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Spontaneous tumor rejection is not always due to a complete cellular rejection
Exp. Pathol. 31, 33-38 (1987) VEB Gustav Fischer Verlag Jena Department of Experimental Pathology, State University
Utrec~t,
Utrecht, The Netherlands
Spontaneous tumor rejection is not always due to a complete cellular rejection By H. F. J. DULLENS, S. SCHAKENRAD and W. DEN OTTER With one figure
Address for correspondence: Dr. H. F. J. DULLENS, State University of Utrecht, Department of Experimental Pathology, Pasteurstraat 2, 3511 HX Utrecht, The Netherlands Ke y W 0 r d s: tumor rejection; cellular immune reaction; antibody dependent cytotoxicity; macrophage cytotoxicity; anti-tumor cytotoxicity Ab brevia tions: ADCC = antibody dependent cytotoxicity; BCG = Bacillus Calmette Guerin; CI = cytotoxicity index; FBS = fetal bovine serum; PBS = phosphate buffered saline; ip = intraperitoneal(ly); sc = subcutaneous(ly)
Summary
In this paper we have studied whether there is a correlation between efficacy of hosts to reject tumor ceIls spontaneously and the induction of cytotoxic macrophages in these recipients due to injection (immunization) with tumor cells. Results show that there is no cause-effect relationship between macrophage cytotoxicity and tumor rejection in vivo. This lack of cause-effect relationship is greatly influenced by differences in immunogenicity between the various tumor cell lines as well as by the release of tumor-factors by some of these tumors. Introduction
Nowadays it is widely believed, that tumors are at least potentially immunogenic in the original host (14), This means that immune reactions may be induced, directed against the tumor cells. Such immune reactions in vivo comprise a number of interacting reactions. In this sequence of events among others, macrophages seem to play an important role. This supposition is supported by the finding of VAN LOVEREN et al. (16) who found that adoptive transfer of immune spleen lymphocytes into tumor bearing hosts only resulted in eradication of the tumor cells if the host macrophage population was intact. This suggests that these macrophages are obviously necessary for the killing of the tumor cells. Probably, a specific macrophage arming factor (6, 10) produced by sensitized lymphocytes upon contact with the specific antigen and able to induce macrophage cytotoxicity (3) is involved. The results of VAN LOVEREN et al. (16) further suggest that sensitized lymphocytes cannot only render macrophagcs cytotoxic in vitro (10-12) but, also may activate host macrophages in vivo to become tumor cell killers. Extensive studies during the past decade, both clinical and experimental, have shown that many malignancies can strongly influence cell mediated immune responses as weIl as antibody formation (7), Sevl'ral authors suggest that these effects might be mediated by soluble factors released by tUlllor c('lls (1,9). In this paper we have studil'd whether there is a correlation between efficacy of recipient mice to rejeet tUlllor eells spontaneously and the induction of cytotoxic macrophages due to the injection of these tumor eells. The infJuence(s) of immunogenicity and production of tumor factors by the various tumor cell lines are discussed. 3
33
Table 1. Tumors used in this stillty Tumor
Strain of origin
H-2 type
8L2 lymphosarcoma L5178Y lymphosarcoma P815X2 mastocytoma L1210 lymphosarcoma AK8L2 lymphosarcoma MOPC 195 plasmacytoma WEHI/3 myelomonocytir leukemia TLX9 lymphosarcoma EL4 lymphosarcoma
DBA/2 DBA/:? DBA/2 DBA/:?
d d d d
BALB/c BA LB/c
d
AKR
C57BL C57BL
k d b b
Materials and .11 ethods Mice Inbred DBA/:?, CBA, BALB/c and AKR mice were obtained from CPB-TNO, Zeist, The Netherlands. Inbred C57BL/10ScCr were obtained from GL Bomholtgard, Denmark. Male and female mice were used at an age of 2-3 months. Tumors See table 1. All tumors grow ip as ascitic tumors, and were maintained by weekly ip passage of 106 cells. I'll vitro cultures were prppared in growth medium: Fischer's medium supplemented with 10 % fetal bovine serum (FBS). The initial concentration was 2 x 1Q5 cells/ml in a total volume of 20 ml. Collection of cells from the peritoneal cavity Peritoneal cells werp collected by washing the peritoneal cavity with 5 mt Fischer's medium. Cells were centrifuged and fl'sllspended in Fischer's medium. Rejection expPfiments Groups of 5 CBA mice earh were injected ip with 1Q5, 106 , 107 or 5 x 10 7 tumor cells and the survival time of the mice was established. Experiments were done twice. Immunization CBA mice were immunized against the non-CBA derived tumors by one ip injection with the maximal dose of tumor cells that was rejected in vivo. Peritoneal macrophages were collected 10 d later. Macrophage cytotoxicity Procedures for preparation of macrophage monolayers and the determination of their in vivo cytotoxicity have been described in detail elsewhere (3). The cytotoxicity was measured after 24 h by counting the remaining viable tumor cells and is expressed as: (1 - TIN) x 100, where CI = Cytotoxicity Index; N = number of viable tumor cells in the controls; T = the number of viable tumor cells in the tests. I'll vitro tests were done at least twice in threefold. Tumor factors P815-factor: A low molecular peptide factor released by P815 mastocytoma cells was isolated and used as has been described in detail by our group elsewhere (4,5). WEHI-factor: BALB/c mice were injected ip with 106 WEHI/3 tumor cells. Seven days after injection the peritoneal cavities of the tumor bearing mice were washed with Fischer's medium. The tumor cell suspension was centrifuged for 20 min (15,000 rpm) and the supernatant was decanted, filtered through a millipore filter (pore size 0.45,um) and used in the experiments.
Results Tum 0 r
r e j ec t ion in vivo
CBA mice (haplotype H-2 k ) were able to reject high doses of the DBAj2 (H-2 d ) derived tumors P815 and L5178Y, the BALBjc (H-2 d ) derived tumor MOPC195 and WEHIj3, and the AKR (H-2 k ) derived tumor AK8L2 (table 2). On the other hand the DBAj2 derived tumors 8L2 and \,1210 as well as both C57BL (H-2 b ) derived tumors EL4 and TLX9 were only rejerted when rrlatively low numbers of tumor cells (about lOS) had been injected
34
Exp. Pathol. 31 (1987) 1
Table 2. Rejection of various tumor cell lines by normal non-stimulated CBA mice (haplotype H-2 k ) Number of tumor cells rejected after ip injection
Tumor-origin (haplotype)
8L2 P815 L5178Y L1210 MOPC 195 WEHIj3 AK8L2 EL4 TLX9
+++: ++: +: ±:
DBAj2 (H-2 DBAj2 (H-2 d ) DBAj2 (H-2 d ) DBAj2 (H-2 d ) BALBjc (H-2 d ) BALBjc (H-2 d ) AKR (H-2 k ) C57BL (H-2 b ) C57BL (H-2 b ) d)
105
lOG
+++ +++ +++ +++ +++ +++ +++ ++ ++
±
+++ +++ +++ +++ +++ +++ +
10'
5x10'
+++ +++ ++ +++ +++ +++
+++ +++ ± +++ +++ +++
±
All mice survived 100 d. 100-75 % of the number of mice survived 100 d. 75-50 % of the number of mice survived 100 d. 50-25 % of the number of mice survived 100 d. 25 %-none of the mice survived.
Ten mice were used per observation (the experiment was done twice in fivefold).
(table 2). Since the AKSL2 tumor cells and the CBA mice both have the haplotype H-2 k we would have expected that AKSL2 cells would not be easily rejected by CBA mice. Obviously differences in other antigens than the MHC antigens between CBA and AKSL 2 are strong enough to evoke the complete rejection of 5 x la' tumor cells. Spontaneous anti-tumor cytotoxicity of macrophages in vitro Spontaneous cytotoxicity of normal non-elicited peritoneal CBA macrophages was observed when these macrophages were incubated with the DBAj2 derived P815 and L5178Y tumors or with the BALBjc derived WEHIj3 tumor. No spontaneous macrophage cytotoxicity was found against any of the other tumor lines tested (fig. 1; black bars). It is known from literature that P815 tumor cells (4, 5) produce a factor which is responsible for the induction of macrophage cytotoxicity. To study whether WEHIj3 cells do produce a similar factor the cell free exudate obtained from BALBjc mice bearing the WEHIj 3 tumor ip was tested for its ability to induce macrophage cytotoxicity. Normal CBA macrophages were incubated with the cell free exudate in vitro for 6 h. The exudate was washed off and the macrophages were challenged with a tumor (SL2) against which no spontaneous cytotoxicity was expressed originally. When the macrophages had been incubated with the WEHIj3 cell free exudate a Cytotoxicity Index of 5 ± 1 against SL2 was observed. If the SL2 tumor cells were pre-incubated with the cell free WEHIj3 exudate, washed with medium and then added to the maerophages, a Cytotoxicity Index of 23 ± 2 could be measured. Anti-tumor eytotoxicity of macrophages in vitro after in vivo immunization Groups of CBA miee were immunized against the different tumor eell hnes by one ip injeetion with the maximum number of cells whieh could be rejected spontaneously (see table 2). Peritoneal macrophages were collected 10 days later and their eytotoxicity was tested against tumor eell lines used for immunization. High levels of cytotoxicity were found against the DBAj2 derived SL2, the BALBjc derived MOPC195 and WEHIj3, theAKR derived AKSL2 and the C57BL derived ErA tumor (Cytotoxicity Indices ranged from 59-74; fig. 1, open bars). Significantly lower levels of cytotoxieity (CI ranged from 34-40) were found against the DBAj2 derived P815 and L5178Y tumor as well as against the C57BL derived TLX9 tumor (fig. 1). However it should be stressed that eompared with the spontaneous cytotoxicity expressed against P81 5 and L5178Y tumor cells no signifieant increase in cytotoxicity due to immunization was obtained (fig. 1). No cytotoxieity could be measured against 11210 tumor cells. 3* Exp. Pathol. 31 (1987) 1 35
100
.
L
~
)(
1&1
0
-z
SO
1
>
~
I.)
x 0
~
0
~
>
I.)
0
•
S L2
P81S
LS178Y
-40
•
..
MO PC-19S
-
W Hp
•
AKSL2
•
EL4
.
TLX9
TUMOR CELL L1NES-.
Fig. 1. Cytotoxicity of (a) normal non-elicited peritoneal macrophages (.) and (b) immune-CBA peritoneal macrophages (0) against various tumor cell lines. Mice had been immunized by one ip injection with the maximum number of tumor cells which was sublethal (see also table 2). Peritoneal macrophages were collected 10 days after immunization. The cytotoxicity was measured after incubation of the macrophages with tumor cells for 24 h. In most cases normal macrophages expressed "negative-cytotoxicity" (*) against the various tumor cell lines (CI varied from -40 till -120). The experiments were donr twice in triplicate.
DiscuSS1:0n As shown in this paper the AK8L2 tumor (haplotype H-2 k ) is easily rejected in CBA mice (also haplotype H-2 k ) whereas for instance tumors like TLX9 (H-2 b ) and 8L2 (H-2 d ) are Jess easily rejected despite their allogenicity (table 2). The fact that AK8L2 cells are easily rejected in CBA mice suggests that differences in minor-histocompatibility antigens between the AK8L2 cells and the CBA host may be involved, as well as neoantigens (tumor associated antigens) present either as a consequenee of the malignant transformation or as a consequenee of additional histo(in)compatibilities developing with continued transplantation of the eell line (13). Sueh incompatibilities have been found to occur even when the tumor is eontinuously transplanted in syngeneie mice. An example of weh a tumor with "additional histo(in)compatibility antigens" is the 8L2 tumor: ROBINSON and SCHIRRMACHER (15) have shown that 8L2 cells have "alien" H-2 k antigens on their surfaee. This may explain why 8L2 cells are not easily rejected by CBA miee whereas they arc easily rejected by allogeneic C57BL (H-2 b ) mice (2). As shown in this paper immunization with tumor eells does not always result in highly cytotoxic macrophages. The latter are only indueed if tumor eells are used for immunization against which the macrophages do not express "spontaneous" cytotoxieity (fig. I). When a high spontaneous cytotoxieity of the macrophages is present, obviously many tumor cells are killed directly, leaving onl~- II "mall number of tumor cell" to evoke a specific. immune reaction. Tn other words, when tumor eells are not immediately killed by the macrophages (spontaneous cytotoxic ma("rophages are absent) they might be present for a longer time and divide, which results in an inerease in available antigen. This may lead to a strong immune reaction resulting in c~-totoxi(" maerophages. On the other hand it ("an not bc excluded that the differences in induction of cytotoxic macrophages b~- the \'1uioLl" tumor cell lines might be due to differences in activation of the type of effector ",-"tp111 ". So for instancp. instead of (cytotoxic) macrophages, NK-cells might be activated.
36
Exp. Pathol. 31 (1987) 1
The phenomenon of spontaneous cytotoxicity itself has been described to be due to subclinical infections (12). However in the experiments described here cytotoxicity was not due to infections. In case of the P815 cells the activation of the macrophages is due to the production of a tumor factor: DULLENS and DEN OTTER (4, 5) have described a factor which could be isolated from P815-tumor ascites, supernatant from P815 in vitro cultures as well as from P815-homogenates. The factor has been characterized as a low molecular peptide which could render non-stimulated macrophages non-specifically cytotoxic (5). Similarly, GEMSA et al. (8) have described a product present in the L5178Y induced ascites which also induces cytostatic activity of peritoneal macrophages. This particular product however, could not be shown to be present in in vitro tumor culture supernatants, or in tumor homogenates. This suggests that in the latter case the factor responsible for the activation of the macrophages in their test system as well as in our system may be (tumor)-host derived. Obviously also WEHI/3 cells produce a product resulting in "spontaneous" macrophage tumoricidal activity (this paper). That is, in the WEHI/3 induced ascites fluid a product can be detected which after preincubation with tumor cells leads to an enhanced killing of these tumor cells by macrophages l:n m:tro. One likely possibility to explain this phenomena would be an ADCC mechanism. Experiments to characterize this product and to establish its biological activities are in progress. The significance of spontaneous macrophage cytotoxicity for the induction of specific cytotoxicity is confirmed by previous findings that after previous BCG stimulation (non-specific) only a small increase in cytotoxicity can be induced by immunization with tumor cells (unpublished results). Two tumors are somewhat exceptional. Namely the TLX9 and the L1210 tumor. The reason for these exceptions are: 1. The TLX9 is only weakly immunogenic: This has been tested for the syngeneic situation by immunizing syngeneic C57B1. mice two times with irradiated cells. However after injection of 1-] 0 non-irradiated cells a tumor developed in 100 % of immune mice (unpublished results). Obviously in the allogeneic situation this tumor is also relatively weakly immunogenic (this paper). 2. The 1.1210 tumor line we used appeared to be a cell line which preferably grows in vivo but is difficult to culture in vitro. This means that also in the controls tumor cells die spontaneously. This phenomenon significantly interferes with the measurement of the cytotoxicity of the macrophages. In conclusion, recipient mice may be able to reject tumor cells "spontaneously" but this implies that only low· levels of specific macrophage anti-tumor cytotoxicity can be induced. The spontaneous cytotoxic macrophages may he due to either a) subclinical infection (12) or b) the involvement of tumor factors, or tumor induced-host factors, or c) to a combination of a) and b). Alternatively, tumor cells may be sufficiently immunogenic and induce an immune reaction, resultin~ in cytotoxic macrophages.
Literature 1. CANTARROW, W. D., CHEUNG, H. T., SUNDHARADAS, G.: Modulation of spreading adhesion and migration of peritoneal macrophages by a low molecular weight product extracted from mouse tumors. J. RES. 1978; 24: 657-666. 2. DEN OTTER, W., DE GROOT, J. W., DULLENS, H. F. J.: Eradication of tumor cells after injection into immunized hosts compared with the eradication of tumor cells after transfer of immune peritoneal exudates into tumor-bearing recipients. Cancer Immunol. Immunother. 1983; 16: 72-67. 3. DE WEGER, R. A., PELS, K, DEN OTTER, W.: The induction of lymphocytes with the capacity to render macrophages cytotoxic in an allogeneic murine system. Immunol. 1982; 47: 541-550. 4. DULLENS, H. F. J., DEN OTTER, W.: Murine macrophage cytotoxicity induced by mastocytoma cells, cell-free mastocytoma exudates and extracts. Immunopharmac. 1981a; 3: 241-251. 5. - - A small molecular weight peptide from P815 mastocytoma cells induces macrophage cytotoxicity. Immunopharmac. 1981 b; 3: 309-316. 6. EVANS, R., ALEXANDER, P.: Role of macrophage in tumor immunity. 1. Co-operation of imune lymphoid cells with macrophages in syngeneic tumor immunity. Nature 1970; 228: 620-622. 7. FRIEDMAN, H., SPECTOR, S., KOMA, 1., KASELY, J.: Tumor associated immunosuppressive factors. Ann. .Y. Acad. Sci. 1976; 276: 417-430. Exp. Pathol. 31 (1987) 1
37
8. GEMSA, Do, KRAMER, Wo, NAPIEIlSKI, 1., BARLIN, K, TILL, C., RIESCH, K.: Potentiation of macrophage cytostatis by tumor induced ascites. J. ImmunoI. 1981; 126: 2143-2150. 9. HRSAK, L., MAROTTI, T.: Immunosuppressive effects by Ehrlich ascites fluid. Eur. J. Cancer
1973; 9: 717-724. 10. PELS, K, DE WEGER, R. A., DEN OTTER, W.: Lymphocyte induced macrophage cytotoxicity: Characterization of the cytotoxicity inducing lymphocyte. Immunobiology 1984; 166: 84-95. 11. - DEN OTTER, W.: The role of a cytophylic factor from challenged immune peritoneallymphocytes in specific macrophage cytotoxicity. Cancer Res. 1974; 34: 3089-3094. 12. - - Natural cytotoxic marrophages in the peritoneal cavity of mice. BI. J. Cancer 1979; 40: 856-861. 13. PRAGER, M. D., BAEt'HTH, F. S.: Methods for modification of cancer cells to enhance their antigenicity. In: BUSH, H. (ed.), Methods in cancer research. Acad. Press, New York 1973, p. 347 to 356. 14. PREHN, R. T., Antigenic heterogeneity: A possible base for progression. In: OWENS, A. H., DOFFEY, S. C., BYLIN, S. Bo (eds.), Tumor cell heterogeneity: Origin and implication. Acad. Press, New York 1983, p. 73-82. 15. ROBINSON, P. J., SCHIRHMACHER, V.: Differences in the expression of the histocompatibility antigens on murine lymphocytes and tumor cells: immunochemical studies. Eur. J. ImmunoI. 1979; 9: 61-66. 16. VAN LOVEREN, H., SNOEK, M., DEN OTTEIl, W.: Host maerophages are involved in systemic adoptive immunity against tumors. Experientia. 1982; 38: 488. (Received December 23, 1985)
38
Exp. PathoI. 31 (1987) 1
Utrec~t,
Utrecht, The Netherlands
Spontaneous tumor rejection is not always due to a complete cellular rejection By H. F. J. DULLENS, S. SCHAKENRAD and W. DEN OTTER With one figure
Address for correspondence: Dr. H. F. J. DULLENS, State University of Utrecht, Department of Experimental Pathology, Pasteurstraat 2, 3511 HX Utrecht, The Netherlands Ke y W 0 r d s: tumor rejection; cellular immune reaction; antibody dependent cytotoxicity; macrophage cytotoxicity; anti-tumor cytotoxicity Ab brevia tions: ADCC = antibody dependent cytotoxicity; BCG = Bacillus Calmette Guerin; CI = cytotoxicity index; FBS = fetal bovine serum; PBS = phosphate buffered saline; ip = intraperitoneal(ly); sc = subcutaneous(ly)
Summary
In this paper we have studied whether there is a correlation between efficacy of hosts to reject tumor ceIls spontaneously and the induction of cytotoxic macrophages in these recipients due to injection (immunization) with tumor cells. Results show that there is no cause-effect relationship between macrophage cytotoxicity and tumor rejection in vivo. This lack of cause-effect relationship is greatly influenced by differences in immunogenicity between the various tumor cell lines as well as by the release of tumor-factors by some of these tumors. Introduction
Nowadays it is widely believed, that tumors are at least potentially immunogenic in the original host (14), This means that immune reactions may be induced, directed against the tumor cells. Such immune reactions in vivo comprise a number of interacting reactions. In this sequence of events among others, macrophages seem to play an important role. This supposition is supported by the finding of VAN LOVEREN et al. (16) who found that adoptive transfer of immune spleen lymphocytes into tumor bearing hosts only resulted in eradication of the tumor cells if the host macrophage population was intact. This suggests that these macrophages are obviously necessary for the killing of the tumor cells. Probably, a specific macrophage arming factor (6, 10) produced by sensitized lymphocytes upon contact with the specific antigen and able to induce macrophage cytotoxicity (3) is involved. The results of VAN LOVEREN et al. (16) further suggest that sensitized lymphocytes cannot only render macrophagcs cytotoxic in vitro (10-12) but, also may activate host macrophages in vivo to become tumor cell killers. Extensive studies during the past decade, both clinical and experimental, have shown that many malignancies can strongly influence cell mediated immune responses as weIl as antibody formation (7), Sevl'ral authors suggest that these effects might be mediated by soluble factors released by tUlllor c('lls (1,9). In this paper we have studil'd whether there is a correlation between efficacy of recipient mice to rejeet tUlllor eells spontaneously and the induction of cytotoxic macrophages due to the injection of these tumor eells. The infJuence(s) of immunogenicity and production of tumor factors by the various tumor cell lines are discussed. 3
33
Table 1. Tumors used in this stillty Tumor
Strain of origin
H-2 type
8L2 lymphosarcoma L5178Y lymphosarcoma P815X2 mastocytoma L1210 lymphosarcoma AK8L2 lymphosarcoma MOPC 195 plasmacytoma WEHI/3 myelomonocytir leukemia TLX9 lymphosarcoma EL4 lymphosarcoma
DBA/2 DBA/:? DBA/2 DBA/:?
d d d d
BALB/c BA LB/c
d
AKR
C57BL C57BL
k d b b
Materials and .11 ethods Mice Inbred DBA/:?, CBA, BALB/c and AKR mice were obtained from CPB-TNO, Zeist, The Netherlands. Inbred C57BL/10ScCr were obtained from GL Bomholtgard, Denmark. Male and female mice were used at an age of 2-3 months. Tumors See table 1. All tumors grow ip as ascitic tumors, and were maintained by weekly ip passage of 106 cells. I'll vitro cultures were prppared in growth medium: Fischer's medium supplemented with 10 % fetal bovine serum (FBS). The initial concentration was 2 x 1Q5 cells/ml in a total volume of 20 ml. Collection of cells from the peritoneal cavity Peritoneal cells werp collected by washing the peritoneal cavity with 5 mt Fischer's medium. Cells were centrifuged and fl'sllspended in Fischer's medium. Rejection expPfiments Groups of 5 CBA mice earh were injected ip with 1Q5, 106 , 107 or 5 x 10 7 tumor cells and the survival time of the mice was established. Experiments were done twice. Immunization CBA mice were immunized against the non-CBA derived tumors by one ip injection with the maximal dose of tumor cells that was rejected in vivo. Peritoneal macrophages were collected 10 d later. Macrophage cytotoxicity Procedures for preparation of macrophage monolayers and the determination of their in vivo cytotoxicity have been described in detail elsewhere (3). The cytotoxicity was measured after 24 h by counting the remaining viable tumor cells and is expressed as: (1 - TIN) x 100, where CI = Cytotoxicity Index; N = number of viable tumor cells in the controls; T = the number of viable tumor cells in the tests. I'll vitro tests were done at least twice in threefold. Tumor factors P815-factor: A low molecular peptide factor released by P815 mastocytoma cells was isolated and used as has been described in detail by our group elsewhere (4,5). WEHI-factor: BALB/c mice were injected ip with 106 WEHI/3 tumor cells. Seven days after injection the peritoneal cavities of the tumor bearing mice were washed with Fischer's medium. The tumor cell suspension was centrifuged for 20 min (15,000 rpm) and the supernatant was decanted, filtered through a millipore filter (pore size 0.45,um) and used in the experiments.
Results Tum 0 r
r e j ec t ion in vivo
CBA mice (haplotype H-2 k ) were able to reject high doses of the DBAj2 (H-2 d ) derived tumors P815 and L5178Y, the BALBjc (H-2 d ) derived tumor MOPC195 and WEHIj3, and the AKR (H-2 k ) derived tumor AK8L2 (table 2). On the other hand the DBAj2 derived tumors 8L2 and \,1210 as well as both C57BL (H-2 b ) derived tumors EL4 and TLX9 were only rejerted when rrlatively low numbers of tumor cells (about lOS) had been injected
34
Exp. Pathol. 31 (1987) 1
Table 2. Rejection of various tumor cell lines by normal non-stimulated CBA mice (haplotype H-2 k ) Number of tumor cells rejected after ip injection
Tumor-origin (haplotype)
8L2 P815 L5178Y L1210 MOPC 195 WEHIj3 AK8L2 EL4 TLX9
+++: ++: +: ±:
DBAj2 (H-2 DBAj2 (H-2 d ) DBAj2 (H-2 d ) DBAj2 (H-2 d ) BALBjc (H-2 d ) BALBjc (H-2 d ) AKR (H-2 k ) C57BL (H-2 b ) C57BL (H-2 b ) d)
105
lOG
+++ +++ +++ +++ +++ +++ +++ ++ ++
±
+++ +++ +++ +++ +++ +++ +
10'
5x10'
+++ +++ ++ +++ +++ +++
+++ +++ ± +++ +++ +++
±
All mice survived 100 d. 100-75 % of the number of mice survived 100 d. 75-50 % of the number of mice survived 100 d. 50-25 % of the number of mice survived 100 d. 25 %-none of the mice survived.
Ten mice were used per observation (the experiment was done twice in fivefold).
(table 2). Since the AKSL2 tumor cells and the CBA mice both have the haplotype H-2 k we would have expected that AKSL2 cells would not be easily rejected by CBA mice. Obviously differences in other antigens than the MHC antigens between CBA and AKSL 2 are strong enough to evoke the complete rejection of 5 x la' tumor cells. Spontaneous anti-tumor cytotoxicity of macrophages in vitro Spontaneous cytotoxicity of normal non-elicited peritoneal CBA macrophages was observed when these macrophages were incubated with the DBAj2 derived P815 and L5178Y tumors or with the BALBjc derived WEHIj3 tumor. No spontaneous macrophage cytotoxicity was found against any of the other tumor lines tested (fig. 1; black bars). It is known from literature that P815 tumor cells (4, 5) produce a factor which is responsible for the induction of macrophage cytotoxicity. To study whether WEHIj3 cells do produce a similar factor the cell free exudate obtained from BALBjc mice bearing the WEHIj 3 tumor ip was tested for its ability to induce macrophage cytotoxicity. Normal CBA macrophages were incubated with the cell free exudate in vitro for 6 h. The exudate was washed off and the macrophages were challenged with a tumor (SL2) against which no spontaneous cytotoxicity was expressed originally. When the macrophages had been incubated with the WEHIj3 cell free exudate a Cytotoxicity Index of 5 ± 1 against SL2 was observed. If the SL2 tumor cells were pre-incubated with the cell free WEHIj3 exudate, washed with medium and then added to the maerophages, a Cytotoxicity Index of 23 ± 2 could be measured. Anti-tumor eytotoxicity of macrophages in vitro after in vivo immunization Groups of CBA miee were immunized against the different tumor eell hnes by one ip injeetion with the maximum number of cells whieh could be rejected spontaneously (see table 2). Peritoneal macrophages were collected 10 days later and their eytotoxicity was tested against tumor eell lines used for immunization. High levels of cytotoxicity were found against the DBAj2 derived SL2, the BALBjc derived MOPC195 and WEHIj3, theAKR derived AKSL2 and the C57BL derived ErA tumor (Cytotoxicity Indices ranged from 59-74; fig. 1, open bars). Significantly lower levels of cytotoxieity (CI ranged from 34-40) were found against the DBAj2 derived P815 and L5178Y tumor as well as against the C57BL derived TLX9 tumor (fig. 1). However it should be stressed that eompared with the spontaneous cytotoxicity expressed against P81 5 and L5178Y tumor cells no signifieant increase in cytotoxicity due to immunization was obtained (fig. 1). No cytotoxieity could be measured against 11210 tumor cells. 3* Exp. Pathol. 31 (1987) 1 35
100
.
L
~
)(
1&1
0
-z
SO
1
>
~
I.)
x 0
~
0
~
>
I.)
0
•
S L2
P81S
LS178Y
-40
•
..
MO PC-19S
-
W Hp
•
AKSL2
•
EL4
.
TLX9
TUMOR CELL L1NES-.
Fig. 1. Cytotoxicity of (a) normal non-elicited peritoneal macrophages (.) and (b) immune-CBA peritoneal macrophages (0) against various tumor cell lines. Mice had been immunized by one ip injection with the maximum number of tumor cells which was sublethal (see also table 2). Peritoneal macrophages were collected 10 days after immunization. The cytotoxicity was measured after incubation of the macrophages with tumor cells for 24 h. In most cases normal macrophages expressed "negative-cytotoxicity" (*) against the various tumor cell lines (CI varied from -40 till -120). The experiments were donr twice in triplicate.
DiscuSS1:0n As shown in this paper the AK8L2 tumor (haplotype H-2 k ) is easily rejected in CBA mice (also haplotype H-2 k ) whereas for instance tumors like TLX9 (H-2 b ) and 8L2 (H-2 d ) are Jess easily rejected despite their allogenicity (table 2). The fact that AK8L2 cells are easily rejected in CBA mice suggests that differences in minor-histocompatibility antigens between the AK8L2 cells and the CBA host may be involved, as well as neoantigens (tumor associated antigens) present either as a consequenee of the malignant transformation or as a consequenee of additional histo(in)compatibilities developing with continued transplantation of the eell line (13). Sueh incompatibilities have been found to occur even when the tumor is eontinuously transplanted in syngeneie mice. An example of weh a tumor with "additional histo(in)compatibility antigens" is the 8L2 tumor: ROBINSON and SCHIRRMACHER (15) have shown that 8L2 cells have "alien" H-2 k antigens on their surfaee. This may explain why 8L2 cells are not easily rejected by CBA miee whereas they arc easily rejected by allogeneic C57BL (H-2 b ) mice (2). As shown in this paper immunization with tumor eells does not always result in highly cytotoxic macrophages. The latter are only indueed if tumor eells are used for immunization against which the macrophages do not express "spontaneous" cytotoxieity (fig. I). When a high spontaneous cytotoxieity of the macrophages is present, obviously many tumor cells are killed directly, leaving onl~- II "mall number of tumor cell" to evoke a specific. immune reaction. Tn other words, when tumor eells are not immediately killed by the macrophages (spontaneous cytotoxic ma("rophages are absent) they might be present for a longer time and divide, which results in an inerease in available antigen. This may lead to a strong immune reaction resulting in c~-totoxi(" maerophages. On the other hand it ("an not bc excluded that the differences in induction of cytotoxic macrophages b~- the \'1uioLl" tumor cell lines might be due to differences in activation of the type of effector ",-"tp111 ". So for instancp. instead of (cytotoxic) macrophages, NK-cells might be activated.
36
Exp. Pathol. 31 (1987) 1
The phenomenon of spontaneous cytotoxicity itself has been described to be due to subclinical infections (12). However in the experiments described here cytotoxicity was not due to infections. In case of the P815 cells the activation of the macrophages is due to the production of a tumor factor: DULLENS and DEN OTTER (4, 5) have described a factor which could be isolated from P815-tumor ascites, supernatant from P815 in vitro cultures as well as from P815-homogenates. The factor has been characterized as a low molecular peptide which could render non-stimulated macrophages non-specifically cytotoxic (5). Similarly, GEMSA et al. (8) have described a product present in the L5178Y induced ascites which also induces cytostatic activity of peritoneal macrophages. This particular product however, could not be shown to be present in in vitro tumor culture supernatants, or in tumor homogenates. This suggests that in the latter case the factor responsible for the activation of the macrophages in their test system as well as in our system may be (tumor)-host derived. Obviously also WEHI/3 cells produce a product resulting in "spontaneous" macrophage tumoricidal activity (this paper). That is, in the WEHI/3 induced ascites fluid a product can be detected which after preincubation with tumor cells leads to an enhanced killing of these tumor cells by macrophages l:n m:tro. One likely possibility to explain this phenomena would be an ADCC mechanism. Experiments to characterize this product and to establish its biological activities are in progress. The significance of spontaneous macrophage cytotoxicity for the induction of specific cytotoxicity is confirmed by previous findings that after previous BCG stimulation (non-specific) only a small increase in cytotoxicity can be induced by immunization with tumor cells (unpublished results). Two tumors are somewhat exceptional. Namely the TLX9 and the L1210 tumor. The reason for these exceptions are: 1. The TLX9 is only weakly immunogenic: This has been tested for the syngeneic situation by immunizing syngeneic C57B1. mice two times with irradiated cells. However after injection of 1-] 0 non-irradiated cells a tumor developed in 100 % of immune mice (unpublished results). Obviously in the allogeneic situation this tumor is also relatively weakly immunogenic (this paper). 2. The 1.1210 tumor line we used appeared to be a cell line which preferably grows in vivo but is difficult to culture in vitro. This means that also in the controls tumor cells die spontaneously. This phenomenon significantly interferes with the measurement of the cytotoxicity of the macrophages. In conclusion, recipient mice may be able to reject tumor cells "spontaneously" but this implies that only low· levels of specific macrophage anti-tumor cytotoxicity can be induced. The spontaneous cytotoxic macrophages may he due to either a) subclinical infection (12) or b) the involvement of tumor factors, or tumor induced-host factors, or c) to a combination of a) and b). Alternatively, tumor cells may be sufficiently immunogenic and induce an immune reaction, resultin~ in cytotoxic macrophages.
Literature 1. CANTARROW, W. D., CHEUNG, H. T., SUNDHARADAS, G.: Modulation of spreading adhesion and migration of peritoneal macrophages by a low molecular weight product extracted from mouse tumors. J. RES. 1978; 24: 657-666. 2. DEN OTTER, W., DE GROOT, J. W., DULLENS, H. F. J.: Eradication of tumor cells after injection into immunized hosts compared with the eradication of tumor cells after transfer of immune peritoneal exudates into tumor-bearing recipients. Cancer Immunol. Immunother. 1983; 16: 72-67. 3. DE WEGER, R. A., PELS, K, DEN OTTER, W.: The induction of lymphocytes with the capacity to render macrophages cytotoxic in an allogeneic murine system. Immunol. 1982; 47: 541-550. 4. DULLENS, H. F. J., DEN OTTER, W.: Murine macrophage cytotoxicity induced by mastocytoma cells, cell-free mastocytoma exudates and extracts. Immunopharmac. 1981a; 3: 241-251. 5. - - A small molecular weight peptide from P815 mastocytoma cells induces macrophage cytotoxicity. Immunopharmac. 1981 b; 3: 309-316. 6. EVANS, R., ALEXANDER, P.: Role of macrophage in tumor immunity. 1. Co-operation of imune lymphoid cells with macrophages in syngeneic tumor immunity. Nature 1970; 228: 620-622. 7. FRIEDMAN, H., SPECTOR, S., KOMA, 1., KASELY, J.: Tumor associated immunosuppressive factors. Ann. .Y. Acad. Sci. 1976; 276: 417-430. Exp. Pathol. 31 (1987) 1
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8. GEMSA, Do, KRAMER, Wo, NAPIEIlSKI, 1., BARLIN, K, TILL, C., RIESCH, K.: Potentiation of macrophage cytostatis by tumor induced ascites. J. ImmunoI. 1981; 126: 2143-2150. 9. HRSAK, L., MAROTTI, T.: Immunosuppressive effects by Ehrlich ascites fluid. Eur. J. Cancer
1973; 9: 717-724. 10. PELS, K, DE WEGER, R. A., DEN OTTER, W.: Lymphocyte induced macrophage cytotoxicity: Characterization of the cytotoxicity inducing lymphocyte. Immunobiology 1984; 166: 84-95. 11. - DEN OTTER, W.: The role of a cytophylic factor from challenged immune peritoneallymphocytes in specific macrophage cytotoxicity. Cancer Res. 1974; 34: 3089-3094. 12. - - Natural cytotoxic marrophages in the peritoneal cavity of mice. BI. J. Cancer 1979; 40: 856-861. 13. PRAGER, M. D., BAEt'HTH, F. S.: Methods for modification of cancer cells to enhance their antigenicity. In: BUSH, H. (ed.), Methods in cancer research. Acad. Press, New York 1973, p. 347 to 356. 14. PREHN, R. T., Antigenic heterogeneity: A possible base for progression. In: OWENS, A. H., DOFFEY, S. C., BYLIN, S. Bo (eds.), Tumor cell heterogeneity: Origin and implication. Acad. Press, New York 1983, p. 73-82. 15. ROBINSON, P. J., SCHIRHMACHER, V.: Differences in the expression of the histocompatibility antigens on murine lymphocytes and tumor cells: immunochemical studies. Eur. J. ImmunoI. 1979; 9: 61-66. 16. VAN LOVEREN, H., SNOEK, M., DEN OTTEIl, W.: Host maerophages are involved in systemic adoptive immunity against tumors. Experientia. 1982; 38: 488. (Received December 23, 1985)
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