Original Papers Int. Archs Allergy appl. Immun. 59: 1-12 (1979)
Rejection of Tumor Cells in vitro: Morphological Studies on Killer T Cells and Damaged Tumor Cells' /. Berczi, K. Kovacs, E. Horvath and A. H. Sehon Department of Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Man. and Department of Pathology. St. Michael's Hospital, University of Toronto. Toronto. Ont.
Introduction The study of tumor immunity in various systems revealed the important role of thy mus-derived (T) lymphocytes in host def ence [9, 12, 16, 22, 31, 32]. Previous ex periments from this laboratory showed that 1 This work was supported in part by grants from the Medical Research Council of Canada (MA-4745), the National Cancer Institute of Ca nada and the National Institute of Health (CA13192). We are grateful to Mrs. Geziiui Use and Mr. N. J. Huzel for their excellent technical help.
a methylcholanthrene-induced sarcoma (MC-D) of inbred strain 13 guinea pigs is infiltrated by cytotoxic effector cells capable of proliferating and rapidly destroying tu mor cells in vitro [4], Similar cytotoxic cells were induced by co-cultivation of guinea pig lymphoid cells from the thymus, lymph nodes, spleen, bone marrow and blood with MC-D cells. This cytotoxic reaction was in hibited by pretreatment of the lymphoid cell populations with a rabbit antiserum specific for guinea pig thymus-derived lymphocytes in the presence of guinea pig complement
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Abstract. The in vitro destruction of a methylcholanthrene-induced guinea pig sarcoma (MC-D) by killer T lymphocytes was investigated by light and electron microscopy. Various degrees of cell damage ranging from slight to extensive were observed. In cells with slight injury, dilatation, vesiculation, fragmentation and degeneration of the rough surfaced endo plasmic reticulum were the most characteristic findings. In cells with extensive injury, widespread nuclear and cytoplasmic alterations were evident and many of these cells were fragmented into smaller portions and finally transformed into granular membranous and amorphous debris. Cytoplasmic vacuoles filled with electron-lucent material were frequent in extensively damaged cells. Killer lymphocytes resembled closely antibody-forming plasma cells when examined with light microscopy but lacked the extensive network of rough surfaced endoplasmic reticulum, and did not produce immunoglobulin. It is sug gested that these extensively differentiated T-derived killer cells are end cells similar to those of B lymphocyte-derived plasma cells. Viral particles resembling closely C-type viruses were observed in mixed killer cell MC-D cultures.
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Materials and Methods Animals. A colony of inbred. Sewall-Wright strain 13 guinea pigs (Gp. 13) was produced with breeders generously provided in 1969 by Dr. M. W. Chase of the Rockefeller University, New York. MC-D Tumor. This tumor had been induced originally with 3-mcthylcholanthrene in a female strain 13 guinea pig by Oettgen et al. [28]; two tu mor-bearing animals were donated in 1969. An exchange of skin grafts between the tumor-bearing guinea pigs and members of the strain 13 guinea pig colony developed at the University of Manito ba did not reveal histocompatibility differences, inasmuch as the grafts were retained in a viable state for over 2 years [3J. Induction of Cytotoxic Lymphoblasts (LHC). Cultures of cytotoxic LBC were obtained in two different ways: (i) MC-D tumors were trypsinized and cultured in Eagle’s minimal essential medium (Difco) which was completed with nonessential amino acids (Difco), penicillin (100/tg/ml) and streptomycin (100 ng/ml) and with 15"/o heat-inac tivated fetal calf serum. In about two-thirds of the cultures LBC emerged spontaneously as reported earlier |4J; (ii) established tissue culture cell lines of MC-D were co-cultivated with thymus cells as described earlier [5]. Light Microscopy. Mixed MC-D-LBC cultures were grown on glass coverslips in Leighton tubes. At various stages of interaction the coverslips were stained with Wright stain or with methyl green-pyronin [29]. Preparation for Electron Microscopy. Cultured cells were fixed with glutaraldehyde and osmium tetroxide, and embedded in Araldite resin as de scribed by Nelson and Flaxman [26]. Ultrathin sections were cut with a Porter-Blum Mt-2 ul tramicrotome, stained with uranyl acetate and lead citrate and investigated with a Philips 300 electron microscope. Serological Procedures. Cells grown on glass
coverslips and fixed with cold acetone were exam ined by indirect immunofluorescence for immu noglobulin that may be produced by LBC. Specif ic rabbit antibodies against guinea pig lgG2 and F(ab')2 dimer were used on separate slides as the first treatment, which was followed, after wash ing. by staining the slides with fluorescein-tagged sheep antibodies to rabbit lgG. The mixed agglu tination technique was carried out also on glass slides for the detection of surface Ig with nonfixed cells as described by Eagraetts et al. 114). For inhi bition of hemagglutination, sheep red blood cells (SRBC) were coated with a subagglutinating dilu tion of guinea pig anti-SRBC antibodies and were used as antigen. Rabbit antiserum to guinea pig F(ab'), was used as agglutinating agent. Superna tants of LBC cultures were examined for the pres ence of guinea pig Ig by inhibition test using four hemagglutinating units of antiserum in the Takatsy microtiter system 134].
Results In agreement with our earlier findings [4], killer lymphoblast cells usually emerged between day 5 and 7, when MC-D tissue culture cell lines were co-cultivated with lymphoid cells, and between day 10 and 20 from about 70°/» of primary MC-D cultures. Lymphoblasts first appeared in small foci on the monolayer, which exhibited window like discontinuity in many cases in the cen ter of foci indicating the cytotoxic action of LBC (fig. 1). During the following days nu merous other foci appeared with identical morphology, the windows grew continuous ly with proliferating LBC on their margin, and the monolayer was destroyed complete ly within 48-72 h after the appearance of first LBC foci. This rapid distribution of cy totoxic cells together with the observation that some of the LBC were always floating in the culture supernatant and these floating cells were cytotoxic if transferred to new
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[5]. In this paper we present the morpholo gy of killer cells and of MC-D cells which had been damaged to various extents by T cells in vitro.
Morphology of Killer T Cell-Tumor Interaction
Fig. 1. Destruction of MC-D sarcoma cells in vitro by lym phoblast cells (LBC). Photograph of a viable primary tumor culture shows a focus of destruction by LBC. Bound-shaped LBC at tached themselves to tumor cells while lysing the latter and thus creating discontinuity in the monolayer (arrows). X 300.
monolayers, indicated that the killer cells were extremely mobile. LBC never adhered to the plastic tissue culture vessels but only to the spindle-shaped ‘target’ (MC-D) cells. This made it possible for us to recognize
target and cytotoxic cells easily during ul trastructural examination. Once all the MC-D cells were killed, LBC floated freely in the supernatant, stopped proliferating and lost their viability gradually as judged
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Fig. 2. Photograph of stained LBC preparation. Note the round cells with plasma cell morpholo gy (LBC) aggregated around spindle-shaped tumor cells (ar rows). Wright stain. X 450. Fig. 3. Late culture of LBC. Note vacuolation of large round cells with plasma cell morpholo gy (open arrows). Rounding and disintegration of tumor cells is also noticeable (solid arrows). Wright stain. X 450.
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Tabic I. Viability of LBC in stimulated and unstimulated cultures1
Days3
1 4 7 10 12 14
Number2 of LBC in cultures stimulated with MC-D cells
unstimulated
viable
dead
viable
0.0010
0.1000
0.0060 ± 0.003 0.0070 ± 0.005 0.0810 ± 0.090 0.0100 ± 0.010 0.1220 ± 0.050
2.4500 2.1500 0.4600 0.0096 0.0009
0.1000
4.0000 4.9000 54.6700 85.0000 426.6700
± 0.86 ±1.21 ±20.14 ± 28.58 ± 152.0
dead 0.0010
± ± ± ± ±
l.l00 1.300 0.320 0.008 0.000
0.0550 ± 0.0100 ± 0.0340 ± 0.0060 ± 0.0460 ±
0.055 0.011
0.030 0.003 0.002
by the eclusion of 0.2% trypan blue dye (ta ble I). Some of the LBC, especially those of small size, remained viable in the absence of stimulation up to 3 weeks and could be reactivated at any time during this period simply by adding MC-D target cells to the medium. Upon restimulation proliferation occurred. Proliferation and cytotoxicity were parallel phenomena always in our nu merous experiments. Histological examination of mixed MC-D-LBC cultures grown on glass coverslips and stained according to Wright re vealed that LBC resembled plasma cells (fig. 2). The cytoplasm was abundant around the eccentric nucleus. The nucleolus was prominent. In some of the cultures, at a late stage of cytotoxic reaction, vacuolation of LBC along with the rounding up and dis integration of MC-D cells were observed with light microscopy (fig. 3). When stained with methyl green-pyronin LBC showed
strong pyroninophilia indicating an active RNA synthesis. Surface immunoglobulin could not be shown on LBC with two differ ent methods, e.g. by indirect immunofluo rescence and by a Coombs-type mixed ag glutination. Supernatants of LBC cultures did not contain immunoglobulin as judged by inhibition of hemagglutination. Ultrastructural examination showed that LBC had the cytoplasm arranged around the eccentric nucleus to form a uropod-like structure, which exhibited cytoplasmic pro cesses. The Golgi apparatus was well devel oped, with abundant vesicles and microfila ments in the cytoplasm. Ribosomes were mostly free and only short segments of the rough endoplasmic reticulum were visible (fig- 4). Rarely, if ever, MC-D target cells showed signs of degeneration while killer LBC were adhering to them. In general, MC-D cells were spindle shaped with cen-
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1 Quadruplicates. Viability was estimated by dye exclusion (0.2% trypan blue). 2 The numbers indicate viable and dead cells per culture x 10 '• ± SD. Nonadherent lymphoblast cells were counted only. Intact but stained cells were considered dead. Cell debris is not included. 3 Each culture was started on day I by co-cultivating IO’ LBC with IO'1MC-D cells. After each cell count the medium was changed for all cultures and MC-D cells were added to the stimulated cultures in increasing numbers keeping an approximate ratio of LBC: MC-D = 1 :1 . Cell concentration was maintained between 5 x i0M0»/ml.
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Fig. 4. Electron micrograph of LBC adhering to a tumor cell. The nucleus is eccentric with large nucleolus. The cytoplasm forms a uropod with finger-like processes. The Golgi apparatus is well developed, vesicles are nu merous in the cytoplasm. Ribo somes are numerous; only short segments of rough endoplasmic reticulum (RER) are visible. The target cell is flattened on the plastic surface, contains abundant RER and a dense body appears in the cytoplasm adjacent to the killer cell. X 8,800.
trally located nuclei and well-developed nu cleoli. The cytoplasm contained a well-dev eloped endoplasmic reticulum, abundant microfilaments, mitochondria, some free ri bosomes and occasionally vacuoles, which appeared empty.
In cytotoxic cultures swelling and signs of degeneration occurred. Cells showing slight injury seemed to be round with a few swollen processes on their surface. In the cytoplasm the most conspicuous finding was the dilation and vésiculation of rough-sur-
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Fig. 5. Rounded target cell. The cytoplasm contains vacuoles varying in size and filled with electron-lucent flaky material. Plasma membranes and nucleus show no disruption. X 4,600.
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faced endoplasmic reticulum (RER) mem branes. In some cells, the dilation of the RER membranes was very pronounced, the cisternae transformed into cyst-like struc tures with an electron-lucent content and with partial degranulation of their walls.
Another prominent change was the appear ance of cytoplasmic vacuoles. They were varying in size, and were randomly distrib uted and filled with an electron-lucent flaky content. Free ribosomes were still numer ous. Mitochondria and lysosomes failed to
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Fig. 6. Extensively damaged cell with disrupted plasma mem brane and spilling of cytoplasm. A large cytoplasmic vacuole re sembling phagosome is visible. X 6,000.
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show major abnormalities. The plasma membranes showed no disruptions (fig. 5). Cells exhibiting extensive injury were fragmented into smaller portions and finally transformed into granular membranous and amorphous debris in which the various cell organelles were not possible to recognize. Before this final phase, widespread nuclear and cytoplasmic alterations were evident. Margination of chromatin was prominent. Inside the nucleoplasm chromatin granules aggregated into larger clumps and empty spaces were formed. Later the nuclear enve lopes became disrupted. The nuclei became fragmented and dark. In the cytoplasm the RER showed cyst-like dilation, vésiculation.
fragmentation and degranulation. Loss of membrane-bound ribosomes was quite pronounced in many cells. Free ribosomes were, however, still apparent even in those cells which showed extensive and advanced damage. Vacuolation of cytoplasm became a conspicuous finding. The vacuoles varied in size. Some of them coalesced and became quite large. They were filled with an elec tron-lucent material (fig. 6). The origin of the vacuoles could not be revealed with cer tainty. They might have derived from RER membrane by dilatation, vesiculation and shedding their ribosomes. Some vacuoles might have originated from mitochondria by the loss of their internal compartment and
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Fig. 7. A virus-infected LBC still adhering to the target cell. Numerous immature viral parti cles are visible in the nucleus and mature particles are budding off the cytoplasmic membrane. The RER is dilated and vesiculated, with partial degranulation. X 5,000.
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Fig. 8. Various maturation stages of viral particles closely resembling C-type viruses. X 26,200.
rounded by an electron-lucent halo and by a double membrane wall, the one on the out side containing a few small spikes. These viral particles were detected in intact cells and also in those cells which showed slight or extensive injury.
Discussion Based on the results of previous studies [4, 5] and on those presented in this paper, LBC may be characterized as follows: They are round cells with plasma cell morphology and are adherent to target cells (MC-D) but not to plastic tissue culture vessels. Upon interaction with target cells LBC proliferate and readily incorporate radiolabelled thymi dine and uridine and stain with pyronin. The target cells are destroyed as a result of interaction with viable LBC. This cytotoxic action is only partially specific. Because of their rapid multiplication and mobility. LBC arc able to destroy excess amounts of target cells in vitro. A factor capable of inhibiting the migration of normal guinea pig macro phages is present in supernatants of LBC cultures [5], but no immunoglobulin or tox ic material could be detected. The induction of LBC in vitro by co-cultivation of synge neic lymphocytes with MC-D tumor cells
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the persistence of the limiting membranes. It was remarkable that mitochondria ap peared to be intact even in those cells which exhibited marked and advanced impair ment. In some cells, however, mitochondria became darker, the cristae disappeared and the limiting membranes became fragmented. Ultimately mitochondria disintegrated into dark, amorphous granules. In the final phase lysosomes seemed to accumulate and in crease in size. Lysosomal alterations were, however, not striking and they occurred only in the later stage. There were many in termediate forms between these two de scribed types showing various extent of cel lular injury. Many cells contained mature and imma ture viral particles resembling closely C-type viruses (fig. 7. 8). They were present in the cytoplasm, nuclei and also in the ex tracellular space. Some of them were inside the cisternae of RER membranes. Those not confined to RER cisternae were ran domly distributed in the cytoplasm, often accumulating adjacent to the cell membrane and budding off into the extracellular space. In some cells they were very numerous. Their size varied between 60 and 200 nm, the majority of them measuring between 80 and 120 nm in diameter. They were spheri cal and had an electron-dense core sur
could be inhibited by pretreatment of the lymphoid cell populations with a rabbit an tiserum specific for guinea pig thymus-der ived lymphocytes in the presence of comple ment. All of these characteristics are com patible with the conclusion that the killer cells studied in this system are derived from T lymphocytes. Light microscopic examination revealed that LBC resembled plasma cells, however, ultrastructurally they did not show the abundant RER, which is very characteristic of antibody-producing plasma cells (fig. 4, 6). The eccentric nucleus with a large nu cleolus and the formation of a uropod by the cytoplasm which contained a well-devel oped Golgi apparatus, but only short seg ments of RER. microfilaments, microtu bules, some vacuoles and free ribosomes, was described by others to be characteristic of activated T cells [8, 21, 24, 25]. There fore, it appears that plasma cells may arise from bone marrow derived (B) lympho cytes, which form antibodies; however, thy mus-derived killer lymphocytes which do not form antibodies may also reach a state during their differentiation in which they re semble B cell derived plasma cells when ex amined by light microscopy. The existence of T associated plasma cells’ in man has been suggested recently by Lennert et al. [23], although they could not reveal the ex act origin of these cells. We showed earlier with the aid of cytotoxic rabbit antibodies specific for guinea pig T lymphocytes that LBC originated from T cells [5], therefore, the T-cell origin of LBC exhibiting plasma cell morphology seems certain in our case. B-derived plasma cells are known to be end cells [17] which die at the end of fulfillment of their function, i.e., the production of spe cific antibodies against the antigen. This
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does not apply to less differentiated B •memory’ cells. Our studies suggest that T-derived ‘plasma cells’ behave the same way, since they died rapidly in the absence of antigenic stimulation with MC-D. Small blast cells, which did not exhibit plasma cell morphology tended to survive for long er periods in culture without stimulation, e.g. the culture could be restimulated by MC-D up to 3 weeks. It is not likely that this death was due to the artificial condi tions in culture, since LBC could be main tained for 6 months in a state of prolifera tion and cytotoxicity if MC-D target cells were supplied continuously. Supernatants of MC-D cultures did not stimulate LBC, nei ther did cultured guinea pig hepatoma or mouse cells [5]. The repopulation of synge neic X-irradiated animals with radioactively labelled small and large killer cells cultured in vitro would reveal their life span in vivo. Such experiments are currently underway. Sprent and Miller [33] also showed that ac tivated thymus-derived cells are short lived on the basis of their survival in vivo. The mechanism of T cell-mediated target cell lysis is obscure [6, 19]. The results pre sented in this paper do not permit us to draw a definite conclusion regarding the mechanism of T cell-mediated killing. It ap pears, however, that the initial changes oc cur in the cytoplasm. The RER was affected in most cases and vacuolation was a conspi cuous finding (fig. 5). Destruction of mem branes occurred only at final stages of cell injury. Some vacuolation was also seen in control MC-D cultures, which is known to occur in tissue culture media containing high amounts of bicarbonate (in our case 2 g/1), and is believed to be reversible. Be sides the empty vacuoles, no significant cell damage was observed in established cul
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These viruses do not seem to have direct cytopathic effect on MC-D cells, since the incubation of MC-D with frozen and thawed LBC cultures did not result in cell damage [5]. It is suggested by our results that these type-C viruses became activated somehow during the interaction of LBC with MC-D.
References 1 André-Schwartz, J.; Schwartz, R. S.; Hirsch, M. S.; Phillips, S. M.. and Black. P. H.: Acti vation of leukemia viruses by graft-versus-host and mixed lymphocyte-culture reactions: elec tron microscopic evidence of C-typc particles. J. natn. Cancer Inst. 51: 507-518 (1973). 2 Basombrio, M. A.: Lymphomas in BALB/C mice inoculated with supernatants from chemi cally induced sarcomas. J. natn. Cancer Inst. 51: 1157-1162 (1973). 3 Berczi. I.: Studies in tumor immunology: thesis Manitoba (1972). 4 Berczi, I.: Strausbauch. P., and Sehon. A. H.: Rejection of tumor cells in vitro. Science 180: 1289-1291 (1973). 5 Berczi. I. and Sehon, A. H.: Rejection of tu mor cells in vitro: a T-cell-mcdiated reaction. Int. J. Cancer 16: 665-674 (1975). 6 Berke, G. and Amos, D. B.: Mechanism of lymphocyte-mediated cytolysis. The LMC cy cle and its role in transplantation immunity. Transplantn Rev. 17: 71-107 (1973). 7 Bernstein, I. D.; Cohen, E. F.. and Wright. P. W.: Relationship of cellular proliferation and the generation of cytotoxic cells in an in vitro secondary immune response to syngeneic rat lymphoma cells. J. Immun. 118: 1090-1094 (1977). 8 Binet, J. L. and Mathé. G.: Optical and elec tron microscope studies of ‘immunologically competent cells’ in graft reactions. Nature, Lond. 193: 992-993 ( 1962). 9 Cerottini, J.-C. et Brunner, K. T.: Lympho cytes cytotoxiques et immunité antitumorale. Annls Inst. Pasteur, Paris 122: 659-668 (1972). 10 Cerottini. J.-C. and Brunner, K. T.: Cell-me-
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tures. While DNA synthesis is known not to be a prerequisite of T cell-mediated target cell destruction, as shown by others [10], it is indicated by our data that cell prolifera tion is always initiated by prolonged stimu lation of cytotoxic cells. Similar results were obtained by Bernstein et at. [7] during the in vitro stimulation of spleen cells of rats, immunized previously to the syngeneic lym phoma (C 58 NT)D, with mitomycin-C treated tumor cells. Elimination of prolifer ating cells by exposure to high specific ac tivity 3H-thymidine at appropriate intervals impaired the generation of cytotoxic activ ity, but the remaining cells could generate cytotoxic effectors to a second set of anti gens. Viral particles were seen in cultures where T cell-mediated target cell killing took place (fig. 7, 8) but not in control MC-D cultures. Both MC-D and LBC ap peared to be infected. Immature viral parti cles, together with fully mature particles, were present in the cytoplasm and nuclei of affected cells. Morphologically they resem bled closely C-type viruses of other mam malian species [18] and of guinea pigs [20], Other investigators detected also type-C vi ruses in chemically induced tumors [2, 11, 13, 15, 30, 35], and it was hypothesized that these viruses are of endogenous origin, which play a co-carcinogenic role during chemical oncogenesis [36], However. Odcika [27] found that the appearance of endo genous viruses was independent of the ac tion of chemical carcinogens in various mouse strains. An alternate possibility is that these type-C viruses became activated as a result of the immune reaction. Murine type-C viruses were shown to be specifically associated with graft-versus-host reaction and with mixed lymphocyte cultures [1 ].
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18
19
20
21
22
23
24
25
26
27
28
29 30
31
32
E.: Morphological observations on the contactinduced lysis of target cells. Eur. J. Immunol. 3: 32-37 (1973). Leclerc, J. C.; Gomard, E.; Plata, F., and Levy, J. P.: Cell mediated immune reaction against tumors induced by oncornaviruses. 11. Nature of the effector cells in tumor-cell cyto lysis. Int. J. Cancer 11: 426-432 (1973). Lennert. K.; Kaiserling, E., and MiillerHermelink. H. K.: T-associated plasma cells. Lancet/; 1031-1032 (1975). Matter, A.; Lisowska-Bernstein, B.; Ryser, J. E.; Lamelin, J.-P., and Vassalli. P.: Mouse thy mus-independent and thymus-derived lymphoid cells. II. Ultra-structural studies. J. exp. Med. 136: 1008-1030 (1972). Matter, A. and Simpson. E.: The differentia tion of cytotoxic T lymphocytes in vitro. An ultrastructural study. Cell Tissue Res. 166: 475-488 (1976). Nelson, B. K. and Flaxman. B. A.: In situ embedding and vertical sectioning for electron microscopy of tissue cultures grown on plastic Petri dishes. Stain Technol. 47: 261-265 (1972) . Odaka, T.: Strain-dependent expression of endo genous mouse-tropic leukemia viruses in chem ically induced murine leukemias. Int. J. Cancer 16: 622-628 (1975). Oettgen, H. F.; Old. L. J.: McLean, E. P.. and Carswell, E. A.: Delayed hypersensitivity and transplantation immunity elicited by soluble antigens of chemically induced tumors in inbred guinea pigs. Nature, Lond. 220: 295-297 (1968). Parker, R. C.: Methods of tissue culture, pp. 309-310 (Harper & Row, New York 1961). Rhim. J. S.; Duh. F. G.; Cho, H. Y.; Edler. E., and Vernon. M. L.: Activation of a type-C RNA virus from tumors induced by rat kidney cells transformed by a chemical carcinogen. J. natn. Cancer inst. 50: 255-261 (1973). Rollinghoff, M. and Wagner, H.: In vitro in duction of tumor specific immunity: Require ments for T lymphocytes and tumor growth in hibition in vivo. Eur. J. Immunol. 3: 471-476 (1973) . Rouse, B. T.: Rollinghoff, M.. and Warner, N. L.: Tumor immunity to murine plasma cell tu mors. II Essential role of T lymphocytes in
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dialed cytotoxicity, allograft rejection and tu mor immunity. Adv. Immunol. IS: 67-132 (1974). Chan. P. L. and Sinclair. N. R. St. C.: Immu nologic and virologic properties of chemically and j'-irradiation-induced thymic lymphomas in mice. J. natn. Cancer Inst. 48: 1629-1640 (1972). Djeu, J. Y.; Glaser, M.; Kirchncr, H.; Huang, K. Y„ and Herberman, R. B.: The effect of specific anti-rat thymocyte serum on cell-me diated tumor immunity and other lymphocyte functions. Cell. Immunol. 12: 164-169 (1974). Dodge, A. H.: Fine structural HaLV gs anti gen, and reverse transcription study of the Syr ian hamster stilbestrol-induced renal carcinoma. Lab. Invest. 31: 250-257 (1974). Fagraeus, A.; Espmark, J. A., and Jonsson, J.: Mixed haemadsorption: a mixed antiglobulin reaction applied to antigens on a glass surface. Preparation and evaluation of indicator red cells; survey of present applications. Immunol ogy 9: 161-175 (1965). Freeman. A. E.: Kclloff. G. J.; Gilden. R. V.; Lane, W. T.; Swain. A. P., and Huebner, R. J.: Activation and isolation of hamster-specific C-type RNA viruses from tumors induced by cell cultures transformed by chemical carcino gens. Proc. natn. Acad. Sci. USA 6S: 2386-2390 (1971). Gorczynski, R. M.: Evidence for in vivo pro tection against murine-sarcoma virus-induced tumors by T lymphocytes from immune ani mals. J. Immun. 112: 533-539 (1974). Harris, T. N.; Hummeler, K., and Harris, S.: Electron microscopic observations on antibody producing lymph node cells. J. exp. Med. 123: 161-172 (1966). Harvcn, E. de: Morphology of murine leuke mia viruses, in Rich, Experimental leukemia pp. 97-129 (Appleton Century Crofts, New York 1968). Henney, C. S.: On the mechanism of T-cell mediated cytolysis. Transplantn Rev. 17: 37-70 (1973). pig leukemia: an overview with reference to Hsiung. G. D.: Virological studies of guinea herpesvirus and oncornavirus. Fed. Proc. Fed. Am. Socs exp. Biol. 36: 2285-2289 (1977). Koren. H. S.; Ax, W„ and Frcund-Mollbert,
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immune response. Eur. J. Immunol. 3: 218-224 (1973). 33 Sprent, J. and Miller, J. F. A. P.: Interaction of thymus lymphocytes with histoincompatible cells. 111. Immunological characteristics of re circulating lymphocytes derived from activated thymus cells. Cell. Immunol. 3: 213-230 (1972). 34 Takatsy, G.: The use of spiral loops in sero logical and virological micro-methods. Acta microbiol. hung. 3: 189-191 (1955). 35 Weinstein. 1. B.; Gebert, R.; Stadler, U. C.: Orenstein, J. M., and Axel, R.: Type C virus from cell cultures of chemically induced rat hepatomas. Science, N.Y. 178: 1098-1100 (19-2).
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36 Whitmire. C. E.: Virus-chemical carcinogene sis: a possible viral immunologic influence on 3-methylcholanthrene sarcoma induction. J. natn. Cancer Inst. 51: 473-478 (1973).
Received: May 11, 1978 Correspondence to: Dr. 1. Berczi. Department of Immunology. Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E OW3 (Canada)
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Rejection of Tumor Cells in vitro: Morphological Studies on Killer T Cells and Damaged Tumor Cells' /. Berczi, K. Kovacs, E. Horvath and A. H. Sehon Department of Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Man. and Department of Pathology. St. Michael's Hospital, University of Toronto. Toronto. Ont.
Introduction The study of tumor immunity in various systems revealed the important role of thy mus-derived (T) lymphocytes in host def ence [9, 12, 16, 22, 31, 32]. Previous ex periments from this laboratory showed that 1 This work was supported in part by grants from the Medical Research Council of Canada (MA-4745), the National Cancer Institute of Ca nada and the National Institute of Health (CA13192). We are grateful to Mrs. Geziiui Use and Mr. N. J. Huzel for their excellent technical help.
a methylcholanthrene-induced sarcoma (MC-D) of inbred strain 13 guinea pigs is infiltrated by cytotoxic effector cells capable of proliferating and rapidly destroying tu mor cells in vitro [4], Similar cytotoxic cells were induced by co-cultivation of guinea pig lymphoid cells from the thymus, lymph nodes, spleen, bone marrow and blood with MC-D cells. This cytotoxic reaction was in hibited by pretreatment of the lymphoid cell populations with a rabbit antiserum specific for guinea pig thymus-derived lymphocytes in the presence of guinea pig complement
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Abstract. The in vitro destruction of a methylcholanthrene-induced guinea pig sarcoma (MC-D) by killer T lymphocytes was investigated by light and electron microscopy. Various degrees of cell damage ranging from slight to extensive were observed. In cells with slight injury, dilatation, vesiculation, fragmentation and degeneration of the rough surfaced endo plasmic reticulum were the most characteristic findings. In cells with extensive injury, widespread nuclear and cytoplasmic alterations were evident and many of these cells were fragmented into smaller portions and finally transformed into granular membranous and amorphous debris. Cytoplasmic vacuoles filled with electron-lucent material were frequent in extensively damaged cells. Killer lymphocytes resembled closely antibody-forming plasma cells when examined with light microscopy but lacked the extensive network of rough surfaced endoplasmic reticulum, and did not produce immunoglobulin. It is sug gested that these extensively differentiated T-derived killer cells are end cells similar to those of B lymphocyte-derived plasma cells. Viral particles resembling closely C-type viruses were observed in mixed killer cell MC-D cultures.
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Materials and Methods Animals. A colony of inbred. Sewall-Wright strain 13 guinea pigs (Gp. 13) was produced with breeders generously provided in 1969 by Dr. M. W. Chase of the Rockefeller University, New York. MC-D Tumor. This tumor had been induced originally with 3-mcthylcholanthrene in a female strain 13 guinea pig by Oettgen et al. [28]; two tu mor-bearing animals were donated in 1969. An exchange of skin grafts between the tumor-bearing guinea pigs and members of the strain 13 guinea pig colony developed at the University of Manito ba did not reveal histocompatibility differences, inasmuch as the grafts were retained in a viable state for over 2 years [3J. Induction of Cytotoxic Lymphoblasts (LHC). Cultures of cytotoxic LBC were obtained in two different ways: (i) MC-D tumors were trypsinized and cultured in Eagle’s minimal essential medium (Difco) which was completed with nonessential amino acids (Difco), penicillin (100/tg/ml) and streptomycin (100 ng/ml) and with 15"/o heat-inac tivated fetal calf serum. In about two-thirds of the cultures LBC emerged spontaneously as reported earlier |4J; (ii) established tissue culture cell lines of MC-D were co-cultivated with thymus cells as described earlier [5]. Light Microscopy. Mixed MC-D-LBC cultures were grown on glass coverslips in Leighton tubes. At various stages of interaction the coverslips were stained with Wright stain or with methyl green-pyronin [29]. Preparation for Electron Microscopy. Cultured cells were fixed with glutaraldehyde and osmium tetroxide, and embedded in Araldite resin as de scribed by Nelson and Flaxman [26]. Ultrathin sections were cut with a Porter-Blum Mt-2 ul tramicrotome, stained with uranyl acetate and lead citrate and investigated with a Philips 300 electron microscope. Serological Procedures. Cells grown on glass
coverslips and fixed with cold acetone were exam ined by indirect immunofluorescence for immu noglobulin that may be produced by LBC. Specif ic rabbit antibodies against guinea pig lgG2 and F(ab')2 dimer were used on separate slides as the first treatment, which was followed, after wash ing. by staining the slides with fluorescein-tagged sheep antibodies to rabbit lgG. The mixed agglu tination technique was carried out also on glass slides for the detection of surface Ig with nonfixed cells as described by Eagraetts et al. 114). For inhi bition of hemagglutination, sheep red blood cells (SRBC) were coated with a subagglutinating dilu tion of guinea pig anti-SRBC antibodies and were used as antigen. Rabbit antiserum to guinea pig F(ab'), was used as agglutinating agent. Superna tants of LBC cultures were examined for the pres ence of guinea pig Ig by inhibition test using four hemagglutinating units of antiserum in the Takatsy microtiter system 134].
Results In agreement with our earlier findings [4], killer lymphoblast cells usually emerged between day 5 and 7, when MC-D tissue culture cell lines were co-cultivated with lymphoid cells, and between day 10 and 20 from about 70°/» of primary MC-D cultures. Lymphoblasts first appeared in small foci on the monolayer, which exhibited window like discontinuity in many cases in the cen ter of foci indicating the cytotoxic action of LBC (fig. 1). During the following days nu merous other foci appeared with identical morphology, the windows grew continuous ly with proliferating LBC on their margin, and the monolayer was destroyed complete ly within 48-72 h after the appearance of first LBC foci. This rapid distribution of cy totoxic cells together with the observation that some of the LBC were always floating in the culture supernatant and these floating cells were cytotoxic if transferred to new
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[5]. In this paper we present the morpholo gy of killer cells and of MC-D cells which had been damaged to various extents by T cells in vitro.
Morphology of Killer T Cell-Tumor Interaction
Fig. 1. Destruction of MC-D sarcoma cells in vitro by lym phoblast cells (LBC). Photograph of a viable primary tumor culture shows a focus of destruction by LBC. Bound-shaped LBC at tached themselves to tumor cells while lysing the latter and thus creating discontinuity in the monolayer (arrows). X 300.
monolayers, indicated that the killer cells were extremely mobile. LBC never adhered to the plastic tissue culture vessels but only to the spindle-shaped ‘target’ (MC-D) cells. This made it possible for us to recognize
target and cytotoxic cells easily during ul trastructural examination. Once all the MC-D cells were killed, LBC floated freely in the supernatant, stopped proliferating and lost their viability gradually as judged
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Fig. 2. Photograph of stained LBC preparation. Note the round cells with plasma cell morpholo gy (LBC) aggregated around spindle-shaped tumor cells (ar rows). Wright stain. X 450. Fig. 3. Late culture of LBC. Note vacuolation of large round cells with plasma cell morpholo gy (open arrows). Rounding and disintegration of tumor cells is also noticeable (solid arrows). Wright stain. X 450.
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Tabic I. Viability of LBC in stimulated and unstimulated cultures1
Days3
1 4 7 10 12 14
Number2 of LBC in cultures stimulated with MC-D cells
unstimulated
viable
dead
viable
0.0010
0.1000
0.0060 ± 0.003 0.0070 ± 0.005 0.0810 ± 0.090 0.0100 ± 0.010 0.1220 ± 0.050
2.4500 2.1500 0.4600 0.0096 0.0009
0.1000
4.0000 4.9000 54.6700 85.0000 426.6700
± 0.86 ±1.21 ±20.14 ± 28.58 ± 152.0
dead 0.0010
± ± ± ± ±
l.l00 1.300 0.320 0.008 0.000
0.0550 ± 0.0100 ± 0.0340 ± 0.0060 ± 0.0460 ±
0.055 0.011
0.030 0.003 0.002
by the eclusion of 0.2% trypan blue dye (ta ble I). Some of the LBC, especially those of small size, remained viable in the absence of stimulation up to 3 weeks and could be reactivated at any time during this period simply by adding MC-D target cells to the medium. Upon restimulation proliferation occurred. Proliferation and cytotoxicity were parallel phenomena always in our nu merous experiments. Histological examination of mixed MC-D-LBC cultures grown on glass coverslips and stained according to Wright re vealed that LBC resembled plasma cells (fig. 2). The cytoplasm was abundant around the eccentric nucleus. The nucleolus was prominent. In some of the cultures, at a late stage of cytotoxic reaction, vacuolation of LBC along with the rounding up and dis integration of MC-D cells were observed with light microscopy (fig. 3). When stained with methyl green-pyronin LBC showed
strong pyroninophilia indicating an active RNA synthesis. Surface immunoglobulin could not be shown on LBC with two differ ent methods, e.g. by indirect immunofluo rescence and by a Coombs-type mixed ag glutination. Supernatants of LBC cultures did not contain immunoglobulin as judged by inhibition of hemagglutination. Ultrastructural examination showed that LBC had the cytoplasm arranged around the eccentric nucleus to form a uropod-like structure, which exhibited cytoplasmic pro cesses. The Golgi apparatus was well devel oped, with abundant vesicles and microfila ments in the cytoplasm. Ribosomes were mostly free and only short segments of the rough endoplasmic reticulum were visible (fig- 4). Rarely, if ever, MC-D target cells showed signs of degeneration while killer LBC were adhering to them. In general, MC-D cells were spindle shaped with cen-
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1 Quadruplicates. Viability was estimated by dye exclusion (0.2% trypan blue). 2 The numbers indicate viable and dead cells per culture x 10 '• ± SD. Nonadherent lymphoblast cells were counted only. Intact but stained cells were considered dead. Cell debris is not included. 3 Each culture was started on day I by co-cultivating IO’ LBC with IO'1MC-D cells. After each cell count the medium was changed for all cultures and MC-D cells were added to the stimulated cultures in increasing numbers keeping an approximate ratio of LBC: MC-D = 1 :1 . Cell concentration was maintained between 5 x i0M0»/ml.
Morphology of Killer T Cell-Tumor Interaction
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Fig. 4. Electron micrograph of LBC adhering to a tumor cell. The nucleus is eccentric with large nucleolus. The cytoplasm forms a uropod with finger-like processes. The Golgi apparatus is well developed, vesicles are nu merous in the cytoplasm. Ribo somes are numerous; only short segments of rough endoplasmic reticulum (RER) are visible. The target cell is flattened on the plastic surface, contains abundant RER and a dense body appears in the cytoplasm adjacent to the killer cell. X 8,800.
trally located nuclei and well-developed nu cleoli. The cytoplasm contained a well-dev eloped endoplasmic reticulum, abundant microfilaments, mitochondria, some free ri bosomes and occasionally vacuoles, which appeared empty.
In cytotoxic cultures swelling and signs of degeneration occurred. Cells showing slight injury seemed to be round with a few swollen processes on their surface. In the cytoplasm the most conspicuous finding was the dilation and vésiculation of rough-sur-
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Fig. 5. Rounded target cell. The cytoplasm contains vacuoles varying in size and filled with electron-lucent flaky material. Plasma membranes and nucleus show no disruption. X 4,600.
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faced endoplasmic reticulum (RER) mem branes. In some cells, the dilation of the RER membranes was very pronounced, the cisternae transformed into cyst-like struc tures with an electron-lucent content and with partial degranulation of their walls.
Another prominent change was the appear ance of cytoplasmic vacuoles. They were varying in size, and were randomly distrib uted and filled with an electron-lucent flaky content. Free ribosomes were still numer ous. Mitochondria and lysosomes failed to
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Fig. 6. Extensively damaged cell with disrupted plasma mem brane and spilling of cytoplasm. A large cytoplasmic vacuole re sembling phagosome is visible. X 6,000.
Morphology of Killer T Cell-Tumor Interaction
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show major abnormalities. The plasma membranes showed no disruptions (fig. 5). Cells exhibiting extensive injury were fragmented into smaller portions and finally transformed into granular membranous and amorphous debris in which the various cell organelles were not possible to recognize. Before this final phase, widespread nuclear and cytoplasmic alterations were evident. Margination of chromatin was prominent. Inside the nucleoplasm chromatin granules aggregated into larger clumps and empty spaces were formed. Later the nuclear enve lopes became disrupted. The nuclei became fragmented and dark. In the cytoplasm the RER showed cyst-like dilation, vésiculation.
fragmentation and degranulation. Loss of membrane-bound ribosomes was quite pronounced in many cells. Free ribosomes were, however, still apparent even in those cells which showed extensive and advanced damage. Vacuolation of cytoplasm became a conspicuous finding. The vacuoles varied in size. Some of them coalesced and became quite large. They were filled with an elec tron-lucent material (fig. 6). The origin of the vacuoles could not be revealed with cer tainty. They might have derived from RER membrane by dilatation, vesiculation and shedding their ribosomes. Some vacuoles might have originated from mitochondria by the loss of their internal compartment and
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Fig. 7. A virus-infected LBC still adhering to the target cell. Numerous immature viral parti cles are visible in the nucleus and mature particles are budding off the cytoplasmic membrane. The RER is dilated and vesiculated, with partial degranulation. X 5,000.
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Fig. 8. Various maturation stages of viral particles closely resembling C-type viruses. X 26,200.
rounded by an electron-lucent halo and by a double membrane wall, the one on the out side containing a few small spikes. These viral particles were detected in intact cells and also in those cells which showed slight or extensive injury.
Discussion Based on the results of previous studies [4, 5] and on those presented in this paper, LBC may be characterized as follows: They are round cells with plasma cell morphology and are adherent to target cells (MC-D) but not to plastic tissue culture vessels. Upon interaction with target cells LBC proliferate and readily incorporate radiolabelled thymi dine and uridine and stain with pyronin. The target cells are destroyed as a result of interaction with viable LBC. This cytotoxic action is only partially specific. Because of their rapid multiplication and mobility. LBC arc able to destroy excess amounts of target cells in vitro. A factor capable of inhibiting the migration of normal guinea pig macro phages is present in supernatants of LBC cultures [5], but no immunoglobulin or tox ic material could be detected. The induction of LBC in vitro by co-cultivation of synge neic lymphocytes with MC-D tumor cells
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the persistence of the limiting membranes. It was remarkable that mitochondria ap peared to be intact even in those cells which exhibited marked and advanced impair ment. In some cells, however, mitochondria became darker, the cristae disappeared and the limiting membranes became fragmented. Ultimately mitochondria disintegrated into dark, amorphous granules. In the final phase lysosomes seemed to accumulate and in crease in size. Lysosomal alterations were, however, not striking and they occurred only in the later stage. There were many in termediate forms between these two de scribed types showing various extent of cel lular injury. Many cells contained mature and imma ture viral particles resembling closely C-type viruses (fig. 7. 8). They were present in the cytoplasm, nuclei and also in the ex tracellular space. Some of them were inside the cisternae of RER membranes. Those not confined to RER cisternae were ran domly distributed in the cytoplasm, often accumulating adjacent to the cell membrane and budding off into the extracellular space. In some cells they were very numerous. Their size varied between 60 and 200 nm, the majority of them measuring between 80 and 120 nm in diameter. They were spheri cal and had an electron-dense core sur
could be inhibited by pretreatment of the lymphoid cell populations with a rabbit an tiserum specific for guinea pig thymus-der ived lymphocytes in the presence of comple ment. All of these characteristics are com patible with the conclusion that the killer cells studied in this system are derived from T lymphocytes. Light microscopic examination revealed that LBC resembled plasma cells, however, ultrastructurally they did not show the abundant RER, which is very characteristic of antibody-producing plasma cells (fig. 4, 6). The eccentric nucleus with a large nu cleolus and the formation of a uropod by the cytoplasm which contained a well-devel oped Golgi apparatus, but only short seg ments of RER. microfilaments, microtu bules, some vacuoles and free ribosomes, was described by others to be characteristic of activated T cells [8, 21, 24, 25]. There fore, it appears that plasma cells may arise from bone marrow derived (B) lympho cytes, which form antibodies; however, thy mus-derived killer lymphocytes which do not form antibodies may also reach a state during their differentiation in which they re semble B cell derived plasma cells when ex amined by light microscopy. The existence of T associated plasma cells’ in man has been suggested recently by Lennert et al. [23], although they could not reveal the ex act origin of these cells. We showed earlier with the aid of cytotoxic rabbit antibodies specific for guinea pig T lymphocytes that LBC originated from T cells [5], therefore, the T-cell origin of LBC exhibiting plasma cell morphology seems certain in our case. B-derived plasma cells are known to be end cells [17] which die at the end of fulfillment of their function, i.e., the production of spe cific antibodies against the antigen. This
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does not apply to less differentiated B •memory’ cells. Our studies suggest that T-derived ‘plasma cells’ behave the same way, since they died rapidly in the absence of antigenic stimulation with MC-D. Small blast cells, which did not exhibit plasma cell morphology tended to survive for long er periods in culture without stimulation, e.g. the culture could be restimulated by MC-D up to 3 weeks. It is not likely that this death was due to the artificial condi tions in culture, since LBC could be main tained for 6 months in a state of prolifera tion and cytotoxicity if MC-D target cells were supplied continuously. Supernatants of MC-D cultures did not stimulate LBC, nei ther did cultured guinea pig hepatoma or mouse cells [5]. The repopulation of synge neic X-irradiated animals with radioactively labelled small and large killer cells cultured in vitro would reveal their life span in vivo. Such experiments are currently underway. Sprent and Miller [33] also showed that ac tivated thymus-derived cells are short lived on the basis of their survival in vivo. The mechanism of T cell-mediated target cell lysis is obscure [6, 19]. The results pre sented in this paper do not permit us to draw a definite conclusion regarding the mechanism of T cell-mediated killing. It ap pears, however, that the initial changes oc cur in the cytoplasm. The RER was affected in most cases and vacuolation was a conspi cuous finding (fig. 5). Destruction of mem branes occurred only at final stages of cell injury. Some vacuolation was also seen in control MC-D cultures, which is known to occur in tissue culture media containing high amounts of bicarbonate (in our case 2 g/1), and is believed to be reversible. Be sides the empty vacuoles, no significant cell damage was observed in established cul
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Morphology of Killer T Cell-Tumor Interaction
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These viruses do not seem to have direct cytopathic effect on MC-D cells, since the incubation of MC-D with frozen and thawed LBC cultures did not result in cell damage [5]. It is suggested by our results that these type-C viruses became activated somehow during the interaction of LBC with MC-D.
References 1 André-Schwartz, J.; Schwartz, R. S.; Hirsch, M. S.; Phillips, S. M.. and Black. P. H.: Acti vation of leukemia viruses by graft-versus-host and mixed lymphocyte-culture reactions: elec tron microscopic evidence of C-typc particles. J. natn. Cancer Inst. 51: 507-518 (1973). 2 Basombrio, M. A.: Lymphomas in BALB/C mice inoculated with supernatants from chemi cally induced sarcomas. J. natn. Cancer Inst. 51: 1157-1162 (1973). 3 Berczi. I.: Studies in tumor immunology: thesis Manitoba (1972). 4 Berczi, I.: Strausbauch. P., and Sehon. A. H.: Rejection of tumor cells in vitro. Science 180: 1289-1291 (1973). 5 Berczi. I. and Sehon, A. H.: Rejection of tu mor cells in vitro: a T-cell-mcdiated reaction. Int. J. Cancer 16: 665-674 (1975). 6 Berke, G. and Amos, D. B.: Mechanism of lymphocyte-mediated cytolysis. The LMC cy cle and its role in transplantation immunity. Transplantn Rev. 17: 71-107 (1973). 7 Bernstein, I. D.; Cohen, E. F.. and Wright. P. W.: Relationship of cellular proliferation and the generation of cytotoxic cells in an in vitro secondary immune response to syngeneic rat lymphoma cells. J. Immun. 118: 1090-1094 (1977). 8 Binet, J. L. and Mathé. G.: Optical and elec tron microscope studies of ‘immunologically competent cells’ in graft reactions. Nature, Lond. 193: 992-993 ( 1962). 9 Cerottini, J.-C. et Brunner, K. T.: Lympho cytes cytotoxiques et immunité antitumorale. Annls Inst. Pasteur, Paris 122: 659-668 (1972). 10 Cerottini. J.-C. and Brunner, K. T.: Cell-me-
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tures. While DNA synthesis is known not to be a prerequisite of T cell-mediated target cell destruction, as shown by others [10], it is indicated by our data that cell prolifera tion is always initiated by prolonged stimu lation of cytotoxic cells. Similar results were obtained by Bernstein et at. [7] during the in vitro stimulation of spleen cells of rats, immunized previously to the syngeneic lym phoma (C 58 NT)D, with mitomycin-C treated tumor cells. Elimination of prolifer ating cells by exposure to high specific ac tivity 3H-thymidine at appropriate intervals impaired the generation of cytotoxic activ ity, but the remaining cells could generate cytotoxic effectors to a second set of anti gens. Viral particles were seen in cultures where T cell-mediated target cell killing took place (fig. 7, 8) but not in control MC-D cultures. Both MC-D and LBC ap peared to be infected. Immature viral parti cles, together with fully mature particles, were present in the cytoplasm and nuclei of affected cells. Morphologically they resem bled closely C-type viruses of other mam malian species [18] and of guinea pigs [20], Other investigators detected also type-C vi ruses in chemically induced tumors [2, 11, 13, 15, 30, 35], and it was hypothesized that these viruses are of endogenous origin, which play a co-carcinogenic role during chemical oncogenesis [36], However. Odcika [27] found that the appearance of endo genous viruses was independent of the ac tion of chemical carcinogens in various mouse strains. An alternate possibility is that these type-C viruses became activated as a result of the immune reaction. Murine type-C viruses were shown to be specifically associated with graft-versus-host reaction and with mixed lymphocyte cultures [1 ].
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15
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17
18
19
20
21
22
23
24
25
26
27
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29 30
31
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E.: Morphological observations on the contactinduced lysis of target cells. Eur. J. Immunol. 3: 32-37 (1973). Leclerc, J. C.; Gomard, E.; Plata, F., and Levy, J. P.: Cell mediated immune reaction against tumors induced by oncornaviruses. 11. Nature of the effector cells in tumor-cell cyto lysis. Int. J. Cancer 11: 426-432 (1973). Lennert. K.; Kaiserling, E., and MiillerHermelink. H. K.: T-associated plasma cells. Lancet/; 1031-1032 (1975). Matter, A.; Lisowska-Bernstein, B.; Ryser, J. E.; Lamelin, J.-P., and Vassalli. P.: Mouse thy mus-independent and thymus-derived lymphoid cells. II. Ultra-structural studies. J. exp. Med. 136: 1008-1030 (1972). Matter, A. and Simpson. E.: The differentia tion of cytotoxic T lymphocytes in vitro. An ultrastructural study. Cell Tissue Res. 166: 475-488 (1976). Nelson, B. K. and Flaxman. B. A.: In situ embedding and vertical sectioning for electron microscopy of tissue cultures grown on plastic Petri dishes. Stain Technol. 47: 261-265 (1972) . Odaka, T.: Strain-dependent expression of endo genous mouse-tropic leukemia viruses in chem ically induced murine leukemias. Int. J. Cancer 16: 622-628 (1975). Oettgen, H. F.; Old. L. J.: McLean, E. P.. and Carswell, E. A.: Delayed hypersensitivity and transplantation immunity elicited by soluble antigens of chemically induced tumors in inbred guinea pigs. Nature, Lond. 220: 295-297 (1968). Parker, R. C.: Methods of tissue culture, pp. 309-310 (Harper & Row, New York 1961). Rhim. J. S.; Duh. F. G.; Cho, H. Y.; Edler. E., and Vernon. M. L.: Activation of a type-C RNA virus from tumors induced by rat kidney cells transformed by a chemical carcinogen. J. natn. Cancer inst. 50: 255-261 (1973). Rollinghoff, M. and Wagner, H.: In vitro in duction of tumor specific immunity: Require ments for T lymphocytes and tumor growth in hibition in vivo. Eur. J. Immunol. 3: 471-476 (1973) . Rouse, B. T.: Rollinghoff, M.. and Warner, N. L.: Tumor immunity to murine plasma cell tu mors. II Essential role of T lymphocytes in
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dialed cytotoxicity, allograft rejection and tu mor immunity. Adv. Immunol. IS: 67-132 (1974). Chan. P. L. and Sinclair. N. R. St. C.: Immu nologic and virologic properties of chemically and j'-irradiation-induced thymic lymphomas in mice. J. natn. Cancer Inst. 48: 1629-1640 (1972). Djeu, J. Y.; Glaser, M.; Kirchncr, H.; Huang, K. Y„ and Herberman, R. B.: The effect of specific anti-rat thymocyte serum on cell-me diated tumor immunity and other lymphocyte functions. Cell. Immunol. 12: 164-169 (1974). Dodge, A. H.: Fine structural HaLV gs anti gen, and reverse transcription study of the Syr ian hamster stilbestrol-induced renal carcinoma. Lab. Invest. 31: 250-257 (1974). Fagraeus, A.; Espmark, J. A., and Jonsson, J.: Mixed haemadsorption: a mixed antiglobulin reaction applied to antigens on a glass surface. Preparation and evaluation of indicator red cells; survey of present applications. Immunol ogy 9: 161-175 (1965). Freeman. A. E.: Kclloff. G. J.; Gilden. R. V.; Lane, W. T.; Swain. A. P., and Huebner, R. J.: Activation and isolation of hamster-specific C-type RNA viruses from tumors induced by cell cultures transformed by chemical carcino gens. Proc. natn. Acad. Sci. USA 6S: 2386-2390 (1971). Gorczynski, R. M.: Evidence for in vivo pro tection against murine-sarcoma virus-induced tumors by T lymphocytes from immune ani mals. J. Immun. 112: 533-539 (1974). Harris, T. N.; Hummeler, K., and Harris, S.: Electron microscopic observations on antibody producing lymph node cells. J. exp. Med. 123: 161-172 (1966). Harvcn, E. de: Morphology of murine leuke mia viruses, in Rich, Experimental leukemia pp. 97-129 (Appleton Century Crofts, New York 1968). Henney, C. S.: On the mechanism of T-cell mediated cytolysis. Transplantn Rev. 17: 37-70 (1973). pig leukemia: an overview with reference to Hsiung. G. D.: Virological studies of guinea herpesvirus and oncornavirus. Fed. Proc. Fed. Am. Socs exp. Biol. 36: 2285-2289 (1977). Koren. H. S.; Ax, W„ and Frcund-Mollbert,
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immune response. Eur. J. Immunol. 3: 218-224 (1973). 33 Sprent, J. and Miller, J. F. A. P.: Interaction of thymus lymphocytes with histoincompatible cells. 111. Immunological characteristics of re circulating lymphocytes derived from activated thymus cells. Cell. Immunol. 3: 213-230 (1972). 34 Takatsy, G.: The use of spiral loops in sero logical and virological micro-methods. Acta microbiol. hung. 3: 189-191 (1955). 35 Weinstein. 1. B.; Gebert, R.; Stadler, U. C.: Orenstein, J. M., and Axel, R.: Type C virus from cell cultures of chemically induced rat hepatomas. Science, N.Y. 178: 1098-1100 (19-2).
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36 Whitmire. C. E.: Virus-chemical carcinogene sis: a possible viral immunologic influence on 3-methylcholanthrene sarcoma induction. J. natn. Cancer Inst. 51: 473-478 (1973).
Received: May 11, 1978 Correspondence to: Dr. 1. Berczi. Department of Immunology. Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E OW3 (Canada)
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