CLL cells is similar to that of nonmalignant statistically significant, based in part on the small number of sam- human B cells. The Journal of Immunology 2203

Table I. CLL cell viabilitya

Culture Circumstance Viable % (day 3) Viable % (day 6)

Media only 82 45 Anti-CD154 mAb 41 20 Anti-CD40 mAb 84 51 Dexamethasone (10Ϫ7 M) 60 22

a CLL B cells (case 28, as in Fig. 5) were placed in culture and examined after 3 and 6 days for viability by hemocytometry and trypan blue exclusion. The results shown indicate the percentage of viable cells at each time point relative to the initial number of viable cells.

CD40-CD154 interaction promotes CLL B cell survival To determine whether CD40-mediated NF-␬B induction was associated with CLL cell survival, we measured apoptosis in cells exposed to the anti-CD40 mAb, or to anti-CD154 mAb, by annexin V binding and fluorescence flow cytometry (Fig. 6). In this ␬ FIGURE 4. Comparison of NF- B proteins in human tonsillar and CLL case, there was some baseline expression of CD154 in the CD19ϩ B cells. Nuclear extracts were prepared from purified human tonsillar or B cells (9%) which was augmented after exposure to anti-CD40 Downloaded from CLL B cells after exposure for 12 h to anti-CD40 mAb and analyzed for specific NF-␬B proteins by supershift EMSA using polyclonal rabbit Abs mAb (17%). This result is similar to what we have reported pre- to p50, p52, p65, RelB, c-Rel, or control rabbit Ig, as indicated. viously regarding CD40-mediated induction of CD154 in CLL B cells (17). As shown, there was only a modest survival benefit in CLL cells exposed to anti-CD40 mAb, which was typical of the results for similar experiments using cells of other cases. In our Impedance to CD154 interaction in a CD154ϩ case results in

laboratory, we have observed much more dramatic prosurvival ef- http://www.jimmunol.org/ ␬ inhibition of NF- B activity and reduced survival in vitro fects of CD40 ligation in CLL B cells when the stimulus to CD40 CLL B cells express CD154 in only a subset of cases (17, 19). To is cell-bound CD154 (Fig. 7, below) as compared with anti-CD40 investigate the consequences of blocking CD154-CD40 interaction mAb. However, there was a dramatic reduction in survival among in a CD154ϩ case, we purified the B cells from the peripheral cells placed in culture with an Ab to CD154 (TRAP clone), which blood of a patient whose cells we had previously characterized in blocks intercellular CD40-CD154 interaction, such that 77% of the our laboratory and determined to be CD154ϩ. After isolation and CLL cells bound annexin V at this time point. Strikingly, the de- purification of the cells, CD154 expression was evaluated by gree of cell death induced by anti-CD154 mAb was similar to that FACS analysis. In this case, Ͼ99% of the purified cells coex- observed in cells exposed to dexamethasone, which is concordant pressed CD5 and CD19, and 16% of the B cells expressed CD154 with the inhibition of NF-␬B activity upon exposure to anti-CD154 by guest on September 27, 2021 (data not shown). As was evident by EMSA (Fig. 5), NF-␬B ac- mAb documented for another case in Fig. 5. Taken together, these tivity in the CLL cells of this case was inhibited by anti-CD154 data suggest that CD40-CD154 interactions are crucial in CLL B mAb to a similar extent as it was upon exposure of the cells to cell survival, and that soluble Ab to CD154 can accelerate CLL dexamethasone. In this particular case, mAb to CD154 also fa- cell death in cases that express CD154. vored CLL cell death in vitro (Table I). Finally, to examine the effect of cell-bound CD154 on CLL cell survival, we employed two Jurkat T cell mutant lines that constitutively express (clone D1.1) or cannot express (clone B2.7) CD154. Fig. 7A shows a representative experiment in which we evaluated apoptosis in CLL cells after coculture with irradiated CD154-expressing T cells in the presence or absence of blocking Ab to CD154. In this case, there was considerable baseline apoptosis in the cells cultured with media alone, such that only 45% of the cells were viable at 48 h. Survival was improved (60%) after exposure to CD154-deficient Jurkats, possibly due to soluble factors such as IL-4 or other cytokines provided by these T cells. However, the proportion of surviving CLL cells was greatest after coculture with CD154-expressing T cells (72%). This prosurvival effect of the T cells was completely abrogated in the presence of Ab to CD154 (14%), but not in the presence of an isotype-matched control Ab to CD23 (62%). Fig. 7B indicates the compiled results for this and three similar experiments, which demonstrate that CD154-expressing T cells promote the survival of CLL B cells in coculture via a CD154-dependent mechanism. The mean survival ␬ FIGURE 5. Inhibition of NF- B activity in a CD154-positive case by value among CLL cells exposed to irradiated CD154ϩ T cells in anti-CD154 mAb. CLL cells were isolated from the peripheral blood of this system was 71%, as compared to 31% among cells exposed patient 28. After the purified cells were evaluated by FACS for coexpres- only to media ( p ϭ 0.0028, Student’s t test). In cells exposed to sion of CD5 and CD19 (99% double positive) and CD154 (16% positive), ϩ nuclear extracts were prepared immediately (“unstimulated”) or after ex- CD154 T cells in the presence of anti-CD154 mAb, there was a significant reduction in the mean value for the surviving cells posure for 12 h to media alone, anti-CD154 mAb (murine IgG1␬), anti- Ϫ7 ϭ CD40 mAb (murine IgG1␬), or dexamethasone (10 M) and examined for (38%, p 0.039), as compared with the results for the same cells NF-␬B-binding activity by EMSA, as in Fig. 1. without blocking mAb. 2204 CONSTITUTIVE AND INDUCIBLE NF-␬B ACTIVITY IN CLL

FIGURE 6. Impact of CD40-CD154 interaction on CLL cell survival in vitro. Puriﬁed B cells from the peripheral blood of a patient with CLL were exposedtomedia,anti-CD40mAb,anti- CD154 mAb, or dexamethasone. A, Flow cytometric analysis for coexpression of CD5 and CD19. B, After 48 h of culture in each circumstance, the cells were evaluated with CD19-FITC (x-axes, top and middle rows) and CD154-PE (y-axis, top row), or as a control, CD3-PE (y-axis, middle row). The percentage of double-positive cells in each dot plot is indicated. Ap-

optosis of the same cells was measured Downloaded from (bottom row) using annexin V-FITC and CD19-PE, and the percentage of annexin V-positive (apoptotic) B cells in each circumstance is indicated. http://www.jimmunol.org/

We have reported that CD154 expression in CLL B cells dimin- Fig. 6. In our laboratory, we have observed that CD154 expression ishes over the initial days in culture, such that it is usually unde- is readily apparent at the cell surface in ϳ15% of CLL cases and tectable within 72 h after isolation of the cells in a positive case can be detected at some level in up to one-third of cases (17, 19). (17). In the example shown in Fig. 7A, the degree of apoptosis that The data included in Figs. 6 and 7 suggest that anti-CD154 mAb occurred in the stimulated cells exposed to anti-CD154 mAb ex- can block endogenous CD154 expressed by tumor B cells. In cases ceeded that which occurred in cells exposed to media only, and that express CD154, impedance to CD154 results in inhibition of by guest on September 27, 2021 was similar in degree to that observed in the experiment shown in NF-␬B activity and CLL cell death. In cases that do not express

FIGURE 7. Engagement of CLL B cells by CD154-expressing T cells promotes CLL B cell survival. A, CLL B cells were puriﬁed and exposed to media alone, irradiated (2000 rad) CD154-deﬁcient mutant Jurkat T cells Ϫ (clone B2.7, CD154 Txr), or to irradiated CD154-positive Jurkat T cells (clone D1.1, ϩ CD154 Txr) in a ratio of 4:1 (B:T cells) in the absence of Ab, with anti-CD154 mAb (3 ␮g/ml, TRAP clone), or with an isotype- matched control Ab (3 ␮g/ml anti-CD23). After 48 h, the B cells were analyzed for apoptosis by FACS after exposure to a PE-con- jugated Ab to CD19 and annexin V-FITC. The percentage of annexin V-negative (viable) B cells in each circumstance is indicated. B, Similar experiments were con- ducted using CLL cells from three additional cases. The columns represent the mean results for the percentage of viable B cells in each circumstance, and the error bars indicate the SD. The Journal of Immunology 2205

CD154, anti-CD154 mAb might impede survival signals conferred nearby CLL cell, would result in NF-␬B activation in vivo. In to CLL cells via CD154-expressing T cells or possibly by other cases that do not express CD154, CD40 ligation might occur due cells such as endothelial cells in the host (35). to tumor-infiltrating CD4ϩ T cells. Such a mechanism for NF-␬B induction and CD40 engagement via CD154 on T cells would be consistent with clinical observations regarding the efficacy of Discussion adenosine analogues in this disease. These chemotherapeutic We have demonstrated that unstimulated CLL B cells have high agents effectively deplete the host’s CD4ϩ T cells for prolonged levels of NF-␬B activity relative to nonmalignant human B cells periods (43) and could function, at least in part, by the elimination and that this activity is significantly induced by CD40 engagement. of T cell “help” that drives the B cell tumor (19). The dominant NF-␬B components in CLL cells appear to be p50, Finally, these results offer two possible targets for new therapies p65, and c-Rel, which is similar to the results reported by Romano in this disease. First, we have demonstrated that in some cases et al. (36). Augmented NF-␬B activity upon CD40 ligation was blocking the CD154-CD40 interaction results in inhibition of associated with improved CLL cell survival. In a CD154ϩ case, NF-␬B activity and CLL cell death. Therefore, new approaches the addition of anti-CD154 mAb resulted in dramatic inhibition of might include the use of Ab to CD154, such as is being tried in NF-␬B activity in the CLL cells to a similar degree as did dexa- patients with autoimmune diseases such as systemic lupus ery- methasone exposure. In the majority of cases examined, enhanced thematosus and idiopathic thrombocytopenic purpura. This treat- NF-␬B activity upon CD40 engagement was associated with CLL ment might be targeted to cases in which the CLL cells express cell survival, and blocking CD40-CD154 interactions by anti- CD154, or cases in which the patients suffer from associated au-

CD154 mAb resulted in accelerated CLL cell death in vitro. Taken toimmunity. Second, our results also support the investigation and Downloaded from together, our results indicate that the CD40-CD154 interaction can trial of specific inhibitors to NF-␬B, which might in themselves have profound effects on transcriptional regulation and tumor cell cause apoptosis of the malignant cells, or would facilitate death in survival in CLL. CLL cells exposed to conventional chemotherapy agents. Previous investigators have identified NF-␬B activity in lymphocytic tumors associated with transforming viruses. For exam- Acknowledgments ple, the Tax protein of the human T cell leukemia virus-1, which http://www.jimmunol.org/ occurs in certain human T cell leukemias and lymphomas, interacts We thank Dr. Morton Coleman, his office staff, and in particular Lucretia Kleinman, who expedited this work by referral of patient samples, and the with the I␬B kinase complex, resulting in enhanced I␬B phosphor- CLL patients who donated blood samples for this research. ylation, degradation, and induction of NF-␬B (37). Recently, high levels of NF-␬B activity were detected in EBV-associated post- transplant B cell lymphoproliferative disorders (38). Other inves- References tigators have observed NF-␬B activity in cell lines derived from B 1. Kipps, T. J., and D. A. Carson. 1993. Autoantibodies in chronic lymphocytic leukemia and related systemic autoimmune diseases. Blood 81:2475. cell tumors (39) and Hodgkin’s disease (40, 41). Our observation 2. Caligaris-Cappio, F. 1996. 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