Int. J . Cancer: 22, 405-412 (1978)

INTERFERON AND SPONTANEOUS CYTOTOXICITY IN MAN. I. ENHANCEMENT OF THE SPONTANEOUS CYTOTOXICITY OF PERIPHERAL LYMPHOCYTES BY HUMAN LEUKOCYTE INTERFERON Stefan EINHORN, Henric BLOMGREN and Hans STRANDER Radiumhemmet, Karolinska Hospital, S-104 01 Stockholm 60, Sweden

A purified preparation of human leukocyte interferon used a t this hospital in the treatment of malignant diseases was tested for i t s ability t o modify the spontaneous cytotoxicity of peripheral lymphocytes from healthy donors. The inhibitory effect of allogeneic lymphocytes on the [3H]thymidine incorporation of a lymphoblastoid cell line, Raji, was augmented by the presence of interferon or by pretreatment of the lymphocytes with interferon. This form of pretreatment also increased lymphocytes' capacity for reducing the number of surfaceadherent tumor cells in a microassay. Moreover, lymphocytes treated with interferon exhibited an enhanced cytotoxic capacity for target cells on incubation with such cells labelled with 5'Cr.

Interferons (IF) are released from cells exposed to virus, certain non-viral microorganisms and a number of natural and synthetic substances (Finter, 1973). IF are characterized by their ability to inhibit viral replication in cells (Isaacs and Lindenmann, 1957). Preparations of IF, however, also display other types of biological activity, including an inhibitory effect on the multiplication of normal and neoplastic cells in vitro and in vivo (Paucker et al., 1962; Frayssinet et al., 1973). Since I F are released by lymphocytes activated by antigens and polyclonal mitogens (Epstein, 1976), they fall within the group of substances known as lymphokines, which are considered to be regulators of immune responses (Waksman and Namba, 1976). It has been established that I F preparations can interfere with immune reactions. They can suppress humoral antibody responses (Gisler et al., 1974) and proliferation of lymphocytes in response to phytomitogens and allogeneic cells (LindahlMagnusson et a/., 1972; Blomgren et al., 1974), delay the rejection of allografts (Mobraaten et al., 1973), and inhibit delayed hypersensitivity responses (De Maeyer et al., 1975). IF preparations can also augment immunological functions; for instance, they can intensify antibody responses at a late stage of sensitization (Gisler et al., 1974) and enhance the cytotoxic activity of allosensitized lymphocytes (Lindahl et a/., 1972). The purpose of the present study was to examine whether the preparations of I F currently in use at this hospital for the treatment of neoplastic diseases (Strander, 1977) are capable of modifying the spontaneous non-specific cytotoxicity of peripheral leukocytes. The IF preparations were found to increase the cytotoxic capacity of non-sensitized

lymphocytes from healthy donors for killing allogeneic tumor cells in vitro. MATERIAL AND METHODS

Lymphocyte preparations Lymphoid cells were separated from heparinized venous blood, drawn from healthy donors of both sexes, by centrifugation on a layer of FicollIsopaque (Jondal et a/., 1972). The cells were then washed twice by centrifugation in Eagle's minimal essential medium supplemented with Earle's salts (MEM). Approximately 90% of the cells were classified as lymphocytic-as determined by crystal violet staining-the rest being classified as monocytic or granulocytic. Target cell lines The following cell lines were used: a lymphoblastoid cell line (Raji) derived from a Burkitt lymphoma (Pulvertaft, 1965) ; the U-cell line derived from human amnion cells (Pohjanpelto, 1961); the 393 T (Ponttn, 1975) and 2 T ( P o n t h and Saksela, 1967) lines, both from osteosarcoma; and Chang cells from human liver. The Raji cell line was grown as a suspension culture in RPMI 1640 medium with 8 % of heatinactivated fetal calf serum (FCS), 125 IU of penicillin and 125 pg of streptomycin per ml. The other cell lines were grown as monolayers in MEM supplemented with 10% of FCS. Penicillin and streptomycin were added to give the same concentrations as in the Raji cultures. All the cell lines were maintained at 37" C in a humidified 5% C0,-air atmosphere. Interferon preparations Preparations of purified human leukocyte interferon (PIF) were derived from peripheral blood leukocytes exposed to Sendai virus (Cantell et al., 1974). This type of preparation, containing approximately 6 x los units of IF per ml, is being used at Radiumhemmet in the treatment of malignant diseases (Strander et al., 1977). The anti-viral activities of the preparations were determined by assaying inhibition of plaques induced by vesicular stomatitis virus in U-cells (Strander and Cantell,

Received: May 23, 1978 and in revised form July 31, 1978.

406

EINHORN ET AL.

1966). The anti-viral activity was measured by comparison with the international reference preparation 69/19, and expressed in international units. Mirotnycin treatment of lymphocytes

Lymphocytes, at a concentration of 2 x 10s cells per ml, were incubated in MEM containing 2.5 pg of mitomycin-C (Nutritional Biochemicals Corp., Cleveland, Ohio). After I h of incubation at 37" C the cells were washed three times by centrifugation and resuspended in MEM. Interferon treatment of lymphocytes

Lymphocytes, at concentrations of 5 x lo610 x IOg/ml, were incubated for various times at 37" C or 4" C in MEM containing various concentrations of IF. The cells were then washed twice by centrifugation and counted. Viability, as tested by trypan-blue exclusion, was not changed by the IF treatment. Control lymphocytes were incubated in MEM without IF, and otherwise treated identically with the test lymphocytes. Determination of D N A synthesis of cells

A microculture technique described elsewhere was used (Lilliehook and Blomgren, 1974). Briefly, 2,OOO-5,000Raji cells were cultured either alone or together with approximately 200,000 mitomycintreated allogeneic lymphocytes in the wells of microtest plates containing 0.2 ml of MEM supplemented with 10% of heat-inactivated human serum (HS), penicillin and streptomycin, In some experiments the lymphocytes were pretreated with IF, and in

because of their low sensitivity for IF (Adams et al., 1975). Micro-assay of cytotoxicity

This technique was applied as described by Takasugi and Klein (1970). Approximately 100 tumor cells were allowed to attach themselves to the bottom of the wells of micro-test plates (No. 3034, Falcon Plastics, Oxnard, Calif., USA) after 24 h incubation at 37" C in 20 ,141 of MEM containing 10% of FCS. The medium was then discarded. To the cultures were added various numbers of lymphocytes suspended in 20 p1 of MEM containing 10% of HS, and also penicillin and streptomycin. Control cultures received medium only or medium containg IF. The cultures were then incubated for 48 h at 37" C in a humidified 5 % C0,-air atmosphere. The wells were then washed three times with a balanced salt solution, fixed and stained by the May-Griinwald-Giemsa method. The number of target cells remaining in the wells were then counted. The reduction in the number of target cells in the experimental cultures was compared to the number in the control cultures incubated without lymphocytes or IF. The mean for 3-6 determinations was calculated. Assuy of

'Cr release

One million target cells suspended in 0.5 ml of MEM containing 10% of HS were labelled by incubation for 1 h at 37" C with 0.1 ml of N a p C r 1 O 4 (75 pCi, specific activity 100-350 /tCi/pg Cr) (The

TABLE I PERCENTAGE INHIBITION OF [SH]THYMIDINE UPTAKE BY 2 x los RAJl CELLS CULTURED IN THE PRESENCE OF VARIOUS CONCENTRATIONS OF INTERFERON (IF) OR 2 x lo6 MITOMYCIN-TREATED ALLOGENEIC LYMPHOCYTES, OR BOTH Inhibition of [3H]thyrnidine uptake (per cent) ~

Raji cells Raji cellsi 10 I F units/ml Raji cellsi 100 IF units/ml Raji cells+ 1000 IF units/ml Raji cells + lymphocytes Raji cells+lymphocytesi-10 IF units/ml Raji cells+lymphocytes+lOO IF units/ml Raji cells tlymphocytes+1000 IF units/ml '

Day 2

Day 4

(9,390) 0 I

(44,340) 0

-4

1 24 42

Day 6

3 12

23 39

47

53 61

48

64

(170,770)

0 19 36 46

55 70 76 78

Uptake expressed in counts per minute.

others IF was present in the cultures throughout the incubation period. The cultures were kept at 37" C in a humidified 5 % C0,-air atmosphere. Twenty-four hours before termination each culture received 1 pCi of [3H]thymidine (5 Ci/mmole. The Radiochemical Centre, Amersham, Bucks., England). The radioactivity incorporated by quadruplicate cultures was determined by liquid scintillation counting and expressed as counts per minute (CPM). Raji cells were used for these types of experiments

Radiochemical Centre, Amersham, England). They were then washed four times by centrifugation and re-suspended in MEM with 10% of HS. Ten thousand labelled cells were transferred to Ependorf reaction tubes (No. 3810, Eppendorf Geratebau Netheler+ Hinz GmbH, Hamburg, West Germany) together with various numbers of lymphocytes; the volume was adjusted to 0.6 ml with medium. To some tubes, intended for measurements of spontaneous release, only target cells and medium

407

EFFECT OF INTERFERON ON CYTOTOXICITY TABLE 11

capacity to inhibit proliferation of Raji cells. As shown in Table 11, the inhibitory activity of the lymphocytes was increased by treating them with IF. The effect was IF dose-dependent.

CAPACITY OF VARIOUS CONCENTRATIONS OF IF OR 2 x lo6 IF-TREATED, MITOMYCIN-BLOCKED ALLOGENEIC LYMPHOCYTES FOR INHIBITING THE PROLIFERATION OF 5 x los RAJI CELLS ' Inhibition of I'H]thymidine uptake (per cent)

Raji cells Raji cells t-10 IF units/nil Raji c e l l s + l O O IF units/ml Raji cellsf1000 IF units/nil Raji cells+lymphocytes Raji cells+lymphocytes pretreated with 10 IF units/ml Raji cells+lymphocytes pretreated with 100 IF units/ml Raji cells+ lymphocytes pretreated with 1,000 units/ml Raji cellsflymphocytes pretreated with 10,000 IF units/ml

(29,620)

0 2 10 15 20

32 43 49 63

The ['Hlthymidine uptake was determined on day 4. The lymphocytcs were pre-incubated for 24 h at 37" C in medium with and without various concentrations of IF. - Uptake expressed in counts per minute.

were added. After centrifugation at 200 g for I min, the cells were incubated for 4 h at 37" C. The radioactivity of 0.2 ml of the supernatant and the remaining 0.4 ml was determined with a gamma counter and expressed as CPM. A cytotoxic index was calculated as: % release with lymphocytes - % spontaneous release 100- % spontaneous release The spontaneous release did not exceed 28%. All the cultures were set up in duplicate. The variability for the duplicate determination did not exceed 7%. RESULTS

Proliferation of lymphoblastoid cells in the presence of allogeneic lymphocytes andlor IF

The extent to which mitomycin-treated allogeneic lymphocytes and/or IF inhibited the proliferation of Raji cells was examined over a culture period of 6 days. The addition of either IF or lymphocytes to cultures of Raji cells inhibited their DNA synthesis (Table I); this effect was most pronounced at the end of the culture period. The addition of both IF and lymphocytes had a stronger inhibitory effect on proliferation than would be expected from the aggregate effects of each when tested separately. This synergism was most evident on the second day of culture. One possible interpretation of these results is that IF activates lymphoid cells in such a way that they become more cytotoxic for or inhibit the growth of the Raji cells. To examine this possibility lymphocytes were incubated for 24 h at 37" C in medium either alone or together with IF in various concentrations. The cells were then washed thoroughly, treated with mitomycin and examined for their

Reduction in the number of surface-adherent tumor cells by IF-treated Iymphocytes

IF concentration and incubation period. The above experiments did not indicate whether IF activates lymphocytes in such a way as to render them more cytotoxic with respect to the target cells or whether they merely become more cytostatic. To ascertain whether the tumor target cells were damaged, lymphocytes were incubated for 24 h in medium with and without IF and then examined for the extent to which they reduced the number of 393 T cells growing on the floor of micro-wells. The lymphocytes that had been preincubated with 10 or 100 IF units per ml exhibited a greater capacity for reducing the number of tumor cells over a wide range of 1ymphocyte:target cell ratios than did the untreated lymphocytes (Fig. 1). In this respect there was no major difference between the two IF concentrations employed.

s- 100

t;

50

ar

2 L

0

z IU

3

0

w

" 0

/ 4 -------{

I

1

1.S:l

I

3:l

t

6:l

I

I

I

12:l

25:l

50:l

LYMPHOCYTE- T A R G E T CELL RATIO FIGURE 1 - Reduction in the number of 393 T target cells by allogeneic lymphocytes. The lymphocytes were incubated for 24 h in medium without IF (X-x)); with 10 units of IF/ml (0-0); with 100 units of IF/ml (0-0). The horizontal line shows the effect of adding 100 units of IF to the target cells. Means*sE are calculated from six determinations.

To determine whether the reduction in the number of target cells might have been due to IF remaining after washing the lymphocytes, 100 IF units were added to some of the wells, together with target

408

EINHORN ET AL. TABLE I11 SENSITIVITY OF FOUR CELL LINES TO INTERFERON-TREATED LYMPHOCYTES, EXPRESSED AS THE PERCENTAGE REDUCTION IN THE NUMBER OF TARGET CELLS ' Reduction in number of target cells (per cent)

Lymphocyte : target cell ratio

Pretreatment of lymphocytes

1OO:l

Incubated in medium Incubated with 10 IF units/ml Incubated with 100 IF units/ml Incubated in medium Incubated with 10 IF units/ml Incubated with 100 IF units/ml

200:l

2 T

393 T

U-cells

Chang

15 35

14 23 41 43 51 55

10 31 15 34 55 70

12 35 46 31 48 65

33 17 49 40

' The lymphocytes were pretreated by incubation with 10 and 100 IF unitsiml for 24 h.

cells. The IF alone reduced the number of target cells by only about 10%. The experiments were repeated using three other cell lines as targets. All three types of target cell were reduced to a greater extent by the IF-treated lymphocytes than by the control lymphocytes (Table 111). Incubation of lymphoid cells for 24 h in medium containing as little as 0.001 units of IF per ml increased their capacity for reducing the

O ' 0I

A

loo

F

w LL

m

z z 3

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u

+

E

Y -

O

100-

0 01

10

1000

O

0 001 10 100 10,000

rY

D

--I

____-___-___-_

0- 4 0001 01

10

0-2 1000 0

0001 01

10 1000

INTERFERON CONCENTRATION ( U N I T S I M L I

FIGURE 2 - Reduction in the number of 393 T target cells by allogeneic lymphocytes treated for 24 h with various concentrations of IF. Three 1ymphocyte:target cell ratios were used: 50:l (0-0), 1OO:l (O--O) and 200:l (A-A). The horizontal lines show the effects of adding IF without lymphocytes: 10 units/inl - - _ _ _ _ , 1000 units/rnl - - - - - - , 10,000 units/ml . Four separate experiments using different cell donors are presented. Symbols denote means calculated from triplicate determinations.

number of target cells (Fig. 2) in some experiments. Notwithstanding the large inter-test variability, it would seem that a further increase in the IF concentration usually resulted in only a moderate increase in the lymphocytes' activity. The time required for IF to activate the lymphocytes was also examined. The capacity of these cells to reduce the number of 393 T cells decreased steeply following incubation for 48 h (Fig. 3). The activity of the IF-treated lymphocytes was higher than for untreated cells for all times tested. Capacity of IF to activate lymphocytes at low temperature. To test whether the activation of lymphocytes by IF is dependent on cell metabolism, lymphocytes were incubated with IF for 24 h at either 37" C or 4" C and compared with the corresponding control lymphocytes for capacity to reduce the number of 393 T cells. The activity of lymphocytes incubated with IF at 37" C was higher than that of the corresponding control lymphocytes (Table IV). For the lymphocytes treated at 4" C there was, however, no such increase. The activity of the control lymphocytes was higher after incubation at 4" C than at 37" C. Capacity of IF to activate mitomycin-treated lymphocytes. Experiments were conducted to ascertain whether the manifestation of enhanced activity following IF treatment requires a stage of DNA synthesis in the lymphocytes. Such synthesis might occur either during incubation with IF or during the subsequent 48 h of incubation with the target cells. IF treatment increased the ability of both normal and mitomycin-treated lymphocytes to reduce the number of 393 T target cells (Table V). Cytotoxicity of IF-treated lymphocytes for labelled target cells With the above technique for measuring the activity of lymphocytes with respect to target cells it is impossible to distinguish between cytotoxicity and detachment of viable target cells. Experiments were therefore conducted to ascertain whether IFtreated lymphocytes are cytotoxic for 51Cr-labelled target cells and thus cause them to release the isotope. In two experiments lymphocytes were incubated for 1 h or 24 h at 37" C in medium with and without

409

EFFECT OF INTERFERON ON CYTOTOXICITY TABLE IV ACTIVATION OF CYTOTOXIC CELLS I N LYMPHOID CELL PREPARATIONS INCUBATED WITH 10 UNITS OF IF PER ML FOR 24 h AT 37" C A N D 4" C

'

Reduction in target cells (per cent)

Lymphocyte :target cell ratio

1oo:l

50:l

Experiment No. 1 Incubation in medium at 37" C Incubation with IF at 37" C Incubation in medium at 4" C Incubation with IF at 4" C Experiment No. 2 Incubation in medium at 37" C Incubation with IF at 37" C Incubation in medium at 4"C Incubation with IF at 4" C

12 34 42 39

32 51 48 51

22 (4) 41 (6) 32 (4) 31 ( 5 )

' 393 T target cells were used. The activation is expressed as the percentage reduction in the number of target cells. 1 Means from three. determinations. - a Means from six determinations. - 3 SE.

and in vivo. I F is capable of inhibiting the multiplication of both malignant and non-malignant cells in vitro (Paucker et al., 1962; Frayssinet et al., 1973). By injecting I F and administering IFinducing agents in animals it is possible to prevent or delay the development of chemically induced (Salerno et al., 1972; Gelboin and Levy, 1970) and virus-induced tumors (Sarma et al., 1969; Regelson and Foltyn, 1966; Atanasiu and Chany, 1960; Gresser et al., 1967). Moreover, the administration of TF or I F inducers to animals may inhibit growth of several types of transplanted tumor cells (Zeleznick and Bhuyan, 1969; Bart et al., 1973; Gresser et al., 1969, 1970). Attempts have recently been made to evaluate the anti-tumor activity of I F preparations in man (Strander, 1977). In 1971 a study of the effect of exogenous human leukocyte IF in patients with osteogenic sarcoma of the long bones was started at Radiumhemmet (Strander et al., 1977).

-

100 units of IF per ml. The cells were then examined for their cytotoxicity toward 51Cr-labelled Chang cells during incubation for 4 h. At all lymphocyte: target ratios tested, the IF-treated lymphocytes exerted a stronger cytotoxic action on the target cells than did the corresponding control lymphocytes. Interferon alone did not damage the target cells (Fig. 4). An experiment was also conducted to determine whether the cytotoxicity of lymphocytes incubated with target cells increased in the presence of IF. The activity of the lymphocytes was considerably greater in the presence of 100 units of IF per ml. IF alone had no effect on the viability of the target cells (Table VI). DISCUSSION

Apart from its anti-viral activity, I F has been shown to exert an anti-tumor effect both in vitro

L, 1 0 [L W

TIME OF INCUBATION

12

24

40

OF LYMPHOCYTES (HOURSI

FIGURE 3 - Reduction in the number of 393 T target cells by allogeneic lymphocytes incubated for various periods at 37" C with or without various concentrations of IF. Lymphocyte:target cell ratio 50:l (left) and 1OO:l (right). Medium without IF, 0-0; 10 units/ml, 0-0; 100 units/ml, A-A. Symbols denote means calculated from triplicate determinations.

TABLE V ACTIVATION OF CYTOTOXIC CELLS IN UNTREATED A N D MITOMYCIN-TREATED LYMPHOID CELL PREPARATIONS INCUBATED WITH 10 UNITS OF IF PER ML FOR 24 h AT 37' C ' Reduction in number of target cells (per cent) Mean

SE

Untreated lymphocytes incubated without IF 9.3 Untreated lymphocytes incubated with IF 20.3 Mitomycin-treated lymphocytes incubated

8.9 5.5

5.8

6.2

without IF

Mitomycin-treated lymphocytes incubated with IF 26.5

2.8 ~

The lymphoid cells were cultured with 393 T target cells, at a ratio of 50:l. Percentage reduction in the number of target cells is shown. - a Means from six determinations.

Theoretically, there are several ways in which IF might inhibit tumor growth in vivo. One possibility is that the proliferation of tumor cells is directly inhibited, another that I F enhances the immunological host response to the tumor. The latter possibility finds support in the observation that IF increases the specific cytotoxic activity of allosensitized murine spleen cells (Lindahl et al., 1972) and enhances the generation of specific killer cells in mixed lymphocytes cultures (Heron et al., 1976). It has also been reported that unsensitized mouse lymphocytes display an increased capacity for lysing allogeneic target cells in vitro in the presence of preparations containg I F (Slavina et al., 1974; Svet-Mo!davsky and Chernyakhovskaya, 1967). These investigators showed that pre-treatment of lymphocytes with I F did not increase their cytotoxic activity, whereas such pre-treatment of the target cells rendered them more susceptible to lysis by

410

EINHORN ET AL.

lymphocytes. Lindahl et al., too, found that treatment of normal mouse lymphocytes with IF preparations did not increase their spontaneous killer activity (Lindahl et al., 1972). It has also been demonstrated that injection of allogeneic lymphocytes in mice raises the serum level of IF and that the nonspecific killer activity of the host's lymphocytes coincides with the peak level of interferon in the serum. It was suggested that the lymphocytes continue to release interferon in vitro and that this renders the target cells more susceptible to lysis by lymphocytes (Svet-Moldavsky et a/., 1973).

25:l

50.1 1OO:l 200.1

50.1 100.1 200 1

LYMPHOCYTE-TARGET CELL RATIO FIGURE 4 - Isotope release by slCr-labelled Chang cells incubated with allogeneic lymphocytes pre-incubated for I h (left) and 24 h (right) in medium alone x -- x , and in medium containing 100 units of IF/ml o--o. The horizontal broken lines show the effect of incubating the target cells in medium containing 100 units of IF/ml. lsotope release is expressed as a cytotoxic

index.

The purpose of this investigation was to examine whether the IF preparation used in the treatment of cancer patients can enhance the capacity of periph-

eral lymphoid cells from healthy donors to damage tumor target cells in vitro. Lymphocytes incubated with IF consistently exhibited a greater cytotoxic capacity for allogeneic target cells than did lymphocytes cultured for the same time without IF. Although the IF preparations used in this study are purified (6 x lo6 unitslmg of protein) it cannot be decided whether the effect was due to IF or to some other constituent of the preparation. However, preliminary experiments have shown that the substance activating the lymphocytes is still present in more highly purified preparations but not in " mock " preparations, and that it has the physiochemical properties of I F (unpublished observations). The observations presented in this article are in agreement with recent results obtained by Oehler et al. (1978) using 1F inducers in mice and rats and by Trinchieri and Santoli (1978) in man. The IF concentration required to increase the non-specific cytotoxic activity of lymphocytes may be very low. The results presented in Figure 2 indicate that IF concentrations as low as O.OOO1 units per ml can have this effect. The test procedure employed may, however, measure not only the cytotoxic capacity toward target cells but also the capacity for their detachment. It is, however, of interest to note that the IF concentration seen in the serum after an IF injection (10-100 units per ml) is far above the IF concentration needed to augment the spontaneous cytotoxicity in vitro. A crucial question in this connection is whether the IF preparation actually activated cytotoxic cells or whether it prevented the rapid decline of killer activity occurring during incubation in medium at 37" C (see Fig. 3). Admittedly, the results do not permit definitive conclusions, but the observed increase in the killer activity of lymphocytes pretreated for only 1 h with IF and of lymphocytes incubated with target cells in the presence of IF strongly indicates that IF not only preserves but also enhances the cytotoxic activity of the cells. The effect of IF therapy on the spontaneous killer activity of the patient's lymphocytes is being examined. The mechanism by which I F preparations increase cytotoxicity is not known. However, it is evident from the study reported here that active cell metab-

TABLE V I

CYTOTOXICITY OF LYMPHOID CELLS FOR 51Cr-LABELLEDCHANG CELLS Lymphocyte: target cell ratio

Pretreatment of lymphocytes

Incubation mixture

50: I

Incubated in medium Incubated in medium lncubated with IF Incubated in medium Incubated in medium Incubated with IF

Lymphocytes +target cells Lymphocytes+ target cells+IF Lymphocytes target cells Lymphocytes +target cells Lymphocytes+target cellsi IF Lymphocytes+ target cells Target cells+IF

1OO:l

+

I

Cytotoxic index

0.07 0.29 0.31 0.16 0.90 0.51 -0.03

-

The lymphoid cells were pre-incubated in medium or with 100 I F units per ml for I h and then incubated with Chany cells in the presence and absence of 100 I F units per ml.

EFFECT OF INTERFERON ON CYTOTOXICITY

o h m is required, since there was no sign of an increase in activity when the lymphocytes were incubated with IF at low temperature. Moreover, our results have shown that DNA synthesis of the lymphocytes is not necessary for the manifestation of augmented activity. It is not known whether IF exerts its effect by increasing the number of cytotoxic cells or if it acts by enhancing the activity of already existing cytotoxic cells. A possible way to answer this question is to study whether the proportion of cells exposing different surface markers is altered by IF treatment. Which cell type is activated by the I F preparation in this system is not clear. However, we have found that T-cell preparations separated by a rosette sedimentation technique are not activated to a major extent by IF (to be published). It is conceivable that the anti-tumor activity of IF is due partly to a direct growth-inhibiting effect of the IF and partly to activation of certain lymphocytes capable of killing the tumor cells. Support for an activation of lymphocytes is provided by

41 1

the observation that a sub-line of Li2IO cells resistant to I F in v i m was inhibited when animals inoculated with these cells were given exogenous IF (Gresser et al., 1972, 1974). This strongly suggests that I F not only exerts a direct effect on the tumor cells but also acts indirectly via immunological mechanisms of the tumor-bearing host. In this context it is open to question whether the activation of non-specific killer cells induced by IF is of any significance for the inhibition of tumor growth in vivu. ACKNOWLEDGEMENTS

We wish to thank Dr. Kari Cantell for a generous supply of IF. The technical assistance of Mrs. Waltraut Szczerba is gratefully acknowledged, We also wish to thank Dr. Jan Ponten and Dr. Georg Klein for providing some of the cell lines used in this study. This investigation was supported by a grant from the Cancer Society of Stockholm, Sweden.

INTERFERON ET CYTOTOXICITE SPONTANEE CHEZ L’HOMME. I. AUGMENTATION DE LA CYTOTOXICITE SPONTANEE DES LYMPHOCYTES PERlPHfiRIQUES SOUS L’EFFET DE L’INTERFERON LEUCOCYTAIRE HU MAIN Une preparation purifike d’interfkron leucocytaire humain utiliske dans notre hapita1 pour le traitemmt des affections malignes a dt6 testte du point de vue de sa capacitt de modifier la cytotoxicite spontanbe des lymphocytes peripheriques de donneurs sains. L’effet inhibiteur des lymphocytes allogkniques sur I’incorporation de thymidine tritiBe d’une lignee lymphoblastolde, Raji, a ete intensifit par la presence d’interftron ou par pretraitement des lymphocytes avec l’interferon. Cette forme de pretraitement a aussi accru la capacite des lymphocytes de rtduire le nombre de cellules tumorales adherentes dans un micro-essai. De plus, les lymphocytes trait& A I’interfkron avaient une cytotoxicite accrue envers les cellules cibles lorsqu’ils Ctaient incubes avec ces cellules marquees au Crl‘.

REFERENCES

FINTER,N. B., Interferon and interferon inducers. NorthADAMS,A., STRANDER. H., and CANTELL, K., Sensitivity of Holland Publ. Co., Amsterdani (1973). the Epstein-Barr virus transformed human lymphoid cell lines to interferon. J. gen. Virol., 28, 207-217 (1975). I., TOVEY,M., and LINDAHL, P., FRAYSSINEI., C., GRESSER, ATANASIU, P., and CHANY,C., Action d’un interfeton Letter: Inhibitory effect of potent interferon preparations provenant de cellules malignes sur I’infection expkrimentale on the regeneration of mouse liver after partial hepatectomy. du hamster nouveau-n6 par ie virus du polyome. C.R. Acad. Nature (Lond.), 245, 146-147 (1973). Sci. (Paris), 251. 1687-1689 (1960). H. V., and LEVY,H. B., Polyinosinic-polycytidylic GELBOIN, J. T.,and LAM,S., Role BART,R. S., KOPF,A. W., VILCEK, acid inhibits chemically induced tumorigenesis in mouse skin. of interferon in the anti-mzlanoma effects of poly (I), poly (C) Science, 167, 205-207 (1970). and Newcastle disease virus. Nature New Biol., 245, 229-230 GISLER,R. H., LINDAHL,P., and GRESSER,I., Effects of (1973). interferon on antibody synthesis in vitro. J. Immunol., 113, K.. Effect of BLOMGREN, H., STRANDER, H., and CANTELL, 438-444 (1974). human leukocyte interferon on the response of lymphocytes to mitogeneic stimuli in vitro. Scand. J . Immunol., 3, 697-705 GRESSER, I., RANDIJ,M. R., and BROUTY-BOY& D., Inter(1974). feron and cell division. IX.Interferon resistant L1210 cells: K., HIRVONEN, S., MOGENSEN, K. E., and PvHALA, Characteristics and origin. J. nut. Canrer Inst., 52, 5.53-559 CANTELL, ( I 974). L. Human leukocyte interferon: production, purification, stability and animal experiments. In: C. Waymouth (ed.), I., BOURALI,C., CHOUROULINKOV, I., FONTAINEGRESSER, The production and use of interferon f o r the treatment and BROUTY-BOYD,D., and THOMAS,M. R., Treatment of prevention of human virris injectious in vitro, p. 35-38, Culture neoplasia in mice with interferon pieparations. Ann. N. Y. Association Workshop, Tiswe Culture Association, RockAcad., Sci., 173, 694-707 (1970). ville (1974). DEMAEYER, E., DE MAEYER-GUIGNARD. J., and VANDEPUTTE, GRESSER,I., BOURALI,C., Ldvu, J. P., FONTAINE-BROUTYBoyd, D., and THOMAS, M. R., Increased survival in mice M., Inhibition by interferon of delayed-type hypersensitivity in the mouse. Proc. nat. Acad. Sci. (Wash.), 72, 1753-1757 inoculated with tumor cells and treated with intecferon preparations. Proc. nut. Acad. Sci. (Wash.), 63, 51-57 (1975). (1969). EPSTEIN,B., The ability of macrophages to augment in vitro GRESSER,I., COPPEY,J., FONTAINE-BROUTY-BOY& D., mitogen and antigen stimulated production of interferon FALCOFZ,R., and FALCOFF,E., Interferon and murine and other meaiators of cellular immunity by lymphocytes. 111. Efficacy of interferon preparations adminleukaemia. In: David S . Nelson (ed.), Immunobiology of the macrophage, istered after inoculation of Friend virus. Nature (Lond.), p. 201-233, Academic Press, New York, San Francisco, 215, 174-175 (1967). London (1976).

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GRESSLR, I., MAURY,C., and BROUTY-BOY& D., Mechanism of the antiturnour effect of interfelon in mice. Nature ( L o n d . ) , 239, 167-168 (1972). HERON,J., BERG,K., and CANTELL,K., Regulatory effect of interferon on T cells in vitro. J. Immunol., 117, 1370-1373 (1976). ISAACS, A., and LINDENMANN, J., Virus interference. I. The interferon. Proc. roy. Soc. B., 147, 258-267 (1957). JONDAL,M., HOLM,G., and W~CZELL, H., Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells. J . exp. Med., 136, 207-215 (1972). LILLIEHOOK,B.. and BLOMGREN, H., Strong stimula~ionof CBA lymphocytes in the mixed lymphocyte interaction with cells from the H-2 identical strain C3H. Scand. J. Immunol., 3, 627-635 (1974). LINDAHL, P., LEARY,P.,and GRESSER, I., Enhancement by interferon of the specific cytotoxicity of sensitized lymphocytes. Proc. nut. Acad. Sci. (Wash.), 69, 721-725 (1972). LINDAHL-MAGNUSSON. P., LEARY,P., and GRESSER,I., Interferon inhibits DNA synthesis induced in mouse lymphocyte suspensions by phytohaemsgglutinin or by allogmeic cells. Nature New Biol., 273, 120-121 (1972). MOBRAATEN, L. E., Dt MAEYER,E., and DE MAEYERGUIGNARD, J., Prolongation of allograft survival in mice by inducers of interferon. Tranrplatztafion. 16, 415-420 (1973). OEHLER, J . R., LINDSAY, L. R., NUNN,M. E., HDLDEN, H. T., R. B., Natural cell-m:diated cytoroxicity and HERBERMANN, in rats. 11. In vivo augmentation of NK-cell activity. Inr. J. Cancer, 21, 210-220 (1978). PAUCKER, K., CANTELL, K., and HENLF,W., Quantitative studies on vital interference i n suspended L cells. 111. Effect of interfering viruses and interferon on the growth rate of cells. Virology, 17, 324-334 (1962). POHJANPELTO, P., Two different thermostable variants of poiio\irus. Virology, 15, 231-236 (1961). P O N T ~ NJ.,, Neoplastic human glia cells in culture. In: J. Fogh (ed.), Human tumour cells in vitro, p. 175-206, Plenum Press, New York and London (1975). PONTBN,J., and SAKSELA, E., Two established in virro cell lines from human mesenchymal tumouis. lilt. J. Cancer, 2, 434-447 (1967). PULVERTAFT, J. V., A study of malignant turnour$ in Nigeria by short-term tissuc culture. J. elin. Path.. 18, 261-273 (1965). REGELSON, W., and FOLTYN,0.. Prevention and treatment uf Friend leukemia virus (FLV) infection by polyanions and

phytohaeniagglutinin. Proc. Amer. Ass. Cancer Res., 7, 58 (1966). SALERNO.R. A., WHITMIRE,C. E., GARCIA,I. M., and R. J., Chemical carcinogenesis in mice inhibited HUEBNER, by inteifecon. Nature New Biol., 239, 31-32 (1972). SARMA, P. S., SHIU,G., NEUBAUER, R. H.,BARON,S.. and HUEBNER, R. J., Virus-induced sarcoma of mice: inhibition by a synthetic polyribonucleotide complex. Proc. nat. Acad. Sci. (Wash.), 62, 1046-1051 (1969). SLAVINA, E. G., LENEVA, N. V., and SVET-MOLDAVSKY, G. J., Sensitivity of target cells to the cytotoxic effects of lymphocytes induced by interferon. Folia Biol. (Praha), 20, 231-237 (1 974). STRANDER, H.,Interfei on: anti-neoplastic drugs? Blut, 35, 277-288 (1977). STRANDER, H., and CANTELL,K., Production of interferon by human leukocytes in vitro. Ann. med. exp. Fenn., 44. 265-273 (1966). STRANDFR, H.,CANTELL, K.. INGIMARSSON, S., JAKOBSSON, U., and SODEREERG, G., Interferon treatP. A., NILSONNE, ment of osteogenic sarcoma-a clinical tiial. Conference on: Modulation of host immune resistance in the prevention or rreatment OF induced neoplasias, Dee. 9-11, 1974, Fogarty Int. Center Proc., US Government Printing Office, Washington D.C., No. 28, 377-381 (1977). SVET-MOLDAVSKY. G. J., and CHERNYAKHOVSKAYA, 1.. Interferon and interaction of allogeneic not ma1 and immune lymphocytes with L-cells. Nature (Lond.), 215, 1299-1300 (1967). SVET-MOLDAVSKY, G. J., NEMIROVSKAYA, B. M., OSIPOVA, T. V., SLAVIUA, E. G., ZINZAR,S. N., KARMONOVA, N. V., and M ~ R O Z O VL. A , F., Interferonogenicity of antigens and ‘‘ early cytotoxicity of lymphocytes. Folia Biol. (Prahn). 20, 255-230 (1973). TAKASUGI,M., and KLEIN, E., A microassay for cellmediated immunity. T,ansplantation, 9, 219-227 (1970). TRINCHIERI, G., and SANTOLI, D., Anti-viral aclivity induced by culturing lymphocytes with tumor-derived or virustransformed cells. Enhancement of human natural killer cell activity by interferon and anragonistic inhibition of susceptibility of target cells t o lysis. J . exp. Med., 147, 1314-1333 (1978). WAKSMAN, B. H.,and NAMBA,Y.,On soluble mediators of immunologic regulation. Cell. Immunol., 21, 161-176 (1976). ZELEZNICK, L. D., and BHUYAN, 8.K., Treatment ofleukemic (L1210) mice with double stranded polyribonucleotides. Proc. SOC.exp. Biol. ( N . Y . ) , 130, 126-128 (1969).