Nonspecific cytotoxic effects of antigen-transformed lymphocytes

CELLULAR

7, 357-369

IMMUNOLOGY

Nonspecific

Cytotoxic Kinetics,

(1973)

Effects

Cell-requirements

of

Antigen-transformed

Lymphocytes

and the Role of Recruitment

A. E. BUTTERWORTH I~~~rrrunoloyy Division, Department of Pathology, Tennis Court Road, Cambridge,

University England

of Cambridge,

Received July 17, 1972 PPD-stimulated human peripheral blood lymphocytes have been shown to exert a nonspecific cytotoxic effect on allogeneic and xenogeneic target cells labeled with VXr. Chromium release induced by washed transformed lymphocytes was linear with time. At lymphocyte to target cell ratios of 120:1, significant killing could be demonstrated as early as + hr. At 8 hr, killing occurred with ratios as low as 3:l. The cytotoxic effect was related to the ability of the lymphocytes to transform to PPD, but reached an earlier peak than either DNA or RNA synthesis. Supernatants from lymphocyte cultures were not cytotoxic: instead, viable transformed cells were required. Partial removal of macrophages from the original cultures increased the cytotoxic effect. Lymphocytes could also be nonspecifically recruited to exert a cytotoxic effect by a mitogenic-like factor produced in transforming cultures. Preliminary evidence suggested that this factor acted independently of PPD and of histocompatibility antigens. These results are discussed with reference to possible amplification mechanisms for late cytotoxic effects, and to delayed hypersensitivity reactions in viva.

INTRODUCTION It is now widely accepted that lymphocytes can exert a nonspecific cytotoxic effect after stimulation with agents which induce transformation. Such agents may themselves be nonspecific, including phytohaemagglutinin ( 1) , anti-lymphocyte serum (2), antigen-antibody complexes (2) and streptolysin 0 (3). Alternatively, lymphocytes may be specifically stimulated with either soluble (2, 4, 5,) or cellbound (4, 6, 7) antigen. It is also clear that lymphocytes, during their specific response to antigen, release mitogenic factors which recruit other lymphocytes to transform (S-11). This phenomenon of recruitment has also been demonstrated indirectly, by quantitation of the transformation response obtained in mixed cultures of antigen-reactive and unreactive cells (12, 13). It has not previously been emphasized, however, that a combination of these two processes might represent an important mechanism by which cell-mediated cytotoxic effects may be amplified. This amplification process would depend on two events, occurring in the later stages of the lymphocyte response to antigen. First, antigen-sensitive cells transform, and in doing so acquire a nonspecific cytotoxic potential. Secondly, other cells are recruited to transform, and these in turn exert a cytotoxic effect. The main aim of this study was to investigate the part played 357

Copyright0 1973by AcademicPress.Inc. All rights of reproductionin any form reserved.

by recruitment in the development of nonsl)ecific cytotoxicity. It first seemed netessary, however, to examine two aspects of mitogen-induced cytotosicity which are still controversial. The first problem is the relationshil) between blast transformation and the development of cytotoxicity. Holm and Perlmann (14) have claimed that PHAinduced cytotoxicity is not solely caused by aggregation 0I lymphocytes and target cells, on the grounds that other agglutinating sul)stances, including polylysine, do not induce rytotoxicity. On the other hand, Perlmann, Nilsson and Leon (IS) have also sl~o\vn that Concanavalin A, itself a mitogcnic agent, inhibits PHA-induced cytotoxicity without affecting subsequent transformation. It is therefore difficult to exclude the possibility that PHA induces cytotoxicity I)); causing changes in the lymphocyte cell surface, and that transformation is in this ca5e an irrelevant secondary phenomenon. Similar problems occur in experiments in which antigen and target cells are mixed together, in the presence of reactive lymphocytes (2, 5). In this case, antigen may be binding nonspecifically to the target cell, and subsequently interacting with the lymphocyte. The most convincing approach to this difficulty has been the demonstration (4) that tllherculin-transformed human lymphocytes exert a cytotoxic effect on Chang cells after extensive washing. This effect was related to the ability of the lymphocytes to transform to tuberculin. The second problem is the question of \~l~etller nolispecific cytotoxicity in zifro is mediated by stable tosic products released from stimulated lymphocytes, or whether the continued presence nf lymphocytes, in the immediate vicinity nf the target cell, is required. Granger’s laboratory has produced evidence’ of factors in cultures, transformed the supernatants of human (16) or mouse (17) lymphocyte with PHA (17) or antigen (16)) which are to.xic for mouse L cells. The cytotosic effect, however, only appears after long periods of incubation of target cells with lymphotoxin. Furthermore, other workers have failed to find evidence of lymphotoxin-like materials in similar systems (6, 7, 18). of nonspecific cytotoxicity to In the present study, therefore, the relationship transformation and to stable soluble toxic factors has been studied, and the role of recruitment has been investigated. MATERIALS

AND

R’lETHODS

Human peripheral blood lymphocytes from healthy donors were prepared by the gelatine sedimentation method of Cnnlson and Chalmers (19). In some cases, partial purification of these cells was carried out by incubation of the leukocyterich supernatant with 200 mg cat-bony1 iron for 10 min at 37°C. followed hy extraction of the phagocytic cells with a magnet. Cells were cultured at 1-2 X 1OF cells/ml in Eagle’s Minimal Essential Medium (Wellcome) (MEM). buffered with 0.08 M bicarbonate and 0.003 A4 HEPES, and containing penicillin, 100 U/ml and streptomycin, 100 pg/ml, together with 209, obtained from the sedimentation supernatant after autologous gelatin?-sernm centrifugation. Cells were counted, before and after culture, on a Model R Coulter Counter. All cultures were carried out in 1.5 or 2 ml volumes in sterile plastic stoppered tubes. Cultures were maintained for 1 to 6 days, and were stimulated with 10 pg acetone-extracted PPD (Weybridge) , or with 10 ~1 phytohaemagglutinin ( Wellcome) (PHA) .

Trarcsforlnation

Assay

Transformation was assayed by the incorporation of labeled precursors into UK.1 or RKA. At the end of the culture period, 2 ,&i “H-thymidine (thymidine-6-H3, 5 Ci/mmole, Radiochemical Centre, Amersham) or 2 &i 3H-uridine (uridine-.5H3, 5 Ci/mmole, Radiochemical Centre, Amersham) were added to each culture. Cultures were incubated for a further 75 min, and uptake was then stopped by cooling in ice. Cultures were harvested by suction onto Whatman’s GF/A discs, and were washed once with 15 ml isotonic phospllate-buffered saline, $1 7.2 (F’BS), t\vice with 10 ml 10% trichloracetic acid and three times \vith 5 ml methanol. Discs were dried ;at room temperature overnight, and were counted without solubilisation in a Nuclear Enterprises Liquid Scintillain a toluene-based scintillator (NE233) tion Counter, NE 8312. No quenching coultl be detected by a channels ratio method at the cell concentrations used. Between-sample variation \vas greater than withinsample variation, justifying the use of this relatively inaccurate counting technique. Cytotoxicity

Assay

The cytotosic effect of lymphocytes on target cells was assessed by the release of 5’Cr from labeled cells, The target cells used in various experiments were : induced by butter yellow, and main(1) au August rat hepatoma, originally tained in the ascitic form in this laboratory by Lveekly passage in August rats, and (2) cultured Detroit 6, a human cell line originally of bone marrow origin. Target cells were washed three times in Hanks’ balanced salt solution (Hanks’ BSS) and were resuspended at approximately 5 x 10: cells/ml. Aliyuots were labeled \vith “‘Cr-sodium chromate (100-300 mCi/mg Cr, Radiochemical Centre, ,&iiersham), at concentrations of SO-100 &i/lOY cells, for 1 hr at 37°C. Samples 1, ere washed four times in Hanks’ RSS before resusl)ension at 2.5 X 10’ cells/ml in TC 199 containing 107~ inactivated foetal calf serum. One millilitre aliquots were dispensed into 2.2 ml sterile plastic tubes (Sterilin). To each tube \vas added 0.5 ml of the transformed lymphocyte suspension under test. This had been washed once in Hanks’ BSS and resuspcntled at varying concentrations in TC 199 containing 10% inactivated foetal calf serum. These cultures, now containing lymphocytes and target cells, were incubated for a further J-24 hr. Cultures were then centrifuged, and 0.5 ml of the supernatant was removed. Cells and supernatant were counted for “‘Cr, and the percent isotope releze was calculated on an IBM 360 computer by means of a program written by D. Franks. Stutistiral

Anolys,is oj Cytotoxicity

Interpretation

of cptotoxicity

Data data is complicated

by two factors :

(a) the considerable and variable release of 5’Cr in control tubes, either medium alone or with untransformed cells, and (b) the increase in variance with increasing isotope release.

with

Such data, therefore, require adequate statistical analysis. In the present study, R. G. Carpenter has helped extensively with this analysis. Individual experiments have been subjected to an analysis of variance for multifactorial design with random

360

IXUTTBRWORTII

and nested variables. These analyses were carried out on the log transformation of the release values, by means of a standard analysis of variance program (I~hl1102V) on an JHM 360 computer. Further coml)arisons were made by means of Duncall’s multiple range tests‘on effects or interactions that were significant in the analysis of variance. Similar tests have been applied to the lymphocyte transformation data. The results of these tests are presented here. In addition, all experiments reported here have been repeated three to five times. Each time, identical conclusions could be drawn from the analysis of variance. Quantitative differences occurred, which were due to variation in release of isotope from medium controls: this release ranged from 11 to 36% at 6 hours, in the case of August hepatoma cells. In each experiment, however, the effect of transformation was assessed by comparing the cytotoxicity produced by PPD-stimulated cultures with that produced by unstimulated controls. The difference between stimulated and unstimulated cultures, which will be referred to as the “transformation effect,” was independent of the medium release value and was entirely reproducible from experiment to experiment. lz”Iodine-Labeling

of PPD

PPD was labeled by the Chloramine T method (21). One pg PPD in 10 ~1 PBS was added to 1.2 mCi carrier-free l”“iodine (Radiochemical Centre, Amersham) in 10 $. One hundred pg Chloramine T in PBS was then added with shaking, and the mixture was left at room temperature for 15 min. The reaction was stopped with 0.2 ml of sodium metabisulphite, 1.25 mg/ml, followed by 1 mg bovine serum albumin. The iodinated protein was separated by Sephadex G25 column chromatography, using a 50 ml column previously equilibrated with bovine serum albumin. A specific activity of approximately 70 &i/pg PPD was obtained.

0 0

1

2

3 4 T,me (hours1

5

6

7

FIG. 1. Time course of “Cr release from August hepatoma cells after incubation with PPDtransformed or untransformed human peripheral blood lymphocytes. Lymphocytes were cultured for 4 days at 2 X 10” cells/ml, with or without 10 fig PPD. Cultures were washed, and added to labeled hepatoma cells at a lymphocyte to target cell ratio of 12O:l. The difference between transformed and untransformed cells was significant at the 1% level at all times.

CYTOTOXJC

EI’l:I~(“I‘S

OF

7‘11ANS1’0K7\11:1~

1,YhlI’I

361

IOCYTl3

RESULTS Nonspecific

Killing

by Antigen-Trczlzsfon~c~

Cells

Early experiments showed that such nonspecific killing can occur. Cells from unpurified J-day human PPD cu!tures were washed and added to labeled hepatoma cells for various times at various lymphocyte to target cell ratios. A significant transformation effect, that is, a significant difference between transformed and untransformed cells, could be demonstrated with lymphocyte to target cell ratios as low as 3 : I, when release was measured at S hr. At higher ratios (120: 1) a significant effect could consistently be demonstrated as early as 4 hr (Fig. 1). Isotope release was linear with time, and extrapolated back to the medium release value at zero time. This implied that there was no lag phase in the development of the cytotoxic effect, in contrast to the direct killing of xenogeneic cells by allergised but untransformed cells (,Franks, Sanderson, Waldmann, Kelly and Butterworth ; in preparation). bIasimum isotope release, as estimated by the total amount of isotope t.hat could be released from the cells by repeated freezing and thawing, had occurred by 7 hr. Extensive washing of the effector cell preparation, sufficient to reduce the amount of antigen to less than 0.25% of the starting concentration, did not reduce its cytotoxic potential. lielutianslzip

to Transformation

The development of the cytotoxic activity of human lymphocytes after culture with PPD was correlated with their ability to transform in response to that antigen. This is shown in Table 1. Two individuals with positive skin reactions to PPD showed a significant transformation effect, both for incorporation of thymidine and

I’ercent isotope release from August hq’ntoma Cf?llSb

Thymidine incorporation (CPS) c

Transformed Not transformed Significance of difference Medium release Transformed Not transformed Significance of difference

48.0 34.6

32.2 32.7

P < 0.001

P >

0.05

35.7 25.0 P < 0.001

22.7 582.5 15.2 P < 0.001

6.2 6.8 P > 0.05

383.3 11.1 P < 0.001

a Lymphocytes cultured with or without 10 rg PPD for 5 days at 1 X lo6 cells/ml. b Sample cultures were washed, resuspended and added to target cells at a ratio of 20: 1. Isotope release was measured after 74 hr. c Other cultures assayed for transformation by the incorporation of 2 pCi 3H-thymidine for 7.5 min.

362 fur

ISUTTERWORTII

cytotosicity

August hepatoma

cells. One individual who did not respond to showed neither increased thymidine uptake nor increased cytotoxic activity after culture of cells with antigen. On the other hand, the time course of the development of the cytotoxic effect did not correspond with that of thymidine or uridine incorporation. Figure 2 sho\vs that in the cytotoxic system a significant difference between stimulated and mnstimulatetl cells could be seen as early as 1 day. This difference reached its peak at 3 days. In contrast, thymidine incorporation was not marked ~lntil 3 days. and did not reach its peak until the 5th or 6th day. Uridine incorporation showed a high early to

PPD on skin-testing

background level, toxicity therefore

but did not reach its maximum until the 4th represented an early event in the transformation

to 6th days. response,

Cytowhich

M He~oJ_ma-

-_--__--------

5 2 3 4 Days of transformot~on

6

2. Relationship between development of cy:otoxici:y and incorporation of nucleic acid FIG. precursors by PPD-transformed human peripheral blood lymphocytes. Lymphocytes were cultured at 2 X 10” cells/ml for 1 to 6 days. Some cultures were washed, and added to labeled August hepatoma or Detroit 6 cells at a lymphocyte to target rell ratio of 4O:l. Release was measured at 8 hr. Other cultures \vere assayed for transformalion by the incorporation of 2 pCi tritiated thymidine or uridine for 75 min. Release : O-O w-----m e-0 * -* Transformation

Transformed lymphocytes tested on Detroit 6 cells Untransformed lymphocytes tested on Detroit 6 cells Transformed lymphocytes tested on August hcpatoma cells Untransformed lymphocytes tested on August hepatoma cells :

0-q n -W l - - - - -0

W- - - - -B

Thymidine incorporation Thymidine incorporation Uridine incorporation by Uridine incorporation by

by transformed cells by untransformed cells transformed cells untransformed cells

The transformation effect (cytotoxicity induced by transformed lymphocytes--that induced by untransformed lymphocytes) was significant for both target cells on all days (P < 0.01). The transformation effect on day 3 was significantly greater than that on all other days (P < 0.01).

is regarded first day.

as a continuous

Relationslzip

process,

to Cell Pvcsence,

Attempts

starting

Viability

\vith

induction

by antigen

during

the

and Type

of transHuman peripheral blood lymphocytes \vere cultured in the presence or absence of PPD for 4 (lays, and the supernatants (A) \\-ere removed. Cultures bvere then washed and inculcated for a further 6 lx before removal oi a second supernatant (B) Cultures were again washed, and the supernatants (A and B) and the cells themselves (C ) were added to labeled August hepntoma cells for a further 6 hr. Isotope release at the end of this period is shown in Table 2. All supernatants gave less release than the medium control. Cells which were transformed with antigen, however, gave significantly more killing than untransformed cells, and more than snpernatants either from transformed or from untransformed cultures. No toxic supernatant factor iorllletl

coultl

were made to demonstrate

cultures,

tllerefore

comparable

to that

3 tosic

clescril)etl

factor

in the supernatants

by Granger

zmd Kolb

(17).

be clem0nstratef.l.

Another possible, though unlikely event, \vas that supernatatit protlucts from stimulated cultures could confer on unstimulated cells an immediate ability to kill. In other words, a supernatant factor might require the presence of normal cells to exert its cytotoxic effect. This was tested as follows : Human peripheral blood cultures were incubated for 4 days in the presence or absence of antigen. Cultures were then centrifuged, and resuspended in fresh medium or in supcrnatnnts from stimulated or unstimulated cultures. Supernatants alone were also taken. Samples were tested for 6 hr on labeled hepatoma cells. The percent release observetl is sho~vn in Tatle 3. Stimulated cells caused significantly

‘yc isotopcl

in original

PPD

relensrn

cultures” diffcrcllcc

+ ~-____ A.

Supcrnatants from 4-day cultures

31.8

33.7

1.1 (dX)

31.8

31.2

0.6

(CIB)

50.3

38.1

12.2

(dC)

13. Supernatants from cultures washed on

4th day, then incubated for further C. Cells

from

Medium

6 hr I-day

cultures

r&ase

35.7 dC - dA dC - d13

a Kelease of %hromium from August superrrntant or cell pxparations. h Human

peripheral

blood

lymphocytes

hepatoma cultured

11.1 11.6

P < 0.001 P < 0.001

cells, measured after 6 hr of incubation with

or without

10

ry PPD for

4 days.

with

364

BUTTERWORTII ‘I-ABLE

3

CYT~TOXIC L%FECTS OP MIXED SWEHNATANT ANI) CELL PIWPAKATIUBS I~IWM PPD-TRANSFORMED HUMAN I,YMPHOCYTE CULTURES e/e isotope release” Cellsa : Transformed (A) Supernatant”: Transformed Untransformed Fresh medium

37.7 NS 37.7 NS 41.6

Untransformed (B)

**d ** **

23.4 NS 21.3 NS 23.9

Nil (0

** ** **

16.2 NS 16.3 NS 17.8

a Lymphocytes were cultured with or without 10 pg PPD for 4 days. b Samples were centrifuged, and the supernatants were removed. Transformed (A) or untransformed (B) cells were then resuspended in supernatants from transformed or untransformed cultures, or in fresh medium. c These preparations, together with supernatants and medium alone (C) were added to 6’Crlabelled August hcpatoma cells, and isotope release was measured after 6 hr. d Differences between adjacent figures: NS = P > 0.05 : not significant; ** = P < 0.01.

greater release than unstimulated cells, whether they were resuspended in fresh medium or in supernatants from stimulated or unstimulated cultures. Unstimulated cells gave no greater killing in supernatants from stimulated cultures than in fresh medium. Supernatants alone again showed no killing. It was therefore unlikely that stimulated supernatants contained a factor which would confer an immediate cytotoxic potential on untransformed cells. Although nonspecific cytotoxicity in this system therefore does not depend on soluble supernatant factors, it may be caused simply by particulate debris released iron1 dead cells. If this is so, cytotoxicity should be obtained with disrupted transformed lymphocytes as well as with live cells. To test this possibility, lymphocytes from stimulated and unstimulated 4-day cultures were washed, and disrupted by repeated freezing and thawing, before addition to target cells without further washing. This procedure abolished the cytotoxic activity of the stimulated cells. Viable cells were therefore required. The cytotoxic cell in these experiments has not been clearly identified. The possibility that activated macrophages may be involved is difficult to exclude, since macrophages are required for the induction of transformation (20). If macrophages are the cells responsible for cytotoxicity, however, their partial removal at the beginning of culture should diminish the cytotoxic effect without affecting transformation as judged by thymidine incorporation. This possibility was tested by partial purification of the starting lymphocyte suspension by the carbonyl iron technique, followed by culture with PPD in the usual way. No adjustment of cell count was made at any stage, and thymidine incorporation by unpurified and partially purified cultures was not significantly different. Isotope release induced by cells from the purified cultures, however, was significantly greater than that induced by unpurified cells (60.6% release at 8 hr, as compared with 35.2% : P < 0.001). The reason for this increase is not clear, but the findings would definitely suggest that macrophages were not the major cell involved in the expression of cytotoxicity.

CYTOTOXIC

Cytotoxic

Effects

EFFECTS

uf ‘Xecruited”

OF

TRANSPORMEI)

LYMI’IIOCYTES

365

Cells

Since it has been shown tliat transformed cells du acquire a nonspecific cytotoxic potential, it should be possible, if transformation involves recruitment of bystander cells, to demonstrate that such recruited cells can exert a cytotoxic effect. Several approaches to this problem are available. Supernatants from stimulated cultures of a PPD-reactive individual can be used to stimulate cultures of a non-reactive individual. This approach, although open to criticism, has proved successful with human cultures. Cells from a PPD-reactive individual were cultured for 24 hr in the presence or absence of PPD. Supernatants from such cultures were then added to fresh cells from a PPD-negative individual. Other fresh cells were stimulated directly with antigen. After a further 3 days of incubation, thymidine incorporation was measured, and cultures were washed and tested on August hepatoma cells. The results are given in Table 4. Fresh PPD did not induce DNA synthesis in cells from the PPD-negative individual, and caused only a marginal increase in cytotoxic effect. Such cells could be induced both to transform and to exert a cytotosic effect, however, by supernatants from 24-hr PPD-stimulated cultures of the positive individual. Supernatants from unstimulated cultures produced no such effect. These data show that cells from an unreactive individual can be recruited to exert a cytotoxic effect, as well as to transform, by supernatants from stimulated cultures of PPD-reactive cells, but give no information about the agent responsible for recruitment. Three possibilities must be considered : (1) (2) (3)

art antigen-dependent factor, including plexes, histocompatibility antigen, and an antigen-independent factor.

particularly

antigen-antibody

com-

The classical way of excluding antigen-antibody complexes involves “reconstitution” of the unstimulated supernatants with antigen (10). In several experiments, reconstitution with antigen produced no change either in cytotoxicity or in transiormation of cells treated with unstimulated supernatants. Equally, addition of further antigen to stimulated supernatants produced no increase in cytotoxicity or transformation of supernatant-stimulated cells. This approach, however, is open to the criticism that unstimulated cultures would not necessarily secrete antibody, and therefore would not form antigen-antibody complexes on reconstitution with antigen. A better way of excluding antigenantibody complexes is therefore to remove antigen from the stimulated supernatant. This approach has the added advantage that since the supernatants produced are antigen-free, they can be used to stimulate cells from the same individual. The possibility that histocompatibility antigens may be involved can therefore also be eliminated. This technique has been applied in experiments of the following type. Cells from a -PPD-reactive individual were cultured with 1’61-labeled PPD of known specific activity for 3 hr. Cultures were then washed thoroughly and transferred to new tubes, before incubation for a further 24 hr. Supernatants from these cultures were then taken and counted for W, and the amount of PPD left in the supernatant was calculated. New cultures from the same individual were set up, and were stimulated either with these supernatants or with an amount of fresh antigen equivalent to

366

RIITTERWORTFI

TABLE

4

TRANSFORMATIONAND CYTOTOXICEFFECTSOF ANTIGEN OK SUPEKNATANT-STIMULATED LYMPHOCYTES

Fresh antigen Nil Fresh antigen Nil Supernatants from stimulated (2) cultures Supernatants f;om unstimulated (2) cultures Medium release

29.6 24.9 40.9 21.2

6.5 11.5 123.7 19.3

52.8

34.3

22.2 17.4

2.1

a Lymphocytes from a PPD-positive donor (2) were cultured for 24 hr in the presence or absence of 10 rg PPD. Cultures of fresh cells from the same individual, and of cells from a PPD-negative individual (1) were then prepared. Cells from the PPD-negative individual were stimulated either with fresh antigen or with 24-hr supernatants from stimulated or unstimulated cultures of the positive individual. * After a further 3 days of culture, these cells were washed and added to 61Cr-labclled August hepatoma cells. Isotope release was measured after 7; hr. c Other cultures were assayed for transformation by incorporation of 2 HCi 3H-thymidine over 75 min.

TABLE

5

CYTOTOXICEFFECTSOF LYMPHOCYTESSTIMULATEDWITH ANTIGEN-DEPLETED SUPEKNATANTSORLow DOSES OF FKESHANTIGEN :I isotope releaser PPD in original

culture --

Lymphocytes

stimulated

_

+

--.

difference

..._~.

with :

n Antigen-depleted supernatant from 24-hr PPD culture

45.8

16.0

29.8 (dS)

P < 0.001 ***

b Fresh antigen, equivalent to that in supernatant

17.6

16.6

1.0 (dp)

P < 0.05 *

28.8

P < 0.001

Medium

release

9.4 (ds) -

(W

***

(1Cells were cultured with or without 10 pg *QI-PPD for 3 hr. Cultures were washed 6 times and transferred to new tubes, before incubation for a further 24 hr. Supernatants were then taken, and counted for rz61. These supernatants were added to fresh cells from the same individual. *Other fresh cells were cultured with or without 0.01 pg PPD, equivalent to the amount of rz51-PPD left in supernatant.” Both groups of cells were cultured for a further 3 days. c Cultures were then washed, and added to KLCr-labelled August hepatoma cells. Isotope release was measured after 41 hr.

that remaining in the supernatant. These fresh preparations were cultured for 3 days, and cytotosicity was assayed in the usual way. Results from suc11 an experiment are given in Table 5. A significantly greater transformation effect was seen in those cultures receiving antigen-depleted supernatants than in those receiving an equivalent amount of fresh antigen. The possibility that “superantigen” could have been produced cannot be excluded, but it sllould be remembered that the full dose of antigen was only present for 3 hr in the original culture. It therefsore seems reasonable to suggest that the recruitment observed was caused by a factor independent both of histocompatihility antigen and of antigenantibody complexes. DISCUSSION Two distinct points arise from the results presented here. First, it is clear that antigen-transformed cells can exert a nonspecific cytotoxic effect. This effect does not depend on the continued presence of free antigen in the cytotoxicity assay system. Supernatants from stimulated cultures do not exert a an immetlicytotoxic effect, nor do such supernatants confer on normal lymphocytes ate cytotoxic potential. Instead, cytotoxicity depends on the continued presence of viable stimulated cells, of which lymphocytes are probably the most important. The cytotoxic efiect is correlated with the ability of the cells to transform, bnt reaches its maximum at an earlier stage than the peak thymidine or uridine incorporation. These results may be compared with those obtained by other authors. They are clearly in contrast with those of Granger and his colleagues ( 16, 17) who ha\,e described a heat-stable lymphotoxin in supernatants from transformed cultures. The main difference in technique is that Granger’s test involves inhibition of protein synthesis in long-term target cell cultures (24-72 hr) Transformed lymphocytes effect requiring could therefore exert two types of cytotoxic effect: a short-term the presence of transformed cells in the immediate vicinity of the target cell, and perhaps in\Tolving a mediator which is either rapidly inactivated or rapidly taken ~13 by neighhouring cells, and a long-term effect produced by a stable soluble mediator. It is possible that both phenomena may be artefacts of the in vifro conditions, although this seems unlikely in the case of the direct cytotoxicity described here, since this is rapidly produced by washed cells at low lymphocyte to target cell ratios. Evaluation of the relative biological importance of the two processes must await the development of methods for in Z&O study. In the PHA-induced lymphocyte cytotoxicity described by Perlmami and his colleagues (l!, 4, 14)) as well as in most of the experiments involving PPD-stimulated lymphocytes described by Ruddle and Waksman (5), the stimulating agent was present throughout the period of the cytotoxicity assay. The comment could therefore be made that the stimulating agent was simply binding non-specifically to the target cell, and that the lymphocytes interact primarily with this hound material, although there is evidence suggesting that this is not the case. Criticisms of this sort cannot be applied either to the experiments of Holm and Perlmann (4), who showed that PPD-transformed lymphocytes exert a nonspecific cytotoxic effect even after extensive washing, or to the results presented here. Several workers have examined the cytotoxic effects of lymphocytes stimulated with histocompatibility antigens, either on fibroblast monolayers or in mixed leucocyte reactions (4, 6, 7, 27). Although they interpret their results in different ways,

368

BUTTEKWOHTIX

it is clear from their data tllat thtp observe a specific effect superimposed on a marked nonspecific effect. It is possible that the mechanism of killing it1 both cases depends on a change in the lymphocyte associated with transformation, and that the increased killing of the spccilic target cell merely represents a more efficient link between the killer cell and the target cell, whereby the interactinn of receptor and antigen holds the two cells together for a period of time sufficient to allow killing to take place. Alternatively, it could be suggested from the results presented here that nonspecific cytotoxicity represents an early event in the continuous process of lymphocyte transformation, rlecrcasing when DNA synthesis and division begin. This might be followed by proliferation of specifically responding cells, and the appearance of specific cytotoxicity. This possibility is currently under investigatioll. The part played by the macrophage in the present experiments has not been clearly established, but partial removal of macrophagcs at the beginning of culture increased the cytotoxic effect of cells after subsequent transformation. Activated macrophages (22, 23) therefore did not seem to be the major cell responsible for cytotoxicity. The second aspect of this study to be discussed is the role of recruitment. Most of the work in this field has been carried out on the transformation response as measured by cell division, and mitogenic factors have been described in several situations (8-13, 24). Early work on recruitment suffered from problems of interpretation, and it was difficult to decide whether the phenomenon was due to histocompatibility antigen, to an antigen-dependent factor such as transfer factor or antigen-antibody complexes, or to an antigen-independent mitogenic factor. Later work with identical siblings (12, 13) and with nrltologous cells (11) has suggested that histocompatibility antigen is not involved. Two facets of this phenomenon have been studied here. In the first place, it has been shown that mitogenic factor, acting in the classical way across a histocompatibility barrier, will induce nonspecific cytotoxicity as well as transformation. In the second place, thr transfer of antigen-&$letcd supernatants to autologous cells has shown that recruitment can take place in the absence of antigen, and that such recruited cells can exert a nonspecific cytotoxic effect. Such recruitment is presumably due to an al7tigen-indepentlent factor. These results show that it is possible that recruitment may provide an amplification mechanism in the intermediate stages of the cytotoxic response: that is, once transformation has started but before DNA synthesis occurs. Furthermore, although it is unwise to extrapolate directly from in vitro experiments to in viva conditions, a testable prediction might be made. This is, that in the delayed hypersensitivity reaction, relatively few lymphocytes would be transforming in direct response to antigen. The remainder would be nonspecifically recruited. Both specific and nonspecific groups would acquire, at least temporarily, a nonspecific cytotoxic potential, which would be exerted both on the cells eliciting the response and on host cells. ,Finrlings from passive transfer experiments in animals (25), and from studies on leprosy in man (26)) might sulqlort this 1”edictioll, wllich provitles a groundwork for future

in z&m experiments.

ACKNOWLEDGMENTS I thank Dr. D. Franks for his continual advice and encouragement, Mr. R. G. Carpenter for extensive help with the statistical analysis, and Miss B. MacFadzean and Miss B. Knott for

CYTOTOXIC

EFlXCTS

OF

TKAi’GSl~OKRIEL)

LYMI’IIOCYTliS

369

expert technical assistance. This work was carried out during the tenure of a Medical Research Council Scholarship for Training in Research hlethods, and was supported by a grant from the Cancer Campaign for Research.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. ‘24. 25. 26. 27.

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