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Nonspecific cytotoxic effects of antigen-transformed lymphocytes
CELLULAR
IMMUNOLOGY
Nonspecific
16, 74-81 (1975)
Cytotoxic Ill.
Effects of Antigen-Transformed Relationship
to Specific
Lymphocytes
Cytotoxicity
A. E. BUTTERWORTH ’ AND D. FRANKS Immunology
Division,
Department of Pathology, Cambridge, England Received August
University
of Cambridge,
8,1974
Human peripheral blood lymphocytes, cultured either with PPD or in one-way mixed leucocyte reactions, develop a nonspecific cytotoxic potential which can be expressed on autologous as well as on unrelated xenogeneic target cells. This nonspecific cytotoxicity develops at an early stage in the mixed leucocyte reaction, before specific cytotoxic effects are demonstrable, and decreases as specific cytotoxicity increases.
INTRODUCTION It is now clearly established that antigen-transformed lymphocytes can exert a direct “nonspecific” cytotoxic effect: that is, a cytotoxic effect on target cells antigenitally unrelated to the stimulating agent (l-3). In this paper, two further aspects of this nonspecific cytotoxicity will be considered. First, it will be shown that PPD-stimulated human lymphocytes can exert a cytotoxic effect on autologous target cells, as well as on unrelated xenogeneic cells. This finding suggests that the cytotoxic effect of stimulated cells is genuinely nonspecific, in the sense that it does not depend on the interaction of a lymphocyte receptor with a specific antigen on the target cell. Some problems about this interpretation, related to the use of PHA-stimulated autologous lymphocytes as target cells, will be discussed. The second point is that, if nonspecific cytotoxicity represents a general property of transforming lymphocytes, then cells cultured in one-way mixed leucocyte reactions (MLR) should also show this effect. Evidence on this point is conflicting, Some workers (4-7) have claimed that cytotoxicity after MLR is entirely specific, affecting only target cells of the stimulator type, and that any apparent nonspecificity that may be observed is attributable to cross-reactions between the stimulator lymphocyte and the third-party cell used as target cell. Others (1, 8, 9), in contrast, have reported that cytotoxicity to target cells unrelated to the stimulating lymphocyte can also occur. One possible explanation for this discrepancy is the finding that nonspecific cytotoxicity develops relatively early in transformation, reaching its peak after 3 days of culture with PPD and thereafter declining (2), and in this paper it will be shown that specific cytotoxicity after human MLR appears at a later stage in culture than nonspecific cytotoxicity, increasing as the nonspecific effect decreases. 1 Present address : Wellcome Trust Research Laboratories, P.O. Box 43640, Nairobi, Kenya.
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
CYTOTOSIC
EFFECTS
OF TRANSFORMEI)
MATERIALS
III
AND METHODS
The techniques described in the accompanying the following modifications. Lym2phocqte
LYNPIIOC’YTES.
paper (3 ) \\WY atlopted, wit11
Cultures
Human peripheral blood lymphocytes, prepared by defibrillation and gelatine sedimentation, and purified by extraction with carbonyl iron, were cultured in Eagle’s 11inimal Essential Medium containing 2O’k autologous gelatine-serum (MEM/AS) (3). For PPD-induced transformation, lymphocytes were cultured at 1 X 10’ cellsin in 2 ml volumes in flat-bottomed polystyrene tubes (keywart! T*aboratories) , &her with or without 10 pg/ml PPD (Weybridge) . For one-way MLRs, stimulator-type lymphocytes from a second individual were inactivated by incubation with 25 pg/ml mitomycin C (Sigma) for 30 min at 37°C. Inactivated cells were washed three times in RIESl, and resuspended in MEhl/AS at 1.5 X 10” cells/ml. Responder-type cells (1.5 x 10F/ml) were mixed with an equal number of stimulator-type cells, and cultured in flat-bottomed tubes for varying periods of time. Cyfoto.dcitg
Assay
Cytotoxicity was assayed by measuring the release of “‘Cr from xenogeneic August rat hepatoma cells (3) and from PH.\-stimulated human lymphoc?;tes. Lymphocytes prepared by gelatine sedimentation and carbonyl iron purification contained a variable number of red blood cells, and for use as target cells were therefore purified further by centrifugation over Ficoll-Hypaque (Ficoll, Pharmacia, 6.3Sc/o, and Hypaque, Bayer Products, loch, in distilled water) at 5009 for 5 min. Interface cells were washed twice in RIEM/AS and cultured at 0.5 X 10” cells/ml. Phytohaemagglutinin (Wellcome) (PHA), 10 pi/ml, was added on day 0 for target cells used on day 3 of culture, and on day 3 for target cells used on tlav 6 of culture, as recommended by Lightbody et al. (IO). The measurement of 51Cr release and the statistical analysis of the data obtained are described in the accompanying paper (3). All analyses were carried out on the original percent isotope release data, but in Table 2, for the sake of clarity. percent cytotoxicity values are given. These have been calculated using the equation s release in test - $% release in medium ‘A cytntoxicitv I = x 100. total 74 releasable - 54~release in medium RESULTS A. Cytotoxicity
Towards
Autologous
Target
Cells
Initial experiments were designed to test whether lymphocytes stimulated by PPD or in one-way MLRs would exert a cytotoxic effect on autologous target cells, as well as on unrelated xenogeneic cells. The results of one such experiment are shown in Table 1. Lymphocytes from two individuals (labelled .4 and R in Table 1) were cultured for 3 days, either with PPD (A-PPD and TWT’D) or with mitomvcin-treated
76
BUTTERWORTH
AND
TABLE
FRANKS
1
NONSPECIFIC CYTOTOXIC EFFECTS OF LYMPHOCYTES CULTURED IN ONE-WAY MIXED LEUCOCYTE REACTIONS OR WITH PPDa 7s isotope release*
Thymidine
uptake
(CPS) c
A-PHA Effector
B-PHA
AH
lymphocytes” AAm ABm BAm BBm AmBm A-PPD B-PPD Mediumb
Statistical
49 67 67 46 35 67 62 18
49 63 58 41 37 68 57 26
39 62 54 34 35 63 52 29
10 645 417 15 8 302 172 -
summary
(i) Analysis (ii) Multiple Target cell A-PHA
of variance:
interaction “target cell type” X “effector lymphocyte P < 0.001 (F = 72.3: Fo.oor;r~,r~ = 3.02).
type”
range tests: Effector lymphocyted Medium AmBm BBm AAm B-PPD
B-PHA
Medium
AmBm
BBm AAm B-PPD
AH
Medium
BBm AmBm
AAm B-PPD
BAm A-PPD
ABm
BAm ABm A-PPD BAm ABm A-PPD
a Lymphocytes from two individuals (A and B) were prepared and were cultured for 3 days either with PPD (A-PPD and B-PPD), or with mitomycin-inactivated stimulator lymphocytes (Am and Bm). Stimulated (ABm and BAm) and control (AAm, BBm and AmBm) mixtures were set up. “After 3 days of culture, washed effector cells were tested for cytotoxicity towards PHAstimulated lymphocytes from each individual (A-PHA and B-PHA) and towards August hepatoma cells (,AH). Percent release of %hromium from the target cells was measured after 8 hr of incubation, Release in the presence of medium alone, with no added lymphocytes, is also shown. c Thymidine uptake was measured after 5 and 6 days of culture for PPD- and MLR-stimulated cells, respectively. d Means that are grouped by underlining are not significantly different at the 5% level.
lymphocytes from the other individual (ABm and BAm). Two types of control culture were set up: First, normal lymphocytes were cultured with mitomycintreated cells from the same individual (AAm and BBm) and, secondly, cultures were set up in which both cell types had been treated with mitomycin (AmBm). On the third day of culture, cells were tested for cytotoxicity towards three types of target cell : August hepatoma cells (AH) and PHA-stimulated lymphocytes from each individual (A-PHA and B-PHA) . Lymphocytes cultured in one-way MLRs (ABm and BAm) showed a marked cytotoxic effect towards August hepatoma cells, as did lymphocytes cultured with PPD (A-PPD and B-PPD) . These lymphocytes were also cytotoxic for autologous as well as for allogeneic PHA-stimulated target cells. There was no evidence, in the case of MLR-stimulated lymphocytes, that the effect on stimulator-type target cells was greater than that on responder-type cells, and it was concluded at this
stage that there was no evidence for the development of specific cvtotoxicity 1lLR. U. Specific ix- tions
nnd Nonspecific
Cytotoxicity
affer
One-Wq
Mixed
Lurcocute
during
Kc-
One possible explanation for the lack of a specific cytotoxic effect in the previous experiment was that cytotoxicity was assayed after a relatively short period, nameI! 3 days, of culture. This period had previously been shown (2) to be optimal for the development of nonspecific cytotoxicity by PPD-stimulated lymphocytes. In contrast, workers who have reported specific cytotoxicity after MLR have usually taken cells from relatively late cultures of 5-10 days (4, 5, 11) . In subsequent experiments, therefore, the specific and nonspecific cytotoxic effects of cells taken from early and late one-way MLRs were compared, and the results of one sucl~ experiment are shown in Table 2. Lymphocytes from two individuals (labelled A and 1’: in Table 2) were cultured either with mitomycin-treated lyn~hocytcs from the same individual (AAm and RP,m, respectively). or with mitomycintreated lymphocytes from the second individual ( ARm and I:Am, respectively). Two-way mitomycin controls (Am&n) were also set up. After 3 and 6 days of culture, washed lymphocytes from these mixtures were tested for cytotoxicity towards three types of target cell : August hepatoma cells (AH) and PHA-stimulated lymphocvtes from each individual (A-PHA and H-PHA). Thvmitline incorporation by replicate cultures was measured after 7 tlavs. For the sake of clarity, percent cytotoxicity values only- are shown in Table 2. Calculation of percent cytotoxicity necessarily inrolves a degree of distortion of the data ; the medium release values from which the cytotoxicity values are derived are .shown at the bottom of each column, and it sl~ould again be stressed that the stntistical analysis was carried out on the log transformation of the original percent release figures. A summary of the analysis of variance and multiple range tests is givrn in the table, and the conclusions that could be drawn may be stated as follows. ’ (i) As in th e previous experiment, stimulated cells (ABm and RAm ) exerted a marked nonspecific cytotoxic effect on August hepatoma (AH ) cells on day 3, when compared with unstimulatecl controls (AAm and l?Bm) and with two-wa\. mitemycin controls (AmBm) . (ii) This nonspecific effect on AH cells was much less marked on day 6 than on day 3. (iii) ABm cells exerted a significantly greater cytotoxic effect on A-PHA cells than did AAm or AmBm cells, both on day 3 and day 6. This represented an effect on responder cells, and was therefore nonspecific. (iv) Similarly, BAm cells exerted a significantly greater cytotoxic effect on B-PHA target cells than did BBm or AmBm cells, again both on day 3 and day 6. (v) RAm cells also showed a specific cytotoxic effect on A-PHA target cells on day 6, but not on day 3. This specific effect was reflected by a greater difference between BAm cells and AmBm controls when tested on A-PHA cells than when tested on B-PHA cells, (vi) There was a significant increase in specificity of RAm cells on da\- 6, as compared with day 3. (vii) ABm cells showed evidence of a specific cytotoxic effect on B-PHA target cells, as compared with A-PHA target cells, both on dav 3 and on day 6. In this
78
BUTTERWORTH
AND
TABLE
FRANKS
2
SPECIFIC AND NONSPECIFIC CYTOTOXIC EFFECTS OF LYMPHOCYTES FKOY EARLY AND LATE ONE-WAY MIXED LEUCOCYTE REACTIONS 7; cytotoxicity against target cell b
Day of cultures 3 A-PHA
Effector
B-PHA
Thymidine uptake (cps) c
6 AH
A-PHA
B-PHA
AH
11 35 42 2
26 77 11 1
4 26 9 -1
lymphocytesa
AAm ABm BAm BBm AmBm Medium releaseb Statistical
23 35 28 3 (4:)
59 68 39 7
-1 75 84 -14
6 79 112 8
(ii)
summary
(i) Analysis of variance: interaction “day of culture” X “target cyte type” P < 0.001 (F = 69.8; Fo.oor;ro,r,~ = 3.54).
cell type”
X “effector
lympho-
(ii) Multiple range tests: given in form (1 - 2)3, where 1 = release in presence of test effector lymphocytes, 2 = release in presence of control effector lymphocytes, 3 = target cell and day of culture. Test
P
Represents nonspecific effect of ABm cells on day 3
- AAm)A-PHA day 6 - AAm)AH day 6
-co.05
- BBm)B-PHA day 6 - BBm)AH day 6
<0.05
nonspecific effect of BAm cells on day 6
(ABm - AmBm)B-PHA day 3 - (ABm - AmBm)A-PHA day 3
specific effect of ABm cells on day 3
(BAm - AmBm)A-PHA day 3 - (BAm - AmBm)B-PHA day 3
>0.05 NS
specific effect of BAm cells on day 3
(ABm - AmBm)B-PHA day 6 - (ABm - AmBm)A-PHA day 6
specific effect of ABm cells on day 6
(BAm - AmBm)A-PHA day 6 - (BAm - AmBm)B-PHA day 6
specific effect of BAm cells on day 6
(ABm (ABm
- AAm)A-PHA day 3 - AAm)AH day 3
(BAm (BAm
- BBm)B-PHA day 3 - BBm)AH day 3
(ABm (ABm (BAm (BAm
nonspecific effect of BAm cells on day 3 nonspecific effect of ABm cells on day 6
a Lymphocytes from two individuals were prepared. Combinations of responder lymphocytes (A or B) and of mitomycin-inactivated stimulator lymphocytes (Am or Bm) were cultured as shown. After 3 and 6 days of culture, respectively, washed effector cells from these mixtures were tested for cytotoxicity towards PHA-stimulated lymphocytes from each individual (A-PHA and B-PHA) and towards August hepatoma cells (AH) over a 7.5hr period. b Percent cytotoxicity values for each target cell are shown. These figures were derived from percent isotope release using the medium release value shown in parentheses at the foot of each column, and a total releasable isotope value of 80%. All statistical analyses were carried out on the original percent release data (see Materials and Methods, and Ref. 3). c Thymidine uptake was measured after 7 days of culture.
case, there was no evidence of increased specificity on day 6. This could be attributed to the high “natural” cytotoxicity of A lymphocytes for B target cells, as shown by the high levels of isotope release from B-PHA cells in the presence of unstimdatcd AAm cells. These rather complicated findings may be sumtnarized more briefly. First, there was again evidence for a marked nonspecific cytotoxic effect after BILK, both on xenogeneic target cells and on autologous responder-type target cells. This effect was apparent on day 6 as well as day 3, but in most cases the effect on day 6 was less marked. Secondly, there was also evidence for a specific effect superimpnsed on this nonspecific effect. This specific effect, on stimulator-type target cells, ~1s more marked on day 6 than on day 3. In other words, specific cytotoxicity appeared later in transformation than the nonspecific effect, and increased as the nonspecific effect decreased. DISCUSSION Several possible interpretations of direct “nonspecific” cytotoxic effects may be offered. \2’hen cytotoxicity towards target cells unrelated both to the responding lymphocyte and to the stimulating antigen is observed, it might be argued either that this effect depends on recruitment during transformation of lymphocytes capable of responding specifically against those target cells, or that the effector lymphocytes are pluripotent in the sense that they bear receptors for more than one antigen. In this paper, however, it has been shown that transformed lymphocytes are cytotoxic for autologous as well as for unrelated target cells. In this case, the autologous target cells studied were PHA-stimulated blasts, and it could be argued that the PHA remains bound to the cell surface and acts as a link between the effector lymphocyte and the target cell. This is unlikely, in view of observations ( 12-14 j that PHA and other mitogens are rapidly endocytosed by stimulated lymphocytes. Alternatively, it might be argued that PHA blasts express new antigens on their surfaces, ant1 that during lymphocyte culture (for instance with PPD) there is recruitment of cells responding specifically against these new antigens. The second observation reported here-namely, the dissociation in time between specific and nonspecific cytotoxicity during MI,&-suggests that this is not the correct explanation. If “nonspecific” cytotoxicity reflects nonspecific recruitment of cells which subsequently interact specifically with new antigens on the target blasts, it would be expected that both “nonspecific” and specific effects after MLR would increase in parallel, and it has been shown in other situations that specific cvtotoxicity during RZLR only develops after at least one cell division (11, 15). In the work reported here and elsewhere (2, 3), however, it has been shown that nonspecific effects are greater during the early stages of culture, before the onset of DSA synthesis and the development of specific cytotoxicity. These findings therefore suggest that the cytotoxicity observed during the early stages of culture is genuinely nonspecific, in the sense that it does not depend on the interaction of a specific lymphocyte receptor with antigen. It may be concluded that there are two phases of cytotoxicity in transforming cultures. A nonspecific phase which occurs early in transformation, before the onset of cell division, is followed by mitosis and expansion of the pool of lymphocytes capable of responding specifically. During this later phase, nonspecific cytotoxicit! decreases. 1t is difficult to assess the relative effectiveness of these two phases, ill
80
BUTTERWORTH
AND
FRANKS
terms of efficiency of killing by a single lymphocyte, since the chromium-release system only allows investigations on populations of effector cells. From the present study, however, it can be concluded that the overall population of lymphocytes in a culture can exert a nonspecific effect early in transformation which is quantitatively similar to the specific effect observed in the later stages of the response. A further point concerning the development of nonspecific as well as specific cytotoxicity is the question of the species under study, in that investigators who have failed to find evidence of nonspecific cytotoxicity during MLR have mainly worked with mouse lymphocytes (4, 5, 11, 15). Cultured mouse cells differ from human in two important respects apart from their lack of demonstrable nonspecific cytotoxicity : First, cell viability is low and there is selective survival of stimulated cells ( 11) and, secondly, recruitment of other lymphocytes to transform has not been reported. It is possible that, in the human system described here, nonspecific cytotoxicity is mediated not only by cells reacting directly to antigen, but also by antigenunreactive cells that have been recruited to transform and which can only exert a nonspecific effect (2). In this case, the overall ratio of specific to nonspecific cytotoxicity might be rather low. In mouse cultures, in contrast, those lymphocytes which under perfect conditions would be recruited may die before recruitment can occur. In this case, only those lymphocytes which are specifically stimulated by antigen during the early stages of culture survive, and hence the ratio of specific to nonspecific cytotoxicity may be much higher. If this explanation is correct, a failure to demonstrate nonspecific cytotoxicity in mouse MLRs may simply reflect inadequate culture conditions. Finally, an attempt may be made to point out two events occurring in viva in which nonspecific cytotoxicity may be involved. First, host tissue damage in delayed hypersensitivity reactions may be caused not only by specific cytotoxic effects against host cells coated with antigen ( 16), but also by nonspecific effects mediated by macrophages (17) or lymphocytes. Although at any one time macrophages are the main components of a delayed hypersensitivity reaction (IS), there is evidence that lymphocytes also enter the lesions in large numbers (19). It is possible that such lymphocytes are recruited to transform (2), develop a nonspecific cytotoxic effect and thereby contribute to host tissue damage, and are then destroyed by other nonspecifically cytotoxic cells. Secondly, there is some good evidence that local nonspecific effects may be involved in the rejection of grafts or tumors (20, 21). In this case, it is not suggested that nonspecific cytotoxicity represents the most important mechanism of cytotoxicity, but it is possible that nonspecific effects, particularly of lymphocytes which have been recruited to transform (2), may effectively amplify the action of specifically cytotoxic lymphocytes. ACKNOWLEDGMENTS We thank Dr. C. J. Sanderson for his stimulating comments and advice, and Mrs. B. Tayabali and Mrs. P. O’Brien for their technical assistance. One of the authors (A.E.B.) was supported by a Medical Research Council Scholarship for Training in Research Methods and by a Harrison Watson Studentship at Clare College, Cambridge. The work was supported by a grant from the Cancer Campaign for Research.
REFERENCES 1. Holm, G., and Perlmann, P., J. Exp. Med. 125, 721, 1967. 2. Butterworth, A. E., Cell. Immwzol. 7, 357, 1973.
CYTOTOXIC
3. 4. 5: 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
EFFECTS
OF
TRAiYSFORMEI)
LY.\II’IIOCYTES.
III
Sl
16, 60, 1973. Butterworth, .4. E., Crll I~~zv7zruol. Hayry, P., and Defendi, V., Science 168, 133, 1970. Wagner. H.. J. Inwwxol. 109. 630. 1972. Solliday,‘S., and Bach, F. H., kTw.‘J. Imwur~ol. 2, 68, 1972. Forman, J., and Mb;ller, G., J. Exp. :Vcd. 138, 672, 1973. Cohen, I. R., and Feldman, M., CrU. 1~~wz111zo1. 1, 521, 1970. Hodes, R. J., and Svedmyr, E. A. J., ?‘rawplantatio~z 9, 470, 1970. Lightbody, J., Bcrnoco, D., Miggiano, V. C., and Ceppellini. K., (;. Butt. I’irol. I~wznuol. 64, 243, 1971. Hiyry, P., A4ndersson, L. C., Nordling, S., and Virolainen, SI.. ‘/‘vczusp/a~~t. Xczq. 12. 91. 1972. Kazavi, L., Nature (Lmdon) 210, 444, 1966. Pauli, R. M., DeSalle, L., Higgins, P., Henderson, E., Xorin, -I., and Strauss, H.. J. IVkVkWkOl. 111, 424, 1973. Unanue, E. R., Perkins, W. D., and Karnovsky, M. J., J. Exp. dlcd. 136, 885, 1972. Clark, W., and Nedrud, J., Cell. I~nnzuw~l. 10, 159, 1974. Perlmann, P., and Holm, G., A&xx 1mww~o1. 11, 117, 1969. Alexander, P., and Evans, R., Nature NW Biol. 232, 76, 1971. Bosman, C., and Feldman, J. D., Amrr. J. Patlzol. 58, 201, 1970. Koster, I;. T., McGregor, D. D., and Mackaness, G. B., J. E.rp. 3rd. 133, 400, 1971. Elkins, W. L., and Guttman, R. D., S&VCC 159, 1250, 1968. Zbar, B., Wepsic, H. T., Borsos, T., and Rapp, H. J., .I. ,Vot. CUIICW Inst. 44, 473, 1070.
IMMUNOLOGY
Nonspecific
16, 74-81 (1975)
Cytotoxic Ill.
Effects of Antigen-Transformed Relationship
to Specific
Lymphocytes
Cytotoxicity
A. E. BUTTERWORTH ’ AND D. FRANKS Immunology
Division,
Department of Pathology, Cambridge, England Received August
University
of Cambridge,
8,1974
Human peripheral blood lymphocytes, cultured either with PPD or in one-way mixed leucocyte reactions, develop a nonspecific cytotoxic potential which can be expressed on autologous as well as on unrelated xenogeneic target cells. This nonspecific cytotoxicity develops at an early stage in the mixed leucocyte reaction, before specific cytotoxic effects are demonstrable, and decreases as specific cytotoxicity increases.
INTRODUCTION It is now clearly established that antigen-transformed lymphocytes can exert a direct “nonspecific” cytotoxic effect: that is, a cytotoxic effect on target cells antigenitally unrelated to the stimulating agent (l-3). In this paper, two further aspects of this nonspecific cytotoxicity will be considered. First, it will be shown that PPD-stimulated human lymphocytes can exert a cytotoxic effect on autologous target cells, as well as on unrelated xenogeneic cells. This finding suggests that the cytotoxic effect of stimulated cells is genuinely nonspecific, in the sense that it does not depend on the interaction of a lymphocyte receptor with a specific antigen on the target cell. Some problems about this interpretation, related to the use of PHA-stimulated autologous lymphocytes as target cells, will be discussed. The second point is that, if nonspecific cytotoxicity represents a general property of transforming lymphocytes, then cells cultured in one-way mixed leucocyte reactions (MLR) should also show this effect. Evidence on this point is conflicting, Some workers (4-7) have claimed that cytotoxicity after MLR is entirely specific, affecting only target cells of the stimulator type, and that any apparent nonspecificity that may be observed is attributable to cross-reactions between the stimulator lymphocyte and the third-party cell used as target cell. Others (1, 8, 9), in contrast, have reported that cytotoxicity to target cells unrelated to the stimulating lymphocyte can also occur. One possible explanation for this discrepancy is the finding that nonspecific cytotoxicity develops relatively early in transformation, reaching its peak after 3 days of culture with PPD and thereafter declining (2), and in this paper it will be shown that specific cytotoxicity after human MLR appears at a later stage in culture than nonspecific cytotoxicity, increasing as the nonspecific effect decreases. 1 Present address : Wellcome Trust Research Laboratories, P.O. Box 43640, Nairobi, Kenya.
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
CYTOTOSIC
EFFECTS
OF TRANSFORMEI)
MATERIALS
III
AND METHODS
The techniques described in the accompanying the following modifications. Lym2phocqte
LYNPIIOC’YTES.
paper (3 ) \\WY atlopted, wit11
Cultures
Human peripheral blood lymphocytes, prepared by defibrillation and gelatine sedimentation, and purified by extraction with carbonyl iron, were cultured in Eagle’s 11inimal Essential Medium containing 2O’k autologous gelatine-serum (MEM/AS) (3). For PPD-induced transformation, lymphocytes were cultured at 1 X 10’ cellsin in 2 ml volumes in flat-bottomed polystyrene tubes (keywart! T*aboratories) , &her with or without 10 pg/ml PPD (Weybridge) . For one-way MLRs, stimulator-type lymphocytes from a second individual were inactivated by incubation with 25 pg/ml mitomycin C (Sigma) for 30 min at 37°C. Inactivated cells were washed three times in RIESl, and resuspended in MEhl/AS at 1.5 X 10” cells/ml. Responder-type cells (1.5 x 10F/ml) were mixed with an equal number of stimulator-type cells, and cultured in flat-bottomed tubes for varying periods of time. Cyfoto.dcitg
Assay
Cytotoxicity was assayed by measuring the release of “‘Cr from xenogeneic August rat hepatoma cells (3) and from PH.\-stimulated human lymphoc?;tes. Lymphocytes prepared by gelatine sedimentation and carbonyl iron purification contained a variable number of red blood cells, and for use as target cells were therefore purified further by centrifugation over Ficoll-Hypaque (Ficoll, Pharmacia, 6.3Sc/o, and Hypaque, Bayer Products, loch, in distilled water) at 5009 for 5 min. Interface cells were washed twice in RIEM/AS and cultured at 0.5 X 10” cells/ml. Phytohaemagglutinin (Wellcome) (PHA), 10 pi/ml, was added on day 0 for target cells used on day 3 of culture, and on day 3 for target cells used on tlav 6 of culture, as recommended by Lightbody et al. (IO). The measurement of 51Cr release and the statistical analysis of the data obtained are described in the accompanying paper (3). All analyses were carried out on the original percent isotope release data, but in Table 2, for the sake of clarity. percent cytotoxicity values are given. These have been calculated using the equation s release in test - $% release in medium ‘A cytntoxicitv I = x 100. total 74 releasable - 54~release in medium RESULTS A. Cytotoxicity
Towards
Autologous
Target
Cells
Initial experiments were designed to test whether lymphocytes stimulated by PPD or in one-way MLRs would exert a cytotoxic effect on autologous target cells, as well as on unrelated xenogeneic cells. The results of one such experiment are shown in Table 1. Lymphocytes from two individuals (labelled .4 and R in Table 1) were cultured for 3 days, either with PPD (A-PPD and TWT’D) or with mitomvcin-treated
76
BUTTERWORTH
AND
TABLE
FRANKS
1
NONSPECIFIC CYTOTOXIC EFFECTS OF LYMPHOCYTES CULTURED IN ONE-WAY MIXED LEUCOCYTE REACTIONS OR WITH PPDa 7s isotope release*
Thymidine
uptake
(CPS) c
A-PHA Effector
B-PHA
AH
lymphocytes” AAm ABm BAm BBm AmBm A-PPD B-PPD Mediumb
Statistical
49 67 67 46 35 67 62 18
49 63 58 41 37 68 57 26
39 62 54 34 35 63 52 29
10 645 417 15 8 302 172 -
summary
(i) Analysis (ii) Multiple Target cell A-PHA
of variance:
interaction “target cell type” X “effector lymphocyte P < 0.001 (F = 72.3: Fo.oor;r~,r~ = 3.02).
type”
range tests: Effector lymphocyted Medium AmBm BBm AAm B-PPD
B-PHA
Medium
AmBm
BBm AAm B-PPD
AH
Medium
BBm AmBm
AAm B-PPD
BAm A-PPD
ABm
BAm ABm A-PPD BAm ABm A-PPD
a Lymphocytes from two individuals (A and B) were prepared and were cultured for 3 days either with PPD (A-PPD and B-PPD), or with mitomycin-inactivated stimulator lymphocytes (Am and Bm). Stimulated (ABm and BAm) and control (AAm, BBm and AmBm) mixtures were set up. “After 3 days of culture, washed effector cells were tested for cytotoxicity towards PHAstimulated lymphocytes from each individual (A-PHA and B-PHA) and towards August hepatoma cells (,AH). Percent release of %hromium from the target cells was measured after 8 hr of incubation, Release in the presence of medium alone, with no added lymphocytes, is also shown. c Thymidine uptake was measured after 5 and 6 days of culture for PPD- and MLR-stimulated cells, respectively. d Means that are grouped by underlining are not significantly different at the 5% level.
lymphocytes from the other individual (ABm and BAm). Two types of control culture were set up: First, normal lymphocytes were cultured with mitomycintreated cells from the same individual (AAm and BBm) and, secondly, cultures were set up in which both cell types had been treated with mitomycin (AmBm). On the third day of culture, cells were tested for cytotoxicity towards three types of target cell : August hepatoma cells (AH) and PHA-stimulated lymphocytes from each individual (A-PHA and B-PHA) . Lymphocytes cultured in one-way MLRs (ABm and BAm) showed a marked cytotoxic effect towards August hepatoma cells, as did lymphocytes cultured with PPD (A-PPD and B-PPD) . These lymphocytes were also cytotoxic for autologous as well as for allogeneic PHA-stimulated target cells. There was no evidence, in the case of MLR-stimulated lymphocytes, that the effect on stimulator-type target cells was greater than that on responder-type cells, and it was concluded at this
stage that there was no evidence for the development of specific cvtotoxicity 1lLR. U. Specific ix- tions
nnd Nonspecific
Cytotoxicity
affer
One-Wq
Mixed
Lurcocute
during
Kc-
One possible explanation for the lack of a specific cytotoxic effect in the previous experiment was that cytotoxicity was assayed after a relatively short period, nameI! 3 days, of culture. This period had previously been shown (2) to be optimal for the development of nonspecific cytotoxicity by PPD-stimulated lymphocytes. In contrast, workers who have reported specific cytotoxicity after MLR have usually taken cells from relatively late cultures of 5-10 days (4, 5, 11) . In subsequent experiments, therefore, the specific and nonspecific cytotoxic effects of cells taken from early and late one-way MLRs were compared, and the results of one sucl~ experiment are shown in Table 2. Lymphocytes from two individuals (labelled A and 1’: in Table 2) were cultured either with mitomycin-treated lyn~hocytcs from the same individual (AAm and RP,m, respectively). or with mitomycintreated lymphocytes from the second individual ( ARm and I:Am, respectively). Two-way mitomycin controls (Am&n) were also set up. After 3 and 6 days of culture, washed lymphocytes from these mixtures were tested for cytotoxicity towards three types of target cell : August hepatoma cells (AH) and PHA-stimulated lymphocvtes from each individual (A-PHA and H-PHA). Thvmitline incorporation by replicate cultures was measured after 7 tlavs. For the sake of clarity, percent cytotoxicity values only- are shown in Table 2. Calculation of percent cytotoxicity necessarily inrolves a degree of distortion of the data ; the medium release values from which the cytotoxicity values are derived are .shown at the bottom of each column, and it sl~ould again be stressed that the stntistical analysis was carried out on the log transformation of the original percent release figures. A summary of the analysis of variance and multiple range tests is givrn in the table, and the conclusions that could be drawn may be stated as follows. ’ (i) As in th e previous experiment, stimulated cells (ABm and RAm ) exerted a marked nonspecific cytotoxic effect on August hepatoma (AH ) cells on day 3, when compared with unstimulatecl controls (AAm and l?Bm) and with two-wa\. mitemycin controls (AmBm) . (ii) This nonspecific effect on AH cells was much less marked on day 6 than on day 3. (iii) ABm cells exerted a significantly greater cytotoxic effect on A-PHA cells than did AAm or AmBm cells, both on day 3 and day 6. This represented an effect on responder cells, and was therefore nonspecific. (iv) Similarly, BAm cells exerted a significantly greater cytotoxic effect on B-PHA target cells than did BBm or AmBm cells, again both on day 3 and day 6. (v) RAm cells also showed a specific cytotoxic effect on A-PHA target cells on day 6, but not on day 3. This specific effect was reflected by a greater difference between BAm cells and AmBm controls when tested on A-PHA cells than when tested on B-PHA cells, (vi) There was a significant increase in specificity of RAm cells on da\- 6, as compared with day 3. (vii) ABm cells showed evidence of a specific cytotoxic effect on B-PHA target cells, as compared with A-PHA target cells, both on dav 3 and on day 6. In this
78
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FRANKS
2
SPECIFIC AND NONSPECIFIC CYTOTOXIC EFFECTS OF LYMPHOCYTES FKOY EARLY AND LATE ONE-WAY MIXED LEUCOCYTE REACTIONS 7; cytotoxicity against target cell b
Day of cultures 3 A-PHA
Effector
B-PHA
Thymidine uptake (cps) c
6 AH
A-PHA
B-PHA
AH
11 35 42 2
26 77 11 1
4 26 9 -1
lymphocytesa
AAm ABm BAm BBm AmBm Medium releaseb Statistical
23 35 28 3 (4:)
59 68 39 7
-1 75 84 -14
6 79 112 8
(ii)
summary
(i) Analysis of variance: interaction “day of culture” X “target cyte type” P < 0.001 (F = 69.8; Fo.oor;ro,r,~ = 3.54).
cell type”
X “effector
lympho-
(ii) Multiple range tests: given in form (1 - 2)3, where 1 = release in presence of test effector lymphocytes, 2 = release in presence of control effector lymphocytes, 3 = target cell and day of culture. Test
P
Represents nonspecific effect of ABm cells on day 3
- AAm)A-PHA day 6 - AAm)AH day 6
-co.05
- BBm)B-PHA day 6 - BBm)AH day 6
<0.05
nonspecific effect of BAm cells on day 6
(ABm - AmBm)B-PHA day 3 - (ABm - AmBm)A-PHA day 3
specific effect of ABm cells on day 3
(BAm - AmBm)A-PHA day 3 - (BAm - AmBm)B-PHA day 3
>0.05 NS
specific effect of BAm cells on day 3
(ABm - AmBm)B-PHA day 6 - (ABm - AmBm)A-PHA day 6
specific effect of ABm cells on day 6
(BAm - AmBm)A-PHA day 6 - (BAm - AmBm)B-PHA day 6
specific effect of BAm cells on day 6
(ABm (ABm
- AAm)A-PHA day 3 - AAm)AH day 3
(BAm (BAm
- BBm)B-PHA day 3 - BBm)AH day 3
(ABm (ABm (BAm (BAm
nonspecific effect of BAm cells on day 3 nonspecific effect of ABm cells on day 6
a Lymphocytes from two individuals were prepared. Combinations of responder lymphocytes (A or B) and of mitomycin-inactivated stimulator lymphocytes (Am or Bm) were cultured as shown. After 3 and 6 days of culture, respectively, washed effector cells from these mixtures were tested for cytotoxicity towards PHA-stimulated lymphocytes from each individual (A-PHA and B-PHA) and towards August hepatoma cells (AH) over a 7.5hr period. b Percent cytotoxicity values for each target cell are shown. These figures were derived from percent isotope release using the medium release value shown in parentheses at the foot of each column, and a total releasable isotope value of 80%. All statistical analyses were carried out on the original percent release data (see Materials and Methods, and Ref. 3). c Thymidine uptake was measured after 7 days of culture.
case, there was no evidence of increased specificity on day 6. This could be attributed to the high “natural” cytotoxicity of A lymphocytes for B target cells, as shown by the high levels of isotope release from B-PHA cells in the presence of unstimdatcd AAm cells. These rather complicated findings may be sumtnarized more briefly. First, there was again evidence for a marked nonspecific cytotoxic effect after BILK, both on xenogeneic target cells and on autologous responder-type target cells. This effect was apparent on day 6 as well as day 3, but in most cases the effect on day 6 was less marked. Secondly, there was also evidence for a specific effect superimpnsed on this nonspecific effect. This specific effect, on stimulator-type target cells, ~1s more marked on day 6 than on day 3. In other words, specific cytotoxicity appeared later in transformation than the nonspecific effect, and increased as the nonspecific effect decreased. DISCUSSION Several possible interpretations of direct “nonspecific” cytotoxic effects may be offered. \2’hen cytotoxicity towards target cells unrelated both to the responding lymphocyte and to the stimulating antigen is observed, it might be argued either that this effect depends on recruitment during transformation of lymphocytes capable of responding specifically against those target cells, or that the effector lymphocytes are pluripotent in the sense that they bear receptors for more than one antigen. In this paper, however, it has been shown that transformed lymphocytes are cytotoxic for autologous as well as for unrelated target cells. In this case, the autologous target cells studied were PHA-stimulated blasts, and it could be argued that the PHA remains bound to the cell surface and acts as a link between the effector lymphocyte and the target cell. This is unlikely, in view of observations ( 12-14 j that PHA and other mitogens are rapidly endocytosed by stimulated lymphocytes. Alternatively, it might be argued that PHA blasts express new antigens on their surfaces, ant1 that during lymphocyte culture (for instance with PPD) there is recruitment of cells responding specifically against these new antigens. The second observation reported here-namely, the dissociation in time between specific and nonspecific cytotoxicity during MI,&-suggests that this is not the correct explanation. If “nonspecific” cytotoxicity reflects nonspecific recruitment of cells which subsequently interact specifically with new antigens on the target blasts, it would be expected that both “nonspecific” and specific effects after MLR would increase in parallel, and it has been shown in other situations that specific cvtotoxicity during RZLR only develops after at least one cell division (11, 15). In the work reported here and elsewhere (2, 3), however, it has been shown that nonspecific effects are greater during the early stages of culture, before the onset of DSA synthesis and the development of specific cytotoxicity. These findings therefore suggest that the cytotoxicity observed during the early stages of culture is genuinely nonspecific, in the sense that it does not depend on the interaction of a specific lymphocyte receptor with antigen. It may be concluded that there are two phases of cytotoxicity in transforming cultures. A nonspecific phase which occurs early in transformation, before the onset of cell division, is followed by mitosis and expansion of the pool of lymphocytes capable of responding specifically. During this later phase, nonspecific cytotoxicit! decreases. 1t is difficult to assess the relative effectiveness of these two phases, ill
80
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terms of efficiency of killing by a single lymphocyte, since the chromium-release system only allows investigations on populations of effector cells. From the present study, however, it can be concluded that the overall population of lymphocytes in a culture can exert a nonspecific effect early in transformation which is quantitatively similar to the specific effect observed in the later stages of the response. A further point concerning the development of nonspecific as well as specific cytotoxicity is the question of the species under study, in that investigators who have failed to find evidence of nonspecific cytotoxicity during MLR have mainly worked with mouse lymphocytes (4, 5, 11, 15). Cultured mouse cells differ from human in two important respects apart from their lack of demonstrable nonspecific cytotoxicity : First, cell viability is low and there is selective survival of stimulated cells ( 11) and, secondly, recruitment of other lymphocytes to transform has not been reported. It is possible that, in the human system described here, nonspecific cytotoxicity is mediated not only by cells reacting directly to antigen, but also by antigenunreactive cells that have been recruited to transform and which can only exert a nonspecific effect (2). In this case, the overall ratio of specific to nonspecific cytotoxicity might be rather low. In mouse cultures, in contrast, those lymphocytes which under perfect conditions would be recruited may die before recruitment can occur. In this case, only those lymphocytes which are specifically stimulated by antigen during the early stages of culture survive, and hence the ratio of specific to nonspecific cytotoxicity may be much higher. If this explanation is correct, a failure to demonstrate nonspecific cytotoxicity in mouse MLRs may simply reflect inadequate culture conditions. Finally, an attempt may be made to point out two events occurring in viva in which nonspecific cytotoxicity may be involved. First, host tissue damage in delayed hypersensitivity reactions may be caused not only by specific cytotoxic effects against host cells coated with antigen ( 16), but also by nonspecific effects mediated by macrophages (17) or lymphocytes. Although at any one time macrophages are the main components of a delayed hypersensitivity reaction (IS), there is evidence that lymphocytes also enter the lesions in large numbers (19). It is possible that such lymphocytes are recruited to transform (2), develop a nonspecific cytotoxic effect and thereby contribute to host tissue damage, and are then destroyed by other nonspecifically cytotoxic cells. Secondly, there is some good evidence that local nonspecific effects may be involved in the rejection of grafts or tumors (20, 21). In this case, it is not suggested that nonspecific cytotoxicity represents the most important mechanism of cytotoxicity, but it is possible that nonspecific effects, particularly of lymphocytes which have been recruited to transform (2), may effectively amplify the action of specifically cytotoxic lymphocytes. ACKNOWLEDGMENTS We thank Dr. C. J. Sanderson for his stimulating comments and advice, and Mrs. B. Tayabali and Mrs. P. O’Brien for their technical assistance. One of the authors (A.E.B.) was supported by a Medical Research Council Scholarship for Training in Research Methods and by a Harrison Watson Studentship at Clare College, Cambridge. The work was supported by a grant from the Cancer Campaign for Research.
REFERENCES 1. Holm, G., and Perlmann, P., J. Exp. Med. 125, 721, 1967. 2. Butterworth, A. E., Cell. Immwzol. 7, 357, 1973.
CYTOTOXIC
3. 4. 5: 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.
EFFECTS
OF
TRAiYSFORMEI)
LY.\II’IIOCYTES.
III
Sl
16, 60, 1973. Butterworth, .4. E., Crll I~~zv7zruol. Hayry, P., and Defendi, V., Science 168, 133, 1970. Wagner. H.. J. Inwwxol. 109. 630. 1972. Solliday,‘S., and Bach, F. H., kTw.‘J. Imwur~ol. 2, 68, 1972. Forman, J., and Mb;ller, G., J. Exp. :Vcd. 138, 672, 1973. Cohen, I. R., and Feldman, M., CrU. 1~~wz111zo1. 1, 521, 1970. Hodes, R. J., and Svedmyr, E. A. J., ?‘rawplantatio~z 9, 470, 1970. Lightbody, J., Bcrnoco, D., Miggiano, V. C., and Ceppellini. K., (;. Butt. I’irol. I~wznuol. 64, 243, 1971. Hiyry, P., A4ndersson, L. C., Nordling, S., and Virolainen, SI.. ‘/‘vczusp/a~~t. Xczq. 12. 91. 1972. Kazavi, L., Nature (Lmdon) 210, 444, 1966. Pauli, R. M., DeSalle, L., Higgins, P., Henderson, E., Xorin, -I., and Strauss, H.. J. IVkVkWkOl. 111, 424, 1973. Unanue, E. R., Perkins, W. D., and Karnovsky, M. J., J. Exp. dlcd. 136, 885, 1972. Clark, W., and Nedrud, J., Cell. I~nnzuw~l. 10, 159, 1974. Perlmann, P., and Holm, G., A&xx 1mww~o1. 11, 117, 1969. Alexander, P., and Evans, R., Nature NW Biol. 232, 76, 1971. Bosman, C., and Feldman, J. D., Amrr. J. Patlzol. 58, 201, 1970. Koster, I;. T., McGregor, D. D., and Mackaness, G. B., J. E.rp. 3rd. 133, 400, 1971. Elkins, W. L., and Guttman, R. D., S&VCC 159, 1250, 1968. Zbar, B., Wepsic, H. T., Borsos, T., and Rapp, H. J., .I. ,Vot. CUIICW Inst. 44, 473, 1070.