Clin. exp. Immunol. (1977) 28, 542-545.
BRIEF COMMUNICATION
Spontaneous lymphocyte transformation in chronic lymphocytic leukaemia G. SEMENZATO, G. AMADORI, F. TOSATO & G. GASPAROTTO Istituto di Medicina Clinica della Universitd di Padova, Clinica Medica 2 e Cattedra di Immunopatologia, Padova, Italy
(Received 5 January 1977)
SUMMARY
In ten patients with CLL and in eight controls T lymphocytes were separated out by the rosetting method. The in vitro reactivity of these cells and of unseparated lymphocytes was studied in cultures without stimulants. A net increase ofspontaneous lymphocyte transformation was found in the populations enriched with T cells in patients with CLL with respect to analogous populations in the controls. This increase is interpreted as a cell-mediated immunological reaction and due to T-cell sensitization towards the neoplastic B cells.
INTRODUCTION There is general agreement that most chronic lymphocytic leukaemias (CLL) are characterized by a monoclonal proliferation of B lymphocytes with demonstrable surface immunoglobulins (Aisenberg & Bloch, 1972; Preud'homme & Seligman, 1972). The impaired response to phytohaemagglutinin (PHA) of lymphoid cells from CLL patients is due to the fact that the normal PHA-responsive lymphocytes are diluted with a large number of unresponsive leukaemic B cells. Moreover an absolute increase in the number of circulating T lymphocytes was found by some investigators (Catovsky et al., 1974; Macavei & Halmos, 1975; Lang et al., 1975). With different methods of isolating T lymphocytes several authors have shown a normal in vitro reactivity of a T-cell population in CLL (Wybran et al., 1973; Schweitzer et al., 1973; Goldstone et al., 1973; Blomgren et al., 1974). In the present study spontaneous lymphocyte transformation of T-enriched cells from ten patients with CLL was examined in order to obtain further information on the reactivity of T lymphocytes and to determine their significance in this disease. MATERIAL AND METHODS Patients and controls. Lymphocytes from ten patients with CLL were investigated. The diagnoses were based on accepted clinical and laboratory criteria. Their ages ranged from 52-78 years; the total white blood cell counts ranged from 20-160 x 103/mm3 with 70-98%/ lymphocytes. Serum electrophoresis revealed no monoclonal immunoglobulin peak. None of the patients was under treatment when the blood was drawn. As controls lymphocytes from eight volunteers, who showed a normal blood picture and did not have a history of allergy, infection, medications or immunizations, were accepted. These donors were in the same age range. Lymphocyte separation. Lymphocytes were separated from freshly drawn peripheral blood by density-gradient sedimentation (Boyum, 1968) with lymphoprep (Nyegaard & Co. As, Oslo). The cells were washed three times in Hanks's balanced salt solution and resuspended in TC medium 199 (Difco Laboratories, Detroit, U.S.A.). Purification of Tcells. The method of Wybran et al. (1973) with some modifications was employed for obtaining lymphocyte suspensions enriched with T cells. After resetting, the lymphocyte suspensions were layered on Lymphoprep (3 ml cell sqspension to 3 ml flotation medium). The tubes were centrifuged at 400 g for 30 min at 4VC and the pellet was collected
Correspondence: Dr G. Semenzato, Istituto di Medicina Clinica dell'Universita di Padova, Policlinico, 35100 Padova, Italy.
542
Spontaneous lymphocyte transformation in CLL
543
using a Pasteur pipette. SRBC were haemolized (by NH4Cl in Tris Ammonium, at pH 7.38), the cells washed three times in Hanks's solution, and resuspended in TC medium 199. Cellular viability was tested by the trypan-blue exclusion test. Purity of this population was assayed for their capacity to form rosettes with sheep erythrocytes (always more than 80%) and for their ability to react with fluorescein-conjugated antisera (always less that 6%4) by a direct immunofluorescence method (Papamichail et al., 1971). Spontaneous lymphocyte transformation. Triplicate cultures of 1 x 106 lymphocytes in 0 75 ml of TC medium and 0-25 ml of autologous plasma were made in 16 x 95 mm sterile tubes. Neither foetal calf serum nor antibiotics were added. Cultures were incubated at 370 in a 5% CO2 atmosphere for 5 days. The time-response curve for 3, 5 and 7 days was assessed previously (Fig. 1). DNA synthesis was quantified by incorporation of 1 #GCi of 3H-labelled thymidine (CEA-IRE 500
450
400 7 >1
-a
350
0
300
E
N, 0 -c
.E
250 Ai
200
L
"I
150 100
50 r) J
3
7
5 Time
(days)
FIG. 1. Percentage of 3H-labelled thymidine incorporation of lymphocytes at different time intervals. Mean of three experiments. (0) Unseparated lymphocytes from CLL patients; (c) enriched T lymphocytes from CLL patients; (A) unseparated lymphocytes from controls; (A) enriched T lymphocytes from controls. SORIN, specific activity 26 mCi/mM) for the last 12 hr of culture. The radioactive DNA protein was transferred to a scintillation vial with scintillation liquid, and the ct/min were determined in a Packard Liquid Scintillation Counter. The results are expressed as the mean ct/min/ 106 lymphocytes of triplicate tubes + s.e.m.. Student's t-test was used to estimate the significance. The morphologic controls were carried out in order to exclude the presence of granulocyte precursors
(Farnes et al., 1976).
RESULTS In the three cases examined at 3,5 and 7 days, it was observed in both leukaemic patients and controls that the maximum of 3H-labelled thymidine incorporation occurred after 5 days of culture (Fig. 1). The data is summarized in Table 1. Observe that, in patients suffering from CLL, the samples TABLE 1. Spontaneous lymphocyte transformation in separated and unseparated lymphocytes from controls and patients with CLL
Group
CLL Controls
No. studied
Unseparated lymphocytes
Enriched T lymphocytes
10 8
45+ 8 206+ 23
454+48 276+ 31
544
G. Semenzato et al.
enriched with T cells, which underwent spontaneous blastogenesis in unstimulated cultures, demonstrate clearly greater values of 3H-labelled thymidine incorporation than the unseparated lymphocytes from the same patient (454+48 vs 45+8; P
BRIEF COMMUNICATION
Spontaneous lymphocyte transformation in chronic lymphocytic leukaemia G. SEMENZATO, G. AMADORI, F. TOSATO & G. GASPAROTTO Istituto di Medicina Clinica della Universitd di Padova, Clinica Medica 2 e Cattedra di Immunopatologia, Padova, Italy
(Received 5 January 1977)
SUMMARY
In ten patients with CLL and in eight controls T lymphocytes were separated out by the rosetting method. The in vitro reactivity of these cells and of unseparated lymphocytes was studied in cultures without stimulants. A net increase ofspontaneous lymphocyte transformation was found in the populations enriched with T cells in patients with CLL with respect to analogous populations in the controls. This increase is interpreted as a cell-mediated immunological reaction and due to T-cell sensitization towards the neoplastic B cells.
INTRODUCTION There is general agreement that most chronic lymphocytic leukaemias (CLL) are characterized by a monoclonal proliferation of B lymphocytes with demonstrable surface immunoglobulins (Aisenberg & Bloch, 1972; Preud'homme & Seligman, 1972). The impaired response to phytohaemagglutinin (PHA) of lymphoid cells from CLL patients is due to the fact that the normal PHA-responsive lymphocytes are diluted with a large number of unresponsive leukaemic B cells. Moreover an absolute increase in the number of circulating T lymphocytes was found by some investigators (Catovsky et al., 1974; Macavei & Halmos, 1975; Lang et al., 1975). With different methods of isolating T lymphocytes several authors have shown a normal in vitro reactivity of a T-cell population in CLL (Wybran et al., 1973; Schweitzer et al., 1973; Goldstone et al., 1973; Blomgren et al., 1974). In the present study spontaneous lymphocyte transformation of T-enriched cells from ten patients with CLL was examined in order to obtain further information on the reactivity of T lymphocytes and to determine their significance in this disease. MATERIAL AND METHODS Patients and controls. Lymphocytes from ten patients with CLL were investigated. The diagnoses were based on accepted clinical and laboratory criteria. Their ages ranged from 52-78 years; the total white blood cell counts ranged from 20-160 x 103/mm3 with 70-98%/ lymphocytes. Serum electrophoresis revealed no monoclonal immunoglobulin peak. None of the patients was under treatment when the blood was drawn. As controls lymphocytes from eight volunteers, who showed a normal blood picture and did not have a history of allergy, infection, medications or immunizations, were accepted. These donors were in the same age range. Lymphocyte separation. Lymphocytes were separated from freshly drawn peripheral blood by density-gradient sedimentation (Boyum, 1968) with lymphoprep (Nyegaard & Co. As, Oslo). The cells were washed three times in Hanks's balanced salt solution and resuspended in TC medium 199 (Difco Laboratories, Detroit, U.S.A.). Purification of Tcells. The method of Wybran et al. (1973) with some modifications was employed for obtaining lymphocyte suspensions enriched with T cells. After resetting, the lymphocyte suspensions were layered on Lymphoprep (3 ml cell sqspension to 3 ml flotation medium). The tubes were centrifuged at 400 g for 30 min at 4VC and the pellet was collected
Correspondence: Dr G. Semenzato, Istituto di Medicina Clinica dell'Universita di Padova, Policlinico, 35100 Padova, Italy.
542
Spontaneous lymphocyte transformation in CLL
543
using a Pasteur pipette. SRBC were haemolized (by NH4Cl in Tris Ammonium, at pH 7.38), the cells washed three times in Hanks's solution, and resuspended in TC medium 199. Cellular viability was tested by the trypan-blue exclusion test. Purity of this population was assayed for their capacity to form rosettes with sheep erythrocytes (always more than 80%) and for their ability to react with fluorescein-conjugated antisera (always less that 6%4) by a direct immunofluorescence method (Papamichail et al., 1971). Spontaneous lymphocyte transformation. Triplicate cultures of 1 x 106 lymphocytes in 0 75 ml of TC medium and 0-25 ml of autologous plasma were made in 16 x 95 mm sterile tubes. Neither foetal calf serum nor antibiotics were added. Cultures were incubated at 370 in a 5% CO2 atmosphere for 5 days. The time-response curve for 3, 5 and 7 days was assessed previously (Fig. 1). DNA synthesis was quantified by incorporation of 1 #GCi of 3H-labelled thymidine (CEA-IRE 500
450
400 7 >1
-a
350
0
300
E
N, 0 -c
.E
250 Ai
200
L
"I
150 100
50 r) J
3
7
5 Time
(days)
FIG. 1. Percentage of 3H-labelled thymidine incorporation of lymphocytes at different time intervals. Mean of three experiments. (0) Unseparated lymphocytes from CLL patients; (c) enriched T lymphocytes from CLL patients; (A) unseparated lymphocytes from controls; (A) enriched T lymphocytes from controls. SORIN, specific activity 26 mCi/mM) for the last 12 hr of culture. The radioactive DNA protein was transferred to a scintillation vial with scintillation liquid, and the ct/min were determined in a Packard Liquid Scintillation Counter. The results are expressed as the mean ct/min/ 106 lymphocytes of triplicate tubes + s.e.m.. Student's t-test was used to estimate the significance. The morphologic controls were carried out in order to exclude the presence of granulocyte precursors
(Farnes et al., 1976).
RESULTS In the three cases examined at 3,5 and 7 days, it was observed in both leukaemic patients and controls that the maximum of 3H-labelled thymidine incorporation occurred after 5 days of culture (Fig. 1). The data is summarized in Table 1. Observe that, in patients suffering from CLL, the samples TABLE 1. Spontaneous lymphocyte transformation in separated and unseparated lymphocytes from controls and patients with CLL
Group
CLL Controls
No. studied
Unseparated lymphocytes
Enriched T lymphocytes
10 8
45+ 8 206+ 23
454+48 276+ 31
544
G. Semenzato et al.
enriched with T cells, which underwent spontaneous blastogenesis in unstimulated cultures, demonstrate clearly greater values of 3H-labelled thymidine incorporation than the unseparated lymphocytes from the same patient (454+48 vs 45+8; P
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