Immunology, 1975, 29, 719.

Characteristics of the Effector Cells Mediating Cytotoxicity Against Antibody-coated Target Cells I. PHAGOCYTIC AND NON-PHAGOCYTIC EFFECTOR CELL ACTIVITY AGAINST ERYTHROCYTE AND TUMOUR TARGET CELLS IN A 51Cr RELEASE CYTOTOXICITY ASSAY AND [1251]IUdR GROWTH INHIBITION ASSAY A. H. GREENBERG, L. SHEN AND G. MEDLEY Department of Immunology, Middlesex Hospital, London

(Received 20th January 1975; accepted for publication 13th March 1975) Summary. Both phagocytic and non-phagocytic effector cells were able to kill rabbit antibody-coated chicken erythrocytes (CRBC) while only non-phagocytic effector cells were active against alloantibody-coated SL2 lymphoma. In addition to the variation in susceptibility of erythrocyte and tumour target cells to various effector cell populations, it was found that different tumour cells can vary markedly in their ability to be killed by non-immune spleen cells in the presence of antibody. It is postulated that both the type of antibody and certain characteristics of the cell membrane are important in determining whether target cells are susceptible to antibody-dependent cell-mediated cytotoxicity detected by the 51Cr release assay. It was also demonstrated that alloantibody-coated P-815-Y mastocytoma, which showed very little evidence of cytotoxicity in the 5'Cr release assay, was markedly inhibited in its ability to incorporate [1251]IUdR after incubation with antiserum and non-immune spleen cells. This growth inhibition in the absence of cytotoxicity, or cytostasis, is discussed in relation to the potential mechanisms of target cell damage, and in the light of recent observations (Plata, Gomard, LeClerc and Levy, 1974; Newlands and Roitt, 1975) that cytotoxicity and growth inhibition assays detect different effector cell populations in tumour-bearing animals. INTRODUCTION Studies of antibody-dependent cell-mediated cytotoxicity have produced conflicting observations about the nature of the cells that can kill different target cells. Phagocytic effector cells are able to kill the chicken erythrocyte in the mouse (Greenberg, Shen and Roitt, 1973), guinea-pig (Temple, Loewi, Davies and Howard, 1973), and human (Perlmann and Perlmann, 1970), but not the Chang cell in the human or rat (MacLennan, 1972) or the YAC lymphoma in the mouse (Forman and Moller, 1973). Non-phagocytic effector cells appear to be able to kill all of these target cells although even this observation is disputed (Dennert and Lennox, 1973). These differences are partly due to the different Correspondence: Dr A. H. Greenberg, Manitoba Institute of Cell Biology, 700 Bannatyne Avenue, Winnipeg,

Manitoba, Canada R3E OV9.

719

A. H. Greenberg, L. Shen and G. Medley methods used to obtain non-phagocytic cells and to assess their phagocytic properties; however, it may also be true that similar preparations of effector cells can differ in their ability to kill various target cells. In the present study we have tried to resolve these questions by comparing the susceptibility of erythrocyte and tumour target cells to well characterized phagocytic and non-phagocytic populations of effector cells. In addition, we have also examined the susceptibility of different tumour target cells to antibody-dependent cell-mediated cytotoxicity detected in the 5"Cr release assay and an [125I]IUdR growth inhibition assay. The results of these experiments are discussed in relation to the potential mechanisms of target cell damage, and in the light of recent observations that cytotoxicity and growth inhibition assays detect different effector cell populations.

720

MATERIALS AND METHODS

Spleen suspensions Spleens were taken from 8- to 16-week-old BALB/c mice bred in our colony or 8-16week-old C3H mice obtained from a commercial source (S.A.C.I., Ltd, Braintree, Essex). Cells were prepared for further fractionation by gently disrupting the spleens with forceps and allowing the clumps to sediment at 40 for 10 minutes. The cells were then washed twice in Eagle's minimal essential medium (MEM) or Fischer's medium. Antisera Rabbit anti-CRBC antiserum was raised by injecting 2 ml of 10 per cent CRBC intravenously twice with a 14-day interval. The rabbits were bled 12 days after the second

injection. C3H (H-2k) anti-P-815-Y (H-2d) antisera were raised by three intraperitoneal injections of 107 tumour cells at 14-day intervals. The mice were bled out 10 days after the last injection. Sera were absorbed with normal C3H spleen cells at 220 for 1 hour, then heatinactivated at 560 for 30 minutes. Target cells Chicken erythrocytes were obtained from young White Leghorn chickens in a heparinized syringe by cardiac puncture. SL2 lymphoma (kindly supplied by Dr R. Evans, Chester Beatty Institute, Sutton, Surrey) and P-815-Y mastocytoma were carried in either DBA/2 or (DBA/2 x C3H)F1 mice by intraperitoneal passage. Cells were obtained 7 days after passage and placed in culture in Fischer's medium containing 10 per cent FCS at a concentration of 4 x 105/ml. After 24 hours the cells were usually 2-2 times the original concentration. They were diluted to 2 x 105/ml and maintained in culture for up to 72 hours by in vitro passage every 24 hours before use in the cytotoxicity assays. Phagocytosis assay Sheep erythrocytes stored in Alsever's medium were washed three times and a 200-p1 sample of packed cells was incubated with 200 yCi of 51Cr for 1 hour at 37°. The cell suspension was then diluted 1:20 in Eagle's MEM, and rabbit anti-sheep haemolysin (haemagglutination titre 1:500) added to a final concentration of 1:1200. After a 30minute incubation at 370 the cells were washed three times and the pellet resuspended in Eagle's MEM containing a 1: 20 dilution of fresh mouse serum as a source of complement.

Characteristics of Antibody-dependent Cytotoxic Cells. I. 721 After a further 30-minute incubation at 370 the cells (erythrocyte-antibody-complement (EAC) complexes) were washed three times and adjusted to a concentration of 3 x 108/ml in Eagle's MEM containing 10 per cent FCS. A 300-pl sample of spleen cells at a concentration of 107/ml was then placed in a 2-ml siliconized glass tube with 50 pl of the 51Cr-labelled EAC to give a final cell ratio of 5: 1 EAC to spleen cells. The cell suspension was gassed with 5 per cent CO2 and incubated on a rocking platform for 1 hour at 370. An identical set of tubes containing an additional 5 pl (70 pg) of cytochalasin B in DMSO was set up for each sample. At the end of the incubation period 1 ml of 0-83 per cent NH4C1 was added and left at room temperature for 6 minutes causing the lysis of all extracellular EAC. The cells were then washed three times and the pellet counted in a gamma counter. All samples were examined in triplicate.

Carbonyl iron methodfor depleting phagocytes This method is a modification of the procedure described by Lundgren, Zukoski and Moller (1968). Spleen cells were suspended in medium containing 10 per cent foetal calf serum at a concentration of 3 x 106/ml in conical-bottomed 25-ml universal containers and carbonyl iron added to make a final concentration of 4 mg/ml. The suspension was mixed and placed in a 370 water bath for 1 hour, agitating every 10-15 minutes. The containers were then transferred to a 40 room and powerful magnets were placed at the base. After 10 minutes' sedimentation the supernatant was transferred to a new container and the sedimentation repeated, this time applying the magnet to the side of the container. After 10 minutes the cells were again transferred to a fresh vessel and a third sedimentation at the base removed the remaining iron particles. Any residual iron could be detected in the cell pellet during the subsequent wash and, if necessary, a final sedimentation carried out. Cell recovery was 43A4+6-6 per cent (34-6-54.6). Two types of iron were compared during preliminary studies, iron powder (Fe) excarbonyl iron (B.D.H. Ltd), particle size 5-6 jim, and carbonyl iron S.F. (Fe2 (CO)9) (G.A.F. (U.K.) Ltd), particle size 3-4 pm. These experiments showed that at a given concentration iron carbonyl was more efficient at removing phagocytic cells as measured by their ability to ingest EAC complexes. Further studies on the conditions necessary for optimal removal of phagocytes with carbonyl iron revealed that the concentration of both the cell suspension and the iron was critical. Leucocyte suspensions of greater than 3 x 106/ml and iron concentrations less than 4 mg/ml were found to be inadequate. Higher carbonyl iron concentrations were found to be detrimental in that cell loss was greater, resulting both from non-specific adherence of cells other than phagocytes, and from cell death, presumably due to the toxicity of the iron.

Cytotoxicity assays (a) Cytotoxicity of chicken erythrocytes by a 5"Cr release assay. This assay has been described in an earlier publication (Greenberg et al., 1973). Briefly, 200-ul aliquots of effector cells and rabbit anti-CRBC antibody (final dilution 1: 30,000) were mixed with 100 P1 of " Crlabelled CRBC ( 105/ml) and 100 pl of washed unlabelled SRBC (2 x 1 07/ml). The incubation mixture was kept in a 5 per cent CO2 atmosphere for 18 hours, then mixed, spun and the supernatant separated. Both the CRBC and the tumour cytotoxicity assays (see below) were carried out in tubes that allowed the cells to sediment into a pellet. (b) Cytotoxicity of the SL2 lymphoma (H-2d) and P-8 15-r mastocytoma (H-2d) by a 5"Cr release assay. SL2 or P-815-Y cells were taken from in vitro cultures, washed, and then incubated

A. H. Greenberg, L. Shen and G. Medley 722 with 51Cr (100 Ci/ cells) for 30 minutes at 370. The cells were then washed three times and adjusted to a concentration of 5 x 104/ml for use in the assay. The timing of the incubation was such that there was no delay between the final preparation of the antibodycoated target cells and their being distributed into the incubation mixture with an equal volume (200p1) of spleen cells. The assay was carried out in Fischer's medium containing 10 per cent FCS and all samples were examined in triplicate. After 18 hours' incubation in a 5 per cent atmosphere at 370 the supernatants were harvested as previously described (Greenberg et al., 1973) and the cytotoxicity calculated by the following formula: [(percentage "Cr release from antibody-treated tumour-percentage 5tCr release from normal serum-treated tumour) /(percentage 'Cr release from Triton X-treated tumour percentage 51Cr release from normal serum-treated tumour)] x 100. per Maximal release of5'Cr was estimated from samples of tumour incubated with 0-255 Cr of the total label. Spontaneous per cent 95 usually about X and was cent Triton release in the normal serum-treated controls in the presence of non-immune spleen cells was between 25 and 30 per cent. of the procedure described by (c) Growth inhibition assay. This method is a modification from in vitro cultures, washed and were taken cells Tumour Chia and Festenstein (1973). medium. A 200-,ul FCS-Fischer's x in cent 5 a of to concentration 104/ml 10 per adjusted anti-P-815-Y or C3H cells and either of volumes with was incubated spleen equal sample normal C3H serum. Additional controls of spleen cells incubated without target cells, and a 24-hour incubation in a 5 per cent target cells incubated alone were also prepared. After washed once and resuspended in was mixture incubation at the CO2 atmosphere 370, 600 pl of 10 per cent FCS-Fischer's medium containing 0 05 PCi of [1251]IUdR. After an at 370 the cells were washed three times and overnight incubation in 5 per cent CO2 The counted in a well-type gamma counter. growth inhibition was calculated by the tumour - ct/minute in lymphocyte in formula: antibody-coated (ct/minute following/ (ct/minute [1in normal serum-treated tumour - ct/minute in lymphocyte control)] control)

07

-

x 100.

At 100: 1 lymphocyte to tumour cell ratio the lymphocyte control incorporated approximately 5 per cent of the [1251]IUdR of the normal serum-treated tumour cells. RESULTS

POPULATIONS An assay characterizing the phagocytic potential of spleen leucocytes was established to measure the efficiency of various preparations of iron powder and iron carbonyl in depleting phagocytic cells. Erythrocyte-antibody-complement complexes were used as an indicator rather than inert particles, such as polystyrene beads or latex, since we were primarily interested in the ability of the spleen preparations to phagocytose whole cells. Pretreatment with antibody and complement assured that maximal phagocytosis was observed. Preliminary studies indicated that the direct morphological assessment of phagocytosis by mouse peritoneal exudate cells correlated well with their ability to take up 5 'Cr-EAC complexes; however, the isotope method was preferred because of its relative ease in handling and good reproducibility. A phagocytic index was calculated as the ratio between the cell associated 51Cr in the test sample and in a like sample treated with cytochalasin B. In agreement with earlier observations (Klaus, 1973; Temple et al., 1973) the presence of PREPARATION OF NON-PHAGOCYTIC EFFECTOR CELL

Characteristics of Antibody-dependent Cytotoxic Cells.

I.

723

200 i

!I

10

000

5 0_

a,~~~~~~~~~6 0-

2 -0

PEC

Whole

Fe

spleen

splIeen

FIG. 1. Phagocytosis of EAC by mouse cells. The percentage of the total "lCr-EAC which is found in the washed cell pellet after lysis with NH4Cl following incubation at 370 for I hour in 10 per cent foetal calf serum (,&), is compared to the same cell population incubated in the presence of cytochalasin B (A) . The results are shown in triplicate for three cell preparations, mouse peritoneal exudate cells (PEC) induced with proteose peptone, whole mouse spleen cells and spleen cells treated with carbonyl iron powder (Fe spleen). The dashed line represents the percentage of the 5'Cr-EAC incubated without leucocytes which remains after NH4Cl IYSiS.

cytochalasin B completely blocked the uptake of EAC and showed only minimally higher counts than the lysed EAC background (Fig. 1 ). This small increase over background was also noted by Klaus (1973) and may represent pinocytosis of labelled erythrocyte proteins, a phenomenon which is not affected by cytochalasin B. The phagocytic index ignores the pinocytosis and refers only to the phagocytic potential of the leucocytes. A ratio of I -0 indicates the absence of phagocytosis. Comparing spleen cell populations before and after treatment with optimal amounts of carbonyl iron, the phagocytic index falls from 2-91 + 0-72 (+s.d.) in whole spleen to 1-04+0-08 (Table 1). Peritoneal exudate cells usually have an

index of greater than 20-0.

TABLE I THE EFFECT OF CARBONYL IRON TREATMENT ON THE ABILITY OF SPLEEN CELLS TO PHAGOCYTOSE COMPLEXES

S51Cr-EAC

Phagocytic index* Experiment number 1

Whole spleen

Carbonyl irontreated spleen

2-16

099 1-17 1-07 1-04 094

2 3

4*04

4 5 6

2-74 3-52 2-71 2-91+ 0-72

Mean

2-32

1-04+ 0-08

* In the absence of phagocytosis the phagocytic index would be 1 00.

A. H. Greenberg, L. Shen and G. Medley 724 Although spleen cells showed no evidence of phagocytosis immediately after carbonyl iron treatment, the possibility existed that some of the cells had only temporarily lost their phagocytic properties by exposure to the iron which, as mentioned earlier, can be toxic. It was also not clear whether immature cells which were not phagocytic after carbonyl iron treatment could become phagocytic in culture. Spleen cells were therefore treated with carbonyl iron, tested for their ability to phagocytose EAC, and then placed in culture with unlabelled CRBC in the presence of rabbit antiserum, normal antiserum or without serum for 18 hours. At the end of the incubation the cells were washed and retested. In two out of three experiments their ability to phagocytose EAC remained low and in one experiment the phagocytic index was 28 per cent higher than was found immediately after iron treatment and before the incubation period. THE EFFECT OF PHAGOCYTIC AND NON-PHAGOCYTIC SPLEEN CELLS ON ERYTHROCYTE AND TUMOUR TARGET CELLS

Fig. 2 illustrates the cytotoxicity of spleen cells, before and after depletion of phagocytes, for rabbit antibody-coated CRBC and alloantibody-coated SL2 lymphoma. A large pro(a) 80 -(b) 45

.600

40-

50:1

25:1

12:1

100:1

75:1

50:1

Spleen:target cel ratio FIG. 2. Effects of depletion of phagocytic cells on the cytotoxicity of tumour and erythrocyte target cells. The cytotoxicity (± s.d.) of whole spleen cells (circles) and non-phagocytic spleen cells prepared by carbonyl iron treatment (triangles) for rabbit antibody-coated SRBC (a) and for alloantibody-coated SL2 lymphoma (b). Target cell concentration was 5 x 104/ml.

portion of the cells capable of killing the CRBC was removed by iron treatment. In contrast, the SL2 target cells were killed more readily by non-phagocytic spleen cells at most effector to target cell ratios. A COMPARISON OF ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY DETECTED BY 51Cr RELEASE AND [125I]IUdR GROWTH INHIBITION ASSAYS USING THE SL2 (H-2d) LYMPHOMA

AND P-815-Y (H-2d) MASTOCYTOMA

Fig. 3 compares the cytotoxicity of whole non-immune C3H spleen cells for antibodycoated SL2 lymphoma and P-815-Y mastocytoma target cells in a 51Cr release assay. Despite the fact that the same antibody, C3H (H-2k) anti-P-815-Y (H-2d), was used to pretreat both target cells, little or no effect could be detected on the P-815-Y target, while

Characteristics of Antibody-dependent Cytotoxic Cells.

I.

725

24

16

-

1:20

1:200 1:2000 Antiserum dilution

1:20000

FIG. 3. Susceptibility of different tumour target cells to antibody-dependent cell-mediated cytotoxicity. Cytotoxicity of SL2 lymphoma (H-2d) (0) and P-815-Y mastocytoma (H-2d) (0) treated with C3H (H-2k) anti-P-815-Y (H-2d) antiserum and exposed to whole non-immune spleen cells. The effector to target cell ratio was 100:1 with a target cell concentration of 5 x 104/ml.

SL2 cytotoxicity was readily demonstrated. Since the antiserum was raised against the P-81 5-Y it seemed unlikely that the difference could be due to the absence of antibody to the P-815-Y. This possibility was tested, however, by assessing the relative binding of IgG antibody to the two tumours with fluorescein-conjugated purified rabbit anti-mouse IgG (Fig. 4). The proportion of the two target cells reacting with the antibody was virtually identical at all antiserum dilutions in two different experiments. 100

75 Z

500

25-

1:10

1:102

1:103

l:i04

Control

Antiserum dilution

FIG. 4. Titration of alloantigens on SL2 and P-815-Y cells by immunofluorescence. The percentage of SL2 lymphoma cells (H-2d) (0) and P-815-Y mastocytoma cells (H-2d) (0) binding C3H (H-2") antiP-815-Y (H-2d) antibody detected with fluorescein-conjugated, rabbit anti-mouse IgG antibody. Control cells were treated with normal C3H serum.

The P-815-Y target cell was then examined in a growth inhibition assay (Fig. 5). As above, the target cells were exposed to non-immune C3H spleen cells in the presence of C3H anti-P-815-Y antiserum for 24 hours. A pulse of ['25I]IUdR was then delivered to

726

A. H. Greenberg, L. Shen and G. Medley 80

C

60 _

2

40

20

102

103

104

Antiserum dilution

FIG. 5. Percentage growth inhibition ( s.d.) of P-815-Y mastocytoma incubated with various dilutions of C3H anti-P-815-Y antiserum and normal non-immune C3H spleen cells.

measure the ability of these target cells to incorporate the label. Significant inhibition of IUdR uptake was detected at several dilutions of antiserum.

DISCUSSION The analysis of the carbonyl iron method presented in this study shows that under optimal conditions phagocytic cells can be completely eliminated from mouse spleen. These cells appear to regain little, if any, of their phagocytic potential even after prolonged incubation. It should be noted that this carbonyl iron treatment removes cells that have no phagocytic properties by adhering to their cell surface (Golstein and Blomgren, 1973). Our studies (Greenberg, Shen, Walker, Arnaiz-Villena and Roitt, 1975) suggest that up to 25 per cent of the immunoglobulin-bearing cells may be lost by this treatment. The comparison of the effector cells mediating cytotoxicity for the CRBC and the SL2 lymphoma has shown that these target cells differ in their susceptibility to killing by phagocytes (i.e. cells removed by carbonyl iron treatment). Recent electronmicroscopic studies (Penfold, Greenberg and Roitt, 1976) have confirmed the present observations that phagocytic cells, both monocytes and PMN, can readily kill erythrocyte target cells and are in agreement with the findings of Temple et al. (1973) and Melsom and Seljelid (1973), who observed macrophage-mediated erythrocyte cytotoxicity. In contrast, phagocytic cells are much less active against the SL2 target. At low effector cell: target cell ratios the enrichment in the specific activity of cytotoxic cells after carbonyl iron treatment is virtually equivalent to the proportion of cells lost, suggesting that the depleted cells were inactive. This is not the case, however, at high effector cell: target ratios, where no change or a slight reduction in the specific activity is seen. This may be at least partly due to a non-specific inhibition of cytotoxicity by the high concentration of spleen erythrocytes left in the fractionated spleens after carbonyl treatment (Greenberg et al., 1975). A difference in the susceptibility of tumour target cells to antibody-dependent cellmediated cytotoxicity was observed by the comparison of the SL2 lymphoma and P-81 5-Y mastocytoma. The antiserum raised against the P-815-Y contained anti-H-2d IgG antibodies, and possibly other antibodies, which were capable of binding to both tumours.

Characteristics of Antibody-dependent Cytotoxic Cells. I.

727 However, specific 5"Cr release in the presence of non-immune C3H spleen was demonstrated only with the SL2 and not the P-815-Y. The reasons for this are not immediately apparent since the effector cells and antiserum were identical. In addition, the similar binding characteristics of the antiserum to the two tumours suggested an equivalent distribution and density of membrane antigens. One must, therefore, consider some other characteristics of the membranes of these two tumours which could account for their differences. The rate of turnover of the cell surface antigens may be of importance since the binding of the antibody to these antigens provides the recognition site for the effector cells. The P-815-Y may be less susceptible to cytotoxicity if the antibody is cleared more rapidly from its surface than the SL2. This explanation seems less likely with the additional observation that antibody-coated P-815-Y are recognized by non-immune effector cells in the growth inhibition assay, which is also dependent on the presence of membrane-bound antibody. Another characteristic which may be of interest is the rate at which tumours are capable of repairing their cell membrane in response to damage induced by this cytotoxic mechanism. In addition to the characteristics of different tumour membranes, it is probably true that different antibodies are more or less effective in mediating this type of cytotoxicity. It has been observed by ourselves (unpublished data) and others (Zigheboim, Bonavida and Fahey, 1973) that heterologous antibody is more effective in the "Cr release assay than alloantibody using the same effector cell population and target cell. It is not yet established whether syngeneic tumour-specific antibody is capable of inducing killing in this assay. Although there is no explanation for these differences at this time, the correlation of cytotoxocity and membrane antigen density cannot be overlooked. A critical amount of antibody may be required before effector cell-target cell interaction can take place in a way which results in membrane damage. The importance of antigen density in complementdependent cytotoxicity is a well known concept (Anderson, Wigzell and Klein, 1967). The effects of non-immune spleen cells on antibody-coated tumours appear to be of two types, inhibition of growth, or cytostasis, and direct membrane damage leading to cell death. These two phenomena have been demonstrated to be independent of one another in that the P-815-Y can be inhibited from incorporating ['25I]IUdR while failing to release 5Cr using the same antibody and effector cell population. This cannot be explained simply by the sensitivity of the two assays since the same antibody concentration which fails to induce "Cr release in the P-815-Y readily kills the SL2. The explanation most probably lies within the nature of the assays themselves and exactly what they are capable of measuring. The ['25I]IUdR inhibition assay can assess both cytotoxicity and cytostasis since it measures the inability of cells to synthesize DNA, a common feature of both cell death and failure to divide. 51Cr release, on the other hand, is thought to occur only when membrane damage is severe enough to allow large labelled proteins to escape from the cell and, therefore, correlates well with cell death (Henney, 1973). Cytostasis, which is the inhibition of cell growth in the absence of cell death, has been readily demonstrated in various situations, including contact inhibition of tumour cells growing in culture (Burger, 1971), or by treating cells with chalones (Simard, Corneille, Deschamps and Verly, 1973), dibutyryl-3',5'-cyclic AMP or other drugs (Thomas, Medley and Lingwood, 1973). It has been postulated that a membrane event in G, commits tumour cells to a reversible inhibiton of cell division (Thomas et al., 1973). It is possible then that the observed effector cell-target cell interaction which resulted in cessation of tumour cell growth occurred through a similar membrane signal without the concomitant membrane damage

A. H. Greenberg, L. Shen and G. Medley which would have resulted in 5"Cr release. The additional possibility that inhibition of [251I]IUdR incorporation represents the failure of cell membrane nucleotide transport rather than inhibition of DNA synthesis cannot be ruled out. These findings are also relevant to the recent observations of Plata et al. (1974) on the nature of the effector cells that are active in microcytotoxicity and "Cr release assays in tumour-bearing animals. They found that T cells are the only active cell in the "Cr release test, while both T and non-T cells were responsible for the growth inhibition of the microcytotoxicity test. Since it has been suggested that non-T cell detected in this assay is the antibody-dependent cytotoxic cell (O'Toole, Perlmann, Wigzell, Unsgaard and Zetterlund, 1973; Greenberg and Shen, 1973), the lack of activity of this cell in the 5tCr release assay was difficult to understand in the light of our findings and that of many other investigators that tumour cell death can be readily detected by 5 Cr release in the presence of antibody and non-immune spleen cells (MacLennan, 1972; Forman and Mbller, 1973; Zigheboim et al., 1973). The observations reported in this study indicate that not all tumours are susceptible to damage detectable by "Cr release and not all antibodies are capable of inducing killing in this type of assay, but at the same time effector cell activity can be detected in a growth inhibition assay. Since Plata et al. (1974) were unable to find non-T-cell effects in their 51Cr assay it may be that either the MSV-induced tumour they used as a target is not susceptible to this type of effector cell, or that syngeneic tumourspecific antibody is unable to induce 5Cr release by this mechanism. Syngeneic antibody from tumour-bearing mice is active in the colony inhibition assay in the presence of nonimmune lymphoid cells (Pollock, Heppner, Brown and Nelson, 1972), but it has yet to be demonstrated that it is capable of inducing cell death in a "Cr release assay.

728

ACKNOWLEDGMENTS We wish to thank Professor I. M. Roitt for his advice and encouragement and Mrs Pearl Emery for typing this manuscript. This work was supported by the Medical Research Council of Great Britain and the Medical Research Council of Canada (grant number MA-5390).

NOTE ADDED IN PROOF The P-818-Y clone of DBA/2 mastocytoma used in this study was subsequently found to have undergone some antigen simplification when compared to the parent tumour. The present observations have been repeated using the P-8 1 5-X2 clone and a hyperimmune C57B1/6 anti-P-815 antiserum with the ability to induce complement mediated lysis of both the SL2 and P-815-X2 in a similar manner. We have again observed the relatively greater 5'Cr release cytotoxicity of the SL2, with only low levels of activity against the P-815-X2. REFERENCES

ANDERSSON, B., WIGZELL, H. and KLEIN, G. (1967). 'Some characteristics of 19S and 7S mouse isoantibodies in vivo and in vitro.' Transplantation, 5, 11. BURGER, M. M. (1971). Ciba Foundation Symposium (ed by G. E. W. Wolstenholme and J. Knight), p. 45. Churchill Livingstone, Edinburgh.

CHIA, E. and FESTENSTEIN, H. (1973). 'Specific cytostatic effect of lymph node cells from normal and T cell-deficient mice on syngeneic tumour target cells in vitro, and its specific abrogation by syngeneic fluid from tumour-bearing mice.' Europ. J. Immunol., 3, 483.

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