Laboratory of Immunology, Institute of Haematologic Research, National Academy of Medicine, Buenos Aires, Argentina

Correspondence to: R Gamberale, Laboratory of Immunology, National Academy of Medicine, Pacheco de Melo 3081 (1425), Buenos Aires, Argentina; Fax: 54-11-48 03 94 75

Malignant B cells from chronic lymphocytic leukemia (B-CLL) patients have a long survival in vivo, although, in culture, they spontaneously die by apoptosis. Here, we analyzed the capacity of accessory leukocytes to modulate apoptosis of B-CLL cells in vitro. To this end, we performed long-term cultures using total mononuclear cells (TMC) from B-CLL patients and TMC depleted from monocytes, NK cells and T lymphocytes (B-CLL cells). In all the patients studied (n = 25) the presence of accessory leukocytes markedly prolonged the survival of B-CLL cells. The anti-apoptotic effect was exerted by monocytes and, to a lesser degree, NK cells, partially through the release of soluble factors. Indeed, accessory leukocytes separated from leukemic cells by semipermeable membranes were still able to prolong B-CLL cell survival. By flow cytometric analysis we found that the protective effect of non-malignant cells was associated with delayed down-regulation of Bcl-2 expression on leukemic cells. By contrast, the expression of Fas and Fas ligand proteins was unchanged in most samples. Our findings suggest that monocytes and NK cells, by delaying leukemic cell apoptosis, may play a role in B-CLL cell accumulation in vivo. Leukemia (2001) 15, 1860-1867.

B-CLL; apoptosis; Bcl-2; Fas/FasL

Introduction

B cell chronic lymphocytic leukemia (B-CLL) is characterized by the progressive accumulation of monoclonal CD5⁺ B lymphocytes.^1,2 More than 99% of circulating B-CLL cells are arrested in the G₀G₁ phase of the cell cycle, therefore their accumulation in vivo appears to result from the inhibition of apoptosis rather than increased proliferation.^2,3 Apoptosis is a fundamental biological mechanism involved in embryogenesis, morphogenesis and lymphoid cytotoxicity.⁴ Cells dying by apoptosis usually, but not always, undergo characteristic morphological changes which include chromatin condensation, plasma membrane blebbing, cell shrinkage and fragmentation of genomic DNA.^5,6 All of these morphological changes depend on the activities of a family of cysteine proteases known as caspases.^7,8 In turn, the activities of caspases are governed directly or indirectly by a variety of other proteins that either promote or inhibit apoptosis. In B-CLL, defective apoptosis has been attributed to the over-expression of the anti-apoptotic protein Bcl-2, which is present in most B-CLL cells despite the infrequency of the t(14;18) chromosomal translocation, characteristic of follicular lymphoma.^9,10 Bcl-2 belongs to a large family of apoptosis-regulatory proteins, which may either inhibit apoptosis, like Bcl-xL, Bcl-w and Mcl-1 or promote cell death, like Bax, Bak and Bcl-xS. Although B-CLL cells express not only members of the first group but also Bax and Bak, the pattern of expression of these proteins seems to be skewed toward prevention of apoptosis, favoring the accumulation of the leukemic cells.^10,11 Likewise, B-CLL cells express very low levels of the pro-apoptotic molecules Fas^12,13 and Fas ligand (FasL),^14,15 although the former can be up-regulated by culture with CD40 ligand, bryostatin or type-I cytokines.^16,17

Resistance to apoptosis is not an intrinsic property of B-CLL cells. In fact, when cultured in vitro, they usually die in short-term incubations, suggesting the existence in vivo of a survival-promoting microenvironment.^18,19,20 A variety of cellular and humoral factors have been reported to prevent apoptosis of B-CLL cells. Regarding cell-cell and cell-matrix interactions, Lagneaux et al²¹ found that contact with bone marrow stromal cells via 1 and 2 integrins rescues B-CLL cells from apoptosis. Recent evidence suggests that bone microenvironment might play a role in the pathogenesis of B-CLL. Thus, there is an increased bone marrow angiogenesis which correlates positively with the clinical stage of B-CLL patients.²² Moreover, B-CLL cells over-express the receptor for the CXC chemokine, stromal cell-derived factor-1 (SDF-1), which contributes to their tropism for the bone marrow stroma.²³ More importantly, SDF-1 is able not only to induce B-CLL cell migration but also to protect them from spontaneous apoptosis.²⁴

Among soluble factors involved in increased survival of B-CLL cells, different cytokines such as interleukin-4 (IL-4),^25,26 interferon (IFN-),²⁷ interferon (IFN-),²⁸ IL-8²⁹ and lymphotoxin-³⁰ have been shown to inhibit spontaneous or drug-induced apoptosis when added in vitro to B-CLL cell cultures. These cytokines can be released by the leukemic cells themselves, exerting an autocrine control of B-CLL cell survival, or by accessory leukocytes. In fact, non-malignant leukocytes in circulation constitute a major source of regulatory signals capable of modulating activation and survival of leukemic cells. While the reciprocal interaction between B-CLL and T cells has been addressed by a number of studies,^31,32,33 the participation of monocytes and NK cells in the control of B-CLL survival is largely unknown. In this regard, we have recently shown that immune complexes, by stimulating monocytes and NK cells through their receptors for IgG, are able to delay spontaneous and fludarabine-induced apoptosis of B-CLL cells.³⁴ The aim of this study was to analyze the capacity of accessory leukocytes to modulate survival of B-CLL cells in vitro. We found that monocytes and, to a lesser extent NK cells, significantly inhibit spontaneous apoptosis of leukemic cells. This anti-apoptotic effect was associated with delayed down-regulation of Bcl-2 on B-CLL cells.

Materials and methods

Reagents

Acridine orange, ethidium bromide and propidium iodide were purchased from Sigma Chemical (St Louis, MO, USA). Monoclonal antibodies (MoAbs) anti-human CD3 (clone UCHT1, mouse IgG1), anti-human CD14 (clone RM052, mouse IgG2a), anti-human CD16 (clone 3G8, mouse IgG1) and anti-human CD56 (clone C218, mouse IgG1), employed in B-CLL purification, were obtained from Immunotech (Marseille, France). Depletion of T lymphocytes, monocytes and NK cells was evaluated by using fluorescent MoAbs directed to human CD4 (clone SK3, mouse IgG1), CD8 (clone SK1, mouse IgG1) (BD Biosciences, Bedford, MA, USA), CD56 (clone N901 nkh-1, mouse IgG1) and CD19 (clone J4.119, mouse IgG1) (Beckman Coulter, Fullerton, CA, USA). Anti-human IFN (clone 25718.11, mouse IgG2a), anti-human IL-4 (clone 34019.111, mouse IgG2b), anti-human IL-10 (clone 23738.11, mouse IgG2b) were obtained from R&D (Minneapolis, MN, USA). Anti-human CD40 (clone mAb89, mouse IgG1) was obtained from Immunotech. Anti-human Bcl-2 (mouse IgG1) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MoAbs anti-human Fas (clone UB2, mouse IgG₁) and anti-human FasL (clone NOK-2, mouse IgG1) were purchased from BD Biosciences. Anti-mouse IgG-FITC was obtained from Immunotech. Cells were fixed and permeabilized by using Fix & Perm from Caltag Laboratories (Burlingame, CA, USA).

Patients

Sterile heparinized peripheral blood was obtained from 25 patients with B-CLL (age range 62 to 91), who were either untreated or had not received cytoreductive chemotherapy for at least 3 months before investigation. Patient samples were provided by the Institute of Hematologic Research at the National Academy of Medicine, Buenos Aires. B-CLL was diagnosed according to standard clinical and laboratory criteria. At the time of analysis, all patients were free from clinically relevant infectious complications.

Separation procedures

Total mononuclear cells (TMC) were isolated by centrifugation over a Ficoll-Hypaque layer (Lymphoprep; Nycomed Pharma, Oslo, Norway). For purification of B-CLL cells (B-CLL), TMC were incubated with MoAbs specific for CD3, CD14, CD16 and CD56 for 45 min at 4°C. Then, cells were washed twice and treated with magnetic beads coated with antimouse IgG antibodies (Dynabeads M450, Dynal, Oslo, Norway), according to the manufacturer's instructions. The purity of B-CLL population was checked by FACS analysis using anti-CD19 MoAb and was found to be >98%. For selective depletion of T cells (CD3^-) monocytes (CD14^-) or NK cells (CD56^-), TMC were incubated with specific MoAbs and treated as described above for B-CLL purification. The efficacy of selective depletion was checked by FACS analysis employing MoAbs directed to CD4 and CD8 to determine remaining T lymphocytes and remaining monocytes (CD4^low). Depletion of NK cells was checked using an anti-CD56 which recognizes a different epitope from the MoAb employed for purification. We only used depleted samples in which the proportion of the remaining population was less than 0.1%.

Taking into account that separation procedures could decrease cellular viability, for TMC cultures, samples were treated in the same way as for B-CLL purification except that MoAbs were not added.

Cell cultures

TMC or B-CLL cells were resuspended in RPMI 1640 (Life Technologies, Grand Island, NY, USA) supplemented with 100 g/ml streptomycin, 100 U/ml penicillin and 10% of heat-inactivated fetal calf serum (culture medium), all from Gibco Laboratories (Grand Island, NY, USA). Then, aliquots of 0.15 ml of these cells (2 ´ 10⁶/ml) were placed in 96-well flat-bottom microplates and cultured at 37°C in an atmosphere of 5% CO₂.

In experiments aimed at investigating the participation of soluble factors in the prevention of apoptosis of leukemic cells, co-cultures of TMC and B-CLL cells (2 ´ 10⁶ cells/ml) were performed using transwell culture inserts. TMC were placed in the lower compartment of 48-well flat-bottom plates at a total volume of 600 l and autologous B-CLL cells were added into 0.4 m poro size inserts (upper compartment) (Costar, Cambridge, MA, USA). By this method, purified leukemic cells in the upper compartment were physically separated from cells in the lower compartment, while the passage of macromolecules across the semipermeable membrane was allowed. As control, we performed co-cultures of B-CLL cells by placing them both in the upper and in the lower compartments.

Quantitation of cellular apoptosis and viability by fluorescence microscopy

Quantitation was performed as previously described,³⁵ using fluorescent DNA-binding dyes acridine orange (100 g/ml) to determine the percentage of cells that had undergone apoptosis, and ethidium bromide (100 g/ml) to differentiate between viable and nonviable cells. With this method, nonapoptotic cell nuclei show variations in fluorescent intensity that reflect the distribution of euchromatin and heterochromatin. By contrast, apoptotic nuclei exhibit highly condensed chromatin that is uniformly stained by acridine orange. To assess the percentage of cells showing morphologic features of apoptosis, at least 200 cells were scored in each experiment.

Quantitation of cellular apoptosis by propidium iodide staining and flow cytometry

The proportion of cells that displays a hypodiploid DNA peak, ie apoptotic cells, was determined using a modification of Nicoletti et al's protocol.³⁶ Briefly, cell pellets (2.5 ´ 10⁶ cells) were suspended in 400 l of hypotonic fluorochrome solution (propidium iodide, 50 g/ml in 0.1% sodium citrate plus 0.1% Triton X-100) and incubated for 2 h at 4°C. The red fluorescence of propidium iodide of individual nuclei was measured using a FACScan flow cytometer (Becton Dickinson Immunocytometry System, San Jose, CA, USA). The forward scatter and side scatter of particles were simultaneously measured. Cell debris was excluded from analysis by appropriately raising the forward scatter threshold. The red fluorescence peak of cells with normal (diploid) DNA content was set at channel 250 in the logarithmic mode. Apoptotic cell nuclei emitted fluorescence in channels 4-200.

Quantitation of cell death by annexin V staining and flow cytometry

The percentage of overall cell death (apoptosis and necrosis) in the different leukocyte subpopulations was measured by two-color flow cytometry using annexin V-FITC (Immunotech). Briefly, 3 ´ 10⁵ TMC were stained with PE-conjugated anti-CD3, anti-CD56 or anti-CD14, washed twice and incubated with saturating concentrations of annexin V-FITC according to the manufacturer's instructions. Negative controls were performed simultaneously using irrelevant PE-conjugated MoAb of the corresponding Ig isotype.

Flow cytometric analysis

For intracellular detection of Bcl-2 protein, 6 ´ 10⁵ cells (TMC or B-CLL) were fixed and permeabilized by using Fix & Perm, before the addition of anti-bcl-2 (4 g/ml) or isotype-matched MoAb (4 g/ml). Then, the Bcl-2 expression in leukemic cells was revealed with anti-mouse IgG-FITC in the CD19⁺ population.

The expression of Fas and FasL was analyzed by flow cytometry using specific MoAbs anti-human Fas or anti-human FasL and anti-mouse IgG-FITC.

Statistical analysis

The Wilcoxon nonparametric test was used to analyze the statistical significance of the experimental results.

Results

Accesory leukocytes increase the survival of B-CLL cells

In order to determine whether accessory cells were able to modulate the survival of B-CLL cells in vitro, we compared the apoptotic rates of unfractionated TMC and purified leukemic lymphocytes. In agreement with previous results,^19,37,38 we found highly variable rates of spontaneous apoptosis among B-CLL samples. Nevertheless, in all the patients analyzed (n = 25) the presence of accessory leukocytes, even if they were at very low levels (Table 1), markedly prolonged the survival of leukemic cells. The data depicted in Figure 1a correspond to the day of culture in which the percentage of apoptotic B-CLL cells was higher than 80%. In most cases (n = 17), this level of apoptosis was observed between days 4 and 11 of culture. These findings were confirmed by flow cytometric analysis of apoptosis with propidium iodide staining (Figure 1c). Results in Figure 1b show the survival kinetics of two representative patients. As can be seen, the apoptotic rate of TMC from patient 7 was very low, even after 2 weeks in culture. By contrast, around 50% of TMC from patient 11 were already dead after 7 days in vitro. Differences in the survival kinetics of TMC could not be directly associated with higher percentages of accessory leukocytes (see data from patients 7 and 11 in Table 1). Nevertheless, in both cases, the absence of accessory leukocytes markedly increased apoptotic rates.

Having shown that apoptosis was higher in purified B-CLL cells than in TMC cultures, we next determined the levels of non-malignant cells death in TMC samples by two-color flow cytometry using PE-conjugated MoAbs directed to T lymphocytes, monocytes or NK cells, and annexin V-FITC. Results from a representative sample are depicted in Figure 2. As shown, death of B-CLL cells almost completely account for the percentage of apoptosis observed in TMC cultures. In fact, at the time of analysis, less than 5% of annexin V-positive cells corresponded to non-malignant leukocytes in all patients evaluated (n = 7).

Monocytes and NK cells exert anti-apoptotic effects on B-CLL cells

To find out if the protective effect of accessory leukocytes on B-CLL cells could be ascribed to a particular population, we selectively depleted monocytes, NK cells and T lymphocytes from TMC. Then, these depleted suspensions (Mon^-, NK^- and T^-) were cultured and apoptosis was analyzed daily. Results in Figure 3 show that depletion of monocytes and, to a lesser extent NK cells, significantly impaired the anti-apoptotic effect exerted by accessory leukocytes suggesting the involvement of these populations in improving B-CLL cell survival. By contrast, depletion of T lymphocytes did not modify the protective effect of accessory cells.

Soluble factors are partially responsible for the anti-apoptotic effect mediated by accessory leukocytes

In an attempt to determine whether the protective effect mediated by accessory cells depends on the release of soluble factors we carried out incubations using semipermeable cell cultures inserts. As described in Materials and methods, purified leukemic cells placed into the upper compartment, were physically separated from TMC (lower compartment) by a transwell insert, which allows the passage of macromolecules but prevents cell-cell contact. In the same way, purified leukemic cells were placed in both compartments, being the percentages of apoptosis of B-CLL cells in the upper compartment evaluated daily. In all the samples tested (n = 7), purified leukemic cells showed decreased levels of apoptosis when they were co-cultured with TMC (white bars in Figure 4), suggesting that soluble factor(s) released from TMC exerted a protective effect on leukemic cells. However, it should be noted that, in the case of the transwell setting, the anti-apoptotic effect induced by accessory leukocytes was less pronounced than that observed in TMC cultures (data not shown). This might indicate that, not only soluble factors but also cell-cell interactions were involved in the protective effect mediated by accessory leukocytes.

In an attempt to identify these factors, we performed cultures of TMC in the presence of neutralizing antibodies directed to IL-4, IFN, IL-10 or CD40. As shown in Figure 5, there was a marked heterogeneity among different patients. Thus, opposite effects were observed in distinct samples as a consequence of the addition of neutralizing antibodies directed to IFN, IL-4 or IL-10 (Figure 5a, b and c). On the other hand, the addition of blocking antibodies directed to CD40 resulted in increased percentages of apoptosis in only one out of the five patients analyzed (Figure 5d). Taken together, these results strongly suggest that the mechanisms through which accessory leukocytes modulate B-CLL apoptosis in samples from different patients do not involve a unique pathway.

Accessory leukocytes delayed Bcl-2 down-regulation of cultured B-CLL cells

It has been previously reported that the levels of apoptosis of B-CLL cells in vitro correlate with a decrease expression of Bcl-2.^39,40 A number of treatments which prolong the survival of leukemic cells, such as addition of IL-4,²⁵ thioredoxin¹⁹ or contact with bone marrow stromal cells,²¹ are also able to delay Bcl-2 down-regulation. In an attempt to determine whether the anti-apoptotic effect of accessory leukocytes was associated with changes in Bcl-2 levels, we analyzed its expression in permeabilized CD19⁺ cells from TMC and B-CLL cultures. The assays were performed when we observed clear differences in the percentages of apoptosis between TMC and purified B-CLL cells. As expected, Bcl-2 was expressed at a high level (Bcl-2^high) and in a unimodal fashion in fresh isolated cells from the six patients evaluated (data not shown). As apoptotic rate increased during the culture, this constitutive expression of Bcl-2 was down-regulated in a proportion of cells (Bcl-2^low), which was higher in purified B-CLL cultures compared to TMC cultures (mean ± s.e.m. = 32.7 ± 3.3 and 13.5 ± 3.8, respectively, n = 6, P < 0.05). Results from a representative experiment are depicted in Figure 6a and b. Fluorescence histograms (panel b) revealed a bimodal expression of Bcl-2 in B-CLL cultures with a proportion of cells, which coincided with apoptotic cells (panel a), showing decreased levels of Bcl-2. Data in panel c show the level of decrease in Bcl-2 expression for the six B-CLL cultures evaluated. Comparable results were obtained when cultures were performed in the transwell setting described above. Thus, the percentages of Bcl-2^low cells in purified B-CLL (upper compartment) were 30.7 ± 5.8 and 22.1 ± 6.2 for cells co-cultured with purified B-CLL and TMC cells, respectively (n = 3, P < 0.05).

Analysis of Fas and Fas ligand expression on cultured B-CLL cells

Although leukemic cells from B-CLL patients are either negative or only weakly positive for Fas and Fas ligand expression,^12,13,14,15 they can be up-regulated by a number of stimuli such as IL-4, bryostatin or CD40 ligand.^16,17 We asked whether the enhanced apoptosis of purified B-CLL cells in vitro might be associated with an increased expression of Fas and Fas ligand. To this end, we analyzed their expression on leukemic cells cultured in the presence or absence of accessory leukocytes. As in the case of Bcl-2 the analysis was performed when percentages of apoptosis between both cultures (TMC and purified B-CLL) were significantly different. We found low to moderate expression of Fas in all the samples analyzed, the percentage of positive cells being 20-85% (n = 7). In only two samples were these levels higher in cultures of purified B-CLL cells compared to TMC (Figure 7a), while in the other five there were no differences (Figure 7b). In regard to Fas ligand, the very low expression (% of positive cells: 5-10%) observed in two of six samples studied was similar in TMC and B-CLL cells (Figure 7d). Together, these data indicate that the enhanced apoptosis of purified leukemic cells was not associated with Fas/Fas ligand induction.

Discussion

Several observations indicate that signals from the microenvironment in vivo might influence the survival of B-CLL cells.^{19,20,21,41,42,43,44} In fact, when long-life span leukemic cells are cultured in vitro they spontaneously die by apoptosis. The results presented here show that non-malignant leukocytes, particularly monocytes and, to a lesser extent NK cells, are able to prolong survival of B-CLL cells. By contrast, T lymphocytes did not exert any modulatory effect. It is important to note that the inhibition of apoptosis by accessory leukocytes was observed in all the patients analyzed, even in those with very low percentages of monocytes and NK cells (<5%). While accessory leukocytes displayed their protective effect without the addition of specific stimuli, we cannot rule out the possibility that they became activated during culture. In fact, in all the samples analyzed we observed a clear 'spreading' of monocytes as culture progressed.

Over the last decade, a variety of cytokines have been shown to delay apoptosis of B-CLL cells when added in vitro to short-term cultures. Thus, IL-4,^25,26 IFN-,²⁷ IFN-,²⁸ and IL-8²⁹ show noticeable anti-apoptotic activity, while the effects of IL-10^45,46 and TNF-^47,48 remain controversial. We have recently reported that IFN- released from accessory leukocytes in response to immune complexes was partially responsible for inhibiting spontaneous and drug-induced apoptosis of B-CLL cells.³⁴ In an attempt to determine whether this cytokine was also involved in the protecting effects of accessory cells described here, we carried out cultures in the presence of anti-IFN antibodies. We found that neutralization of IFN led to increased levels of apoptosis in five out of 12 samples analyzed, while it had no effect or even decreased apoptotic rates in the rest. Highly variable results were also observed by employment of neutralizing antibodies directed to either IL-4, IL-10 or CD40, suggesting that the mechanisms involved in the regulation of apoptosis might differ among B-CLL samples. In addition, soluble factors other than classical cytokines can also be involved in B-CLL cell protection. Thus, nitric oxide, which has been shown to exert pro- and anti-apoptotic effects in different human neoplastic cells,⁴⁹ protects B-CLL cells from programmed cell death.⁵⁰ Similarly, soluble CD23⁵¹ and thioredoxin, a potent anti-oxidant protein which is present in high quantities on monocytes and macrophages, prolong survival of leukemic cells in long-term cultures.¹⁹ It is, therefore, conceivable that an array of factors rather than a single factor individually may account for the protective effects exerted by monocytes and NK cells on B-CLL lymphocytes.

Not only soluble factors but also cell-cell and cell-matrix interactions play a role in modulating B-CLL cell survival.^21,24,42,44 A recent report²⁰ has shown that adherent cells from peripheral blood of CLL patients supported the survival of leukemic cells in vitro. These so-called 'nurse-like cells', which appear to be related to bone marrow stromal cells, are not present in blood from healthy donors. Taking into account that during the initial 2 to 3 days of culture these cells are not identifiable, the authors suggest that they are functionally immature in the bloodstream. The possibility exists that precursors of these cells are depleted under our experimental conditions, ie by using anti-CD14, CD16 and/or CD56 MoAbs, giving rise to the rapid deterioration in the survival of purified B-CLL cells.

Over-expression of the anti-apoptotic protein Bcl-2 in the absence of the t(14;18) translocation is a well-documented finding in B-CLL.^9,10 It has been proposed that external signals generated via cytokine receptors participate in maintaining high Bcl-2 levels in vivo.^25,40 Accordingly, growth factor removal in vitro results in Bcl-2 down-regulation which correlates with decreased survival of leukemic cells.^19,20,21,25 By using flow cytometric analysis of purified B-CLL cells, we observed the progressive appearance of an expanded population with reduced expression of Bcl-2, which corresponds to the apoptotic population as depicted in dot-plot profiles. Inhibition of apoptosis by accessory leukocytes was associated with a delayed down-regulation of Bcl-2 in all the samples analyzed. These data suggest that signals delivered from monocytes and NK cells may increase the survival of B-CLL lymphocytes by delaying the loss of Bcl-2. It should be mentioned that not only levels of expression of Bcl-2 are relevant for apoptosis regulation. Recent evidence indicates that anti-apoptotic functions of Bcl-2 and other members of Bcl-2 family, such as Bcl-xL and Mcl-1, can be regulated by phosphorylation.⁵² Further experiments will be necessary to determine whether phosphorylation of Bcl-2 proteins is involved in the modulation of B-CLL cell apoptosis by accessory leukocytes.

The presence of Fas (CD95) and Fas ligand (CD154) proteins as well as their potential role in the induction of apoptosis by B-CLL cells are still unclear.⁵³ Thus, there is evidence showing that these proteins are absent from leukemic cells,^14,17 or are expressed at low¹³ to moderate levels.¹² Moreover, Fas expression can be up-regulated by different treatments, such as incubation with bryostatin, CD40 ligand or IL-2.^12,16,17 Finally, B-CLL cells which up-regulate Fas protein have been reported to be susceptible,^12,54 or resistant^13,55 to induction of apoptosis with anti-Fas antibodies. We show here that decreased survival of purified B-CLL cells in culture did not correlate with an increased expression of the Fas/Fas ligand proteins, precluding a relevant role for this system in leukemic cell apoptosis.

While the reciprocal interaction between B-CLL and T cells has been previously addressed by a number of studies,^31,32 the participation of monocytes and NK cells in the control of B-CLL survival is largely unknown. Our study provides evidence that signals from these accessory leukocytes are able to inhibit B-CLL cell apoptosis through a Bcl-2-dependent pathway. The relevance of these mechanisms in vivo remains to be established.

Acknowledgements

We would like to thank Ms Selma Tolosa and Ms Nelly Villagra for their excellent technical assistance and Fundación de la Hemofilia for the use of the FACScan cytometer. This work was supported by grants from Consejo Nacional de Investigaciones Cientificas y Técnicas, Agencia-Foncyt, Buenos Aires University School of Medicine, Ministerio de Salud and Fundación Alberto J Roemmers.

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Figure 1 Accessory leukocytes delay B-CLL cells apoptosis. Unfractionated TMC or purified leukemic cells (B-CLL) (2 ´ 10⁶ cells/ml) were incubated at 37°C and apoptosis was evaluated by fluorescence microscopy (a and b) or flow cytometry (c). (a) The results shown (mean ± s.e.m. of 25 patients) correspond to the day of culture in which the percentage of B-CLL cell apoptosis was higher than 80%. *Statistically significant (P < 0.01) compared to TMC. (b) Kinetics of spontaneous apoptosis of two representative patients. (c) Histograms of a representative experiment showing the percentage of nuclei with hypodiploid DNA content. Apoptosis of TMC and B-CLL cells from patient 3 was evaluated after 6 days of culture.

Figure 2 Cell death of leukemic and accessory leukocytes in TMC cultures by Annexin V. The percentage of cell death in the different cell populations was measured by two-color flow cytometry after staining with PE-conjugated anti-CD3, anti-CD56 or anti-CD14 and annexin V-FITC. Representative dot plots from one out of seven patients are shown.

Figure 3 Role of monocytes and NK cells in prevention of B-CLL cells apoptosis. TMC, purified B-CLL and TMC depleted of T lymphocytes (T^-), monocytes (Mon^-) or NK cells (NK^-) were incubated at 37°C and apoptosis was evaluated daily by fluorescence microscopy. The results shown (mean ± s.e.m., n = 6-8) correspond to the day of culture in which the percentage of apoptosis in B-CLL cultures was higher than 70%. *Statistically significant (P < 0.05) compared to TMC.

Figure 4 Involvement of soluble factors in prevention of B-CLL cells apoptosis by accessory leukocytes. Cultures were performed using the transwell setting. Purified leukemic cells in the upper compartment were co-cultured with TMC (white bars) or the same population of purified leukemic cells (grey bars) in the lower compartment. Apoptosis of B-CLL cells (upper compartment) was evaluated daily by fluorescence microscopy. The results of individual patients are shown.

Figure 5 Role of IFN, IL-4, IL-10 and CD40 in modulation of B-CLL cell apoptosis. TMC were incubated at 37°C in the absence (open bars) or presence (grey bars) of neutralizing Abs (10 g/ml) directed to IFN (a), IL-4 (b) or IL-10 (c), as well as a blocking Ab (10 g/ml) to CD40 (d). All the Abs were added at the beginning of the culture and every 48 h. Apoptosis was evaluated daily by fluorescence microscopy. Shown are percentages of apoptosis of individual patients at the day of culture in which apoptosis of purified B-CLL cells was higher than 80%.

Figure 6 Bcl-2 expression in TMC and B-CLL cultures. TMC and purified leukemic cells (B-CLL) were incubated at 37°C and Bcl-2 expression in CD19⁺ cells was analyzed by flow cytometry. The results shown correspond to dot-plots (a) and histograms (b) from a representative sample (patient 11) evaluated at day 5 of culture. Solid line, Bcl-2 expression in TMC; dotted line, Bcl-2 expression in purified B-CLL cells; grey area, isotype control. (c) Mean fluorescence intensity (MFI) of Bcl-2^high and Bcl-2^low cells from the six B-CLL cultures evaluated. The percentages of Bcl-2^low cells in TMC were <15% at the time of analysis.

Figure 7 Fas and FasL expression in TMC and B-CLL cultures. TMC and B-CLL cells were incubated at 37°C and Fas or FasL expression in CD19⁺ cells was analyzed by flow cytometry at the day of culture in which both populations displayed different apoptotic levels. Histograms from four representative samples (n = 7) are shown. Solid line, Fas or FasL expression in TMC; dotted line, Fas or FasL expression in B-CLL cells; grey area, isotype control.

Table 1 Accessory leukocytes in B-CLL samples