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The role of T and suppressor cells in MTV-directed cellular immunity
Europ. J. Cancer Vol. 13, pp. 261-267. Pergamon Press 1977. Printed in Great Britain
The Role ofT and Suppressor Cells in MTV-Directed Cellular Immunity* PAULA CREEMERS and PETER BENTVELZEN Radiobiological Institute TNO, Rijswijk, The Netherlands A b s t r a c t - - T h e lymphocyte population from mice immunized with mammary tumour virus ( M T V ) which responds in vitro with blastogenesis in the presence of M T V , proves to be T cells. In mice bearing spontaneous MTV-induced mammary tumours, th# T cell reactivity was very often depressed. The depression was partly due to the presence of suppressor cells which have adherentproperties. These suppressor cells appear early during tumour growth and exert their greatest influence when the tumour weighs 0.5-1.5 g. In animals bearing larger tumours less suppressor cells werepresent and the hyporeactivity in the lymphocyte stimulation test must also be due to otherfactors.
plant lectins, can be inhibited by suppressor cells present in the spleen of animals bearing progressively growing tumours: Glaser et al. [3] demonstrated this in animals with Gross Leukemia Virus induced tumours. The same phenomenon was also shown in the Moloney Sarcoma Virus (MSV) system [4, 5]. The suppressor cells were thought to be macrophages. To further study the mechanisms of M T V directed cellular immunity in mice bearing primary m a m m a r y tumours, we again used the LS test. Evidence is presented that T cells are most likely responsible for the reactivity observed, and that the decline in blastogenesis with increasing tumour weight is caused, among others, by the presence of suppressor cells which have adherent properties.
INTRODUCTION WHF.N incubated with m a m m a r y tumour virus (MTV) leukocytes of mice with virally induced m a m m a r y tumours show virus-specific blastogenesis in the leukocyte stimulation (LS) test (preceding paper). Blair and Lane [1] state that MTV-specific cytotoxicity is a property of T cells and null cells which are recruited by an M T V specific factor produced by B cells. These null cells are found in the cell fraction that does not adhere to nylon wool, but they cannot be killed by antitheta serum and complement. However, despite these immune responses, primary m a m m a r y tumours show progressive growth. It is reported by Blair and Lane [1] that, as the tumours of the female mice increase tn size, the null cell cytotoxic activity is no longer detectable, but they do not mention a loss of T cell dependent cytotoxicity in the microcytotoxicity (MC) test. MTV-specific leukocyte stimulation and Leukocyte Adherence Inhibition (LAI) in mice decreased to almost zero when the tumour reached a weight of about 2 g (preceding paper). In human breast cancer, LAI reactivity decreased with increasing tumour burden [2]. It has been shown that some in vitro lymphocyte functions, as for instance stimulation by
MATERIAL AND METHODS BALB/cfC3H mice carrying MTV-S and GR, which spontaneously releases the endogenous MTV-P, were used. Tumours were induced in these mice by forced breeding. Male mice of 12 weeks of age were used as positive controls, they were immunized i.p. with 1 /~g disrupted alum precipitated virus and killed 5 days later. Male mice not older than 10 weeks served as normal controls, because they do not release endogenous M T V at that age. All mice were killed by cervical dislocation.
Accepted 15 October 1976. *This investigation was performed pursuant to N I H Contract NO1 CP 43328 with the National Cancer Institute, Division of Cancer Cause and Prevention, Viral Oncology, Department of Health, Education and Welfare, Bethesda, Md., U.S.A.
Virus
The standard strain of mouse m a m m a r y tumour virus (MTV-S) was isolated from 26/
262
Paula Creemers and Peter Bentvelzen
BALB/cfC3H m a m m a r y tumours, as described in the preceding paper. Again, virus content was estimated on the basis of the amount of protein, as measured by the Folin method. Before use as antigen, the virus was disrupted by repeated freezing and thawing.
LS test This test was carried out as described in the preceding paper. Blast formation was estimated by the incorporation of 2-14C_thymidine" In all experiments 10 . 4 mg M T V / m l was added; purified R L V (10 . 4 mg/ml) was used as a control throughout. The cultures were run in triplicate. Statistical significance was established by means of the two-tailed Student's t-test. Per cent stimulation was calculated according to the formula: a-bib x 100% in which a is the average counts/min (cpm) of the cultures incubated with M T V and b is that irt the cultures incubated with RLV. To detect whether the spleen of animals bearing tumours of different sizes contained suppressor cells, individual animals were tested: A T-cellenriched cell fraction was made (see below) and 0.5 x 106 spleen cells derived from the same animal in 0.2 ml medium supplemented with 20% foetal calf serum were added to each culture. Cultures to which normal spleen cells in the same concentration were added served as controls. All cultures were incubated with M TV. Per cent inhibition was calculated by: ( 1 - c / d ) x l 0 0 % , in which d i s the average cpm of the cultures containing T-cellenriched populations to which spleen cells containing suppressor cells were added and d is that in the cultures to which normal spleen cells in the same concentration were added.
Separation of cell subpopulations B-cell-enriched fractions were obtained by incubating 4 x 106 macrophage-depleted spleen cells with mouse antitheta serum and complement for 1 hr. The anti theta serum was made by injecting 2 x 107 thymocytes derived from C 3 H mice intraperitoneally into A K R mice. This was done six times with intervals of 14 days. Then the A K R mice were bled; the resulting ant tiheta serum killed 70% of C 3 H thymocytes up to a dilution of 1/16. As a source of complement guinea pig serum absorbed with agarose and stored in liquid nitrogen was used. The complement was active up to a dilution of 1/8. After treatment, the cells were washed and used for the LS test. T-cell-enriched cell populations were obtained by adherence on a nylon wool column [6]. After elution about 20% of the original
cell population was recovered. O f the eluted cell population, 60-70% could be killed with antitheta serum and complement, whereas 10% were shown to be Ig-positive B cells in direct membrane fluorescence studies. Adherent ceils were removed by incubation of spleen cells (4x106/ml) in Falcon tissue culture flasks (25 cm 2) during 2-3 hr. When adherent cells in combination with T-cellenriched populations were tested, the adherent cells present in 2.5 x l06 spleen cells were allowed to adhere in the tubes in which the LS-test was performed; the supernatant was then removed and the adherent ceils were washed three times with medium, after which T-cell-enriched cell fractions were added. The approximate number of adherent cells was estimated by subtracting the number of cells that did not adhere from the original concentration.
Membrane fluorescence For the detection of viral antigen that could be attached to the cells, adherent cells were grown in microtitre plates as is described by Sorg [7]. They were washed three times and then incubated for 1 hr with goat polyvalent anti-MTV-S serum at 37°C in a humidified atmosphere. After three washings in PBS, the cells were treated with fluorescein conjugated antigoat Ig serum (Nordic, Tilburg, The Netherlands) which was diluted 1:20 in PBS and incubated for 30 min. They were again washed three times prior to observation. Antibody attached to the cells was determined by treatment with fluorescein conjugated antimouse Ig serum, which was also used in a 1 : 20 dilution. RESULTS
MTV-specific blast forming cells In every experiment, the spleen cells used were derived from at least 3 positive control animals. If lymphocytes, T-cell-enriched and B-cell-enriched cell populations prepared from the same pool of spleen cells were compared, the T-cell-enriched fraction showed the highest stimulation as compared to the RLV-control, whereas the degree of blastogenesis observed with the same number of cells of the B-cellenriched cell population was not noteworthy (Table 1). Even if stimulation of the spleen cells was not significant, significant stimulatiort emerged when T-cell-enriched cell fractions were used. Stimulation of the T-cell-enriched fraction was not increased if adherent cells were added
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as c o m p a r e d to T cells to w h i c h a d h e r e n t cells d e r i v e d f r o m n o r m a l a n i m a l s were a d d e d ( T a b l e 2).
Suppressor cells I n b o t h G R a n d B A L B / c f C 3 H mice b e a r i n g large M T V - i n d u c e d t u m o u r s (2 g or larger), it was r e p e a t e d l y o b s e r v e d t h a t the T cell response was m a r k e d l y depressed if a d h e r e n t cells derived f r o m the s a m e a n i m a l s were a d d e d , as c o m p a r e d to cultures to w h i c h a d h e r e n t cells derived f r o m n o r m a l a n i m a l s w e r e a d d e d ( T a b l e 3). W h e n different concentrations of a d h e r e n t cells derived f r o m mice b e a r i n g large t u m o u r s were a d d e d to T-celle n r i c h e d fractions derived f r o m mice b e a r i n g v e r y small t u m o u r s , blast f o r m a t i o n in the presence o f M T V decreased g r a d u a l l y . T h i s was n o t the case w h e n a d h e r e n t ceils f r o m n o r m a l a n i m a l s w e r e a d d e d (Fig. 1). T o d e t e r m i n e w h e t h e r the cellular h y p o r e a c t i v i t y seen at a t u m o u r w e i g h t exceeding Table 2.
2 g (preceding p a p e r ) c a n be a t t r i b u t e d to suppressor cells, i n d i v i d u a l B A L B / c f C 3 H m i c e b e a r i n g t u m o u r s of v a r y i n g sizes were tested for the presence of such cells. T o T-celle n r i c h e d cell fractions f r o m such animals, spleen cells derived f r o m the s a m e a n i m a l were a d d e d as described u n d e r M a t e r i a l a n d M e t h o d s ; cultures to w h i c h n o r m a l spleen ceils were a d d e d in the s a m e c o n c e n t r a t i o n served as controls. Both sets of cultures were i n c u b a t e d w i t h M T V a n d p e r cent inhibition was calculated. T h e results are s h o w n in Fig. 2. F r o m the results, it is clear t h a t suppressor cells are present early d u r i n g the g r o w t h of the t u m o u r : the first significant i n h i b i t i o n was seen at a t u m o u r weight of 0.5 g. G r e a t e s t i n h i b i t i o n is seen at a t u m o u r weight b e t w e e n 0.5 a n d 1.5 g; also at a b o u t 4 g a considerable i n h i b i t i o n occurs. I n a n i m a l s b e a r i n g large t u m o u r s , spleen cell cultures c o n t a i n i n g suppressor cells sometimes g a v e significantly lower counts t h a n did
Proliferation of T-cell enriched cell populations of positive control animals, cultured with adherent cells derived from the same mice orfrom normal animals Experiment 1
Mouse strain BALB/cfC3H
Cell population
Counts/min± SE* in the presence of RLV
Counts/rain± SE in the presence of MTV
Per cent stimulation
Per cent stimulation
1-991 ± 321
3.047 ± 207
50]'
98~
2" 173 ± 207
3.001 ± 57
46§
74~
1.805 + 134
3.698 ± 325
105;
145~
2.157 ± 288
4.025± 178
87;
136;
T cells and adherent cells T cells and normal adherent cells T cells and adherent cells T cells and normal adherent cells
GR
Exp. 2
*SE: standard error of triplicate cultures.
]'Not significant. ~P < 0.010. §P < 0.025.
Table 3.
Influence of adherent cellsfrom normals or mice bearing tumours on T cell proliferation in the presence of M T V
Turnout weight (g)
T cells and adherent cells Counts/min + SE*
T cells and normal adherent ceils Counts/rain +_SE
Significance
Per cent inhibition
BALB/cfCSH
1.4 3.8
1.428+ 128 1.727 + 256
2.371 + 23 3.579 + 420
P < 0.001 P = 0.025
40 52
GR
2-8 4-2
1.025+ 76 1.810 + 190
2.084+234 3.534 + 337
0.025 < P < 0.050 0.005 < P < 0.010
51 49
Mouse strain
*SE: standard error of triplicate cultures.
T and Suppressor Cells in MTV-Directed Cellular Immunity the RLV-control; removing the suppressor cells gave equal values for M T V and R L V cultures; even then, no MTV-specific proliferation was obtained. This was not seen in animals bearing small tumours. This is demonstrated in Table 4. Average per cent stimulation of T-cell-enriched cell fractions of 9 animals with small tumours (average 0.9_+ 0-2 g) was 83.1, and varied from 23 to 154; for 10 animals tested with large tumours (average 3.5_+0.2g) this value was 15.2 with a variation of 0-62 %. Neither antigen nor antibody could be detected on the surface of adherent suppressor cells by membrane fluorescence studies.
o
x
40-
265
DISCUSSION From results with the fractions enriched for T-cells it can be concluded that the M T V specific blast formation observed in splenic cell cultures derived from BALB/cfC3H and G R mice primed with M T V is due primarily to activities of T cells. Blair and Lane [1] claim that a major part of m a m m a r y tumour cell killing in the MC test is T-cell dependent. T h e y also report on antibody-dependent null cell activity in this system. Such activity could not be detected in our leukocyte stimulation assays. Fractions enriched for B cells which also would contain null cells did not give any significant proliferation upon incubation with MTV. The addition of adherent cells derived from positive control animals did not enhance the blastogenesis of T cells as compared to when normal adherent cells were added. It is therefore not likely that macrophages are required
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20.
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Fig. I. Inhibition of MTV-specific response of T-cellenriched cell populations derived from 3 BALB/cfC3H mice bearing very small tumours (average 0"27 g) when varying amounts of adherent suppressor cells derived from 4 BALB[ cfC3H mice bearing large tumours (average 3 . 1 0 g ) are added. - normal adherent cells added. ..... suppressive adherent cells added. Vertical bars indicate standard error of triplicate cultures. This figure represents one out of two experiments.
tumor weight
in grams
Fig. 2. Percent inhibition of MTV-specific blastforming caused by suppressor cells from individual BALB/cfC3H mice bearing tumours of different sizes. 0 : inhibition is not significant 63 : inhibition is significant (P < 0.025).
Table 4. Inhibition of MTV-specific and of nonspecific T cell response by suppressor cells present in the spleens of BALB/cfC3H mice bearing spontaneous MTV-induced tumours
Tumour weight
Cell p o p u l a t i o n
C o u n t s / r a i n + SE* i n t h e presence of R L V
C o u n t s / r a i n + SE in t h e presence of M T V
Significance
Per cent stimulation
1.40
S p l e e n cells T-cell e n r i c h e d
3"464 + 127 2.650 + 664
4.272 + 106 6.747 + 380
0.010 < P < 0.005 0.005 < P < 0.001
24 155
2.90
Spleen cells T-cell e n r i c h e d
5.611 + 342 2"446 _+ 368
3.856 + 98 2-216 _+929
0.010 < P < 0-005 N.S.I"
-0
* S E : s t a n d a r d e r r o r of triplicate cultures. ~'N.S. : n o t significant.
266
Paula Creemers and Peter Bentvelzen
in large quantities for induction of specific T cell proliferation. In the previous paper the LS and the LAI tests gave parallelar results in the MTV-system. Holt [8] claims that the production of LAIF, which is responsible for the inhibition of adherence of neighbouring leukocytes, is T-cell-dependent. We have confirmed this for the MTV-system (Creemers, unpublished results). It seems that T cells play a major part in different systems of cellular reactivity to M T V . In the present study, it was demonstrated that the M T V specific response in tumour bearing animals was depressed by the presence of adherent cells. This suppression appeared not to be a tissue culture artifact, since increasing the cell density by adding an equal number of normal adherent cells had no inhibitory effect. The suppressive effect was already seen when about 0.5% adherent suppressive spleen cells were added to a responding cell population and about 5% was enough to reach the maximal inhibitory effect. These results are in accord with those of Glaser et al. [3] in rats bearing progressively growing Gross Leukemia Virus induced turnouts. Kirchner et al. [4, 5] found suppression of the P H A response of lymphocytes from mice bearing primary M S V induced tumours. Surprisingly, they found an inverse relationship between the development of suppressor cells and virus-specific cytotoxicity. It is therefore possible that no depression will be found in Tcell-dependent cytotoxicity in the MC test with increasing tumour weights. However, Eggers and Wunderlich [9] reported the presence of adherent suppressor cells in the MC test in methylcholanthrene induced tumour bearing animals. Kirchner et al. [4, 5] observed a good correlation between the growth pattern of MSV-
induced tumours and suppressor ceils were detected before the tumour had reached an appreciable size. In this system, suppressor cells could already be demonstrated at a tumour weight of 0.5 g. The greatest inhibitory effect was seen at a tumour weight between 0.5 and 1.5 g; there was also a considerable effect at 4 g. The decline in spleen cell reactivity when the tumour exceeds one gram in weight can be attributed to increasing suppressor cell activity. At 2 g, when the spleen cell activity in the blastogenesis test is at a minimum (preceding paper), the maximum suppressor cell activity has already disappeared. Removal of adherent cells did not result in a significant T cell reaction at heavier turnout weights in most cases. Thus, suppressor cells cannot be the only cause of the hyporeactivity observed at a large tumour burden; other factors which make the T cell unresponsive must be involved. Studies on this subject are now in progress in our laboratory. The mode of action of suppressor cells remains unclear. Membrane fluorescence studies for the detection of antigen or antibody on the surface of the suppressor cells gave negative results. Eggers and Wunderlich [9] found no evidence for the mediation of suppression by soluble factors. In this study it was not investigated whether the suppressor adherent cells are T lymphocytes [10]. Suppressor cells are found only in cases in which animals are overloaded with antigen and the possibility must be considered that suppressor cells are part of a tolerance inducing mechanism that is activated when the immune system is confronted with excess tumour antigen.
Acknowledgements--I should like to thank Miss A. J. H. den Hollander for technical assistance and Dr. J. Ouwehand for providing the purified virus.
REFERENCES 1. 2. 3.
4.
P.B. BLAIR and M. A. LANE,Non-T-cell killing of mammary tumour cells by spleen cells: secretion of antibody and recruitment of cells, or. Immunol. 115, 184 (1975). N. GROSSERand D. M. P. THOMPSON,Cell mediated antitumour immunity in breast cancer patients evaluated by antigen-induced leukocyte adherence inhibition in test tubes. Cancer Res. 35, 2571 (1975). M. GLASER,H. KIRCHNERand R. B. HERBERMAN,Inhibition of in vitro lymphoproliferative responses to tumour-associated antigens by suppressor ceils from rats bearing progressively growing Gross leukemia virus-induced tumours. Int. J. Cancer 16, 384 (1975). H. KIRCHNER, T. M. CHUSED, R. B. HERBERMANN, H. T. HOLDEN and D. H. LAVRIN, Evidence of suppressor cellactivityin spleens of mice bearing primary tumours induced by Moloney sarcoma virus,d. exp. Med. 139, 1473
(1974).
T and Suppressor Cells in MTV-Directed Cellular Immunity 5.
6. 7. 8. 9. 10.
E
H. KIRCHNER, A. V. MUCI~MORE,T. M. CHUSED, H. T. HOLDENand R. B. HERBRRMAN, Inhibition of proliferation of lymphomacells and T-lymphocytes by suppressor cells from spleens of tumour bearing mice. J. Immunol. 114, 206 (1975). 1V[. H. JuLiUS, E. SIMPSON and L. A. HERZENBERG,A rapid method for the isolation of functional thymus-derived lymphocytes. Europ. J. Immunol. 3, 645 (1973). C. SORG, A rapid micromethod for the detection of membrane-associated antigens on monolayer cells by indirect immunofluorescence. Eur. J. Immunol. 4, 832 (1974). P.G. HOLT, L. M. ROBERTS,P.J. FIMUF.Land D. KEAST,The LAI microtest: a rapid and sensitive procedure for the demonstration of cell-mediated immunity in vitro. J. immunol. Methods 8, 277 (1975). A.E. EGGERSand J. R. WUNDERLICH,Suppressor cells in tumour bearing mice capable of nonspeeific blocking of in vitro immunization against transplant antigens, or. Immunol. 114, 1554 (1975). D . R . WEBB and A. T. JAUIESON, Control of mitogen-induced transformation: characterization of a splenic suppressor cell and its mode of action. Cell. Immunol. 24, 45 (1976).
267
The Role ofT and Suppressor Cells in MTV-Directed Cellular Immunity* PAULA CREEMERS and PETER BENTVELZEN Radiobiological Institute TNO, Rijswijk, The Netherlands A b s t r a c t - - T h e lymphocyte population from mice immunized with mammary tumour virus ( M T V ) which responds in vitro with blastogenesis in the presence of M T V , proves to be T cells. In mice bearing spontaneous MTV-induced mammary tumours, th# T cell reactivity was very often depressed. The depression was partly due to the presence of suppressor cells which have adherentproperties. These suppressor cells appear early during tumour growth and exert their greatest influence when the tumour weighs 0.5-1.5 g. In animals bearing larger tumours less suppressor cells werepresent and the hyporeactivity in the lymphocyte stimulation test must also be due to otherfactors.
plant lectins, can be inhibited by suppressor cells present in the spleen of animals bearing progressively growing tumours: Glaser et al. [3] demonstrated this in animals with Gross Leukemia Virus induced tumours. The same phenomenon was also shown in the Moloney Sarcoma Virus (MSV) system [4, 5]. The suppressor cells were thought to be macrophages. To further study the mechanisms of M T V directed cellular immunity in mice bearing primary m a m m a r y tumours, we again used the LS test. Evidence is presented that T cells are most likely responsible for the reactivity observed, and that the decline in blastogenesis with increasing tumour weight is caused, among others, by the presence of suppressor cells which have adherent properties.
INTRODUCTION WHF.N incubated with m a m m a r y tumour virus (MTV) leukocytes of mice with virally induced m a m m a r y tumours show virus-specific blastogenesis in the leukocyte stimulation (LS) test (preceding paper). Blair and Lane [1] state that MTV-specific cytotoxicity is a property of T cells and null cells which are recruited by an M T V specific factor produced by B cells. These null cells are found in the cell fraction that does not adhere to nylon wool, but they cannot be killed by antitheta serum and complement. However, despite these immune responses, primary m a m m a r y tumours show progressive growth. It is reported by Blair and Lane [1] that, as the tumours of the female mice increase tn size, the null cell cytotoxic activity is no longer detectable, but they do not mention a loss of T cell dependent cytotoxicity in the microcytotoxicity (MC) test. MTV-specific leukocyte stimulation and Leukocyte Adherence Inhibition (LAI) in mice decreased to almost zero when the tumour reached a weight of about 2 g (preceding paper). In human breast cancer, LAI reactivity decreased with increasing tumour burden [2]. It has been shown that some in vitro lymphocyte functions, as for instance stimulation by
MATERIAL AND METHODS BALB/cfC3H mice carrying MTV-S and GR, which spontaneously releases the endogenous MTV-P, were used. Tumours were induced in these mice by forced breeding. Male mice of 12 weeks of age were used as positive controls, they were immunized i.p. with 1 /~g disrupted alum precipitated virus and killed 5 days later. Male mice not older than 10 weeks served as normal controls, because they do not release endogenous M T V at that age. All mice were killed by cervical dislocation.
Accepted 15 October 1976. *This investigation was performed pursuant to N I H Contract NO1 CP 43328 with the National Cancer Institute, Division of Cancer Cause and Prevention, Viral Oncology, Department of Health, Education and Welfare, Bethesda, Md., U.S.A.
Virus
The standard strain of mouse m a m m a r y tumour virus (MTV-S) was isolated from 26/
262
Paula Creemers and Peter Bentvelzen
BALB/cfC3H m a m m a r y tumours, as described in the preceding paper. Again, virus content was estimated on the basis of the amount of protein, as measured by the Folin method. Before use as antigen, the virus was disrupted by repeated freezing and thawing.
LS test This test was carried out as described in the preceding paper. Blast formation was estimated by the incorporation of 2-14C_thymidine" In all experiments 10 . 4 mg M T V / m l was added; purified R L V (10 . 4 mg/ml) was used as a control throughout. The cultures were run in triplicate. Statistical significance was established by means of the two-tailed Student's t-test. Per cent stimulation was calculated according to the formula: a-bib x 100% in which a is the average counts/min (cpm) of the cultures incubated with M T V and b is that irt the cultures incubated with RLV. To detect whether the spleen of animals bearing tumours of different sizes contained suppressor cells, individual animals were tested: A T-cellenriched cell fraction was made (see below) and 0.5 x 106 spleen cells derived from the same animal in 0.2 ml medium supplemented with 20% foetal calf serum were added to each culture. Cultures to which normal spleen cells in the same concentration were added served as controls. All cultures were incubated with M TV. Per cent inhibition was calculated by: ( 1 - c / d ) x l 0 0 % , in which d i s the average cpm of the cultures containing T-cellenriched populations to which spleen cells containing suppressor cells were added and d is that in the cultures to which normal spleen cells in the same concentration were added.
Separation of cell subpopulations B-cell-enriched fractions were obtained by incubating 4 x 106 macrophage-depleted spleen cells with mouse antitheta serum and complement for 1 hr. The anti theta serum was made by injecting 2 x 107 thymocytes derived from C 3 H mice intraperitoneally into A K R mice. This was done six times with intervals of 14 days. Then the A K R mice were bled; the resulting ant tiheta serum killed 70% of C 3 H thymocytes up to a dilution of 1/16. As a source of complement guinea pig serum absorbed with agarose and stored in liquid nitrogen was used. The complement was active up to a dilution of 1/8. After treatment, the cells were washed and used for the LS test. T-cell-enriched cell populations were obtained by adherence on a nylon wool column [6]. After elution about 20% of the original
cell population was recovered. O f the eluted cell population, 60-70% could be killed with antitheta serum and complement, whereas 10% were shown to be Ig-positive B cells in direct membrane fluorescence studies. Adherent ceils were removed by incubation of spleen cells (4x106/ml) in Falcon tissue culture flasks (25 cm 2) during 2-3 hr. When adherent cells in combination with T-cellenriched populations were tested, the adherent cells present in 2.5 x l06 spleen cells were allowed to adhere in the tubes in which the LS-test was performed; the supernatant was then removed and the adherent ceils were washed three times with medium, after which T-cell-enriched cell fractions were added. The approximate number of adherent cells was estimated by subtracting the number of cells that did not adhere from the original concentration.
Membrane fluorescence For the detection of viral antigen that could be attached to the cells, adherent cells were grown in microtitre plates as is described by Sorg [7]. They were washed three times and then incubated for 1 hr with goat polyvalent anti-MTV-S serum at 37°C in a humidified atmosphere. After three washings in PBS, the cells were treated with fluorescein conjugated antigoat Ig serum (Nordic, Tilburg, The Netherlands) which was diluted 1:20 in PBS and incubated for 30 min. They were again washed three times prior to observation. Antibody attached to the cells was determined by treatment with fluorescein conjugated antimouse Ig serum, which was also used in a 1 : 20 dilution. RESULTS
MTV-specific blast forming cells In every experiment, the spleen cells used were derived from at least 3 positive control animals. If lymphocytes, T-cell-enriched and B-cell-enriched cell populations prepared from the same pool of spleen cells were compared, the T-cell-enriched fraction showed the highest stimulation as compared to the RLV-control, whereas the degree of blastogenesis observed with the same number of cells of the B-cellenriched cell population was not noteworthy (Table 1). Even if stimulation of the spleen cells was not significant, significant stimulatiort emerged when T-cell-enriched cell fractions were used. Stimulation of the T-cell-enriched fraction was not increased if adherent cells were added
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Paula Creemers and Peter Bentvelzen
as c o m p a r e d to T cells to w h i c h a d h e r e n t cells d e r i v e d f r o m n o r m a l a n i m a l s were a d d e d ( T a b l e 2).
Suppressor cells I n b o t h G R a n d B A L B / c f C 3 H mice b e a r i n g large M T V - i n d u c e d t u m o u r s (2 g or larger), it was r e p e a t e d l y o b s e r v e d t h a t the T cell response was m a r k e d l y depressed if a d h e r e n t cells derived f r o m the s a m e a n i m a l s were a d d e d , as c o m p a r e d to cultures to w h i c h a d h e r e n t cells derived f r o m n o r m a l a n i m a l s w e r e a d d e d ( T a b l e 3). W h e n different concentrations of a d h e r e n t cells derived f r o m mice b e a r i n g large t u m o u r s were a d d e d to T-celle n r i c h e d fractions derived f r o m mice b e a r i n g v e r y small t u m o u r s , blast f o r m a t i o n in the presence o f M T V decreased g r a d u a l l y . T h i s was n o t the case w h e n a d h e r e n t ceils f r o m n o r m a l a n i m a l s w e r e a d d e d (Fig. 1). T o d e t e r m i n e w h e t h e r the cellular h y p o r e a c t i v i t y seen at a t u m o u r w e i g h t exceeding Table 2.
2 g (preceding p a p e r ) c a n be a t t r i b u t e d to suppressor cells, i n d i v i d u a l B A L B / c f C 3 H m i c e b e a r i n g t u m o u r s of v a r y i n g sizes were tested for the presence of such cells. T o T-celle n r i c h e d cell fractions f r o m such animals, spleen cells derived f r o m the s a m e a n i m a l were a d d e d as described u n d e r M a t e r i a l a n d M e t h o d s ; cultures to w h i c h n o r m a l spleen ceils were a d d e d in the s a m e c o n c e n t r a t i o n served as controls. Both sets of cultures were i n c u b a t e d w i t h M T V a n d p e r cent inhibition was calculated. T h e results are s h o w n in Fig. 2. F r o m the results, it is clear t h a t suppressor cells are present early d u r i n g the g r o w t h of the t u m o u r : the first significant i n h i b i t i o n was seen at a t u m o u r weight of 0.5 g. G r e a t e s t i n h i b i t i o n is seen at a t u m o u r weight b e t w e e n 0.5 a n d 1.5 g; also at a b o u t 4 g a considerable i n h i b i t i o n occurs. I n a n i m a l s b e a r i n g large t u m o u r s , spleen cell cultures c o n t a i n i n g suppressor cells sometimes g a v e significantly lower counts t h a n did
Proliferation of T-cell enriched cell populations of positive control animals, cultured with adherent cells derived from the same mice orfrom normal animals Experiment 1
Mouse strain BALB/cfC3H
Cell population
Counts/min± SE* in the presence of RLV
Counts/rain± SE in the presence of MTV
Per cent stimulation
Per cent stimulation
1-991 ± 321
3.047 ± 207
50]'
98~
2" 173 ± 207
3.001 ± 57
46§
74~
1.805 + 134
3.698 ± 325
105;
145~
2.157 ± 288
4.025± 178
87;
136;
T cells and adherent cells T cells and normal adherent cells T cells and adherent cells T cells and normal adherent cells
GR
Exp. 2
*SE: standard error of triplicate cultures.
]'Not significant. ~P < 0.010. §P < 0.025.
Table 3.
Influence of adherent cellsfrom normals or mice bearing tumours on T cell proliferation in the presence of M T V
Turnout weight (g)
T cells and adherent cells Counts/min + SE*
T cells and normal adherent ceils Counts/rain +_SE
Significance
Per cent inhibition
BALB/cfCSH
1.4 3.8
1.428+ 128 1.727 + 256
2.371 + 23 3.579 + 420
P < 0.001 P = 0.025
40 52
GR
2-8 4-2
1.025+ 76 1.810 + 190
2.084+234 3.534 + 337
0.025 < P < 0.050 0.005 < P < 0.010
51 49
Mouse strain
*SE: standard error of triplicate cultures.
T and Suppressor Cells in MTV-Directed Cellular Immunity the RLV-control; removing the suppressor cells gave equal values for M T V and R L V cultures; even then, no MTV-specific proliferation was obtained. This was not seen in animals bearing small tumours. This is demonstrated in Table 4. Average per cent stimulation of T-cell-enriched cell fractions of 9 animals with small tumours (average 0.9_+ 0-2 g) was 83.1, and varied from 23 to 154; for 10 animals tested with large tumours (average 3.5_+0.2g) this value was 15.2 with a variation of 0-62 %. Neither antigen nor antibody could be detected on the surface of adherent suppressor cells by membrane fluorescence studies.
o
x
40-
265
DISCUSSION From results with the fractions enriched for T-cells it can be concluded that the M T V specific blast formation observed in splenic cell cultures derived from BALB/cfC3H and G R mice primed with M T V is due primarily to activities of T cells. Blair and Lane [1] claim that a major part of m a m m a r y tumour cell killing in the MC test is T-cell dependent. T h e y also report on antibody-dependent null cell activity in this system. Such activity could not be detected in our leukocyte stimulation assays. Fractions enriched for B cells which also would contain null cells did not give any significant proliferation upon incubation with MTV. The addition of adherent cells derived from positive control animals did not enhance the blastogenesis of T cells as compared to when normal adherent cells were added. It is therefore not likely that macrophages are required
I I
t
80-
u
3o
g .'z ..o
60
20.
®
40. 10.
®
®
® ®
o-
20. per cent adherent cells added
Fig. I. Inhibition of MTV-specific response of T-cellenriched cell populations derived from 3 BALB/cfC3H mice bearing very small tumours (average 0"27 g) when varying amounts of adherent suppressor cells derived from 4 BALB[ cfC3H mice bearing large tumours (average 3 . 1 0 g ) are added. - normal adherent cells added. ..... suppressive adherent cells added. Vertical bars indicate standard error of triplicate cultures. This figure represents one out of two experiments.
tumor weight
in grams
Fig. 2. Percent inhibition of MTV-specific blastforming caused by suppressor cells from individual BALB/cfC3H mice bearing tumours of different sizes. 0 : inhibition is not significant 63 : inhibition is significant (P < 0.025).
Table 4. Inhibition of MTV-specific and of nonspecific T cell response by suppressor cells present in the spleens of BALB/cfC3H mice bearing spontaneous MTV-induced tumours
Tumour weight
Cell p o p u l a t i o n
C o u n t s / r a i n + SE* i n t h e presence of R L V
C o u n t s / r a i n + SE in t h e presence of M T V
Significance
Per cent stimulation
1.40
S p l e e n cells T-cell e n r i c h e d
3"464 + 127 2.650 + 664
4.272 + 106 6.747 + 380
0.010 < P < 0.005 0.005 < P < 0.001
24 155
2.90
Spleen cells T-cell e n r i c h e d
5.611 + 342 2"446 _+ 368
3.856 + 98 2-216 _+929
0.010 < P < 0-005 N.S.I"
-0
* S E : s t a n d a r d e r r o r of triplicate cultures. ~'N.S. : n o t significant.
266
Paula Creemers and Peter Bentvelzen
in large quantities for induction of specific T cell proliferation. In the previous paper the LS and the LAI tests gave parallelar results in the MTV-system. Holt [8] claims that the production of LAIF, which is responsible for the inhibition of adherence of neighbouring leukocytes, is T-cell-dependent. We have confirmed this for the MTV-system (Creemers, unpublished results). It seems that T cells play a major part in different systems of cellular reactivity to M T V . In the present study, it was demonstrated that the M T V specific response in tumour bearing animals was depressed by the presence of adherent cells. This suppression appeared not to be a tissue culture artifact, since increasing the cell density by adding an equal number of normal adherent cells had no inhibitory effect. The suppressive effect was already seen when about 0.5% adherent suppressive spleen cells were added to a responding cell population and about 5% was enough to reach the maximal inhibitory effect. These results are in accord with those of Glaser et al. [3] in rats bearing progressively growing Gross Leukemia Virus induced turnouts. Kirchner et al. [4, 5] found suppression of the P H A response of lymphocytes from mice bearing primary M S V induced tumours. Surprisingly, they found an inverse relationship between the development of suppressor cells and virus-specific cytotoxicity. It is therefore possible that no depression will be found in Tcell-dependent cytotoxicity in the MC test with increasing tumour weights. However, Eggers and Wunderlich [9] reported the presence of adherent suppressor cells in the MC test in methylcholanthrene induced tumour bearing animals. Kirchner et al. [4, 5] observed a good correlation between the growth pattern of MSV-
induced tumours and suppressor ceils were detected before the tumour had reached an appreciable size. In this system, suppressor cells could already be demonstrated at a tumour weight of 0.5 g. The greatest inhibitory effect was seen at a tumour weight between 0.5 and 1.5 g; there was also a considerable effect at 4 g. The decline in spleen cell reactivity when the tumour exceeds one gram in weight can be attributed to increasing suppressor cell activity. At 2 g, when the spleen cell activity in the blastogenesis test is at a minimum (preceding paper), the maximum suppressor cell activity has already disappeared. Removal of adherent cells did not result in a significant T cell reaction at heavier turnout weights in most cases. Thus, suppressor cells cannot be the only cause of the hyporeactivity observed at a large tumour burden; other factors which make the T cell unresponsive must be involved. Studies on this subject are now in progress in our laboratory. The mode of action of suppressor cells remains unclear. Membrane fluorescence studies for the detection of antigen or antibody on the surface of the suppressor cells gave negative results. Eggers and Wunderlich [9] found no evidence for the mediation of suppression by soluble factors. In this study it was not investigated whether the suppressor adherent cells are T lymphocytes [10]. Suppressor cells are found only in cases in which animals are overloaded with antigen and the possibility must be considered that suppressor cells are part of a tolerance inducing mechanism that is activated when the immune system is confronted with excess tumour antigen.
Acknowledgements--I should like to thank Miss A. J. H. den Hollander for technical assistance and Dr. J. Ouwehand for providing the purified virus.
REFERENCES 1. 2. 3.
4.
P.B. BLAIR and M. A. LANE,Non-T-cell killing of mammary tumour cells by spleen cells: secretion of antibody and recruitment of cells, or. Immunol. 115, 184 (1975). N. GROSSERand D. M. P. THOMPSON,Cell mediated antitumour immunity in breast cancer patients evaluated by antigen-induced leukocyte adherence inhibition in test tubes. Cancer Res. 35, 2571 (1975). M. GLASER,H. KIRCHNERand R. B. HERBERMAN,Inhibition of in vitro lymphoproliferative responses to tumour-associated antigens by suppressor ceils from rats bearing progressively growing Gross leukemia virus-induced tumours. Int. J. Cancer 16, 384 (1975). H. KIRCHNER, T. M. CHUSED, R. B. HERBERMANN, H. T. HOLDEN and D. H. LAVRIN, Evidence of suppressor cellactivityin spleens of mice bearing primary tumours induced by Moloney sarcoma virus,d. exp. Med. 139, 1473
(1974).
T and Suppressor Cells in MTV-Directed Cellular Immunity 5.
6. 7. 8. 9. 10.
E
H. KIRCHNER, A. V. MUCI~MORE,T. M. CHUSED, H. T. HOLDENand R. B. HERBRRMAN, Inhibition of proliferation of lymphomacells and T-lymphocytes by suppressor cells from spleens of tumour bearing mice. J. Immunol. 114, 206 (1975). 1V[. H. JuLiUS, E. SIMPSON and L. A. HERZENBERG,A rapid method for the isolation of functional thymus-derived lymphocytes. Europ. J. Immunol. 3, 645 (1973). C. SORG, A rapid micromethod for the detection of membrane-associated antigens on monolayer cells by indirect immunofluorescence. Eur. J. Immunol. 4, 832 (1974). P.G. HOLT, L. M. ROBERTS,P.J. FIMUF.Land D. KEAST,The LAI microtest: a rapid and sensitive procedure for the demonstration of cell-mediated immunity in vitro. J. immunol. Methods 8, 277 (1975). A.E. EGGERSand J. R. WUNDERLICH,Suppressor cells in tumour bearing mice capable of nonspeeific blocking of in vitro immunization against transplant antigens, or. Immunol. 114, 1554 (1975). D . R . WEBB and A. T. JAUIESON, Control of mitogen-induced transformation: characterization of a splenic suppressor cell and its mode of action. Cell. Immunol. 24, 45 (1976).
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