lmmunochemlstry. 1975, Vol 12, pp 585-588 Pergamon Press
Printed m Great Britain
CELL-SURFACE MARKERS O N M U R I N E LYMPHOMAS* MARIANA LINKER-ISRAELI and NECHAMA HARAN-GHERA Department of Chemical Immunology, The Welzmann Institute of Sctence, Rehovot, Israel {Recetved 15 November 1974) Abstract--Studies on cells of murlne lymphatic leukemtas, reduced by a system devised in this laboratory, have been carried out. The leukemic cells were found to share with normal B cells three markers surface ~mmunoglobulins, and receptors for the Fc fragment and the C3 component, as assayed by cells forming EA or EAC rosettes The surface ~mmunoglobuhns of the B leukemic cells were sensitive to incubation with trypsin, as m&cated by the drop In lmmunoglobuhns following enzymatic treatment, but were resynthetlzed by the leukemic cell 24hr later, when kept in culture
Some data have been accumulating a b o u t cell populations in b o t h h u m a n a n d a m m a l leukemias a n d lymphomas. The trend has been to look, in these leukenuas, for the same categories of cells found m lymphocytes derived from non-neoplastic t~ssues, namely T cells (thymus a n d thymus-derwed) a n d B cells (Bursa or Bursa-equwalent-derived) A m o n g h u m a n leukemias, several cases of C L L have been reported as bearing B-cell surface markers, such as l m m u n o g l o b u l i n s a n d C3 receptors (Fouhs et al., 1973; Bentwich a n d Kunkel, 1973) a n d m a case of Burkitt L y m p h o m a the Fc receptor has been found o n cells m culture by Shevach et al. (1972a). Similarly, a cell passage hne of a spontaneously occurring leukemm in guinea-pigs, a n d some murlne leukemias were shown to possess B cell surface markers by Shevach et al. (1972b, 72c), Chne et al. (1972) Shevach et al (1973). In this l a b o r a t o r y data concernmg cell p o p u l a u o n s in induced leukemias of SJL/J mice have been reported ( H a r a n - G h e r a a n d Peled, 1972). These leukemias bore either 0 antigens, or l m m u n o g l o b u h n s , or neither of them. The present studies have been carried o u t in order to ascertain the 'true' nature of the i m m u n o g l o b u l i n identified o n these chemtcal carcinogen induced murine leukemic cells a n d whether other B markers, such as receptors for Fc or C3 would be observed o n these cells.
MATERIALS A N D M E T H O D S
Mtce. SJL/J females, bred and reared at The Welzrnann InsUtute of Science, Rehovot. Kept m metal cages at 22°C room temperature, fed Purme Chow Pellets and tap water Induction of tumors Lymphatic leukemia was induced m intact, or T-cell depleted S J L / J mice, by a chemical carcinogen--7,12-&methylbenz(a) anthracene (DM BA). The carcinogen was made up as a lY,o solution in polyethyleneglycol-400, and administered to the mice four times at weekly intervals through a polyethylene stomach tube of about 1 mm bore size. A dose of 1 ml of the DMBA solution (1 mg DMBA) was given at each feeding. Two experimental groups, namely, 8-week-old intact female *This work was supported in part by a grant from the Leukemia Research Foundation, Chicago, Illinois 60601, U S.A
SJL/J mice and SJL/J mice of corresponding age and sex, depleted of T cells, were treated with DMBA The depletion of the T-cell population m the lymphoid tissues was obtained using the following method 4-week-old female SJL/J mice were thymectomlzed and, 20 days thereafter, exposed to 750r whole body irra&atlon W~thln I hr after lrradlaUon they were reconstituted with 5 x l 0 6 isologous spleen cells Incubated previously w~th ant1 0 antiserum and complement Two weeks later, these mice were fed with DMBA as described above Incidence and latency of leukemia development were recorded. All dead nuce were routinely autopsled Unless lymphoid leukemia could be &agnosed unequwolcally, the relevant tissues were examined histologically (fixation m Bouln's fluid and staining with hematoxydln and costa) Leukemic tissue was inoculated intramuscularly into syngene~c recipients, and lines of tumors were kept in the laboratory by further, serial, intramuscular transplantation Into syngenelc recipients. Tumor cell-suspenslons Tumors were gently teased wtth the aid of two scalpels into cold Eagle's medium, then passaged once through a syringe with a 27 gauge needle This procedure yielded suspenstons of 85-95 per cent viable cells (as measured by exclusion of 0-16°o trypan blue) Immunofluorescence 5 x 10 6 tumor cells were washed m 1°/ogelatine in tyrode, then pelleted down and incubated m 005 ml of goat anti-mouse lmmunoglobulln, dduted 1 10 m PBS, at 37°C for 30 mm Cells were washed twice m lY/o gelatine in tyrode, then pelleted down again and relncubated wlth 0'05 ml of FITC (fluorescem lsothlocyanate) conjugated rabb~t anti-goat lgG at 37°C for 20 mln Cells were washed twice agam m 1°o gelatme tyrode, then resuspended m 50% glycerine m PBS and kept at 4°C until read under u v. hght Antlsera were bought from Meloy Laboratories, Inc, Springfield, Virginia Ant~ 0 C3H serum. Was prepared by lmmumzing AKR/J mice with C3H/eb thymocytes (10 x 10 6 cells rejected mtrapentoneally at weekly intervals for 7 weeks). The lmmumzed mice were bled one week after the last thymocyte moculaUon Cell suspensions of the various leukemxas were prepared in cold Tyrode medium containing 0 1°,o BSA. The suspensions were washed twice and adjusted to 5-10 x 10 6 cells ml To 0.l ml of cell suspenston, an equal volume of anti C3H serum in 1 16-1 32 dilution was added The mixture was incubated for 20 mm at 37cC, then cells were spun down, and 0-1 ml of guinea-pig complement (adsorbed on agarose and dduted 1:9 m Kolmer's Saline) was added to the pellet This mixture was incubated for another 30mm at 37°C Index of cytotoxlclty was esttmated by the dye exluslon of trypan blue Trypsm~zat~on 5 x 10 7 tumor cells in suspension were washed twice in Eagle's medium (Dulbecco's modlficauon), 585
MARIANA LINKER-ISRAELI and NECHAMA HARAN-GHERA
586
Table 1.
Surface 0a a n t i g e n and tmsunoglobultns b i n DMBA-tnduced lymphatic leukemias c in SJL/J mice
~or
Mode of tumor
L a t e n t period
induction J-15-16
Intact * DHBA x 4 d
% c e l l s with
Index ~ b e a r i n g
(days)
s u r f a c e ]G
lymphocyCes a 11%
151
36%
J-15-58
158
25%
J-15-48
116
70%
0%
J-15-I07
199
10%
10%
5%
J-15-128
142
55%
12%
J-15-195
163
48%
21%
J-19-242
128
50%
2%
J-19-188
102
68%
5%
J-19-A-215
124
IS%
5%
FR-I-g2
112
10%
12% 1%
95
58%
243
60%
2%
J-16-55
100
74%
4%
J-16-63
i00
52%
I%
J-16-77
92
60%
4%
J-16-78
109
75%
0%
J-16-2 J-16-52
Thymex T depleted~DMBAx4 "
a Index expressed as % dead c e l l s i n a n t i 0 serum - % dead c e l l s i n n o r ~ l % v i a b l e c e l l s in normal serum. b
serum x 100
IG assayed by i n d i r e c t l . ~ u n o f l u o r e s c e n c e .
c H i s t o l o g i c s e c t i o n s checked on a l l tumors
By t h i s c r i t e r i o n a l l t h e surveyed
tumors were lylnpbattc leukemtas. d Mice fed with 7 , 1 2 - d i l m t h y l b a n z ( e ) a n t h r a c e n e 4 t i m e s , a t weekly i n t e r v a l s , 1 mg
per f e e d i n g . • Mice thymectumized a t 4 weeks o f age, whole body i r r a d i a t e d with 750 R 20 days l a t e r , and t ~ R e d i a t e l y i n o c u l a t e d with S x 106 syngeneic spleen c e l l s pretreatad with a n t i * 0 serum and complement.
then incubated at 37°C, for 20tmn, m 0'3% trypsin m Puck's solution Following enzymatic treatment, the ceils were washed three nines in cold 20 per cent hear-inactivated calf serum in Eagle's medmm (Dulbecco's modification), resuspended m the same medium at a concentranon of 5 x 106 cells/ml and incubated at 37°C m 10% CO2 for up to 3 days. Surface immunoglobuhns were checked, by the method for redirect lmmunofluorescence described above, at &fferent mtervals after trypsm~zatlon Rosettes E' Sheep red blood cells m a 1 25 per cent suspension in PBS EA' E, incubated with equal volumes of rabbit anti-sheep red blood cell antmerum, diluted 1.1500, at 37°C for 30min. Three washings in PBS followed, then blood cells were resuspended, in tbelr initial concentration, in Eagle's medium EAC: EA, incubated at 37°C for 30mln with equal volumes of fresh serum from C3H/eb mice m a 1'10 ddution. Three washings m PBS followed, then blood cells were resuspended, m their initial concentration, m Eagle's reed]urn. After three washings tumor cells were resuspended m Eagle's medmm, at a concentration of 2 x 106/rrd. Tumor cells were rmxed w]th equal volumes of EA or EAC. centrifuged for 3 mm at 300 g, and incubated at 37°C for 30 ram. The pellet was gently resuspended, and rosettes (nucleated cells attaching more than 4 sheep red blood cells) were counted m a counting chamber. Percentage of rosettes was calculated as the number of rosette forming cells out of 200 total nucleated cells counted RESULTS
The data summarized in Table 1 in&cate that many of the lymphomas surveyed here exhibit surface immunoglobulins--mamly IgG, while only a negligible fraction of their cells carried the theta antigen. This would confirm previous results from this laboratory
(Haran-Ghera and Peled, 1973). DMBA-mduced lym-' phatic leukemia developed in 65 per cent intact SJL mice fed with DMBA at an average latent period of 154 days, and in 70 per cent of T cell depleted SJL/J mice, at an average latent period of 130 days. Although both groups treated with DMBA yielded lymphatic leukemlas, some of the leukemias developmg in the intact mice originated from thymus derived lymphocytes and some were of B origin--their leukemic cells bearing surface immunoglobuhns, whereas all the leukemias developing in the T-cell depleted mice were of bone marrow derived lymphocytes. The T-cell depleted group displayed a higher and more uniform level of immunoglobuhns (range 52-75 per cent) than the intact one (10-70 per cent). Thus, although it is assumed that a transplanted tumor consists of a homogeneous population of cells, only a fraction of these (ranging from 10-75 per cent) were positive when assayed in the immunofluorescent test for surface immunoglobuhns. The same was true for these lymphoma cells, when assayed for rosette formation (Table 2) with EA or EAC. While spleen cells of normal SJL/J mice had a percentage of 24 rosette forming cells with EA and 29 rosette forming cells with EAC, lymphoma cells formed rosettes in a percentage ranging from 9 to 100. The percentage of rosette forming cells in a given tumor was not always correlated wlth the percentage of ceils positive for immunoglobulins. None of the assayed tumors gave any rosettes with SRBC unsensitized with antibody (E) and they were not killed by anU 0 serum 1:16 (Kills 70--90 per cent thymocytes). A 0 posiuve tumor (J-22-42) formed neither EA nor EAC rosettes.
Printed m Great Britain
CELL-SURFACE MARKERS O N M U R I N E LYMPHOMAS* MARIANA LINKER-ISRAELI and NECHAMA HARAN-GHERA Department of Chemical Immunology, The Welzmann Institute of Sctence, Rehovot, Israel {Recetved 15 November 1974) Abstract--Studies on cells of murlne lymphatic leukemtas, reduced by a system devised in this laboratory, have been carried out. The leukemic cells were found to share with normal B cells three markers surface ~mmunoglobulins, and receptors for the Fc fragment and the C3 component, as assayed by cells forming EA or EAC rosettes The surface ~mmunoglobuhns of the B leukemic cells were sensitive to incubation with trypsin, as m&cated by the drop In lmmunoglobuhns following enzymatic treatment, but were resynthetlzed by the leukemic cell 24hr later, when kept in culture
Some data have been accumulating a b o u t cell populations in b o t h h u m a n a n d a m m a l leukemias a n d lymphomas. The trend has been to look, in these leukenuas, for the same categories of cells found m lymphocytes derived from non-neoplastic t~ssues, namely T cells (thymus a n d thymus-derwed) a n d B cells (Bursa or Bursa-equwalent-derived) A m o n g h u m a n leukemias, several cases of C L L have been reported as bearing B-cell surface markers, such as l m m u n o g l o b u l i n s a n d C3 receptors (Fouhs et al., 1973; Bentwich a n d Kunkel, 1973) a n d m a case of Burkitt L y m p h o m a the Fc receptor has been found o n cells m culture by Shevach et al. (1972a). Similarly, a cell passage hne of a spontaneously occurring leukemm in guinea-pigs, a n d some murlne leukemias were shown to possess B cell surface markers by Shevach et al. (1972b, 72c), Chne et al. (1972) Shevach et al (1973). In this l a b o r a t o r y data concernmg cell p o p u l a u o n s in induced leukemias of SJL/J mice have been reported ( H a r a n - G h e r a a n d Peled, 1972). These leukemias bore either 0 antigens, or l m m u n o g l o b u h n s , or neither of them. The present studies have been carried o u t in order to ascertain the 'true' nature of the i m m u n o g l o b u l i n identified o n these chemtcal carcinogen induced murine leukemic cells a n d whether other B markers, such as receptors for Fc or C3 would be observed o n these cells.
MATERIALS A N D M E T H O D S
Mtce. SJL/J females, bred and reared at The Welzrnann InsUtute of Science, Rehovot. Kept m metal cages at 22°C room temperature, fed Purme Chow Pellets and tap water Induction of tumors Lymphatic leukemia was induced m intact, or T-cell depleted S J L / J mice, by a chemical carcinogen--7,12-&methylbenz(a) anthracene (DM BA). The carcinogen was made up as a lY,o solution in polyethyleneglycol-400, and administered to the mice four times at weekly intervals through a polyethylene stomach tube of about 1 mm bore size. A dose of 1 ml of the DMBA solution (1 mg DMBA) was given at each feeding. Two experimental groups, namely, 8-week-old intact female *This work was supported in part by a grant from the Leukemia Research Foundation, Chicago, Illinois 60601, U S.A
SJL/J mice and SJL/J mice of corresponding age and sex, depleted of T cells, were treated with DMBA The depletion of the T-cell population m the lymphoid tissues was obtained using the following method 4-week-old female SJL/J mice were thymectomlzed and, 20 days thereafter, exposed to 750r whole body irra&atlon W~thln I hr after lrradlaUon they were reconstituted with 5 x l 0 6 isologous spleen cells Incubated previously w~th ant1 0 antiserum and complement Two weeks later, these mice were fed with DMBA as described above Incidence and latency of leukemia development were recorded. All dead nuce were routinely autopsled Unless lymphoid leukemia could be &agnosed unequwolcally, the relevant tissues were examined histologically (fixation m Bouln's fluid and staining with hematoxydln and costa) Leukemic tissue was inoculated intramuscularly into syngene~c recipients, and lines of tumors were kept in the laboratory by further, serial, intramuscular transplantation Into syngenelc recipients. Tumor cell-suspenslons Tumors were gently teased wtth the aid of two scalpels into cold Eagle's medium, then passaged once through a syringe with a 27 gauge needle This procedure yielded suspenstons of 85-95 per cent viable cells (as measured by exclusion of 0-16°o trypan blue) Immunofluorescence 5 x 10 6 tumor cells were washed m 1°/ogelatine in tyrode, then pelleted down and incubated m 005 ml of goat anti-mouse lmmunoglobulln, dduted 1 10 m PBS, at 37°C for 30 mm Cells were washed twice m lY/o gelatine in tyrode, then pelleted down again and relncubated wlth 0'05 ml of FITC (fluorescem lsothlocyanate) conjugated rabb~t anti-goat lgG at 37°C for 20 mln Cells were washed twice agam m 1°o gelatme tyrode, then resuspended m 50% glycerine m PBS and kept at 4°C until read under u v. hght Antlsera were bought from Meloy Laboratories, Inc, Springfield, Virginia Ant~ 0 C3H serum. Was prepared by lmmumzing AKR/J mice with C3H/eb thymocytes (10 x 10 6 cells rejected mtrapentoneally at weekly intervals for 7 weeks). The lmmumzed mice were bled one week after the last thymocyte moculaUon Cell suspensions of the various leukemxas were prepared in cold Tyrode medium containing 0 1°,o BSA. The suspensions were washed twice and adjusted to 5-10 x 10 6 cells ml To 0.l ml of cell suspenston, an equal volume of anti C3H serum in 1 16-1 32 dilution was added The mixture was incubated for 20 mm at 37cC, then cells were spun down, and 0-1 ml of guinea-pig complement (adsorbed on agarose and dduted 1:9 m Kolmer's Saline) was added to the pellet This mixture was incubated for another 30mm at 37°C Index of cytotoxlclty was esttmated by the dye exluslon of trypan blue Trypsm~zat~on 5 x 10 7 tumor cells in suspension were washed twice in Eagle's medium (Dulbecco's modlficauon), 585
MARIANA LINKER-ISRAELI and NECHAMA HARAN-GHERA
586
Table 1.
Surface 0a a n t i g e n and tmsunoglobultns b i n DMBA-tnduced lymphatic leukemias c in SJL/J mice
~or
Mode of tumor
L a t e n t period
induction J-15-16
Intact * DHBA x 4 d
% c e l l s with
Index ~ b e a r i n g
(days)
s u r f a c e ]G
lymphocyCes a 11%
151
36%
J-15-58
158
25%
J-15-48
116
70%
0%
J-15-I07
199
10%
10%
5%
J-15-128
142
55%
12%
J-15-195
163
48%
21%
J-19-242
128
50%
2%
J-19-188
102
68%
5%
J-19-A-215
124
IS%
5%
FR-I-g2
112
10%
12% 1%
95
58%
243
60%
2%
J-16-55
100
74%
4%
J-16-63
i00
52%
I%
J-16-77
92
60%
4%
J-16-78
109
75%
0%
J-16-2 J-16-52
Thymex T depleted~DMBAx4 "
a Index expressed as % dead c e l l s i n a n t i 0 serum - % dead c e l l s i n n o r ~ l % v i a b l e c e l l s in normal serum. b
serum x 100
IG assayed by i n d i r e c t l . ~ u n o f l u o r e s c e n c e .
c H i s t o l o g i c s e c t i o n s checked on a l l tumors
By t h i s c r i t e r i o n a l l t h e surveyed
tumors were lylnpbattc leukemtas. d Mice fed with 7 , 1 2 - d i l m t h y l b a n z ( e ) a n t h r a c e n e 4 t i m e s , a t weekly i n t e r v a l s , 1 mg
per f e e d i n g . • Mice thymectumized a t 4 weeks o f age, whole body i r r a d i a t e d with 750 R 20 days l a t e r , and t ~ R e d i a t e l y i n o c u l a t e d with S x 106 syngeneic spleen c e l l s pretreatad with a n t i * 0 serum and complement.
then incubated at 37°C, for 20tmn, m 0'3% trypsin m Puck's solution Following enzymatic treatment, the ceils were washed three nines in cold 20 per cent hear-inactivated calf serum in Eagle's medmm (Dulbecco's modification), resuspended m the same medium at a concentranon of 5 x 106 cells/ml and incubated at 37°C m 10% CO2 for up to 3 days. Surface immunoglobuhns were checked, by the method for redirect lmmunofluorescence described above, at &fferent mtervals after trypsm~zatlon Rosettes E' Sheep red blood cells m a 1 25 per cent suspension in PBS EA' E, incubated with equal volumes of rabbit anti-sheep red blood cell antmerum, diluted 1.1500, at 37°C for 30min. Three washings in PBS followed, then blood cells were resuspended, in tbelr initial concentration, in Eagle's medium EAC: EA, incubated at 37°C for 30mln with equal volumes of fresh serum from C3H/eb mice m a 1'10 ddution. Three washings m PBS followed, then blood cells were resuspended, m their initial concentration, m Eagle's reed]urn. After three washings tumor cells were resuspended m Eagle's medmm, at a concentration of 2 x 106/rrd. Tumor cells were rmxed w]th equal volumes of EA or EAC. centrifuged for 3 mm at 300 g, and incubated at 37°C for 30 ram. The pellet was gently resuspended, and rosettes (nucleated cells attaching more than 4 sheep red blood cells) were counted m a counting chamber. Percentage of rosettes was calculated as the number of rosette forming cells out of 200 total nucleated cells counted RESULTS
The data summarized in Table 1 in&cate that many of the lymphomas surveyed here exhibit surface immunoglobulins--mamly IgG, while only a negligible fraction of their cells carried the theta antigen. This would confirm previous results from this laboratory
(Haran-Ghera and Peled, 1973). DMBA-mduced lym-' phatic leukemia developed in 65 per cent intact SJL mice fed with DMBA at an average latent period of 154 days, and in 70 per cent of T cell depleted SJL/J mice, at an average latent period of 130 days. Although both groups treated with DMBA yielded lymphatic leukemlas, some of the leukemias developmg in the intact mice originated from thymus derived lymphocytes and some were of B origin--their leukemic cells bearing surface immunoglobuhns, whereas all the leukemias developing in the T-cell depleted mice were of bone marrow derived lymphocytes. The T-cell depleted group displayed a higher and more uniform level of immunoglobuhns (range 52-75 per cent) than the intact one (10-70 per cent). Thus, although it is assumed that a transplanted tumor consists of a homogeneous population of cells, only a fraction of these (ranging from 10-75 per cent) were positive when assayed in the immunofluorescent test for surface immunoglobuhns. The same was true for these lymphoma cells, when assayed for rosette formation (Table 2) with EA or EAC. While spleen cells of normal SJL/J mice had a percentage of 24 rosette forming cells with EA and 29 rosette forming cells with EAC, lymphoma cells formed rosettes in a percentage ranging from 9 to 100. The percentage of rosette forming cells in a given tumor was not always correlated wlth the percentage of ceils positive for immunoglobulins. None of the assayed tumors gave any rosettes with SRBC unsensitized with antibody (E) and they were not killed by anU 0 serum 1:16 (Kills 70--90 per cent thymocytes). A 0 posiuve tumor (J-22-42) formed neither EA nor EAC rosettes.
