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Suppression of in vitro primary immune response by L1210 cells and their culture supernatant: Evidence for cytotoxic effects
cEr.r.ur.AR
II\tMuN0r,0GY 29, 210-215 (1977)
Suppression of in Vitro Primary Immune Cells and Their Culture Supernatant: Cytotoxic Effects R. P. HUGET, IIrparfmxt P.O.
Response by L1210 Evidence for
H.-D. FLAD,~ AND H. G. OPITZ
of Clhical PkJuiology, U~aizwsitg Box 4066, D-7900 Ulm, Germrl.v Rec&vd
August
of
C/m,
11, 19i6
L1210 cells and their culture supernatants were found to inhibit the generation of PFC in the i+z vitro primary immune response of spleen cells to SRBC. As few as 1% of L1210 cells and 1% of culture fluid were inhibitory. Inhibition of DNA or protein synthesis of L1210 cells did not abolish their immunosuppressive activity, excluding exhaustion of culture medium as a possible mechanism of inhibition of PFC. Heating of the supernahnt completely abrogated the suppressive effect and resulted in a marked increase of PFC. Daily evaluation of cell viability in the cultures revealed that, in the presence of L1210 and supernatants, the fraction of surviving cells is markedly reduced. We conclude that a direct cytotoxic effect on splenic lymphocytes and macrophages is the predominant immunosuppressive mechanism of L1210 cells and their culture supernatants.
INTRODUCTION Tumor cells or their culture supernatants have been shown to depress immune responsesin vifro (l-5). Thus, addition of mastocytoma cells leads to a reduction of plaque-forming cells in Mishell-Dutton-type cultures (2, 6). Furthermore, a diminution of rosette-forming cells after incubation of lymphocytes with supernatants from MCV tumor cell cultures and a suppression of DNA synthesis of mitogen-stimulated lymphocytes by tumor cells have been reported (7, 11) . In all of these experiments, it was claimed that the suppressive effect of tumor cells or their supernatants was not due to cytotoxicity, since cell viability was said to be comparable to control cultures. Rather, an inactivation of subpopulations of cells participating in the immune response or, alternatively, an activation of suppressor cells was proposed to be responsible for the suppression observed (6). We present evidence that L1210 mouse lymphoma cells or their culture supernatants markedly reduce cell viability of spleen cells. Cytotoxicity of both lymphocytes and macrophageswas observed. MATERIALS
AND
METHODS
AnimuZs. Mice of the inbred strains DBA/2, CBA/CA, weighing 20-25 g were used in different experiments. 1 Requests D-7900 Ulm,
for reprints Germany.
should
be addressed
to H.-D.
Flad,
University
C57B1/6, and Balb/c of Ulm,
P.0.
Box
4066,
210 Copyright All rights
@ 1977 by of reproduction
Academic Press, Inc. in any form reserved.
ISSK
0008
8749
SHORT
211
COMMUNICATIONS
Antigen. Sheep red blood cells (SRBC) obtained from Behringwerke, Marburg, Germany were stored in sterile Alsever’s solution. SRBC (10’) in 20 ~1 of balanced salt solution (BSS) were added to each culture. Spleen cell suspension. Spleens were removed under sterile conditions and disrupted by a loose-fitting glass homogenizer. After brief sedimentation, the suspension was washed three times with BSS and subsequently suspended in culture medium [RPM-1640 medium (Flow Laboratories) supplemented with penicillin (100 units/ml), streptomycin (100 rg/ml), L-glutamine (2 mM), 5% FCS (Seromed, Munich, Lot. Ko. 108)) and 2-mercaptoethanol (5 X 1e5 M) 1. Cell tiabilit~. Cell counts and viability tests were performed in Neubauer hemocytometers. For the determination of cell viability, 50 ~1 of the cell suspension in RPM-5% FCS was diluted with 1 ml of 0.5% trypan blue solution and the cells were immediately counted. Tuwzor cells. L1210 cells, a permanent lymphoblast cell line, originally obtained from Associated Biomedic Systems, Buffalo, New York, were maintained in RPMI1640 medium containing 157% FCS, penicillin, and streptomycin. Suspensions were grown in 30-ml Polystyrene flasks (Falcon Plastics, Oxnard, California). Under these conditions, the cells had a mean doubling time of 9-12 hr and reached a final density of about 2 x 10F cells/ml. Cells of this concentration were used throughout. L1210 cells were X-irradiated (Siemens Stabilipan, 0.5~mm Cu filter) with doses of 2000, 5000, and 8000 rad, respectively. In some experiments, the cells were treated with mitomycin C (50 pg/ml) or puromycin (30 rg/ml) for 30 min at 3/‘“C, washed three times with RPM, and used in the cultures. Sztpcrnatant fyonz L1210 cells. L1210 cells were allowed to grow for 48 hr to a density of about 2 X lo6 cells/ml. The 4S-hr culture medium was collected, centrifuged at 8OOg to remove cells and debris, and filtered through a Millipore filter TABLE Eifect
of Viable,
Expt.
1
2
3
0 L1210 b L1210
Irradiated, L1210
1
and I’uromycin-Treated I’FC culture
per f SD
4325 4380 3345 125
f 143 zt 848 f. 447 dz 87
None (control) 104, mite” 105, mite” 106, mite”
1800 1050 700 20
f f f f
None 106, 106, 106, 106,
None 104 105 106
cells treated cells treated
cells added
L1210
(control)
(control) 2000 rad 5000 rad 8000 rad puromycin with with
treatedb
50 pg/mI 30 pg/ml
Cells
on I’FC
per Culture
Percentage of control response
Percentage of viable cells (Day 5)
101.0 77.0 3.0
35 31 26 4
158 148 28 10
58.0 38.0 1.1
27 18 10 6
3942 f 244 40 f: 24 15f 7 27 f 14 0
1.0 0.3 0.7 0
30 9 10 9 7
of mitomycin C for 30 min at 37’C. for 30 min at 37°C.
212
SHORT
COMMUNICATIONS
Fmal concentration
of supernatant
(%I
FIG. 1. The effect of various L1210 culture and control supernatants on PFC response of SRBC-stimulated mouse spleen cell cultures. PFC were determined on Day 5 of culture and are expressed as percentage of PFC of control cuiture without supernatants (100%). The mean values of two cultures are given. X-X, native; A---& dialysed; O-0, uv light treated; O-O, L1210 supernatant heated at 56°C for 30 min; n-0, native supernatant from syngeneic spleen cell cultures (control).
(0.45 pm) before use. Supernatants of spleen cells were treated in the same way and served as controls. Cell culture. For the induction of a primary immune responsein vitro, the method of Mishell and Dutton (7) was used. The cultures were kept stationary. Assay of antibody synthesis. A modified hemolytic plaque assay was used to quantitate cellular antibody production in vitro (8). RESULTS Suppressive effect of LlZlO cells on in vitro PFC response. When L1210 cells are added to spleen cells, the PFC response of spleen cells against SRBC is inhibited (Table 1). As few as lo5 L1210 cells, i.e., 1% of the cells in culture, were able to reduce the number of PFC at Day 5. To exclude the possibility that proliferating L1210 cells deplete the tissue culture medium of essential nutrient ingredients, the proliferation of L1210 cells was inhibited by treatment of the cells with either X irradiation or mitomycin C. L1210 cells treated in this way were still able to suppressthe PFC (Table 1). Furthermore, inhibition of protein synthesis of L1210 cells by treating the cells with puromycin did not abolish the immunosuppressive activity of the cells. These findings suggested that the exhaustion of the culture medium by L1210 cells or the de nova synthesis of an inhibitory pro-
SHORT
COMMUNICATIONS
213
FIG. 2. The effect of L1210 culture supernatant on cell viability in Mishell-Dutton cultures. The mean values of two cultures are given. X-X, control (no supernatant) ; O-0, 1% ; A-A, 10% ; O-O, 205% supernatant (final concentration).
tein was not the mechanism by which L1210 cells exerted their inhibitory effect on PFC response. The inhibitory effect of L1210 cells on PFC was not restricted to cells of the same H2 type. Mitomycin-treated L1210 cells suppressed the generation of both syngeneic (DBA/2) and allogeneic PFC. Allogeneic or syngeneic mitomycin-treated spleen cells did not inhibit the PFC formation. Suppressive efect of L1210 culture supernatants. When L1210 cell culture supernatants were added to the spleen cultures in increasing concentrations, a marked decrease of PFC was observed (Fig. 1). Heating of L1210 supernatants for 30 min at 56°C not only completely abolished the PFC inhibitory activity, but rather increased the PFC response. The inhibitory activity was also found to be thermolabile when supernatants were stored for 5 days at room temperature. Treatment of culture supernatants with uv light overnight did not abolish their immunosuppressive activity on PFC responses.Furthermore, the activity was found to be nondialysable. Reduction of cell Viability by L1210 cells or LlZlO-derived culture supernatants. Cell viability of the spleen cultures was tested at daily intervals by trypan blue exclusion. Increasing the number of L1210 cells or the volume of L1210 culture supernatants lead to a marked reduction of cell viability (Table 1 and Fig. 2). It should be mentioned that, during the culture period, macrophages lost their adherence to the petri dishes and disintegrated so that no viable macrophages were found in these cultures on Day 5. Efiect of L1210 cells added at various days of culture on PFC. Attempts were made to define the culture period during which the cultured spleen cells are most sensitive to a suppressive action of L1210 cells on PFC response. When L1210 cells were added at 0, 24, 48, 72, and 96 hr of culture, a marked decreaseof PFC responseswas found during the first 48 hr (Table 2). Addition of L1210 cells at 72 hr or later reduced the PFC responseto a lesser degree, indicating that the tumor
214
SHORT
COMMUNICATIONS
TABLE Inhibition
Expt.
of PFC
Responses Effects
2
by L1210 Cells and Supernatants: and Effect of Preincubation
Condition
Time-Dependent
PFC”
Percentage of viable cells per culture (Day
1
2
Control (no I,1210 L1210 cells addeda 0 1
cells added) at day:
1820 f
147
30
10f 7 38 f 12 208 f 74 812 h 125
3 7 12 15 21
1560 rt 265 30f 17
40 28
0
Control (no preincubation) Preincubation”
5)
a PFC were determined on Day 5 of culture. The mean values of two cultures zt SD are given. b lo6 viable L1210 cells were added to lo7 SRBC-stimulated mouse spleen cells. c Spleen cells (lO?/ml) were incubated in 207, L1210 culture supernatant (final concentration) for 1 hr at 37’C, washed twice, and cultured with SRBC.
cells exerted their inhibitory effect either on the proliferative phase of antigensensitive cells or, possibly, on a population of helper cells. Effect of a preincubation of spleen cells m’tlz L1210 supcrnatant on PFC. To further define the target cell population, spleen cells were preincubated with 20% L1210 culture supernatant for 1 hr at 37°C washed, and then stimulated with SRBC. The PFC response on Day 5 was virtually abolished, despite the fact that the cell viability (on Day 5) was only moderately affected (Table 2). DISCUSSION The results reported here indicate that L1210 lymphoma cells or their culture supernatants markedly suppress the ilz vitro primary immune response of spleen cells against SRBC. The presence of 1% L1210 in the culture system or 1% of tumor cell supernatant markedly depressed the response, suggesting a potent inhibitory activity of tumor cells. Recently, suppressive activity of tumor cells and of their culture supernatants has been described for in vitro antibody responses (2, 46, 9-l 1) and for cytotoxic activity of lymphocytes ( 12). In these reports, it has been claimed that the cell viability of cultures containing tumor cells or their supernatants was not different from that of control cultures, or the cell viability was not mentioned at all. In contrast, our findings indicate that tumor cells or their supernatants are toxic for both lymphocytes and macrophages, although it must be considered that this may be a special property of L1210 cells compared to mastocytoma and other virus-transformed cells. However, the decrease of cell viability alone does not account for the marked decrease of PFC, since a preincubation of spleen cells with tumor cell supernatants for 1 hr resulted in an appreciable reduction of PFC without a marked loss of cell viability,
SHORT
COMMUNICATIONS
215
suggesting that a low number of nonproliferating cells, presumal)ly T cells, represented the primary target for the tumor cell supernatant. With regard to the nature of the supernatant factor, we found that mitomycintreated as well as X-irradiated and puromycin-treated L1210 cells were able to generate immunosuppressive effects. Since the factor was also nondialysable and heat labile (56°C for 30 min) , it may be assumed that a membrane constituent or several components with enzymatic activity could be the active factor. A similar factor which contains marker enzymes of membranous origin and fatty acids and which is toxic for thymocytes has recently been described in the ascites fluid of TA3-Ha tumor-bearing mice (13). Our findings that treatment of supernatant with uv light was not able to abolish the immunosuppressive potential of the factor would argue against a virus released from the transformed cells as being responsible for the immunosuppressive effect, as has been proposed for spleen cells of FLV-infected mice (5). Further studies to elucidate the nature of the immunosuppressive factor of L1210 cells are in progress. ACKNOWLEDGMENTS This work was supportedby Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 112). We thank Mrs. M. Ludwig and H. Savikurki for excellent technical assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Wong, A., Mankovitz, R., and Kennedy, J. C., Int. J. Cancer 13, 530, 1974. Kamo, I., Patel, Ch., Kateley, J., and Friedman, H., J. Ivzmuml. 114, 1749, 1975. DeLustro, F., and Argyris, B. F., Cell. I+~+mrnol. 21, 117, 1976. Roman, J. M., and Golub, E., J. Erfi. Med. 143, 482, 1976. Specter, S., Patel, Ch., and Friedman, H., J. iVat. Cancer Iusf. 56, 143, 1976: Kamo, I., Patel, Ch., Patel, N., and Friedman, H., J. Im+~unol. 115, 382, 1975. Mishell, R., and Dutton, R. W., J. Erfi. Med. 126, 423, 1967. Bullock, W. W., and Mijller, E., Ew. J. Im~zztrtol. 2, 514, 1972. Takada, A., Takada, Y., and Minowada, J., J. E.Q. Med. 140, 538, 1974. Cerny, J., and Stiller, R., J. 1+mmom1. 115, 943, 1975. Bendinelli, M., Kaplan, G. S., and Friedman, H., J. Xat. Caxrer Itz~t. 55, 1425, 1975. Eggers, A. E., and Wunderlich, J. R., J. I~r+mrrzoZ. 114, 1554, 1975. Nowotny, A., Grohsman, J., Abdelnoor, A., Rote, N., Yang, C., and Waltersdorff, Eur. J. I~wtzr~~ol. 4, 73, 1974.
R.,
II\tMuN0r,0GY 29, 210-215 (1977)
Suppression of in Vitro Primary Immune Cells and Their Culture Supernatant: Cytotoxic Effects R. P. HUGET, IIrparfmxt P.O.
Response by L1210 Evidence for
H.-D. FLAD,~ AND H. G. OPITZ
of Clhical PkJuiology, U~aizwsitg Box 4066, D-7900 Ulm, Germrl.v Rec&vd
August
of
C/m,
11, 19i6
L1210 cells and their culture supernatants were found to inhibit the generation of PFC in the i+z vitro primary immune response of spleen cells to SRBC. As few as 1% of L1210 cells and 1% of culture fluid were inhibitory. Inhibition of DNA or protein synthesis of L1210 cells did not abolish their immunosuppressive activity, excluding exhaustion of culture medium as a possible mechanism of inhibition of PFC. Heating of the supernahnt completely abrogated the suppressive effect and resulted in a marked increase of PFC. Daily evaluation of cell viability in the cultures revealed that, in the presence of L1210 and supernatants, the fraction of surviving cells is markedly reduced. We conclude that a direct cytotoxic effect on splenic lymphocytes and macrophages is the predominant immunosuppressive mechanism of L1210 cells and their culture supernatants.
INTRODUCTION Tumor cells or their culture supernatants have been shown to depress immune responsesin vifro (l-5). Thus, addition of mastocytoma cells leads to a reduction of plaque-forming cells in Mishell-Dutton-type cultures (2, 6). Furthermore, a diminution of rosette-forming cells after incubation of lymphocytes with supernatants from MCV tumor cell cultures and a suppression of DNA synthesis of mitogen-stimulated lymphocytes by tumor cells have been reported (7, 11) . In all of these experiments, it was claimed that the suppressive effect of tumor cells or their supernatants was not due to cytotoxicity, since cell viability was said to be comparable to control cultures. Rather, an inactivation of subpopulations of cells participating in the immune response or, alternatively, an activation of suppressor cells was proposed to be responsible for the suppression observed (6). We present evidence that L1210 mouse lymphoma cells or their culture supernatants markedly reduce cell viability of spleen cells. Cytotoxicity of both lymphocytes and macrophageswas observed. MATERIALS
AND
METHODS
AnimuZs. Mice of the inbred strains DBA/2, CBA/CA, weighing 20-25 g were used in different experiments. 1 Requests D-7900 Ulm,
for reprints Germany.
should
be addressed
to H.-D.
Flad,
University
C57B1/6, and Balb/c of Ulm,
P.0.
Box
4066,
210 Copyright All rights
@ 1977 by of reproduction
Academic Press, Inc. in any form reserved.
ISSK
0008
8749
SHORT
211
COMMUNICATIONS
Antigen. Sheep red blood cells (SRBC) obtained from Behringwerke, Marburg, Germany were stored in sterile Alsever’s solution. SRBC (10’) in 20 ~1 of balanced salt solution (BSS) were added to each culture. Spleen cell suspension. Spleens were removed under sterile conditions and disrupted by a loose-fitting glass homogenizer. After brief sedimentation, the suspension was washed three times with BSS and subsequently suspended in culture medium [RPM-1640 medium (Flow Laboratories) supplemented with penicillin (100 units/ml), streptomycin (100 rg/ml), L-glutamine (2 mM), 5% FCS (Seromed, Munich, Lot. Ko. 108)) and 2-mercaptoethanol (5 X 1e5 M) 1. Cell tiabilit~. Cell counts and viability tests were performed in Neubauer hemocytometers. For the determination of cell viability, 50 ~1 of the cell suspension in RPM-5% FCS was diluted with 1 ml of 0.5% trypan blue solution and the cells were immediately counted. Tuwzor cells. L1210 cells, a permanent lymphoblast cell line, originally obtained from Associated Biomedic Systems, Buffalo, New York, were maintained in RPMI1640 medium containing 157% FCS, penicillin, and streptomycin. Suspensions were grown in 30-ml Polystyrene flasks (Falcon Plastics, Oxnard, California). Under these conditions, the cells had a mean doubling time of 9-12 hr and reached a final density of about 2 x 10F cells/ml. Cells of this concentration were used throughout. L1210 cells were X-irradiated (Siemens Stabilipan, 0.5~mm Cu filter) with doses of 2000, 5000, and 8000 rad, respectively. In some experiments, the cells were treated with mitomycin C (50 pg/ml) or puromycin (30 rg/ml) for 30 min at 3/‘“C, washed three times with RPM, and used in the cultures. Sztpcrnatant fyonz L1210 cells. L1210 cells were allowed to grow for 48 hr to a density of about 2 X lo6 cells/ml. The 4S-hr culture medium was collected, centrifuged at 8OOg to remove cells and debris, and filtered through a Millipore filter TABLE Eifect
of Viable,
Expt.
1
2
3
0 L1210 b L1210
Irradiated, L1210
1
and I’uromycin-Treated I’FC culture
per f SD
4325 4380 3345 125
f 143 zt 848 f. 447 dz 87
None (control) 104, mite” 105, mite” 106, mite”
1800 1050 700 20
f f f f
None 106, 106, 106, 106,
None 104 105 106
cells treated cells treated
cells added
L1210
(control)
(control) 2000 rad 5000 rad 8000 rad puromycin with with
treatedb
50 pg/mI 30 pg/ml
Cells
on I’FC
per Culture
Percentage of control response
Percentage of viable cells (Day 5)
101.0 77.0 3.0
35 31 26 4
158 148 28 10
58.0 38.0 1.1
27 18 10 6
3942 f 244 40 f: 24 15f 7 27 f 14 0
1.0 0.3 0.7 0
30 9 10 9 7
of mitomycin C for 30 min at 37’C. for 30 min at 37°C.
212
SHORT
COMMUNICATIONS
Fmal concentration
of supernatant
(%I
FIG. 1. The effect of various L1210 culture and control supernatants on PFC response of SRBC-stimulated mouse spleen cell cultures. PFC were determined on Day 5 of culture and are expressed as percentage of PFC of control cuiture without supernatants (100%). The mean values of two cultures are given. X-X, native; A---& dialysed; O-0, uv light treated; O-O, L1210 supernatant heated at 56°C for 30 min; n-0, native supernatant from syngeneic spleen cell cultures (control).
(0.45 pm) before use. Supernatants of spleen cells were treated in the same way and served as controls. Cell culture. For the induction of a primary immune responsein vitro, the method of Mishell and Dutton (7) was used. The cultures were kept stationary. Assay of antibody synthesis. A modified hemolytic plaque assay was used to quantitate cellular antibody production in vitro (8). RESULTS Suppressive effect of LlZlO cells on in vitro PFC response. When L1210 cells are added to spleen cells, the PFC response of spleen cells against SRBC is inhibited (Table 1). As few as lo5 L1210 cells, i.e., 1% of the cells in culture, were able to reduce the number of PFC at Day 5. To exclude the possibility that proliferating L1210 cells deplete the tissue culture medium of essential nutrient ingredients, the proliferation of L1210 cells was inhibited by treatment of the cells with either X irradiation or mitomycin C. L1210 cells treated in this way were still able to suppressthe PFC (Table 1). Furthermore, inhibition of protein synthesis of L1210 cells by treating the cells with puromycin did not abolish the immunosuppressive activity of the cells. These findings suggested that the exhaustion of the culture medium by L1210 cells or the de nova synthesis of an inhibitory pro-
SHORT
COMMUNICATIONS
213
FIG. 2. The effect of L1210 culture supernatant on cell viability in Mishell-Dutton cultures. The mean values of two cultures are given. X-X, control (no supernatant) ; O-0, 1% ; A-A, 10% ; O-O, 205% supernatant (final concentration).
tein was not the mechanism by which L1210 cells exerted their inhibitory effect on PFC response. The inhibitory effect of L1210 cells on PFC was not restricted to cells of the same H2 type. Mitomycin-treated L1210 cells suppressed the generation of both syngeneic (DBA/2) and allogeneic PFC. Allogeneic or syngeneic mitomycin-treated spleen cells did not inhibit the PFC formation. Suppressive efect of L1210 culture supernatants. When L1210 cell culture supernatants were added to the spleen cultures in increasing concentrations, a marked decrease of PFC was observed (Fig. 1). Heating of L1210 supernatants for 30 min at 56°C not only completely abolished the PFC inhibitory activity, but rather increased the PFC response. The inhibitory activity was also found to be thermolabile when supernatants were stored for 5 days at room temperature. Treatment of culture supernatants with uv light overnight did not abolish their immunosuppressive activity on PFC responses.Furthermore, the activity was found to be nondialysable. Reduction of cell Viability by L1210 cells or LlZlO-derived culture supernatants. Cell viability of the spleen cultures was tested at daily intervals by trypan blue exclusion. Increasing the number of L1210 cells or the volume of L1210 culture supernatants lead to a marked reduction of cell viability (Table 1 and Fig. 2). It should be mentioned that, during the culture period, macrophages lost their adherence to the petri dishes and disintegrated so that no viable macrophages were found in these cultures on Day 5. Efiect of L1210 cells added at various days of culture on PFC. Attempts were made to define the culture period during which the cultured spleen cells are most sensitive to a suppressive action of L1210 cells on PFC response. When L1210 cells were added at 0, 24, 48, 72, and 96 hr of culture, a marked decreaseof PFC responseswas found during the first 48 hr (Table 2). Addition of L1210 cells at 72 hr or later reduced the PFC responseto a lesser degree, indicating that the tumor
214
SHORT
COMMUNICATIONS
TABLE Inhibition
Expt.
of PFC
Responses Effects
2
by L1210 Cells and Supernatants: and Effect of Preincubation
Condition
Time-Dependent
PFC”
Percentage of viable cells per culture (Day
1
2
Control (no I,1210 L1210 cells addeda 0 1
cells added) at day:
1820 f
147
30
10f 7 38 f 12 208 f 74 812 h 125
3 7 12 15 21
1560 rt 265 30f 17
40 28
0
Control (no preincubation) Preincubation”
5)
a PFC were determined on Day 5 of culture. The mean values of two cultures zt SD are given. b lo6 viable L1210 cells were added to lo7 SRBC-stimulated mouse spleen cells. c Spleen cells (lO?/ml) were incubated in 207, L1210 culture supernatant (final concentration) for 1 hr at 37’C, washed twice, and cultured with SRBC.
cells exerted their inhibitory effect either on the proliferative phase of antigensensitive cells or, possibly, on a population of helper cells. Effect of a preincubation of spleen cells m’tlz L1210 supcrnatant on PFC. To further define the target cell population, spleen cells were preincubated with 20% L1210 culture supernatant for 1 hr at 37°C washed, and then stimulated with SRBC. The PFC response on Day 5 was virtually abolished, despite the fact that the cell viability (on Day 5) was only moderately affected (Table 2). DISCUSSION The results reported here indicate that L1210 lymphoma cells or their culture supernatants markedly suppress the ilz vitro primary immune response of spleen cells against SRBC. The presence of 1% L1210 in the culture system or 1% of tumor cell supernatant markedly depressed the response, suggesting a potent inhibitory activity of tumor cells. Recently, suppressive activity of tumor cells and of their culture supernatants has been described for in vitro antibody responses (2, 46, 9-l 1) and for cytotoxic activity of lymphocytes ( 12). In these reports, it has been claimed that the cell viability of cultures containing tumor cells or their supernatants was not different from that of control cultures, or the cell viability was not mentioned at all. In contrast, our findings indicate that tumor cells or their supernatants are toxic for both lymphocytes and macrophages, although it must be considered that this may be a special property of L1210 cells compared to mastocytoma and other virus-transformed cells. However, the decrease of cell viability alone does not account for the marked decrease of PFC, since a preincubation of spleen cells with tumor cell supernatants for 1 hr resulted in an appreciable reduction of PFC without a marked loss of cell viability,
SHORT
COMMUNICATIONS
215
suggesting that a low number of nonproliferating cells, presumal)ly T cells, represented the primary target for the tumor cell supernatant. With regard to the nature of the supernatant factor, we found that mitomycintreated as well as X-irradiated and puromycin-treated L1210 cells were able to generate immunosuppressive effects. Since the factor was also nondialysable and heat labile (56°C for 30 min) , it may be assumed that a membrane constituent or several components with enzymatic activity could be the active factor. A similar factor which contains marker enzymes of membranous origin and fatty acids and which is toxic for thymocytes has recently been described in the ascites fluid of TA3-Ha tumor-bearing mice (13). Our findings that treatment of supernatant with uv light was not able to abolish the immunosuppressive potential of the factor would argue against a virus released from the transformed cells as being responsible for the immunosuppressive effect, as has been proposed for spleen cells of FLV-infected mice (5). Further studies to elucidate the nature of the immunosuppressive factor of L1210 cells are in progress. ACKNOWLEDGMENTS This work was supportedby Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 112). We thank Mrs. M. Ludwig and H. Savikurki for excellent technical assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Wong, A., Mankovitz, R., and Kennedy, J. C., Int. J. Cancer 13, 530, 1974. Kamo, I., Patel, Ch., Kateley, J., and Friedman, H., J. Ivzmuml. 114, 1749, 1975. DeLustro, F., and Argyris, B. F., Cell. I+~+mrnol. 21, 117, 1976. Roman, J. M., and Golub, E., J. Erfi. Med. 143, 482, 1976. Specter, S., Patel, Ch., and Friedman, H., J. iVat. Cancer Iusf. 56, 143, 1976: Kamo, I., Patel, Ch., Patel, N., and Friedman, H., J. Im+~unol. 115, 382, 1975. Mishell, R., and Dutton, R. W., J. Erfi. Med. 126, 423, 1967. Bullock, W. W., and Mijller, E., Ew. J. Im~zztrtol. 2, 514, 1972. Takada, A., Takada, Y., and Minowada, J., J. E.Q. Med. 140, 538, 1974. Cerny, J., and Stiller, R., J. 1+mmom1. 115, 943, 1975. Bendinelli, M., Kaplan, G. S., and Friedman, H., J. Xat. Caxrer Itz~t. 55, 1425, 1975. Eggers, A. E., and Wunderlich, J. R., J. I~r+mrrzoZ. 114, 1554, 1975. Nowotny, A., Grohsman, J., Abdelnoor, A., Rote, N., Yang, C., and Waltersdorff, Eur. J. I~wtzr~~ol. 4, 73, 1974.
R.,