(Division of Tumor Immunology, Sidney Farber Cancer. Institute, Harvard Medical School, Boston, MA) (15) and also by rabbit antiserum anti-Gp-140, given to us ...
Proc. Natl. Acad. Sci. USA Vol. 82, pp. 5470-5474, August 1985
Immunology
Complement-dependent cellular cytotoxicity: Lymphoblastoid lines that activate complement component 3 (C3) and express C3 receptors have increased sensitivity to lymphocyte-mediated lysis in the presence of fresh human serum (spontaneous cytotoxicity)
OSCAR F. RAMOS*, GABRIELLA SARMAYt, EVA KLEIN*, EITAN YEFENOFt, AND JANOS GERGELYt *Department of Tumor Biology, Karolinska Institute, S-104 01 Stockholm, Sweden; tDepartment of Immunology, Eotvos Lorknd University, God, Hungary; and MThe Lautenberg Center of Immunology, Hebrew University-Hadassah Medical School, Jerusalem, Israel
Communicated by George Klein, April 3, 1985
appropriate plasma membrane structures. Once the covalent bond is established it is stable (7-9). With the contribution of factors H and I present in the serum, the reaction sequence proceeds and a small peptide, C3f, is split off from C3b. The residual fragment, iC3b, remains on the cell surface. The fragments that result after further degradation steps are C3d, C3dg, and C3c (6). A proportion of lymphocytes with cytotoxic function [killer (K) and NK] express receptors for these-i.e., CR1, CR2, and CR3 (10, 11). In the present work we have analyzed the lymphocyte-target interaction in the presence of human serum.
ABSTRACT Lymphocyte-mediated lysis of cells of the Raji, Daudi, Jijoye, and Bjab lines was elevated when fresh human serum was added to the assay. A higher proportion of effector-target conjugates was observed in the presence of human serum. In similar experiments lysis of 1301, Rael, and P3HRr1 cells was unaltered. All cell lines activated the alternative pathway of complement but they varied in the expression of receptors for complement component 3 (C3) and in the ability to fix the C3 cleavage products on their membrane. The enhancement of lysis in the presence of human serum occurred only with those cells that bound C3. This characteristic was correlated to the expression of C3 receptors. Analysis of the nature of the deposited C3 was performed with Raji cells. Raji cells exposed to human serum bound C3b as indicated by the immunoadherence test. The C3b was further processed to C3bi, because the immunoadherence declined with time and conjugate formation increased with Daudi cells, which carry the C3 receptors CR2 and CR3. This suggests that in the lytic assay lymphocytes with C3bi receptors are recruited in the presence of human serum. We assume that the bridge of C3 molecules between targets and effectors increases the avidity of their interaction.
MATERIALS AND METHODS Human Lymphocytes. Lymphocytes obtained from healthy blood donors were separated on Ficoll/Isopaque gradients and incubated in plastic bottles in the presence of iron at 370C in 5% CO2 for 60 min to deplete adherent cells. Medium. RPMI 1640 medium was supplemented with 20% heat-inactivated fetal calf serum (FCS) (GIBCO), streptomycin at 10 jig/ml, penicillin at 120 jxg/ml, and 2 mM glutamine. In some experiments Iscove's medium was used. Targets. All target cells were of human origin. K562 is an erythroleukemia cell line. Molt-4 and 1301 were derived from T-cell leukemias. Daudi, Rael, Jijoye, and P3HR-1 are Epstein-Barr virus (EBV)-positive Burkitt lymphoma lines. They have been shown to activate C3, but not all the lines express complement receptors (12, 13). Bjab is an EBVnegative Burkitt lymphoma cell. Bjab/B958 and Bjab/HR1K are its EBV-positive sublines converted in vitro with B958 and P3HR-1 virus, respectively. Their capacity to activate the alternative complement pathway and bind C3b has been studied previously (14). All cells were maintained in RPMI 1640 medium containing 20% heat-inactivated FCS. Assay for Alternative Pathway of Complement Activation. Cells (2 x 106) were incubated at 370C for 30 min in freshly collected normal human serum (NHS) in the presence of 0.01 M EGTA and 1.0 mM MgCl2. The reaction was stopped by adding 50 ,ul of 0.02 M EDTA. The supernatant was removed and tested for C3 conversion by two-dimensional crossed immunoelectrophoresis using rabbit antiserum to human C3c. Control samples of serum/EGTA/Mg2+ without cells were always included in the assays. Complement Binding Detected by Immunofluorescence. Cells (106) were incubated for 60 min at 370C with 0.1 ml of NHS. The cells were washed three times in phosphate-buf-
The cell surface moieties on the lymphoblastoid cell lines that are recognized in the natural killer (NK) assay are still undefined. We have previously proposed that plasma membrane properties of the target cells are important in the interaction with lytic cells (1), A phenomenon that can serve as an example for this mechanism is the increased cytotoxic activity of human lymphocytes towards Raji cells in the presence of human serum. Since it occurred with serum that was hypogammaglobulinemic but not with serum that was depleted of complement component 3 (C3), we proposed that C3 activation by Raji cells contributes to the cytotoxicity (2, 3). Activation of C3 by cultured cell lines was demonstrated in 1974 by Okada and Baba (4). Activation of the alternative pathway of complement is a complex process with amplifying and feedback loops (5, 6). The first split products of the C3 molecule are C3a and C3b. The latter is a bivalent ligand at the moment of the activation. It can establish a covalent bond with cell membranes. If it does not enter into reaction, this capacity decays within microseconds. The other binding site determines the interaction with C3 receptors (CR1, CR2, and CR3) on cell surfaces. This is a stable property of the molecule. Thus, after activation the C3 molecule can be bivalent or monovalent, depending on time and on confrontation with cells carrying
Abbreviations: C, complement; CR, complement receptor; ADCC, antibody-dependent cytotoxicity; NK, natural killer; FCS, fetal calf serum; NHS, normal human serum; FITC, fluorescein isothiocya-
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nate.
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Immunology: Ramos et al. fered saline and incubated at 4°C for 60 min with fluorescein isothiocyanate (FITC)-conjugated rabbit antiserum against human C3c (DAKOPATTS, Copenhagen) diluted 1:20. Anti-C3 Receptor Reagents. CR1 was detected by the monoclonal mouse antibody (DAKOPATTS). CR2 was detected by the monoclonal antibody given to us by L. Nadler (Division of Tumor Immunology, Sidney Farber Cancer Institute, Harvard Medical School, Boston, MA) (15) and also by rabbit antiserum anti-Gp-140, given to us by Raymond Frade (Laboratoire de Biochemie des Antigenes de Membrane, Creteil, France) (16). CR3 was detected by the OKM1 reagent (Ortho Diagnostics) (17). Immunoadherence. Cells (2 x 106) were mixed with 0.1 ml of NHS and incubated at 37°C for 30 min. Thereafter EDTA was added (0.01 M) and the cells were washed twice in culture medium and mixed with 0.1 ml of 1% 0 Rh+ human erythrocyte suspension (4, 18). After incubation at 37°C for 30 min the proportion of cells binding at least three erythrocytes was measured. Cell-Medi.ted Cytotoxicity Assay. Techniques for target radiolabeling and the cytotoxicity assays were performed as described previously (19). The effector-to-target ratios were 25:1, 8:1, and 3:1. The test period was 18 hr. The kinetics of target lysis was determined by stopping the effector/target interactions at different times by addition of cytochalasin B (2 ,g/ml) and EDTA (10 mM). Supernatants were collected from all the samples after 18 hr of incubation. Lymphocyte-Raji and Daudi-Raji Conjugates. Equal number of Iymphocytes and Raji or Daudi and Raji cells (2 x 105) were mixed in 0.5 ml of Iscove's medium, centrifuged at 500 x g for 2 min, and incubated at 37°C for 10 min. The pellet was resuspended and scored immediately. The target cells could be easily distinguished from the lymphocytes on the basis of their size; 200 lymphocytes were counted. For conjugates with Daudi cells, they were treated with FITC/ phosphate-buffered saline for 30 min at 37°C to distinguish them from Raji cells. Preparation of Serum Depleted in C3, C5, and Immunoglobulins. C3 and C5 were depleted by the use of specific antibodies (DAKOPATTS) coupled to CNBr-activated Sepharose beads (Pharmacia Fine Chemicals, Uppsala). The
5471
human serum samples were made up to contain 0.01 M EDTA. After passage through the columns the sera were dialyzed in phosphate-buffered saline. RESULTS
Characterization of Human Lymphoblastoid Cell Lines with Regard to Their Capacity to Activate the Alternative Pathway of Complement and Expression of C3 Receptors. The characterization results are given in Table 1. With the monoclonal antibody, CR1 was not detected on any of the cell lines. Raji and Daudi cells both expressed CR2. Daudi but not Raji cells reacted with the OKM1 reagent, which detects CR3. No C3 receptors were detected on Rael, P3HR-1, and 1301 cells. Activation of complement was assayed in two ways. Conversion of C3 to C3c was detected in the supernatants of all cell ines when they were incubated in human serum. The binding of C3 products on the plasma membranes of the different cell lines varied as indicated by their staining intensities with FITC-anti-human C3. Strong staining was seen with Raji, Daudi, Bjab/B958, and Bjab/HRlK cells. These four cell lines had also the highest efficiency in the C3 conversion assay. The B cell lines Rael and P3HR1K, the T-cell lines Molt-4 and 1301, and the erythroid cell line K562 also converted C3 though with lower efficiency. They fixed low quantities of C3 fragments. Fresh blood lymphocytes and erythrocytes did not activate C3. There was a positive correlation betwen C3 membrane deposition and the expression of CR2 receptors (correlation coefficient r = 0.94 and P < 0.001). On cell lines without C3 receptors, such as 1301 and Rael, the deposition of C3 did not occur.
Immunoadherence was observed with Raji, Daudi, Jijoye, and the three Bjab lines. A low proportion of adherence was observed with K562, Molt-4, Rael, P3HR-1, and 1301 cells. These results correlated with those obtained with the immunofluorescence and indicated that C3b was bound by the activating cells. The C3b fragments, however, were further processed, because when the Raji cells were kept for longer time in the serum the percentage of immunoadherencepositive cells decreased (Fig. 1A).
Table 1. Complement receptor expression, C3 activation, and deposition on cell surfaces ImmunoAnti-C3t Anti-Ot adherence, Anti-CR2TInten% posi% posi% posiInten% C3 tive cells tive cells tive cells conversion* sity sity Cells 4+ 4+ 4 75 60 26 100 Raji 50 3+ 24 3 100 3+ 40 Daudi 25 2+ 50 2+ 28 15 2 Bjab 35 38 3+ 75 3+ 20 ± 2 Bjab/B958 3+ 36 30 23 ± 3 70 3+ Bjab/HRlK 2+ 35 40 55 2+ 20 ± 2 Jijoye 1+ 1+ 4 5 7 14 ± 2 Rael 1+ 2 0 5 15 ± 2 P3HR1 1+ 1+ 10 30 16 ± 3 8 K562 1+ 1+ 20 14 ± 2 25 16 Molt-4 1+ 0 0 10 15 ± 3 1301 ND ND 0 0 0 PBL ND 0 0 Erythrocytes 4+ ND 100 70 ± 6 Zymosan PBL, peripheral blood lymphocytes; ND, not determined. *C3 conversion to C3c. It demonstrates the capacity of the cell to activate the alternative pathway of complement. The numbers represent mean ± SEM of three experiments. tReactivity with antibodies against C3. Immunofluorescence was evaluated in a fluorescence-activated cell sorter. tAssayed by immunofluorescence using specific antibodies. In addition to the expression of CR2, the expression of CR1 and CR3 was also tested. None of the cell lines capied CR1, and only Daudi cells expressed CR3, at a frequency of positive cells of 25%.
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Proc. Natl. Acad. Sci. USA 82 (1985)
Immunology: Ramos et al. 70-
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80
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FIG. 1. Effect of treatment of Raji cells with human serum. Aliquots of Raji cells were exposed to human serum containing 0.01 M EGTA and 1.0 mM MgCl2 for different lengths of time. Thereafter EDTA was added (0.01 M) and the cells were washed in phosphate-buffered saline and assayed. Results are mean + SEM of three experiments. (A) Immunoadherence (n) or conjugate formation with Daudi cells labeled with FITC (A). (B) Conjugate formation with freshly separated blood lymphocytes (o).
It was possible to prove that the deposited C3b was cleaved. On the plasma membrane of Daudi cells both CR2 and CR3 are expressed (20). Raji cells formed conjugates with Daudi cells after incubation in human serum with a kinetics that indicated the change of C3b to C3bi and perhaps to C3dg. Increased Natural Cytotoxicity in the Presence of Human Serum. The cytotoxic activity of unmanipulated lymphocytes against Raji cells was higher when fresh human serum was added to the assay. Under the same experimental conditions heat-inactivated or C3-depleted human serum did not enhance the lysis (Table 2). Hypogammaglobulinemic and immunoglobulin- or C5-depleted sera were equally effective. A survey of several target cells showed that the increased cytotoxicity in the presence of human serum occurred with those that in addition to activating complement also fixed the C3 split fragments on their plasma membrane (r = 0.91 and P < 0.001) (Fig. 2). The lysis of Raji and Daudi cells was enhanced most conspicuously. These cells had also the
highest C3 conversion potential, the highest intensity in the C3 binding, and the highest C3 receptor expression. Effect of Human Serum Pretreatment of Targets and Effector Cells. Preincubation of the effector cells with human serum did not modify their lytic potential against Raji, Daudi, or Bjab/B958 target cells. In contrast, preincubation of the target cells in human serum containing EGTA/Mg2+ led to a higher cytotoxic sensitivity. The peak of increased sensitivity was reached after 75-105 min (Fig. 3A). Kinetics of Lysis of Raji Cells Pretreated in Human Serum. The kinetics of lysis were similar with untreated and human serum-treated targets until the fourth hour. The differences between the two targets appeared after the sixth hour of interaction with the effectors, and by 18 hr the lysis of human serum-treated Raji cells was more effective than that of controls (Fig. 3B). Effect of Human Serum on Formation of the Effector-Target Conjugate. After 15 min of incubation there was an increase
Table 2. Effect of human serum on lymphocyte-mediated lysis
Cytotoxicity, LU/106 cells He-HS EI*
HSDC5 HSDIg FCS NHS 60 ± 6 55 6 20 ± 2 60 ± 5 2.3 18 ± 1 50 5 15 ± 1 55 ± 5 2.3 58 ± 4 16 ± 1 ND ND ND 26 ± 2 0.4 19 ± 2 42 ± 5 44 ± 3 38 ± 2 0.9 20 ± 2 23 ± 2 Bjab/B958 ND ND 0.8 ND 39 ± 3 21 ± 1 Bjab/HR1K ND ND 0.2 ND 111 ± 15 90 ± 12 Molt-4 ND ND ND 32 ± 3 30 ± 4 0.0 1301 ND 0.1 ND ND 110 ± 12 98 ± 9 K562 ND ND ND 24 ± 2 47 ± 3 0.9 Jijoye 20 ± 3 22 ± 1 20 ± 2 22 ± 1 24 ± 3 0.1 P3HR1 22 ± 2 18 ± 2 18 ± 2 26 ± 2 0.1 Rael 23 ± 2 The cytotoxicity tests were performed in the presence of 20% FCS or 10% FCS supplemented with 10% fresh NHS, heat-inactivated human serum (He-HS), human serum depleted of C5 (HSDC5) or human serum depleted of immunoglobulins (HSDIg). The 51Cr release in the absence of effector cells varied between 10% and 20% of the total isotope incorporation. It is important to note that there were no differences between the background values obtained in the presence of FCS or human serum-i.e., without effector cells. One lytic unit (LU) represents the number of lymphocytes required for 30% lysis of K562 and Molt-4 or for 20% lysis of the other targets. Results are mean ± SEM of four experiments. ND, not determined. *The increase in cytotoxicity is expressed as enhancement index (El) calculated as (LU in NHS - LU in FCS)/LU in FCS.
Target cells Raji Daudi Bjab
Proc. Natl. Acad. Sci. USA 82 (1985)
Immunology: Ramos et al. 2.5 -
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5473
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FIG. 2. Relationship between deposition of C3 on the cell membrane of cultured cell lines and their increased sensitivity to lymphocyte-mediated lysis in the presence of human serum. r = 0.91 and P < 0.001. The increased sensitivity is expressed as the enhancement index as described for Table 2.
in lymphocyte-Raji binders. After 60 min conjugate formation reached a peak (Fig. 1B). The kinetics was thus different from that of the immunoadherence. On the other hand, it was similar to the conjugate formation with Daudi cells and the increased cytolysis (cf. Figs. 1A, 1B, and 3A).
DISCUSSION An increase in natural cytotoxicity in the presence of human serum had been observed previously with Raji cells (2, 3). We show here that the lysis of other lymphoblastoid cell lines is also enhanced when fresh human serum is added to the test mixture. Comparison of the results with various targets suggests that in addition to C3 activation two other properties were necessary for this enhancement. These were the binding of the generated C3 fragments and the expression of C3 receptors on the plasma membrane. Previous reports showed that Raji cells were lysed when exposed to fresh human serum or to purified complement components of the alternative pathway (21-23). In one of these series of experiments lysis occurred if, in addition to serum, cobra venom factor was also present, and only Raji cells but not Daudi cells were affected (21). In the other series of experiments Raji cells were lysed if incubated in 30%o human serum, and the lytic effect was stronger when protein synthesis was inhibited by puromycin (22, 23). At lower human serum concentrations lysis was weaker and 10% serum, a concentration that was used in our experiments, gave about 10% 51Cr release (23). It is important to emphasize that in our experiments the isotope release in human serum did not differ from that in FCS. Therefore the lysis imposed by the presence of human serum was mediated by the
lymphocytes.
In the presence of human serum the number of effector-target conjugates increased, suggesting that effector cells bound to the target due to interaction with the deposited C3 fragments on their plasma membranes. Preincubation of the targets was sufficient; however, its length was of great importance. The kinetics of the human serum pretreatment of Raji cells for the enhanced lysis correlated with their conju-
gate-forming capacity with lymphocytes and Daudi cells but
0)
r. 0
2
4 6 Incubation time, hr
FIG. 3. Increased sensitivity to lymphocyte-mediated lysis by pretreatment of the targets with human serum. (A) 5"Cr-labeled Raji (o) and Daudi (o) cells were exposed to human serum/EGTA/Mg2" for various times. Thereafter EDTA (0.01 M) was added and cells were washed with phosphate-buffered saline. Lymphocytes, to make up 25:1 ratio with the target, were added in Iscove's medium. The lytic assay was 18 hr; results are mean ± SD of triplicate samples. (B) Kinetics of the lymphocyte-mediated lysis. 51Cr-labeled Raji cells were exposed to human serum/EGTA/Mg2+ (o) or to buffer alone (*) for 90 min. Thereafter EDTA (0.01 M) was added and the cells were washed in phosphate-buffered saline. Cytotoxicity was assayed in Iscove's medium at an effector-to-target ratio of 25:1. The effector-target interaction was stopped by addition of EDTA and cytochalasin B. 5'Cr release was measured after 18 hr. Results are mean ± SD of triplicate samples.
not with the immunoadherence test. The latter test, which detects C3b molecules, showed a rapid decline, while the conjugates with the CR2-receptor-carrying Daudi cells increased with time. The kinetics of the enhanced lysis followed that of the lymphocyte and Daudi cell conjugates. These results suggest that the C3b molecules were further processed on Raji cell membranes and the majority of the recruited effector cells probably carry CR3 receptors. This corresponds well to the results described in ADCC with sensitized bovine erythrocytes as targets. In this system C3 fragments were deposited experimentally on the target and the strongest effect was achieved with C3bi fragments (24, 25). In another antibody-dependent cellular cytotoxicity (ADCC) system also, C3 molecules created a bridge between effectors and targets. Concanavalin A-stimulated human lymphocytes were found to activate C3 and covalently fix the generated C3 fragments. Such cells had enhanced lysis of C3b-receptorbearing erythrocytes sensitized with antibodies (26). Contact itself is not sufficient to provide the basis for the
5474
Immunology: Ramos et al.
lymphocyte-mediated lysis (24, 25). In the two ADCC systems mentioned above, antibodies were required and without them the complement bridge did not initiate lysis. For the NK action the nature of the corresponding primary interaction between target and lymphocyte is still unknown. The kinetics of the lytic process indicate that the effector cells that are recruited require a period for the activation of the mechanism responsible for lysis. The experiments with the various cells that demonstrated binding of the C3 by immunofluorescence and C3b by immunoadherence corresponded well. Interestingly, these results correlated with the expression of C3 receptors when detected with the monoclonal antibody reagents. The property of C3 activation by cells was attributed by Theofilopoulos to the presence of C3 receptors on their plasma membranes (27). Our results would lead to a similar conclusion. In our experiments the C3 activation tested in the fluid phase showed that even C3 receptor-negative lines had this function; their efficiency, however, was lower. It is possible that the C3 receptors act as cofactor, and those C3 molecules that are activated with their contribution are sufficiently close to the acceptor sites on the plasma membrane. It was shown previously that the Raji cell membrane requires further serum factors for the activation of C3 (13). In accordance, Raji cells exposed to purified C3 stained considerably weaker with the anti-C3 antibodies than those exposed to human serum, and C3 added to the cytotoxic test did not increase lysis. The phenomenon described here may have biological significance in vivo, since it involves homologous components. Effectors, targets, and the complement source all were derived from the same species. The possible in vivo role of these events is suggested by experiments in which administration of C3 led to more efficient rejection of melanoma grafts in mice (28). We thank Dr. Hidechika Okada (Department of Microbiology, Fukuoka University School of Medicine, Fukuoka, Japan), Dr. Kusuya Nishioka (The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan), Dr. Gosta Eggertsen (Departments of Medical and Physiological Chemistry, Uppsala University, Uppsala, Sweden), and Dr. Bo Nilsson (Department of Clinical Chemistry, Uppsala University) for valuable suggestions. This work was supported by National Cancer Institute Grant 5 Rol CA25250-06. O.F.R. is supported by The Swedish Institute and by The Cancer Research Institute Concern Foundation. 1. Klein, E. (1980) Immunol. Today 1, iv-vi. 2. Yefenof, E., Yron, I. & Klein, E. (1984) Cell. Immunol. 87, 698-702.
Proc. Natl. Acad. Sci. USA 82 (1985) 3. Yefenof, E., Klein, E. & Yron, I. (1984) Mol. Immunol. 21, 1211-1214. 4. Okada, H. & Baba, T. (1974) Nature (London) 248, 521-522. 5. Pangburg, M. K., Schreiber, R. D. & Muller-Eberhard, H. J. (1983) J. Immunol. 131, 1930-1935. 6. Lachman, P. T. & Hughes-Jones, N. G. (1984) Springer Semin. Immunopathol. 7, 143-162. 7. Weigle, W. O., Goodman, M. G., Morgan, E. L. & Hugh, T. E. (1983) Springer Semin. Immunopathol. 6, 159-172. 8. Muller-Eberhard, H. J. & Schreiber, R. D. (1980) Adv. Immunol. 29, 2-25. 9. Fearon, D. T. & Wong, W. M. (1983) Annu. Rev. Immunol. 1, 243-271. 10. Nocera, A., Montesoro, E., Balbo, P., Ferrarini, M., Leprini, A., Zicca, E. & Grossi, C. E. (1983) Scand. J. Immunol. 18,
345-354. 11. Ross, G. D. (1980) J. Immunol. Methods 37, 197-205. 12. Yefenof, E., Klein, G. & Kvarnung, K. (1977) Cell. Immunol. 31, 225-233. 13. Budzco, D. B., Lachman, P. J. & McConnell, I. (1976) Cell. Immunol. 22, 98-109. 14. McConnell, I., Klein, G., Lint, T. F. & Lachmann, P. J. (1978) Eur. J. Immunol. 8, 453-458. 15. Lida, K., Nadler, L. & Nussenzweig, V. (1983) J. Exp. Med. 158, 1020-1033. 16. Frade, R., Barel, M., Krikorian, L. & Charriaut, C. (1984) Eur. J. Immunol. 14, 542-547. 17. Wright, S. D., Rao, P. E., Van Voorhis, W. C., Craigmyle, L. S., lida, K., Talle, M. A., Westberg, E. F., Goldstein, G. & Silverstein, S. C. (1983) Proc. Natl. Acad. Sci. USA 80, 5699-5703. 18. Nishioka, K. (1971) Advances in Cancer Research (Academic, New York), Vol. 14, pp. 231-293. 19. Ramos, 0. F., Masucci, M. G. & Klein, E. (1984) Cancer Res. 44, 1857-1862. 20. Okada, H. & Nishioka, K. (1973) J. Immunol. 111, 1444-1449. 21. Theofilopoulos, A. N., Bokish, V. A. & Dixon, F. J. (1974) J. Exp. Med. 139, 696-703. 22. Baker, P. J., Lint, T. F., Mortensen, R. F. & Gewurz, H. (1977) J. Immunol. 118, 198-205. 23. Schreiber, R. D., Pangburg, M. K., Medicus, R. G. & MullerEberhard, H. J. (1980) Clin. Immunol. Immunopathol. 15, 384-396. 24. Wahlin, B., Perlmann, H., Perlmann, P., Schreiber, R. D. & Muller-Eberhard, H. J. (1983) J. Immunol. 180, 2831-2840. 25. Perlmann, H., Perlmann, P., Schreiber, R. D. & MullerEberhard, H. J. (1981) J. Exp. Med. 153, 1592-1603. 26. Erdei, A., Benczur, M., Fdbry, Z. S., Dierich, M. P. & Gergely, J. (1984) Scand. J. Immunol. 20, 125-131. 27. Theofilopoulos, A. N. & Perrin, L. H. (1976) J. Exp. Med. 143, 271-277. 28. Cooper, P. D. & Sim, R. B. (1984) Int. J. Cancer 33, 683-687.
