Clin. exp. Immunol. (1978) 33, 302-308.
B-cell activation of peripheral blood lymphocytes from patients with chronic lymphatic leukaemia K.-H. ROBLRT, ERNA MOLLER, G. GAHRTON, HARRIET ERIKSSON & B. NILSSON Transplantation Immunology Laboratory, Department ofClinical Immunology, and Section of Oncology and Haematology, Department of Medicine, Huddinge Hospital, Sweden
(Received 12 January 1978) SUMMARY
The mitogenic response of blood lymphocytes from twelve patients with B-cell CLL and seven healthy controls was investigated. All patients were untreated and had a typical B-cell dominance in their peripheral blood. The patients demonstrated a scattered response pattern with a higher variance than in the control group. We demonstrated examples of stimulation of DNA-synthesis by polyclonal B-cell activators (PBA) as well as inhibition. The variable pattern between the patients might result from a malignant transformation of cells originating from different lymphocyte subpopulations in different patients. Thus PBA studies of lymphocytes from CLL patients might be a valuable tool in characterizing further this clinically variable disease at the cellular level.
INTRODUCTION Studies on cell-surface receptors have proved to be useful tools in differentiating various subpopulations of T and B lymphocytes. The presence of identical receptors on a normal lymphocyte subpopulation and on lymphoma cells increases the likelihood of malignancy developing from that specific normal cell subpopulation. Thus in the majority of chronic lymphatic leukaemia (CLL) cases, the malignant lymphocytes are considered to be transformed B-cells, as they generally express surface Ig (S-Ig), Fc and C'3receptors (Mellstedt & Pettersson, 1974). Lately, however, the so-called HP-receptor (Helix pomatia), considered to be a marker of the T-cell population, has proved to be a constituent of the malignant (B-) lymphocytes of CLL (Hellstrom et al., 1976). Thus the presence of HP-receptors indicates a definite difference between CLL cells and most normal B lymphocytes in the blood. However, it is possible that normal B-cells with HP-receptors might be found in other secondary lymphoid organs. The use of markers has, so far, not proved to be of any greater use in characterizing chronic lymphatic B-cell leukemias into different subgroups with different prognoses. The majority of CLL express S-Ig, HP, Fc and C'3-receptors while the clinical features of the disease are highly variable with regard to symptoms, haematological complications and length of survival. The aim of the present study was to activate the malignant B lymphocytes of CLL in order to examine a new possibility of characterizing different subpopulations of malignant lymphocytes. The use of polyclonal B-cell activators (PBA) has led to the demonstration of characteristic and different responses of lymphocytes from different lymphoid compartments in man, which indicate the presence of different B lymphocyte subpopulations (Ringden, 1976). Furthermore, experiments in mice have shown that the polyclonal B-cell activators DxS, LPS and PPD selectively stimulate B-cells belonging to different maturation stages (Gronowicz & Couthino, 1975). The same mitogens have proved capable of inhibiting selectively DNA synthesis in different murine lymphosarcoma cell lines (Ralph, Correspondence: Dr K.-H. Robert, Transplantation Immunology Laboratory, Huddinge Hospital, S-141 86 Huddinge, Sweden. 0099-9104/78/0800-0302$02.00 A) 1978 Blackwell Scientific Publications
302
B-cell activation in CLL
303
Nakointz & Rascke, 1974). It was suggested, that the malignant cells preserve the PBA receptor(s) after malignant transformation, but that activation is specifically inhibited due to an already high spontaneous activity of the cells. If a similar concept could be applied to human lymphoma cells, i.e. if human lymphocytes keep their PBA receptors and capability of responding after a leukaemic transformation, it might raise the possibility ofa functional characterization of the malignant cells. Earlier studies on mitogen-reactivity of lymphocytes from CLL patients, have been concerned with responses to T-cell mitogens such as PHA, Con A and PWM (Wybran, Chantler & Fudenberg, 1973; Epstein & Cline, 1974; Brittinger et al., 1975; Traczyk et al., 1975; Utsinger, 1975; Hunyadi et al., 1976; Schultz, Davis & Rubin, 1976). This approach should at least theoretically serve as an in vitro test of the condition of the non-malignant T-cells, but be of minor importance for the characterization of the malignant B-cells. Thus we have used a battery of polyclonal B-cell activating substances in order to study whether leukaemic cells can respond at all, and if so, whether cells from different patients express different PBA receptors.
MATERIALS AND METHODS Patients. Twelve patients were investigated. They had typical CLL criteria with respect to symptoms, clinical signs and laboratory findings. The patients were untreated and had never received any cytostatic agents, steoids or radiotherapy. Seven healthy volunteers were included as controls. Cells. Peripheral white blood cell suspensions were prepared from heparinized or defibrinated venous blood. Lymphocytes were then separated by flotation on a Ficoll-Isopaque gradient. Cell concentration in the mitogen studies was 2 5 x
10n/ml. Mitogens. Dextran sulphate (DxS) (Pharmacia Fine Chemicals, Uppsala, Sweden) was used in concentrations of 5, 50 and 500 jig/ml. Lipopolysaccharide (LPS) from Escherichia coli (0 55: 85) was prepared by phenolwater extraction (courtesy of Prof. Holme, Department of Bacteriology, Karolinska Institute). Before use in culture, the lyophilized preparation was dissolved in balanced salt solution (BSS) (1 mg/ml). Concentrations in culture were 0 25, 2 5 and 25 jig/ml. PPD from tuberculin was obtained from the State Serum Institute, Copenhagen, Denmark, and used in concentrations of 2 5, 25 and 250 jig/ml. Rabbit IgG anti-human P2 microglobulin (anti-P2m) (lot No. 055, Dakopotts Reagents, Copenhagen, Denmark) was dialysed overnight, heat-inactivated at 560C for 30 min and used in final dilutions of 1: 4, 1 :10 and 1:40. Phytohaemagglutinin (PHA) (Wellcome Ltd., UK) was used in dilutions of 1 : 1000, 1 : 100 and 1 10. PHA was included as a routine control ofthe functional capacity of the T-cells. DxS, LPS and anti-p2m are considered B-cell mitogens. The PPD response is, however, a mitogen with a more complex activity, lower concentrations being antigenic for specifically immunized T-cells with a peak response at day 5-6 and higher concentrations (>250 jig/ml) being mitogenic for B-cells with a peak response on day 3 (Nilsson, 1972). Assayfor T lymphocytes. The capacity of human lymphocytes to bind sheep erythrocytes (E-RFC) was used to determine the relative number of T-cells (E%) (Jondal, Holm & Wigzell, 1972). Assay for B lymphocytes. In order to enumerate the relative number of B-cells, the surface membrane immunoglobulin marker (S-Ig) was used (Moller, 1961). Isolated lymphocytes were incubated for 30 min at room temperature in 0-2 ml of a fluorescein-labelled polyvalent rabbit anti-human immunoglobulin serum (Behringwerke, Germany), diluted 1: 4 in PBS. After washing 3 times in cold PBS, the cells were kept on ice until examination for membrane fluorescence, using a Zeiss microscope with a Ploem type incident UV-light and indirect phase contrast optics. Culture procedure and assay for DNA-synthesis. Cells were suspended in HEPES buffered Eagle's minimal essential medium containing 10% heat-inactivated human AB-serum and nutrients. Cells were cultured in microtiter plates (3040, Falcon, USA) with 0 5 x 106 cells per culture per 0-2 ml. Cultures were incubated for 3 days in a humidified atmosphere of 5% CO2 in air at 370C. 24 hr before harvesting, 1 0 jiCi of 3H-thymidine with a specific activity of 20 Ci/mmol (Radiochemical Centre, Amersham, UK) was added to each culture. Harvesting was performed with a Skatron harvesting machine (Lierbyen, Norway) and radioactivity was measured by scintillation spectrophotometry (Intertechnique, Nanoteknik, Sweden). Statistical analysis. Data were given and analysed as means ofquadruplicate cultures. The s.e.m. values of the quadruplicate cultures were in most instances within 10% of the means. Data were analysed in terms of stimulation index, i.e. division of experimental cpm by the spontaneous cpm-level (background), and net-cpm, i.e. substraction of the spontaneous cpm from the experimental cpm. The mean response to a particular mitogenic dose in the whole patient group was compared to the corresponding mean of the control group. Hereby a two-sided parametric test was applied. When estimating the degrees of freedom, the difference of variance between the 2 groups was accounted for (Satterthwaite, 1946). When comparing the variance of data ofthe 2 groups, a two-sided direct-variance ratio-F-test was used.
K.-H. Robert et al.
304
RESULTS
Surface markersfor T- and B-cells All control bloods were haematologically examined (Hb, white blood cell counts and differential counts) and were considered normal. Haematological data, identification and enumeration of E-RFC positive cells (E%) and surface immunoglobulin positive cells (S-Ig%) were performed and the results for all patients are summarized in Table 1. Controls were within normal range (45 %
B-cell activation of peripheral blood lymphocytes from patients with chronic lymphatic leukaemia K.-H. ROBLRT, ERNA MOLLER, G. GAHRTON, HARRIET ERIKSSON & B. NILSSON Transplantation Immunology Laboratory, Department ofClinical Immunology, and Section of Oncology and Haematology, Department of Medicine, Huddinge Hospital, Sweden
(Received 12 January 1978) SUMMARY
The mitogenic response of blood lymphocytes from twelve patients with B-cell CLL and seven healthy controls was investigated. All patients were untreated and had a typical B-cell dominance in their peripheral blood. The patients demonstrated a scattered response pattern with a higher variance than in the control group. We demonstrated examples of stimulation of DNA-synthesis by polyclonal B-cell activators (PBA) as well as inhibition. The variable pattern between the patients might result from a malignant transformation of cells originating from different lymphocyte subpopulations in different patients. Thus PBA studies of lymphocytes from CLL patients might be a valuable tool in characterizing further this clinically variable disease at the cellular level.
INTRODUCTION Studies on cell-surface receptors have proved to be useful tools in differentiating various subpopulations of T and B lymphocytes. The presence of identical receptors on a normal lymphocyte subpopulation and on lymphoma cells increases the likelihood of malignancy developing from that specific normal cell subpopulation. Thus in the majority of chronic lymphatic leukaemia (CLL) cases, the malignant lymphocytes are considered to be transformed B-cells, as they generally express surface Ig (S-Ig), Fc and C'3receptors (Mellstedt & Pettersson, 1974). Lately, however, the so-called HP-receptor (Helix pomatia), considered to be a marker of the T-cell population, has proved to be a constituent of the malignant (B-) lymphocytes of CLL (Hellstrom et al., 1976). Thus the presence of HP-receptors indicates a definite difference between CLL cells and most normal B lymphocytes in the blood. However, it is possible that normal B-cells with HP-receptors might be found in other secondary lymphoid organs. The use of markers has, so far, not proved to be of any greater use in characterizing chronic lymphatic B-cell leukemias into different subgroups with different prognoses. The majority of CLL express S-Ig, HP, Fc and C'3-receptors while the clinical features of the disease are highly variable with regard to symptoms, haematological complications and length of survival. The aim of the present study was to activate the malignant B lymphocytes of CLL in order to examine a new possibility of characterizing different subpopulations of malignant lymphocytes. The use of polyclonal B-cell activators (PBA) has led to the demonstration of characteristic and different responses of lymphocytes from different lymphoid compartments in man, which indicate the presence of different B lymphocyte subpopulations (Ringden, 1976). Furthermore, experiments in mice have shown that the polyclonal B-cell activators DxS, LPS and PPD selectively stimulate B-cells belonging to different maturation stages (Gronowicz & Couthino, 1975). The same mitogens have proved capable of inhibiting selectively DNA synthesis in different murine lymphosarcoma cell lines (Ralph, Correspondence: Dr K.-H. Robert, Transplantation Immunology Laboratory, Huddinge Hospital, S-141 86 Huddinge, Sweden. 0099-9104/78/0800-0302$02.00 A) 1978 Blackwell Scientific Publications
302
B-cell activation in CLL
303
Nakointz & Rascke, 1974). It was suggested, that the malignant cells preserve the PBA receptor(s) after malignant transformation, but that activation is specifically inhibited due to an already high spontaneous activity of the cells. If a similar concept could be applied to human lymphoma cells, i.e. if human lymphocytes keep their PBA receptors and capability of responding after a leukaemic transformation, it might raise the possibility ofa functional characterization of the malignant cells. Earlier studies on mitogen-reactivity of lymphocytes from CLL patients, have been concerned with responses to T-cell mitogens such as PHA, Con A and PWM (Wybran, Chantler & Fudenberg, 1973; Epstein & Cline, 1974; Brittinger et al., 1975; Traczyk et al., 1975; Utsinger, 1975; Hunyadi et al., 1976; Schultz, Davis & Rubin, 1976). This approach should at least theoretically serve as an in vitro test of the condition of the non-malignant T-cells, but be of minor importance for the characterization of the malignant B-cells. Thus we have used a battery of polyclonal B-cell activating substances in order to study whether leukaemic cells can respond at all, and if so, whether cells from different patients express different PBA receptors.
MATERIALS AND METHODS Patients. Twelve patients were investigated. They had typical CLL criteria with respect to symptoms, clinical signs and laboratory findings. The patients were untreated and had never received any cytostatic agents, steoids or radiotherapy. Seven healthy volunteers were included as controls. Cells. Peripheral white blood cell suspensions were prepared from heparinized or defibrinated venous blood. Lymphocytes were then separated by flotation on a Ficoll-Isopaque gradient. Cell concentration in the mitogen studies was 2 5 x
10n/ml. Mitogens. Dextran sulphate (DxS) (Pharmacia Fine Chemicals, Uppsala, Sweden) was used in concentrations of 5, 50 and 500 jig/ml. Lipopolysaccharide (LPS) from Escherichia coli (0 55: 85) was prepared by phenolwater extraction (courtesy of Prof. Holme, Department of Bacteriology, Karolinska Institute). Before use in culture, the lyophilized preparation was dissolved in balanced salt solution (BSS) (1 mg/ml). Concentrations in culture were 0 25, 2 5 and 25 jig/ml. PPD from tuberculin was obtained from the State Serum Institute, Copenhagen, Denmark, and used in concentrations of 2 5, 25 and 250 jig/ml. Rabbit IgG anti-human P2 microglobulin (anti-P2m) (lot No. 055, Dakopotts Reagents, Copenhagen, Denmark) was dialysed overnight, heat-inactivated at 560C for 30 min and used in final dilutions of 1: 4, 1 :10 and 1:40. Phytohaemagglutinin (PHA) (Wellcome Ltd., UK) was used in dilutions of 1 : 1000, 1 : 100 and 1 10. PHA was included as a routine control ofthe functional capacity of the T-cells. DxS, LPS and anti-p2m are considered B-cell mitogens. The PPD response is, however, a mitogen with a more complex activity, lower concentrations being antigenic for specifically immunized T-cells with a peak response at day 5-6 and higher concentrations (>250 jig/ml) being mitogenic for B-cells with a peak response on day 3 (Nilsson, 1972). Assayfor T lymphocytes. The capacity of human lymphocytes to bind sheep erythrocytes (E-RFC) was used to determine the relative number of T-cells (E%) (Jondal, Holm & Wigzell, 1972). Assay for B lymphocytes. In order to enumerate the relative number of B-cells, the surface membrane immunoglobulin marker (S-Ig) was used (Moller, 1961). Isolated lymphocytes were incubated for 30 min at room temperature in 0-2 ml of a fluorescein-labelled polyvalent rabbit anti-human immunoglobulin serum (Behringwerke, Germany), diluted 1: 4 in PBS. After washing 3 times in cold PBS, the cells were kept on ice until examination for membrane fluorescence, using a Zeiss microscope with a Ploem type incident UV-light and indirect phase contrast optics. Culture procedure and assay for DNA-synthesis. Cells were suspended in HEPES buffered Eagle's minimal essential medium containing 10% heat-inactivated human AB-serum and nutrients. Cells were cultured in microtiter plates (3040, Falcon, USA) with 0 5 x 106 cells per culture per 0-2 ml. Cultures were incubated for 3 days in a humidified atmosphere of 5% CO2 in air at 370C. 24 hr before harvesting, 1 0 jiCi of 3H-thymidine with a specific activity of 20 Ci/mmol (Radiochemical Centre, Amersham, UK) was added to each culture. Harvesting was performed with a Skatron harvesting machine (Lierbyen, Norway) and radioactivity was measured by scintillation spectrophotometry (Intertechnique, Nanoteknik, Sweden). Statistical analysis. Data were given and analysed as means ofquadruplicate cultures. The s.e.m. values of the quadruplicate cultures were in most instances within 10% of the means. Data were analysed in terms of stimulation index, i.e. division of experimental cpm by the spontaneous cpm-level (background), and net-cpm, i.e. substraction of the spontaneous cpm from the experimental cpm. The mean response to a particular mitogenic dose in the whole patient group was compared to the corresponding mean of the control group. Hereby a two-sided parametric test was applied. When estimating the degrees of freedom, the difference of variance between the 2 groups was accounted for (Satterthwaite, 1946). When comparing the variance of data ofthe 2 groups, a two-sided direct-variance ratio-F-test was used.
K.-H. Robert et al.
304
RESULTS
Surface markersfor T- and B-cells All control bloods were haematologically examined (Hb, white blood cell counts and differential counts) and were considered normal. Haematological data, identification and enumeration of E-RFC positive cells (E%) and surface immunoglobulin positive cells (S-Ig%) were performed and the results for all patients are summarized in Table 1. Controls were within normal range (45 %
