Immunology 1978 34 405
Synergistic cytotoxic effects of antibodies directed against different cell surface determinants
E. V. ELLIOTT, A. PINDAR, F. K. STEVENSON & G. T. STEVENSON Tenovus Research Laboratory, General Hospital, Tremona Road, Southampton S09 4XY
Received 12 May 1977; accepted for publication 4 July 1977
chain produce antibodies which react with L2C cells in vitro, and these antibodies can be separated into those reacting with the light chain idiotypic determinants (anti-Id) and those reacting with the light chain C region determinants (anti-A). Using the separated anti-Id and anti-A we showed that both would destroy L2C cells in vitro when in the presence of either rabbit complement or human K cells (Stevenson, Elliott & Stevenson, 1977). One striking feature of these results was that unfractionated antisera would kill L2C cells at far higher dilutions than would the separated anti-Id or anti-A on their own. We thus thought that there might be synergy in the mediating of complement and K-cell cytotoxicities when these two antibodies are present together. In this paper we describe the effects of anti-Id, anti-A and antibodies to the targets recognized by anti-lymphocyte serum (ALS), either singly or in combination, on L2C cells in the presence of rabbit complement or human K cells. The target antigens of anti-lymphocyte sera have not been defined molecularly but have not been described to include surface Ig (see Discussion). We demonstrate that when the antigenic determinants to which the antibodies are directed are on the same molecule, then an appreciable amount of synergy occurs with both killing systems, whereas when the antigenic determinants are on separate molecules much less synergy is apparent.
Summary. Three antibody populations were raised in rabbits against surface antigens on guinea-pig L2C leukaemic lymphocytes: against idiotypic determinants on the A chain of the surface immunoglobulin, against C region determinants on the A chain, and against the surface antigens recognized by conventional anti-lymphocyte sera. Complement and K-cell cytotoxicities effected by the antibodies on L2C cells were studied in vitro. In both cytotoxic systems mixtures of the antibodies revealed synergy, in that the titres of the mixtures exceeded predicted additive titres of their components. The synergy was greater when the mixed antibodies were directed to determinants on the same molecule rather than to determinants on different molecules.
INTRODUCTION
During the growth of the L2C leukaemia in strain 2 guinea-pigs there is excreted in the urine a monoclonal light chain of A class. This is believed to arise from synthesis by the leukaemic cells of a small molar excess of the light chains incorporated into their surface IgM (Stevenson, Mole, Raymont & Stevenson, 1975). Rabbits immunized with this light Correspondence: Dr E. V. Elliott, Tenovus Research Laboratory, General Hospital, Tremona Road, Southampton S09 4XY.
405
406
E. V. Elliott et at. MATERIALS AND METHODS
Lymphocytes Blood was obtained by cardiac puncture from strain 2 guinea-pigs in the terminal stages of the L2C lymphocytic leukaemia (100,000-300,000 by lymphocytes/pl). The course of the L2C lymphocytic leukaemia and the preparation of pure cell populations from citrated blood have been described previously (Stevenson, Eady, Hough, Jurd & Stevenson, 1975).
Antibodies Antisera were raised in New Zealand White rabbits to the leukaemic A chain obtained from the urine of strain 2 guinea-pigs bearing the L2C leukaemia. The rabbit IgG antibodies were fractionated into those directed against idiotypic determinants (antiId) and those directed against determinants present also on other A chains (anti-A) according to previously published methods (Stevenson, Elliott & Stevenson, 1977). Two types of ALS were produced, by injecting 5 x 108 L2C cells or 5 x 108 normal guinea-pig thymocytes twice at 2-week intervals into New Zealand White rabbits. Ten days after the second injection, the rabbits were exsanguinated and the serum was collected. Rabbit IgG was prepared from all the antisera as well as normal rabbit serum by a conventional method (Stevenson & Dorrington, 1970). Assessments of antibody activity L2C cells were labelled with 51Cr for assays of cytotoxicity. 0-1 pCi Na25tCrO4 (100400 mCi/mg Cr; Radiochemical Centre, Amersham) was added to 5 x 107 cells in 3 ml Eagle's minimal essential medium (MEM), and the mixture was incubated for 30 min at 37°. After four washes the cells were made up to 1 x 106/ml in cold MEM. At this stage viability, as judged by exclusion of trypan blue, was > 95 %. Effector cells for K-cell-mediated cytotoxicity (McLennan, Loewi & Howard, 1969; Perlmann & Holm, 1969) were prepared from normal human blood. Ten volumes of venous blood was collected into one volume of 3*5% sodium citrate, pH 7*4. One-third the volume of Dextran T250 (Pharmacia) in PBS was added and the blood was allowed to sediment for 60 min at 37°. The leucocyte-rich supernatant was removed, centrifuged at 250g for
15 min, and the cell pellets were washed three times in cold phosphate-buffered saline, pH 7-4 (PBS) and three times in cold MEM. The cells were finally resuspended in Eagle's MEM supplemented with 10 % foetal calf serum, and 100 iu/ml of both penicillin and streptomycin. In the K-cell experiments reported here, four different blood donors were bled, and the cells obtained were used at concentrations from 6-0 to 12-2 x 106/ml (effector to target ratios varied from 48: 1 to 98: 1). Red blood cell contamination was slight and varied between 2 and 3 red blood cells to one leucocyte, and the viability of the leucocytes as judged by trypan blue exclusion was always greater than 96 %. For both complement and K-cell cytotoxicities, doubling dilutions of rabbit IgG, from antibody preparations or a normal serum control, were made in MEM. To 0-1 ml IgG solution in a 2-5 ml Perspex tube (Staynes Laboratories Ltd) was added 0-1 ml suspension containing 1 x 105 5'Cr-labelled L2C cells. The tubes were incubated at 00 for 90 min. Then either complement (0-8 ml fresh rabbit serum, 1: 4 in MEM) or K cells (4-8-9-8 x 106 leucocytes in 0-8 ml supplemented MEM) were added. The complement tubes were mixed, incubated at 37° for 45 min, and after centrifugation assayed for supernatant 51Cr in a gamma scintillation counter (Wallac). The K-cell tubes were left in ice for 2-5 h, permitting cell settling and thereby good cell contacts, and were then incubated at 370 for 15 h. Finally, the contents were mixed, centrifuged and assayed for 5'Cr in the supernatants. Percentages of specific 5'Cr-release in the cytotoxicity tests were taken as:
Counts released -Counts released by antibody IgG by normal IgG 100 Counts released -Counts released by detergent by normal IgG where the IgG concentration in the antibody preparation was the same as in the control normal IgG preparation, and where detergent lysis was carried out in 1 % Nonidet 40. All points on the graphs represent the means of duplicate determinations. Owing to the complexities of the complement and K-cell systems we cannot predict accurately in either case the additive effects of two different antibody populations. For convenience, however, we define notional additive killing as the sum of specific 51Cr-releases caused by each antibody acting alone X
Synergistic cytotoxic effects of antibodies at the concentration present in the mixture. The notional additive killing can be defined only to a maximum equal to the highest 5tCr-release observed experimentally, as this plateau could represent an inherent limitation of the biological lysis as compared with detergent lysis.
407
80
() 0-
60 40 _-
\'
"a
20
0 -A
RESULTS
-20
80r
Complement cytotoxicity Fig. 1 shows the results of a typical experiment in which anti-Id, anti-A and antibody from ALS were allowed to react with L2C cells in the presence of rabbit complement, either singly or in combination.
0
60
lllJllll
,'si3-'7
( b)
U)
Table 1. Complement cytotoxicity. Observed killing minus notational killing (410g2 titre)
Experiment 1
2
3
2-7
2-2
1-7
09
0-8
0-8
0-1
09
11
Anti-Id +
anti-A Anti-Id +
ALS Anti-A +
ALS
Fig. la shows that when anti-Id and anti-A act in combination they yield a titre for 50% maximum specific 5'Cr-release 4-5 times the notional additive titre. In contrast the anti-Id plus ALS (Fig. lb), and anti-A plus ALS (Fig. 1c) combinations, yield titres only 1-7 and 1 8 respectively times their notional additive titres. Table 1 shows the combined results of three such experiments which confirm the observations shown in Fig. 1. The differences in titres between the observed and notional additive killings, treated as log2 titres, are significantly greater for anti-Id plus anti-A than for the other combinations (00I P< 0 05 by rank sum; Snedecor & Cochran, 1967).
Synergistic cytotoxic effects of antibodies directed against different cell surface determinants
E. V. ELLIOTT, A. PINDAR, F. K. STEVENSON & G. T. STEVENSON Tenovus Research Laboratory, General Hospital, Tremona Road, Southampton S09 4XY
Received 12 May 1977; accepted for publication 4 July 1977
chain produce antibodies which react with L2C cells in vitro, and these antibodies can be separated into those reacting with the light chain idiotypic determinants (anti-Id) and those reacting with the light chain C region determinants (anti-A). Using the separated anti-Id and anti-A we showed that both would destroy L2C cells in vitro when in the presence of either rabbit complement or human K cells (Stevenson, Elliott & Stevenson, 1977). One striking feature of these results was that unfractionated antisera would kill L2C cells at far higher dilutions than would the separated anti-Id or anti-A on their own. We thus thought that there might be synergy in the mediating of complement and K-cell cytotoxicities when these two antibodies are present together. In this paper we describe the effects of anti-Id, anti-A and antibodies to the targets recognized by anti-lymphocyte serum (ALS), either singly or in combination, on L2C cells in the presence of rabbit complement or human K cells. The target antigens of anti-lymphocyte sera have not been defined molecularly but have not been described to include surface Ig (see Discussion). We demonstrate that when the antigenic determinants to which the antibodies are directed are on the same molecule, then an appreciable amount of synergy occurs with both killing systems, whereas when the antigenic determinants are on separate molecules much less synergy is apparent.
Summary. Three antibody populations were raised in rabbits against surface antigens on guinea-pig L2C leukaemic lymphocytes: against idiotypic determinants on the A chain of the surface immunoglobulin, against C region determinants on the A chain, and against the surface antigens recognized by conventional anti-lymphocyte sera. Complement and K-cell cytotoxicities effected by the antibodies on L2C cells were studied in vitro. In both cytotoxic systems mixtures of the antibodies revealed synergy, in that the titres of the mixtures exceeded predicted additive titres of their components. The synergy was greater when the mixed antibodies were directed to determinants on the same molecule rather than to determinants on different molecules.
INTRODUCTION
During the growth of the L2C leukaemia in strain 2 guinea-pigs there is excreted in the urine a monoclonal light chain of A class. This is believed to arise from synthesis by the leukaemic cells of a small molar excess of the light chains incorporated into their surface IgM (Stevenson, Mole, Raymont & Stevenson, 1975). Rabbits immunized with this light Correspondence: Dr E. V. Elliott, Tenovus Research Laboratory, General Hospital, Tremona Road, Southampton S09 4XY.
405
406
E. V. Elliott et at. MATERIALS AND METHODS
Lymphocytes Blood was obtained by cardiac puncture from strain 2 guinea-pigs in the terminal stages of the L2C lymphocytic leukaemia (100,000-300,000 by lymphocytes/pl). The course of the L2C lymphocytic leukaemia and the preparation of pure cell populations from citrated blood have been described previously (Stevenson, Eady, Hough, Jurd & Stevenson, 1975).
Antibodies Antisera were raised in New Zealand White rabbits to the leukaemic A chain obtained from the urine of strain 2 guinea-pigs bearing the L2C leukaemia. The rabbit IgG antibodies were fractionated into those directed against idiotypic determinants (antiId) and those directed against determinants present also on other A chains (anti-A) according to previously published methods (Stevenson, Elliott & Stevenson, 1977). Two types of ALS were produced, by injecting 5 x 108 L2C cells or 5 x 108 normal guinea-pig thymocytes twice at 2-week intervals into New Zealand White rabbits. Ten days after the second injection, the rabbits were exsanguinated and the serum was collected. Rabbit IgG was prepared from all the antisera as well as normal rabbit serum by a conventional method (Stevenson & Dorrington, 1970). Assessments of antibody activity L2C cells were labelled with 51Cr for assays of cytotoxicity. 0-1 pCi Na25tCrO4 (100400 mCi/mg Cr; Radiochemical Centre, Amersham) was added to 5 x 107 cells in 3 ml Eagle's minimal essential medium (MEM), and the mixture was incubated for 30 min at 37°. After four washes the cells were made up to 1 x 106/ml in cold MEM. At this stage viability, as judged by exclusion of trypan blue, was > 95 %. Effector cells for K-cell-mediated cytotoxicity (McLennan, Loewi & Howard, 1969; Perlmann & Holm, 1969) were prepared from normal human blood. Ten volumes of venous blood was collected into one volume of 3*5% sodium citrate, pH 7*4. One-third the volume of Dextran T250 (Pharmacia) in PBS was added and the blood was allowed to sediment for 60 min at 37°. The leucocyte-rich supernatant was removed, centrifuged at 250g for
15 min, and the cell pellets were washed three times in cold phosphate-buffered saline, pH 7-4 (PBS) and three times in cold MEM. The cells were finally resuspended in Eagle's MEM supplemented with 10 % foetal calf serum, and 100 iu/ml of both penicillin and streptomycin. In the K-cell experiments reported here, four different blood donors were bled, and the cells obtained were used at concentrations from 6-0 to 12-2 x 106/ml (effector to target ratios varied from 48: 1 to 98: 1). Red blood cell contamination was slight and varied between 2 and 3 red blood cells to one leucocyte, and the viability of the leucocytes as judged by trypan blue exclusion was always greater than 96 %. For both complement and K-cell cytotoxicities, doubling dilutions of rabbit IgG, from antibody preparations or a normal serum control, were made in MEM. To 0-1 ml IgG solution in a 2-5 ml Perspex tube (Staynes Laboratories Ltd) was added 0-1 ml suspension containing 1 x 105 5'Cr-labelled L2C cells. The tubes were incubated at 00 for 90 min. Then either complement (0-8 ml fresh rabbit serum, 1: 4 in MEM) or K cells (4-8-9-8 x 106 leucocytes in 0-8 ml supplemented MEM) were added. The complement tubes were mixed, incubated at 37° for 45 min, and after centrifugation assayed for supernatant 51Cr in a gamma scintillation counter (Wallac). The K-cell tubes were left in ice for 2-5 h, permitting cell settling and thereby good cell contacts, and were then incubated at 370 for 15 h. Finally, the contents were mixed, centrifuged and assayed for 5'Cr in the supernatants. Percentages of specific 5'Cr-release in the cytotoxicity tests were taken as:
Counts released -Counts released by antibody IgG by normal IgG 100 Counts released -Counts released by detergent by normal IgG where the IgG concentration in the antibody preparation was the same as in the control normal IgG preparation, and where detergent lysis was carried out in 1 % Nonidet 40. All points on the graphs represent the means of duplicate determinations. Owing to the complexities of the complement and K-cell systems we cannot predict accurately in either case the additive effects of two different antibody populations. For convenience, however, we define notional additive killing as the sum of specific 51Cr-releases caused by each antibody acting alone X
Synergistic cytotoxic effects of antibodies at the concentration present in the mixture. The notional additive killing can be defined only to a maximum equal to the highest 5tCr-release observed experimentally, as this plateau could represent an inherent limitation of the biological lysis as compared with detergent lysis.
407
80
() 0-
60 40 _-
\'
"a
20
0 -A
RESULTS
-20
80r
Complement cytotoxicity Fig. 1 shows the results of a typical experiment in which anti-Id, anti-A and antibody from ALS were allowed to react with L2C cells in the presence of rabbit complement, either singly or in combination.
0
60
lllJllll
,'si3-'7
( b)
U)
Table 1. Complement cytotoxicity. Observed killing minus notational killing (410g2 titre)
Experiment 1
2
3
2-7
2-2
1-7
09
0-8
0-8
0-1
09
11
Anti-Id +
anti-A Anti-Id +
ALS Anti-A +
ALS
Fig. la shows that when anti-Id and anti-A act in combination they yield a titre for 50% maximum specific 5'Cr-release 4-5 times the notional additive titre. In contrast the anti-Id plus ALS (Fig. lb), and anti-A plus ALS (Fig. 1c) combinations, yield titres only 1-7 and 1 8 respectively times their notional additive titres. Table 1 shows the combined results of three such experiments which confirm the observations shown in Fig. 1. The differences in titres between the observed and notional additive killings, treated as log2 titres, are significantly greater for anti-Id plus anti-A than for the other combinations (00I P< 0 05 by rank sum; Snedecor & Cochran, 1967).
