HIGHER PROLIFERATIVE RESPONSE IN B-CHRONIC LYMPHOCYTIC LEUKEMIA (B-CLL) AS COMPARED TO
B-MONOCLONAL LYMPHOCYTOSIS OF UNDETERMINED SIGNIFICANCE (B-MLUS) AFTER STIMULATION WITH
STAPHYLOCOCCUS A UREUS AND ANTI-CD40 MONOCLONAL ANTIBODIES
CARLOS A. GARCIA,*t ANDERS ROSI~N,~ MIGUEL AGUILAR-SANTELISES,§ MIKAEL JONDAL§ and H/~KAN MELLSTEDTt
*Department of Biology, Instituto Nacional de Oncologia y Radiobiologia, Habana, Cuba; SDepartment of Cell Biology, Faculty of Health Sciences, University of Link6ping, Sweden; §Department of Immunology, Karolinska Institute, Stockholm, Sweden; and tDepartment of Oncology (Radiumhemmet) and Immunology Research Laboratory, Karolinska Hospital, Stockholm,
Sweden
(Received 24 June 1993. Revision accepted 13 July 1993)
Abstraet--B-CLL is a malignant monoclonal B-cell disorder and B-MLUS is the benign counterpart. The proliferative response and the capacity to secrete IgM was measured in B-CLL and B-MLUS, respectively, and compared to normal B-cells. SAC and a mAb against CD40 were used as stimulatory agents. No cell population responded to anti-CD40 mAb alone. SAC only induced a high DNA synthesis rate in normal B-cells as well as in B-CLL cells, although the magnitude was three-fold lower and delayed for about 48 h in B-CLL. B-MLUS cells did not proliferate in response to SAC. The combination of anti-CD40 and SAC enhanced the proliferative capacity of normal B-cells and produced a more rapid response in B-CLL. B-MLUS ceils were not activated. Normal B-cells and B- MLUS did not secrete IgM after SAC stimulation, while B-CLL cells had a continuous increase in the IgM production during a 6-day culture period. The higher proliferative capacity of B-CLL cells compared with B-MLUS cells may be explained by an increased expression of activation molecules e.g. receptors for various cytokines and growth factors. Moreover, the inertness and inability of B- MLUS cells in comparison to normal B- and B-CLL cells to respond to powerful activation signals might indicate an intrinsic defect of B-MLUS cells in the signal transduction leading to a block of mitosis and a benign course of the disease.
B-CLL IS A monoclonal proliferation of neoplastic B- cells ' frozen' at an early stage of maturation. The differentiation block is however not irreversible. The cells might be induced to maturation under appro- priate conditions in vitro [1]. Tumor promotors e.g. phorbol esters or B-cell mitogens have been shown
Abbreviations: B-CLL, chronic lymphocytic leukemia of B-cell type; B-MLUS, monoclonal lymphocytosis of undetermined significance of B-cell type; BSA, bovine serum albumin; lg, immunoglobulin; mAb, monoclonal antibody; PWM, pokeweed mitogen; SAC, Staphylococcus aureus Cowan I.
Correspondence to: Hhkan Mellstedt, Department of Oncology (Radiumhemmet), Karolinska Hospital, S-104 01 Stockholm, Sweden.
933
to induce B-CLL cells to differentiate to an 'acti- vated' cell with the phenotype of prolymphocytic leukemia [2] and to mature plasma cells [3, 4].
One of the most widely used B-cell mitogens is SAC. It induces a T-cell-independent activation of B-cells through all stages of the cell cycle by cross- linking of surface Ig [5]. In B-CLL a varying response to SAC was observed. This might be due to the heterogeneity of the disease as revealed by the phenotypic characteristics, clinical presentations and response to therapy [1, 6, 7].
CD40 is a 55 kD glycoprotein expressed on normal B-cells as well as on epithelial derived malignancies and is a receptor for B-cell progression signals [8]. A natural ligand for the murine CD40 was recently cloned [9] as well as the human ligand [10-12]. These ligands as well as monoclonal antibodies ($2C6 or
934 C.A. GARCIA et al.
G28-5) against the CD40 receptor deliver powerful proliferative signals [13]. The extracellular domain of the CD40 molecule shows significant homology with a family of cell surface glycoproteins that include the receptor for the nerve growth factor (NGF) and tumor necrosis factor (TNF) [14, 15].
In previous studies we suggested that monoclonal B-cell lymphocytosis might be divided into two clini- cal entities: B-CLL and monoclonal B-lymphocytosis of undetermined significance (B-MLUS). These dis- orders may be distinguished on the basis of dif- ferences in the cell surface phenotype, mainly molecules related to growth signals suggesting that the two diseases represented different stages of the B-cell differentiation/activation pathway [16, 17]. There was also a pronounced imbalance between CD4 and CD8 T-cells. B-CLL had a low CD4/CD8 ratio which was within the upper normal range in B-MLUS. B-CLL patients had a high number of circulating immature NK cells compared to B-MLUS while B-MLUS patients had a higher proportion of NK cells with a high lytic capacity compared to B- CLL [18]. B-CLL cells had a low production of IL-1 while B-MLUS lymphocytes had a normal or high secretion [19].
The proliferative response of B-CLL cells and the ability to differentiate after stimulation with T-cell derived cytokines, phorbol ester, calcium ionophore and anti-IgM antibodies was heterogeneous [3, 20- 33]. There was no clear relationship between the clinical activity and the in vitro response.
A selective increase in soluble CD4 was found in serum of patients with progressive B-CLL [34]. Moreover, there was a difference in the proliferative response to soluble T-cell factors between B-CLL and B-MLUS suggesting that B-CLL cells were more readily activated (data to be published).
The majority of normal B-cells capable of dif- ferentiating to IgG and IgM secreting cells after PWM exposure were found in the Leu8- sub- population [35-37]. It was suggested that cells responsive in vitro to PWM already had received an in vivo signal and lost surface IgD [37]. The pheno- type of the PWM responsive blood lymphocyte subset was Leu8- / s lgD- . In contrast, cells proliferating in response to SAC were mostly found in the Leu8 ÷ fraction of circulating B-cells. This observation indi- cates that the majority of cells responsive to PWM or SAC belonged to distinct, possibly non-overlapping subsets. Moreover, Leu8- cells (PWM-responding cells) were also responding to IL-1 stimulation [38] while Leu8 ÷ cells (SAC-responsive cells) were not responsive to IL-1 [39].
Detailed analysis of B-MLUS and B-CLL cells may be of importance for a better understanding of
benign and malignant B-cell physiology and of the leukemogenic process. In the present report we have studied some aspects of the capacity of B-CLL and B-MLUS cells to proliferate and differentiate.
MATERIALS AND METHODS
Patients Seven B-CLL and four B-MLUS patients were included.
The mean age of the B-CLL patients was 71 yr (range 60-- 80 yr) and 70 yr (range 52-85 yr) for the B-MLUS patients. All patients had a monoclonal B-cell population in per- ipheral blood determined by the restriction for r or X surface Ig light chains. The main criteria for B-CLL was infiltration in the bone marrow of small lymphocytes and -->15 × 109//1 lymphocytes in peripheral blood [40]. B- MLUS was defined as the presence of ->4 × 109/1 lym- phocytes in peripheral blood with a clonal B-cell fraction (see above), without general symptoms (fever, night sweats, weight loss), enlarged lymph nodes, hepato- splenomegaly, anemia (Hb->120g/1), thrombocytopenia (platelet counts ->150 × 109/1) and no increase in lac- todehydrogenase (LD-<8.0 ~tkat/1). Furthermore, during the observation period no significant increase in the blood lymphocyte count (doubling of the lymphocyte count) should occur. No other symptoms or signs related to CLL should be present during follow-up. The median obser- vation time for B-MLUS patients was 68 months (range 35-218 months).
Healthy control donors Six healthy persons between the ages of 20 and 60 yr
were used as controls. No control donors had any signs of infectious disease or were on medication.
Lymphocyte preparation Lymphocytes from patients were enriched by Ficoll-
Isopaque gradient centrifugation of heparinized venous blood. After incubation with iron powder, phagocytic cells were removed [41]. The lymphocyte suspensions contained >98% viable cells. Non-T-(=B) cells were enriched by E- rosette fractionation over a Ficoil-Isopaque gradient as described earlier [16]. Normal B-ceils were purified by Ficoll-Isopaque gradient centrifugation, carbonyl iron treatment, plastic adherence, E-rosetting and panning to deplete CD3 ÷, CD13 + and CD16 + cells [19].
Kinetics of DNA synthesis Non-T-(B-CLL and B-MLUS B-cells) and normal B-
cells were cultured at 2 x 105 cells/200 Ixl of RPMI 1640 (Gibco Ltd, Parseley, Scotland) supplemented with 5% fetal calf serum, penicillin and streptomycin (culture medium). Stimulations were performed by adding SAC (1:5000) (Pansorbin, Calbiochem) and 1 ~tg/ml of $2C6 (anti-CD40) mAb [42, 43]. Cultures were incubated in 96 well microplates (Nunc, Roskilde, Denmark) at 37°C in 5% COE atmosphere and stopped after 24, 48, 72, 96, 120 and 144 h of incubation including an 18 h pulse with 1 ~tCi/
P r o l i f e r a t i o n in B - C L L a n d B - M L U S 9 3 5
5 0 -
4 O
~ g 2 0
1 0
F I [ i ~1
24 48 72 96 120 144 INCUBATION PERIOD (h}
FIG. 1. DNA synthesis (SI) (mean -+ S.E.M.) of normal B- cells (n = 6) after stimulation with SAC (O-----O), mAb $2C6 (anti-CD40) (O O) alone and in combination
(A A).
well of 3H-thymidine (Amersham, U.K.). Stimulation index (S/) was defined as:
DNA synthesis (cpm) of stimulated cells
SI = spontaneous DNA synthesis (cpm)"
of non-stimulated cells
Culture conditions for IgM analyses Non-T-cells from patients and normal B-cells were cul-
tured at 1 x 106 cells/ml in culture medium and stimulated with SAC and mAb $2C6 at the same concentrations as above. Supernatants were collected after 24, 48, 72, 96 and 120 h of incubation. Samples were centrifuged and stored at -70°C until analysis.
ELISA for lgM analyses Supernatants were assayed in ELISA using flat bottomed
microtiter plates (Dynatech, Cambridge, MA, U.S.A.) coated at 4°C overnight with goat anti-human IgM (Dak- opatts A/S, Copenhagen, Denmark) [42, 44]. After block- ing with 0.5% BSA in coating buffer for i h at 37°C, diluted samples were added for 2 h at 37°C. Finally, the plates were reacted for 2 h at 37°C with a rabbit anti-human IgM alkaline phosphatase conjugate (1:1000) (Dakopatts A/ S). The wells were extensively washed, developed by p- nitrophenylphosphate-diethanolamine buffer for 30 rain and read at 405 nm in an automatic ELISA reader (Tit- ertech). The assay sensitivity was <10 ng/ml.
Statistics Comparisons between means were done using the Mann-
Whitney U test.
RESULTS
Normal B-cells responded weakly to stimulation with mAb $2C6 (anti-CD40) alone (Fig. 1, Table 1). However, 48 h activation with SAC induced a marked DNA synthesis, which was maintained dur- ing the remaining culture period. Addition of mAb $2C6 further increased the SAC response.
DNA synthesis was also induced in B-CLL cells after stimulation with SAC. However, the magnitude
of the response was three-fold lower than that of normal B-cells and delayed for about 48 h. In B- MLUS cells only a marginal proliferative response was seen at 72 h (Fig. 2, Table 1).
When mAb $2C6 alone was added to B-CLL cul- tures, no proliferation was induced. However, when combined with SAC, a proliferative response was noted at an earlier time-point as compared to stimu- lation with SAC alone (Fig. 3, Table 1). mAb $2C6 alone or in combination with SAC did not stimulate DNA synthesis in B-MLUS cells (Fig. 4, Table 1).
|gM secretion was also measured after SAC stimu- lation. Normal B-cells and B-MLUS cells did not synthesize IgM in any substantial amount. However, a low IgM secretion was noted in the B-CLL popu- lation. The secretion was markedly augmented after addition of SAC (Fig. 5, Table 1).
DISCUSSION
Leukemic B-cell clones have successfully been used as models to analyze the proliferation and/or differentiation inducing effects of various mito- genic factors [45]. Maturation to lymphoblasts/ plasmablasts without proliferation can be induced by the phorbol ester TPA [46] acting by translocation and activation of protein kinase C. Induction of pro- liferation requires at least two signals: one activation/ triggering signal and one progression signal such as interleukin-2 (IL-2) or the B-cell stimulatory factor, BSF-MP6 [47,481. The latter was recently defined as a novel isoform of thioredoxin (Trx) and named MP6- Trx [49]. Similarly, Wakasugi et al. [50] observed a co-cytokine effect of an adult T-cell leukemia derived factor (ADF), which was shown to be identical to human thioredoxin. Activation signals may be TPA, SAC or anti-~t. The effect of the progression signals depends on the nature of the activation signal. Potent activation/progression signals are SAC + BSF- MP6 + IL-2 [48] or TPA + BSF-MP6 + IL-4 [51]. Signal transduction through the B-cell receptors CD23 and CD40 are powerful signals [5].
In this study we analyzed the most potent of these molecules, the CD40 receptor. We found a clear difference between B-CLL and B-MLUS cells in response to stimulation with SAC and an anti-CD40 mAb [$2C61. B-CLL cells exhibited a higher re- sponsiveness to SAC stimulation measured both as proliferation (thymidine incorporation) and differentiation (IgM production) as compared to B- MLUS cells. B-CLL cells have been shown to respond to factors produced by T-cells [20-29]. Patients with progressive disease had more easily responding leukemic cells than patients with non- progressive disease (data to be published). The high
936 C . A . GARCIA et al.
TABLE 1. STATISTICAL COMPARISONS (p-VALUES) OF D N A SYNTHESIS AND IgM SECRETION IN NORMAL B-CELLS, B - M L U S B-CELLS AND B - C L L B CELLS AFTER IN VITRO STIMULATION USING STAPHYLOCOCCUS AUREUS COWAN 1 (SAC) AND m A b $2C6 (ANTI-CD40) ALONE AND IN
SAC vs without SAC - - NS <0.01 <0.01 <0.01 - - B-CLL vs B-MLUS B-cells
SAC - - NS <0.01 <0.01 <0.01 - - B-CLL vs normal B-cells
SAC - - NS <0.01 <0.01 <0.01 - -
All other comparisons (Figs 1-5) were statistically not significant. NS = not significant.
/ Z A
. . t 4
/
0 I I I I I
24 48 72 96 120 144 INCUBATION PERIOD (h)
FIG. 2. DNA synthesis (SI) (mean - S.E.M.) after stimu- lation with SAC of B-CLL B-cells (n = 5) (O O) and
B-MLUS B-cells (n = 4) (0--------0).
responding capacity to SAC indicates that B-CLL cells may also be triggered in a T-cell independent way. m A b CD40 in combinat ion with SAC induced an earlier response peak in B-CLL cells than did SAC alone, whereas B-MLUS cells showed only a marginal response to these agents. Thus, B-CLL cells might be induced to proliferate in response to T- cel l /monocyte derived cytokines as well as to strong activation signals delivered through the CD40 mol- ecule.
Our results may also reflect the phenotypic dif- ferences between the two lymphoproliferat ive dis- orders [16]. The high surface Ig expression on B- CLL cells compared to B-MLUS might add to the
relatively high sensitivity of B-CLL cells to SAC stimulation (Ig cross-linking). Leu8- B-cells did not respond to SAC, while Leu8 + B-cells did [52] and B- CLL cells had a high expression of the Leu8 antigen. The B-cell subset susceptible to EBV-infect ion is overlapping with those cells which may be activated by SAC and B-CLL cells showed a significant increase in the expression of CD21 (EBV receptor) as compared to B-MLUS. The cell populat ion expressing the IL-2 receptor and the transferrin receptor was also larger in B-CLL than in B-MLUS [16]. In addition, most B-CLL cells are CD5 + and
Proliferation in B-CLL and B-MLUS 937
I 0
B
2
2 4 4 8 7 2 9 6 1 2 0 144 INCUBATION PERIOD (h)
FIG. 4. DNA synthesis (SI) (mean-S .E .M.) of B- MLUS B-cells (n = 4) after stimulation with mAb $2C6 (1~----O), SAC (O O) and mAb $2C6+ SAC
(A A).
1 4 0
120
~ lO0
_.~ 8o ~-{/)
~+' 60 ¢n E
2O
0 l . . . . . . 1 I ] T - - - -~
24 48 72 96 120 144 INCUBATION PERIOD (h}
FIG. 5. In vitro cumulative IgM secretion (ng/ml) (mean - S.E.M.) after stimulation without or with SAC. Normal B-cells (n = 4): without ( 9 ~ - - O ) and with SAC (0 O). B-CLL B-cells (n = 7): without (A &) and with SAC (A A). B-MLUS B-cells (n = 4): without
([] !1) and with SAC ([] F]).
may thus belong to the internally activated pool of B-cells producing low affinity auto-antibodies and have the potential for self-renewal [53]. All these differences in surface receptor structures might con- tribute to the high proliferative capacity of B-CLL cells compared to B-MLUS.
mAb $2C6 mimics the natural ligand for CD40 [8, 13] but a SAC signal was needed for the activation of normal B-cells. In B-MLUS, the combination of mAb $2C6 and SAC, however, did not induce a proliferative response. This might indicate that B- MLUS cells represent a different subset of B-cells than B-CLL not adapted to respond to activation signals, or alternatively display a defect in the signal transduction.
Normal resting B-cells did not differentiate to IgM
secreting cells upon SAC or anti-~t stimulation unless T-helper cell factors as BSF-MP6 were added [46]. In the present study, neither normal B-cells nor B- MLUS cells responded with IgM secretion upon SAC stimulation. However , B-CLL cells produced high amounts of IgM. Thus, B-MLUS cells reacted simi- larly to normal B-cells. B-CLL might represent 'memory ' B-cells, which can rapidly differentiate upon a proper signal.
In conclusion, B-CLL cells have been shown to represent a 'higher activation stage' than B-MLUS cells [16]. This characteristic might contribute to the ability to easily activate B-CLL cells to proliferation and differentiation as compared with B-MLUS. However, B-MLUS cells may also have an intrinsic defect which make the cells inert to proliferative stimuli, a factor which might be of importance for the benign course of the disease. The differences between a benign and malignant clonal expansion of B-cells may also partly be explained by a dynamic interaction between normal T-cells and the clonal B- cells through the action of cytokines and cell-cell contact.
Acknowledgements--This study was supported by grants from The Cancer Society in Stockholm, The Karolinska Institute Foundations. For technical assistance we thank Ms Ingrid Eriksson for technical assistance and Ms Lena Helgesson for excellent secretarial help.
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