Int. 1.Cancer: 17, 448-454 (1976)
CONCANAVALIN A RECEPTORS ON THE SURFACE MEMBRANE OF LYMPHOCYTES FROM PATIENTS WITH AFRICAN BURKITT’S LYMPHOMA AND LYMPHOMA CELL LINES Hannah BEN-BASSAT l, Natan GOLDBLUM l , Stella MITRANI l, George KLEIN and Bo JOHANSSON 3. Chanock Centre for Virology, Hebrew University-Hadassah Medical School, Jerusalem, Israel; Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden; and Kenyatta National Hospital, Nairobi, Kenya SUMMARY
Lymphocytes isolated from the peripheral blooa and from tumor tissues of patients with African Burkitt’s lymphoma have been studied for cap formation and agglutinability by Concanavalin A (Con A ) . Peripheral bloodfrom healthy adult persons served as a normal control and bloodfrom patients with carcinoma served as a non-lymphoma control. These studies included 29 patients with Burkitt ’s lymphoma, 93 with carcinoma, and 105 healthy adult persons, as well as tumor tissues from 13 patients with Burkitt’s lymphoma. The great majority of the carcinomas were from the face and neck regions. Lymphocytes from the blood of the majority of patients with Burkitt’s lymphoma, as well as those from tumor tissues, exhibited a reduced cap-forming ability (2-6 and increased Con-A-induced agglutinability compared to lymphocytes from healthy normal donors and from patients with carcinoma, although some of the lyinphocytes from patients with carcinoma had a somewhat lower range of cap formation than the lymphocytes from healthy donors. No difference was observed in the interaction with Con A of lypmhocytes from the different types of carcinoma studied. Eight lymphoid cell lines were established in our laboratory from the tumor tissues of patients with Burkitt ’s lymphoma. The cap-forming ability and agglutinability by Con A of these lines was examined and compared to those of the ‘‘ classical ” lymphoma lines: Raji, Daudi and P 3 H R I .All cell lines exhibited an increased Con-Ainduced agglutinability and a reduced cap-forming ability compared to normal lymphocytes, except for P,HR1 cells which exhibited a cap-forming ability of 15-20 %. These findings are discussed in relation to the association of the lymphocytes with malignancy and as a possible aid in the differential diagnosis between malignant lymphomas and other diseases.
x)
Concanavalin A (Con A, Sumner and Howell, 1936), has been used as a probe to study changes in the mobility of specific receptors on the cell surface membrane (Inbar et al., 1973 a, b, c ; Ben-Bassat and Goldblum, 1974, 1975). Studies with this probe have shown differences in the structure and function of the surface membrane of normal and malignant cells
(Ben-Bassat et al., 1971 ; Burger, 1969; Gahmberg et al., 1975; Inbar et al., 1972; Yahara et al., 1972). We have previously reported that lymphocytes from patients with chronic lymphocytic leukemia, Hodgkin’s disease and other malignant lymphomas have a reduced cap-forming ability and a higher Con-A-induced agglutinability than normal lymphocytes (Ben-Bassat and Goldblum, 1974, 1975). A significant decrease in the ability of lymphocytes to form caps was also observed in biopsy material from patients with Hodgkin’s disease and other malignant lymphomas. Lymphocytes isolated from tonsils of patients undergoing tonsillectomy and from axillary lymph nodes of breast cancer patients exhibited a cap-forming ability identical to that of normal lymphocytes. The ability of cells from a normal donor or a lymphoma patient to form caps was independent of the source from which the lymphocytes were isolated, e.g. lymph node, spleen or blood (Ben-Bassat and Goldblum, 1975, 1976). The present studies were undertaken in order: (1) to determine whether there are changes in the mobility of Con A receptors on the surface of lymphocytes of patients with African Burkitt’s lymphoma; (2) to compare the receptor mobility of lymphocytes from Burkitt’s lymphoma with that of lymphocytes isolated from other malignancies, e.g. carcinoma; and (3) to test whether lymphoma cells from established Burkitt lymphoma lines exhibit similar Con A receptor mobility to that of the patient’s lymphocytes. MATERIAL AND METHODS
Patients Biopsies and other material for these studies were obtained from ambulatory and hospitalized patients with Burkitt’s lymphoma and carcinoma from the following hospitals: Kenyatta National Hospital in Kenya; Uganda Cancer Institute, Kampala; Hadassah and the Central Hospital of Afula in Received: October 27, 1975, and in revised form January 23, 1976. Permanent address: Radiumhemmet, Karolinska Sjukhuset, Stockholm 60, Sweden.
449
CON A RECEPTORS ON BL AND LYMPHOMA CELLS
Israel. The 13 tumor biopsies were obtained from untreated patients, as well as from treated patients with recurring tumors. Peripheral blood was obtained from 29 patients with Burkitt's lymphoma and 72 with carcinoma. The group of patients with carcinoma included the following types : nasopharyngeal (36); maxilla (9); antrum (8); mandibula (4); cheek (4); tongue (3); larynx (3); nose (3); and neck (2). These included both untreated and treated cases.
Assay for binding of fluorescent Con A
Fluorescein isothiocynate-conjugated Con A (F-Con A) was prepared by Miles-Yeda, Rehovoth, Israel, at a mole ratio of 1.86 fluorescein to protein. For the experiments, 0.5-1.0x 10' cells were incubated with different concentrations of fluorescent Con A up to 100 pglml for 15 min at 37" C. The cells were washed with PBS and the fluorescence was determined on a drop of cells with a fluorescence microscope, Five hundred cells were counted for Preparation of biopsies and blood samples each point. The results of fluorescent Con A were Biopsy material was cut into small pieces with obtained with saturation conditions and only single scissors into medium RPMI 1640 containing 10% cells and cells in small clumps (2-5 cells) were fetal calf serum. The lymphocytes were isolated from counted for the percentage of caps. biopsies that were kept at 4" C and shipped to Israel, a procedure that took 4-5 days. Assay for agglutination Blood was drawn into sterile bottles containing Con A was obtained from Miles-Yeda. One-half heparin, kept at 4 ° C and shipped to Israel under ml of Con A at different concentrations diluted in similar conditions. PBS was mixed with 0.5ml of cell suspension to give a final concentration of 1 x loe cells per ml in a Isolation of lymphocytes 35 mm Petri dish. The density and size of aggregates Lymphocytes were isolated from peripheral blood was scored on a scale from - to after and biopsy material by the Ficoll-Hypaque gradient 30 min incubation at 24" C. centrifugation method (Boyum, 1968), washed twice with phosphate-buffered saline (PBS) (PH 7.2) and RESULTS diluted in PBS to the appropriate concentrations. Viability of the cells was determined by trypan blue Cap formation with fluorescent Concanavalin A of exclusion. The viability of the cells used in the exper- lymphocytes from peripheral blood of patients with iments was 70-95 % due to storage and travel effects. Burkitt 's lymphoma
++++
Lymphocytes from peripheral blood of patients with Burkitt's lymphoma were separated by the Eight cell lines from Burkitt lymphoma tumor Ficoll-Hypaque method and examined for cap forbiopsies were established in our laboratory. The mation with F-Con A. I n addition, we included in biopsy material was washed in medium RPMI 1640 this study a group of healthy donors and a group of with 10% fetal calf serum and a five-fold concen- patients with carcinoma. Altogether 29 patients tration of antibiotics, transferred to a Petri dish with Burkitt's lymphoma, 72 with carcinoma and containing medium and sectioned with scissors in the 45 healthy donors were examined. The blood samples medium. The material was grown in test tubes (150 x of Burkitt's lymphoma and carcinoma patients were 15 mm) in 2 ml RPMI 1640, 30% fetal calf serum examined after 4-5 days of storage at 4" C and travel and antibiotics. Medium was changed every 4-6 from Nairobi to Israel. The blood samples from days, carefully without disturbing the culture, until healthy donors came from Israel and were stored growth was established and active passages were 4-5 days at 4" C. The recovery of lymphocytes and begun. The cultures were kept at 37" C, 5 % CO, in their viability, as determined by morphological air and 80-95 %relative humidity. After establishment observation and vital staining, were satisfactory. of the cell line, the serum was reduced to 20%; the Figure 1 illustrates the results obtained with 100 pg/ lymphoma cells grew in suspension and were ml F-Con A. Lymphocytes from patients with maintained under the same conditions by sub- Burkitt's lymphoma exhibit a significant reduction culturing every 4-6 days. The lag phase before in the ability to form caps with F-Con A compared establishment of the cell lines was 3-8 weeks. to lymphocytes from healthy donors and patients For the experiments, cells were used between with carcinoma. The majority of the patients with passages 5-20, 2 days after refeeding, their viability Burkitt's lymphoma are within the range of 2-6% being 90-100% by trypan.blue exclusion. cap-forming ability. The great majority of patients These cell lines were compared with '' classical " with carcinoma were within a range similar to or Burkitt lymphoma lines: Raji, Daudi and PBHR1, lower than that of the healthy donors and generally obtained from the Department of Tumor Biology, higher than that of patients with Burkitt's lymphoma. Six patients with Burkitt's lymphoma and one with Karolinska Institute. Establishment of Burkitt lymphoma cell lines
450
BEN-BASSAT ET AL.
No difference in the interaction with Con A of lymphocytes from different types of carcinoma could be observed.
2 a
V
20
1
Agglutination of peripheral blood lymphocytes from patients with Burkitt Is lymphoma by Con A oooo0
Like the experiments reported for cap-forming ability, the agglutination experiments were also carried out with cells isolated by the Ficoll-Hypaque method from peripheral blood. The final concentration of Con A was 250,ug/ml. The results are shown in Figure 3. A significant increase in agglutin-
oooo 000
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0 0 00
w
TABLE I
V
CAP FORMATION WITH F-CON A I N LYMPHOCYTES ISOLATED FROM THE SAME PATIENT AT DIFFERENT PERIODS '
NORMAL
CARCINOM, BURKITT' 5 LYMPHOMA
FIGURE1
Cap formation with F-Con A o n the surface membrane of lymphocytes from peripheral blood. Lymphocytes were isolated from blood of patients with African Burkitt's lymphoma ( 0 ) . Lymphocytes from normal adults served as controls (0)and lymphocytes from patients with carcinoma (m) served as non-Iymphomatous controls. Cells were incubated with 1OOpg F-Con A/ml for 15 min at 37" C and the presence of lymphocytes with caps was determined.
carcinoma were examined several times during a period of 3 weeks to 5 months. During this period the patients had already been under treatment. Blood samples were collected and the lymphocytes tested for capforming ability with F-Con A. The results summarized in Table I indicate that the cap formation with F-Con A of several samples of lymphocytes from the same patient is generally very consistent. No obvious differences in absolute numbers of cells from the same patient and from identical blood samples could be observed. In order to determine whether lymphocytes from patients with carcinoma exhibit a reduced ability of cap formation compared to normal lymphocytes or reflect '' storage travel " effects, we examined an additional group of fresh lymphocytes from patients with carcinoma. Twenty-two cases of carcinoma and 60 normal healthy donors were included in the study. Figure 2 shows the results of this study. All healthy donors and patients with carcinoma exhibited the pattern of cap formation of normal lymphocytes, although some of the lymphocytes from carcinoma patients had a somewhat lower range of cap formation than the lymphocytes from healthy donors.
Patient No.
Disease
Date
1
Burkitt's 23. 8.74 lymphoma 9. 9.74
2
Burkitt's 28. 8.74 lymphoma 9. 9.74
3
Burkitt's 30. 8.74 lymphoma 8.11.74 Burkitt's 30. 8.74 lymphoma 8.1 1.74
11.10.74 11.10.74
4
5 6
7
6.12.74 24. 1.74 Burkitt's 31. 1.75 lymphoma 21. 2.75 Burkitt's 24. 1.75 lymphoma 14. 2.75 14. 3.75 Carcinoma 8.11.74 22.11.74
No. of lymphocytes in the sample (I03
Lymphocytes with caps
2.2 3.8 2.7 4.9 8.0 2.1 0.9 I .2 5.5 3. I 4.5 2.0 4.8 4.0 1.8 2.1 2.0 1.8 1.3
5.2 2.2 8.6 5.0 4.3 5.0 6.0 3.6 4.3 3.0 5.7 6.0 5.5 5.0 7.0 4.4 8.6 4.0 5.8
' 1-2 x 10' cells were incubated with IOOpg of F-Con A/ml for 15 min at 37" C, washed with PBS and the fluorescence was determined on a drop of living cells. Five hundred cells were counted for each point and the percentage of cells with caps determined. ability is evident for lymphocytes from patients with Burkitt's lymphoma as compared to healthy donors and patients with carcinoma. We have previously shown that B and T blood lymphocytes from normal healthy persons are agglutinated by Con A to the same extent (Ben-Bassat and Goldblum, 1975). Also, in these cases the agglutinability of lymphocytes from patients with Burkitt's lymphoma was found not to be due to changes ifi the relative proportions of B- and T-lymphocytes in the blood. Agglutinability was specific as determined in each instance by inhibition with 0. I hi-a-methyl-D-mannopyranoside.
45 1
CON A RECEPTORS ON EL AND LYMPHOMA CELLS
35
Cap formation and agglutinability by Con A of I established Burkitt lymphoma Iines Burkitt lymphoma cell lines were examined for cap-forming ability and agglutinability by Con A. The following lines were tested: Raji (Pulvertaft, 1965; Epstein et al., 1966); Daudi (Klein et a/., 1968); P,HR1 (Himuma et a/., 1967) and six out of the eight Burkitt lymphoma lines established in our laboratory: Bu-1, Bu-3, Bu-4, Bu-6, Bu-8 and Bu-10. The cap-forming ability with F-Con of the Raji, Daudi and Bu-1, 3, 4,6, 8, 10 cells was low (2-5 %), whereas that of P,HR1 was 15-20%. P,HR, differs from the other eight lines in that it is a good EBV producer and lacks membrane receptors for EBV
00 00
0 0 0 0
25
O00
ooo 00 0
0 0
++++ z
- +++
0 I-
NORMAL
CARCINOMA
FIGURE 2
Cap formation with F-Con A on the surface membrane of lymphocytes from fresh peripheral blood. Lymphocytes
were isolated from blood of healthy adult donors (0)and from patients with carcinoma (a). Cells were incubated with 1OOpg fluorescent Con A/ml for 15min at 34°C and the presence of lymphocytes with caps was determined.
2 -
l-
3 (3 (3
++
a.
00000
a
+
Cap-forming ability and agglutinability by Con A of cells from tumor tissues of patients with Burkitt's lymphoma Biopsy material was obtained from tumors of patients with Burkitt's lymphoma. The lymphoma cells were separated by the Ficoll-Hypaque method after storage at 4" C and travel for 4-5 days, and examined for cap formation and agglutinability by Con A. Table I1 illustrates the results obtained with lymphoma cells from 13 patients with Burkitt's lymphoma. The lymphoma cells from all cases exhibit a reduced ability to form caps with fluorescent Con A and increased agglutinability by native Con A. The binding of F-Con A and native Con A was inhibited when a-methyl-D-mannopyranoside was added as a hapten inhibitor.
.a
0 000
0000 0000080 0000000
0000
PERIPHERAL BLOOD LYMPHOCYTES FIGURE 3
Agglutinability of lymphocytes from patients with African Burkitt's lymphoma ( O ) , carcinoma ( 0 ) and from healthy adult donors (0).Lymphocytes were isolated from peripheral blood and incubated with 250pg Con A/ml at 1-2 x lo6cells/ml for 30 min at 24" C.
452
BEN-BASSAT ET AL. TABLE I1 CAP FORMATION A N D AGGLUTINABILITY BY CON A O F CELLS FROM TUMOR TISSUES O F PATIENTS WITH AFRICAN BURKITT'S LYMPHOMA '
Patient No.
Sex and age (rears)
1 3 4 5 6 7 8 93 10 12 13 14 15
M-8 M-4 M-6 F-9 M-4 M-9 F-14 F-8 F-10 F-6 F-10 F-7 M-5
Duration of the disease (months)
Interaction with Con A Treatment
2R 3R 7 1.5 2
Cells with caps
0.5
7R 1
Established cell line
-t-
5.4 3.6 6.1 7.0 7.0 7.6 5.3
-t +ti
7.5
++++ + ++++ ++++ +++i.k i--I- + +
++++ ++++ t+ti NT NT NT NT
11.5 7.0 5.0 3.4
4R
Agglutination
( %)
+ ++ + + ~
-
T
.!.
5
+ 5
7.5
6
Lymphocytes were isolated from tumor biopsies of patients with African Burkitt's lymphoma by the Ficoll-Hypaque method. Cells were incubated with 100 pg of F-Con Aim1 for I5 min at 37" C, washed with PBS and the cells with caps were determined on a drop of living cells. For the agglutination experiments, cells were incubated with 250 pg of Con A/ml and the degree of agglutination was scored after not tested. - Still under cultivation. incubation of 30 min at 24" C. - R = recurring tumor. - 'Diagnosis not confirmed. - ' N T
-
(Himuma et al., 1967; Jondal et al., in preparation). The possible association between these characteristics and the increased mobility of Con A surface receptors, as determined by increased capforming ability, is being further investigated.
a very low concentration of Con A (1 ,ug/ml) and with a cell density as low as lo6 per ml. DISCUSSION
The results of these studies clearly indicate that lymphocytes isolated from patients with African Burkitt's lymphoma, the lymphoma cells contained in the tumor, as well as the lymphoma cell lines in vitro from the tumor biopsy, have a grown LYMPHOMA CELLS reduced mobility of Con A receptor sites. We have i= +--I+
CONCANAVALIN A RECEPTORS ON THE SURFACE MEMBRANE OF LYMPHOCYTES FROM PATIENTS WITH AFRICAN BURKITT’S LYMPHOMA AND LYMPHOMA CELL LINES Hannah BEN-BASSAT l, Natan GOLDBLUM l , Stella MITRANI l, George KLEIN and Bo JOHANSSON 3. Chanock Centre for Virology, Hebrew University-Hadassah Medical School, Jerusalem, Israel; Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden; and Kenyatta National Hospital, Nairobi, Kenya SUMMARY
Lymphocytes isolated from the peripheral blooa and from tumor tissues of patients with African Burkitt’s lymphoma have been studied for cap formation and agglutinability by Concanavalin A (Con A ) . Peripheral bloodfrom healthy adult persons served as a normal control and bloodfrom patients with carcinoma served as a non-lymphoma control. These studies included 29 patients with Burkitt ’s lymphoma, 93 with carcinoma, and 105 healthy adult persons, as well as tumor tissues from 13 patients with Burkitt’s lymphoma. The great majority of the carcinomas were from the face and neck regions. Lymphocytes from the blood of the majority of patients with Burkitt’s lymphoma, as well as those from tumor tissues, exhibited a reduced cap-forming ability (2-6 and increased Con-A-induced agglutinability compared to lymphocytes from healthy normal donors and from patients with carcinoma, although some of the lyinphocytes from patients with carcinoma had a somewhat lower range of cap formation than the lymphocytes from healthy donors. No difference was observed in the interaction with Con A of lypmhocytes from the different types of carcinoma studied. Eight lymphoid cell lines were established in our laboratory from the tumor tissues of patients with Burkitt ’s lymphoma. The cap-forming ability and agglutinability by Con A of these lines was examined and compared to those of the ‘‘ classical ” lymphoma lines: Raji, Daudi and P 3 H R I .All cell lines exhibited an increased Con-Ainduced agglutinability and a reduced cap-forming ability compared to normal lymphocytes, except for P,HR1 cells which exhibited a cap-forming ability of 15-20 %. These findings are discussed in relation to the association of the lymphocytes with malignancy and as a possible aid in the differential diagnosis between malignant lymphomas and other diseases.
x)
Concanavalin A (Con A, Sumner and Howell, 1936), has been used as a probe to study changes in the mobility of specific receptors on the cell surface membrane (Inbar et al., 1973 a, b, c ; Ben-Bassat and Goldblum, 1974, 1975). Studies with this probe have shown differences in the structure and function of the surface membrane of normal and malignant cells
(Ben-Bassat et al., 1971 ; Burger, 1969; Gahmberg et al., 1975; Inbar et al., 1972; Yahara et al., 1972). We have previously reported that lymphocytes from patients with chronic lymphocytic leukemia, Hodgkin’s disease and other malignant lymphomas have a reduced cap-forming ability and a higher Con-A-induced agglutinability than normal lymphocytes (Ben-Bassat and Goldblum, 1974, 1975). A significant decrease in the ability of lymphocytes to form caps was also observed in biopsy material from patients with Hodgkin’s disease and other malignant lymphomas. Lymphocytes isolated from tonsils of patients undergoing tonsillectomy and from axillary lymph nodes of breast cancer patients exhibited a cap-forming ability identical to that of normal lymphocytes. The ability of cells from a normal donor or a lymphoma patient to form caps was independent of the source from which the lymphocytes were isolated, e.g. lymph node, spleen or blood (Ben-Bassat and Goldblum, 1975, 1976). The present studies were undertaken in order: (1) to determine whether there are changes in the mobility of Con A receptors on the surface of lymphocytes of patients with African Burkitt’s lymphoma; (2) to compare the receptor mobility of lymphocytes from Burkitt’s lymphoma with that of lymphocytes isolated from other malignancies, e.g. carcinoma; and (3) to test whether lymphoma cells from established Burkitt lymphoma lines exhibit similar Con A receptor mobility to that of the patient’s lymphocytes. MATERIAL AND METHODS
Patients Biopsies and other material for these studies were obtained from ambulatory and hospitalized patients with Burkitt’s lymphoma and carcinoma from the following hospitals: Kenyatta National Hospital in Kenya; Uganda Cancer Institute, Kampala; Hadassah and the Central Hospital of Afula in Received: October 27, 1975, and in revised form January 23, 1976. Permanent address: Radiumhemmet, Karolinska Sjukhuset, Stockholm 60, Sweden.
449
CON A RECEPTORS ON BL AND LYMPHOMA CELLS
Israel. The 13 tumor biopsies were obtained from untreated patients, as well as from treated patients with recurring tumors. Peripheral blood was obtained from 29 patients with Burkitt's lymphoma and 72 with carcinoma. The group of patients with carcinoma included the following types : nasopharyngeal (36); maxilla (9); antrum (8); mandibula (4); cheek (4); tongue (3); larynx (3); nose (3); and neck (2). These included both untreated and treated cases.
Assay for binding of fluorescent Con A
Fluorescein isothiocynate-conjugated Con A (F-Con A) was prepared by Miles-Yeda, Rehovoth, Israel, at a mole ratio of 1.86 fluorescein to protein. For the experiments, 0.5-1.0x 10' cells were incubated with different concentrations of fluorescent Con A up to 100 pglml for 15 min at 37" C. The cells were washed with PBS and the fluorescence was determined on a drop of cells with a fluorescence microscope, Five hundred cells were counted for Preparation of biopsies and blood samples each point. The results of fluorescent Con A were Biopsy material was cut into small pieces with obtained with saturation conditions and only single scissors into medium RPMI 1640 containing 10% cells and cells in small clumps (2-5 cells) were fetal calf serum. The lymphocytes were isolated from counted for the percentage of caps. biopsies that were kept at 4" C and shipped to Israel, a procedure that took 4-5 days. Assay for agglutination Blood was drawn into sterile bottles containing Con A was obtained from Miles-Yeda. One-half heparin, kept at 4 ° C and shipped to Israel under ml of Con A at different concentrations diluted in similar conditions. PBS was mixed with 0.5ml of cell suspension to give a final concentration of 1 x loe cells per ml in a Isolation of lymphocytes 35 mm Petri dish. The density and size of aggregates Lymphocytes were isolated from peripheral blood was scored on a scale from - to after and biopsy material by the Ficoll-Hypaque gradient 30 min incubation at 24" C. centrifugation method (Boyum, 1968), washed twice with phosphate-buffered saline (PBS) (PH 7.2) and RESULTS diluted in PBS to the appropriate concentrations. Viability of the cells was determined by trypan blue Cap formation with fluorescent Concanavalin A of exclusion. The viability of the cells used in the exper- lymphocytes from peripheral blood of patients with iments was 70-95 % due to storage and travel effects. Burkitt 's lymphoma
++++
Lymphocytes from peripheral blood of patients with Burkitt's lymphoma were separated by the Eight cell lines from Burkitt lymphoma tumor Ficoll-Hypaque method and examined for cap forbiopsies were established in our laboratory. The mation with F-Con A. I n addition, we included in biopsy material was washed in medium RPMI 1640 this study a group of healthy donors and a group of with 10% fetal calf serum and a five-fold concen- patients with carcinoma. Altogether 29 patients tration of antibiotics, transferred to a Petri dish with Burkitt's lymphoma, 72 with carcinoma and containing medium and sectioned with scissors in the 45 healthy donors were examined. The blood samples medium. The material was grown in test tubes (150 x of Burkitt's lymphoma and carcinoma patients were 15 mm) in 2 ml RPMI 1640, 30% fetal calf serum examined after 4-5 days of storage at 4" C and travel and antibiotics. Medium was changed every 4-6 from Nairobi to Israel. The blood samples from days, carefully without disturbing the culture, until healthy donors came from Israel and were stored growth was established and active passages were 4-5 days at 4" C. The recovery of lymphocytes and begun. The cultures were kept at 37" C, 5 % CO, in their viability, as determined by morphological air and 80-95 %relative humidity. After establishment observation and vital staining, were satisfactory. of the cell line, the serum was reduced to 20%; the Figure 1 illustrates the results obtained with 100 pg/ lymphoma cells grew in suspension and were ml F-Con A. Lymphocytes from patients with maintained under the same conditions by sub- Burkitt's lymphoma exhibit a significant reduction culturing every 4-6 days. The lag phase before in the ability to form caps with F-Con A compared establishment of the cell lines was 3-8 weeks. to lymphocytes from healthy donors and patients For the experiments, cells were used between with carcinoma. The majority of the patients with passages 5-20, 2 days after refeeding, their viability Burkitt's lymphoma are within the range of 2-6% being 90-100% by trypan.blue exclusion. cap-forming ability. The great majority of patients These cell lines were compared with '' classical " with carcinoma were within a range similar to or Burkitt lymphoma lines: Raji, Daudi and PBHR1, lower than that of the healthy donors and generally obtained from the Department of Tumor Biology, higher than that of patients with Burkitt's lymphoma. Six patients with Burkitt's lymphoma and one with Karolinska Institute. Establishment of Burkitt lymphoma cell lines
450
BEN-BASSAT ET AL.
No difference in the interaction with Con A of lymphocytes from different types of carcinoma could be observed.
2 a
V
20
1
Agglutination of peripheral blood lymphocytes from patients with Burkitt Is lymphoma by Con A oooo0
Like the experiments reported for cap-forming ability, the agglutination experiments were also carried out with cells isolated by the Ficoll-Hypaque method from peripheral blood. The final concentration of Con A was 250,ug/ml. The results are shown in Figure 3. A significant increase in agglutin-
oooo 000
oooo 00 00
0 0 00
w
TABLE I
V
CAP FORMATION WITH F-CON A I N LYMPHOCYTES ISOLATED FROM THE SAME PATIENT AT DIFFERENT PERIODS '
NORMAL
CARCINOM, BURKITT' 5 LYMPHOMA
FIGURE1
Cap formation with F-Con A o n the surface membrane of lymphocytes from peripheral blood. Lymphocytes were isolated from blood of patients with African Burkitt's lymphoma ( 0 ) . Lymphocytes from normal adults served as controls (0)and lymphocytes from patients with carcinoma (m) served as non-Iymphomatous controls. Cells were incubated with 1OOpg F-Con A/ml for 15 min at 37" C and the presence of lymphocytes with caps was determined.
carcinoma were examined several times during a period of 3 weeks to 5 months. During this period the patients had already been under treatment. Blood samples were collected and the lymphocytes tested for capforming ability with F-Con A. The results summarized in Table I indicate that the cap formation with F-Con A of several samples of lymphocytes from the same patient is generally very consistent. No obvious differences in absolute numbers of cells from the same patient and from identical blood samples could be observed. In order to determine whether lymphocytes from patients with carcinoma exhibit a reduced ability of cap formation compared to normal lymphocytes or reflect '' storage travel " effects, we examined an additional group of fresh lymphocytes from patients with carcinoma. Twenty-two cases of carcinoma and 60 normal healthy donors were included in the study. Figure 2 shows the results of this study. All healthy donors and patients with carcinoma exhibited the pattern of cap formation of normal lymphocytes, although some of the lymphocytes from carcinoma patients had a somewhat lower range of cap formation than the lymphocytes from healthy donors.
Patient No.
Disease
Date
1
Burkitt's 23. 8.74 lymphoma 9. 9.74
2
Burkitt's 28. 8.74 lymphoma 9. 9.74
3
Burkitt's 30. 8.74 lymphoma 8.11.74 Burkitt's 30. 8.74 lymphoma 8.1 1.74
11.10.74 11.10.74
4
5 6
7
6.12.74 24. 1.74 Burkitt's 31. 1.75 lymphoma 21. 2.75 Burkitt's 24. 1.75 lymphoma 14. 2.75 14. 3.75 Carcinoma 8.11.74 22.11.74
No. of lymphocytes in the sample (I03
Lymphocytes with caps
2.2 3.8 2.7 4.9 8.0 2.1 0.9 I .2 5.5 3. I 4.5 2.0 4.8 4.0 1.8 2.1 2.0 1.8 1.3
5.2 2.2 8.6 5.0 4.3 5.0 6.0 3.6 4.3 3.0 5.7 6.0 5.5 5.0 7.0 4.4 8.6 4.0 5.8
' 1-2 x 10' cells were incubated with IOOpg of F-Con A/ml for 15 min at 37" C, washed with PBS and the fluorescence was determined on a drop of living cells. Five hundred cells were counted for each point and the percentage of cells with caps determined. ability is evident for lymphocytes from patients with Burkitt's lymphoma as compared to healthy donors and patients with carcinoma. We have previously shown that B and T blood lymphocytes from normal healthy persons are agglutinated by Con A to the same extent (Ben-Bassat and Goldblum, 1975). Also, in these cases the agglutinability of lymphocytes from patients with Burkitt's lymphoma was found not to be due to changes ifi the relative proportions of B- and T-lymphocytes in the blood. Agglutinability was specific as determined in each instance by inhibition with 0. I hi-a-methyl-D-mannopyranoside.
45 1
CON A RECEPTORS ON EL AND LYMPHOMA CELLS
35
Cap formation and agglutinability by Con A of I established Burkitt lymphoma Iines Burkitt lymphoma cell lines were examined for cap-forming ability and agglutinability by Con A. The following lines were tested: Raji (Pulvertaft, 1965; Epstein et al., 1966); Daudi (Klein et a/., 1968); P,HR1 (Himuma et a/., 1967) and six out of the eight Burkitt lymphoma lines established in our laboratory: Bu-1, Bu-3, Bu-4, Bu-6, Bu-8 and Bu-10. The cap-forming ability with F-Con of the Raji, Daudi and Bu-1, 3, 4,6, 8, 10 cells was low (2-5 %), whereas that of P,HR1 was 15-20%. P,HR, differs from the other eight lines in that it is a good EBV producer and lacks membrane receptors for EBV
00 00
0 0 0 0
25
O00
ooo 00 0
0 0
++++ z
- +++
0 I-
NORMAL
CARCINOMA
FIGURE 2
Cap formation with F-Con A on the surface membrane of lymphocytes from fresh peripheral blood. Lymphocytes
were isolated from blood of healthy adult donors (0)and from patients with carcinoma (a). Cells were incubated with 1OOpg fluorescent Con A/ml for 15min at 34°C and the presence of lymphocytes with caps was determined.
2 -
l-
3 (3 (3
++
a.
00000
a
+
Cap-forming ability and agglutinability by Con A of cells from tumor tissues of patients with Burkitt's lymphoma Biopsy material was obtained from tumors of patients with Burkitt's lymphoma. The lymphoma cells were separated by the Ficoll-Hypaque method after storage at 4" C and travel for 4-5 days, and examined for cap formation and agglutinability by Con A. Table I1 illustrates the results obtained with lymphoma cells from 13 patients with Burkitt's lymphoma. The lymphoma cells from all cases exhibit a reduced ability to form caps with fluorescent Con A and increased agglutinability by native Con A. The binding of F-Con A and native Con A was inhibited when a-methyl-D-mannopyranoside was added as a hapten inhibitor.
.a
0 000
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PERIPHERAL BLOOD LYMPHOCYTES FIGURE 3
Agglutinability of lymphocytes from patients with African Burkitt's lymphoma ( O ) , carcinoma ( 0 ) and from healthy adult donors (0).Lymphocytes were isolated from peripheral blood and incubated with 250pg Con A/ml at 1-2 x lo6cells/ml for 30 min at 24" C.
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BEN-BASSAT ET AL. TABLE I1 CAP FORMATION A N D AGGLUTINABILITY BY CON A O F CELLS FROM TUMOR TISSUES O F PATIENTS WITH AFRICAN BURKITT'S LYMPHOMA '
Patient No.
Sex and age (rears)
1 3 4 5 6 7 8 93 10 12 13 14 15
M-8 M-4 M-6 F-9 M-4 M-9 F-14 F-8 F-10 F-6 F-10 F-7 M-5
Duration of the disease (months)
Interaction with Con A Treatment
2R 3R 7 1.5 2
Cells with caps
0.5
7R 1
Established cell line
-t-
5.4 3.6 6.1 7.0 7.0 7.6 5.3
-t +ti
7.5
++++ + ++++ ++++ +++i.k i--I- + +
++++ ++++ t+ti NT NT NT NT
11.5 7.0 5.0 3.4
4R
Agglutination
( %)
+ ++ + + ~
-
T
.!.
5
+ 5
7.5
6
Lymphocytes were isolated from tumor biopsies of patients with African Burkitt's lymphoma by the Ficoll-Hypaque method. Cells were incubated with 100 pg of F-Con Aim1 for I5 min at 37" C, washed with PBS and the cells with caps were determined on a drop of living cells. For the agglutination experiments, cells were incubated with 250 pg of Con A/ml and the degree of agglutination was scored after not tested. - Still under cultivation. incubation of 30 min at 24" C. - R = recurring tumor. - 'Diagnosis not confirmed. - ' N T
-
(Himuma et al., 1967; Jondal et al., in preparation). The possible association between these characteristics and the increased mobility of Con A surface receptors, as determined by increased capforming ability, is being further investigated.
a very low concentration of Con A (1 ,ug/ml) and with a cell density as low as lo6 per ml. DISCUSSION
The results of these studies clearly indicate that lymphocytes isolated from patients with African Burkitt's lymphoma, the lymphoma cells contained in the tumor, as well as the lymphoma cell lines in vitro from the tumor biopsy, have a grown LYMPHOMA CELLS reduced mobility of Con A receptor sites. We have i= +--I+
