CELLULAR
IMMUNOLOGY
Enhancement ANNA
SENIK*,
44, 186-200 (1979)
by Interferon ION
GRESSERt,
ANDERS *Lahorutoire d’lmmunologie Scientifiques sur le Cancer, Uppsulu University,
of Natural
MAuRY,t
CHANTAL
ORN,S
AND
Killer Cell Activity
HANS
MAGNUS
in Mice
GIDLUND,$
WIGZELLS
Celluluire el TLuboratoire d’oncologie Virule, Institut de Recherches B.P. 8, 94800 Villejuif, France, and $Department of Immunology, Biomedical Center, Box 582, S-751, 23 Uppsala, Sweden Received
September
25, 1978
Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for 5’chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as IO3to IO4units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic (nuinu) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro. Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity ofthese cells.
INTRODUCTION It is still a matter of some debate whether the mammalian host possesses an immune surveillance system against neoplastic growth. Until recently this concept had been viewed primarily in terms of thymus (T)-dependent cells as the effecters of cell-mediated anti-tumor responses, especially in virus-induced tumors. However, with time only little evidence was obtained to support the notion that such cells are important in limiting the growth of chemically or spontaneously occurring tumors not expressing surface viral antigens. Moreover, recent studies have demonstrated that athymic (nulnu) nude mice display a very low incidence of spontaneous tumors (1); that they resist some lymphoid tumor grafts better than heterozygote (au/+) 186 OOOS-8749/79/050186-15$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
INTERFERON
ENHANCES
NK
CELL
ACTIVITY
187
mice (2); and that mice having undergone thymectomy, lethal irradiation, and bone-marrow reconstitution resist small tumor inocula, as well, if not better, than untreated mice (3). Findings such as these point toward the operation of an antitumor surveillance mechanism mediated by non-T-dependant noninduced killer” (NK) cells may well represent the principal cytotoxic cells. “Natural cellular component of this defense. Discovered in the mouse (4,5), and in man (6,7) then in the rat (8), NK cells were able to lysein vitro avariety of tumor cells. Studies in mice (9, 10) have characterized these cells as small lymphocytes devoid of adherent properties and lacking the cell surface markers of mature T and B lymphocytes. Able to differentiate into cytotoxic cells from bone-marrow precursors without thymic influence (1 l), NK cells are under polygenic control involving participation of genes of the major histocompatibility complex H-2 (4, 12). Inbred strains of mice have thus been classified as “low” or “high” on the basis of their NK cell activity in vitro. A correlation has been found between the in vivo resistance to the YAC transplantable lymphoma and the level of natural cytotoxicity displayed in vitro against this tumor (12, 13). Although little is known about the mechanism which induces NK-cell cytotoxicity, the following results are germane to the present study. (i) Consistant elevation in levels of NK cell activity have been induced by injecting mice with Corynebacterium parvum, tumor cells, or viruses (14, 15). (ii) Increase of NK-cell-mediated cytotoxicity has been observed in rats injected with double-stranded synthetic polynucleotides (16). It is noteworthy that while viruses (17) and double-stranded polynucleotides (18) are potent interferon inducers, interferon production can also be induced by C. parvum in murine spleen cell cultures (19) and it has recently been shown that certain tumor cell lines on interaction with human lymphocytes are also able to induce in vitro high levels of interferon (20). Furthermore, both interferon and interferon inducers (21-24) exert marked antitumor effects in mice. This effect probably results from direct inhibition of tumor cell multiplication by interferon (23, 25), but the in vivo growth of tumor cells resistant to interferon can also be inhibited in interferon-treated mice (26). It therefore seemed likely that interferon was activating or enhancing some host mechanism involved in tumor cell rejection. Taking into account (i) the enhancement of NK-cell activity by interferon inducers, (ii) the antitumor effect displayed in vivo by interferon and interferon inducers, we have examined the hypothesis that interferon was the responsible factor of enhancement of NK-cell activity. Experiments designed to test this hypothesis show that interferon itself is in fact a potent inducer in vivo and in vitro of splenic cell cytotoxicity for a variety of syngeneic, allogeneic, and xenogeneic target cells when assayed in vitro. The distinctive characteristics of these cytotoxic cells are those associated with NK cells. MATERIALS
AND METHODS
Mice
Fifteen-day- and 2-month-old Balb/c mice were obtained from a specific pathogen-free colony at the Institut du Cancer (Villejuif). Athymic nude Balb/c (H-2d), C3H/He (H-2k), and C57B1/6 (H-2b) mice were obtained from the Bomholgkd laboratories (Denmark) and from the breeding center of the C.N.R.S.
188
SENIK
at Orleans (France). (Uppsala, Sweden).
CBA/H
ET AL.
mice came from the department
of Immunology
Target Cells Characteristics
Cell K-Balb (H-2d) C3H/He (H-2k) L1210 (H-2d) LSTRA (H-2d) YAC (H-2”) MPC 11 (H-2d) P815 (H-2d) K562
Type culture
Kirsten-Moloney sarcoma virus (MSV) transformed nonproducer cell line (established by Dr. S. A. Aaronson) Embryo fibroblast cell line (established by Dr. C. A. Reznikoff) Methylcholanthrene-induced lymphoma
Monolayer
Moloney
virus-induced
lymphoma
Suspension
Moloney
virus-induced
lymphoma
Suspension
Monolayer Suspension
Balb/c myeloma
Suspension
DBA/2 mastocytoma
Suspension
Human
Suspension
myeloid
leukemia
Cells were cultivated in RPM1 1640 medium (Eurobio) containing 10% fetal calf serum (Eurobio), 100 IU/ml penicillin, and 100 pglml streptomycin. Phytohemagglutinin (PHA)-induced lymphoblasts were obtained by cultivating 25 x lo6 mouse spleen cells for 72 hr in Corning 25cm2 flasks containing 20 ml of culture medium supplemented with 5 x 10e5 M 2M-E and 1 puglml PHA (Wellcome Laboratories, England). Preparations
Used to Enhance NK Cell Activity
Interferon. Mouse interferon was prepared from Swiss mouse C-243 cells induced with Newcastle Disease virus (NDV), concentrated, partially purified, as previously described (27). In the control preparation (mock interferon) NDV was either omitted or added for 1 hr immediately before collection of the culture supernatant. All interferon and control preparations were dialyzed further for 24 hr at 4°C against 0.01 M perchloric acid before testing for toxicity on a line of L1210 cells resistant to interferon (28). The specific activity of such mouse C-243 cell interferon preparations was in the range of 2.8 x lo6 to 1.7 x IO’ reference unitslmg of protein. Partially purified human interferon (specific activity 3.5 x IO5 units/mg of protein) prepared in leukocyte suspensions inoculated with SendaI virus was a gift of Dr. K. Cantell. Mouse interferon units referred to in this report equal 4 mouse interferon reference units. Mice were injected intraperitoneally (ip) with 0.2 ml of interferon or control preparation. Interferon inducers. Polyinosinic-polycytidylic acid (poly 1:C) was obtained from C. F. Boehringer and Sohne GMBH (Mannheim, Germany). Mice were injected intravenously (iv) with 100 pg of poly 1:C. Tilorone was provided by Dr.
INTERFERON
ENHANCES
NK
CELL
ACTIVITY
189
G. D. Mayer of Merrell-National Laboratory, Cincinnati, Ohio, and was administrated per OS at a dose of 4 mg/mouse. Newcastle Disease virus (NDV) was propagated in the allantoic cavity of IO-day-old embryonated eggs; lo7 chick embryo fibroblast infectious dose,, in 0.2 ml was injected iv. Assay
of Interferon
Interferon preparations were assayed on mouse L cells in 0.2-ml volumes of Falcon microtest II plates, challenged with 100 TCID,, of vesicular stomatitis virus as previously described (29). The same techniques were used to determine serum or spleen interferon levels or the amount of interferon present in the nutrient medium from cultures of lymphocytes and target cells. Sheep Anti-mouse
Interferon
Globulin
The preparation, partial purification and assay of sheep anti-mouse interferon globulin and normal sheep globulin have been previously described (30). Anti-interferon globulin was diluted 1:3 in phosphate-buffered saline to give a neutralizing titer of 6 x 1O-5 against 8 units of mouse interferon induced by Newcastle Disease virus in mouse C-243 cells. The neutralizing titer of this globulin against serum interferons induced in mice by poly 1:C and tilorone was of similar magnitude (3 1). Mice were injected iv with 0.1 ml of anti-interferon or normal serum globulin (i.e., approximately 2 mg of sheep globulin/mouse). Spleen Cell Fractionation
Procedures
To remove adherent cells and B lymphocytes. Spleen cell suspensions were passed through nylon wool columns according to the method of Julius et al. (32). The cell yield after column passage was approximately 30% and the filtered spleen cells contained 4% Ig-bearing cells, as determined by indirect immunofluorescence using fluorescein-labeled anti-mouse Ig antibodies, and 80% T cells as determined by susceptibility to anti-Thy 1.2 serum and complement. Spleen cells were also fractionated by passage through an anti-mouse Ig column as described by Wigzell et al. (33). In this technique cells are passed through a column of Degalan beads coated with mouse Ig (obtained by 40% ammonium sulfate precipitation) and an excess of rabbit anti-mouse Ig serum. Such anti-Ig column-passed cell populations are known to be depleted of adherent cells, of surface Ig-positive cells and of avid Fc-receptor-bearing cells. To remove T lymphocytes. The spleen cell population was incubated with anti-Thy 1.2 serum and complement. This serum was prepared in AKR mice by eight weekly ip injections of 10’ C3H/He thymocytes, and was absorbed twice on AKR thymus and spleen cells (1 x lo8 cells for 1 ml serum), the first absorption being performed at +4”C and the second at 37°C. When tested on Balbic thymocytes the anti-Thy 1.2 serum had a 50% cytotoxic titer of 1:200. It was used at a final dilution of 1:30 for 10’ spleen cells in 1 ml. Depletion of Fc-receptor-bearing cells. Rosettes were formed between Fc-receptor-bearing spleen cells (FcR+ cells) using sheep red blood cells (SRBC) coated with rabbit anti-Forssman IgG as previously described (34). These rosettes were removed by velocity sedimentation on Ficoll: 20-30 x IO6 spleen cells in 2 ml
190
SENIK
ET AL.
culture medium were layered on 2 ml Ficoll-Urovison (density 1.077) (lymphopreparation Eurobio) in 12 x 75mm plastic tubes. These tubes were centrifuged for 15 min at 1500 rpm, and the cells lying at the interface were recovered, washed twice, and again tested for their rosetting capacity. These steps were performed at +4”C. After these procedures, the remaining 10% of cells did not form rosettes with antibody-coated erythrocytes. In Vitro Treatment
of Spleen Cells with Interferon
Spleen cells (15 x 106) suspended in 1 ml of culture medium were incubated for 2 or 3 hr at 37°C with IO3 units of interferon. After two washings these cells were tested for cytotoxicity on YAC target cells. Cytotoxic
Assay
This assay was based on the technique described by Cerottini and Brunner (35). Target cells (10’) were suspended in serum-free medium and labeled with 100 &i of 51Cr for 1 hr, washed twice, and then adjusted to a concentration of 2 x lo5 cells/ml in culture medium. Target cells (0.1 ml) were dispensed into wells of 6-mm flat-bottomed Falcon TC 3040 microtest plates and mixed with 0.1 ml of spleen cell suspensions obtained from normal or treated mice at an effector target cell ratio varying from 100: 1 to 3: 1 in a total volume of 0.2 ml. Monolayers of K-Balb and C3H were trypsinized and seeded into microtest plates at concentrations of 5 x lo4 cells per well. Sixteen hours later they were labeled with 2 PCi of 51Crfor 2 hr and washed three times before addition of 0.2 ml effector cell suspensions. Prior to addition of effector cells to target cells, anti-mouse interferon globulin was added (titer 5 x 10-3) to the effector cell suspension and left in the culture wells. All these cultures were incubated for 4 hr at 37°C in an humidified atmosphere containing 5% CO, in air. At the end of this incubation period, 0.1 ml of the supernate was transferred into tubes and counted in a gamma counter (SAIP, Malakoff, France). Percentage specific 51Cr release was calculated according to the formula: E-S T-S
x 100,
where E is the 51Cr released from the target cells in the presence of spleen cells, S is the spontaneous release of 51Cr, and T is the maximum release of 51Cr upon addition of 2 N HCl. All determinations were made in triplicate. YAC tumor cells have been previously described as extremely susceptible target cells for natural spleen cell cytotoxicity (4). They were therefore used in most of our assays to examine the effect of interferon on spleen cell cytotoxicity. RESULTS 1. Administration of Interferon Inducers to Mice Causes an Interferon-Mediated Increase in Spleen Cell Cytotoxicity against NK Sensitive Tumor Cells
As previously shown, injection of mice with the interferon inducers, NDV, and tilorone markedly increased spleen cell cytotoxicity for NK-sensitive tumor target cells (36). This effect was abrogated by simultaneous injection of mice with potent sheep anti-mouse interferon globulin, suggesting that the increase in NK cell
INTERFERON
ENHANCES
NK CELL ACTIVITY
191
FIG. 1. Interferon dose-dependent increase of spleen ceII cytotoxicity against YAC tumor cells. Interferon units were injected ip 16 hr before assay. Effector cell:target cell ratios: 0, 1OO:l; 0, 33:l; A, 11:l.
activity was mediated by the production of endogenous interferon. More recently experiments have shown that the increased spleen cell cytotoxicity in mice inoculated with poly 1:C was also inhibited by injection of anti-interferon globulin. Thus, at a 100: 1 spleen cell:target cell ratio, the specific percentage lysis for control mice was -5.4%, for polyI:C-injected mice 17.6%, and for poly I:C-injected mice treated with anti-interferon globulin - 3.2%. 2. Administration of Mouse Interferon to Mice Increases Spleen Cell Cytotoxicity against NK-Sensitive Tumor Cells Znfluence of dose of interferon. Mice were injected with varying amounts of mouse interferon and their spleen cell cytotoxicity was determined 16 hr later. As illustrated in Fig. 1, a marked increase in spleen cell cytotoxic activity was observed in mice injected with lo4 to lo6 interferon units. The lower limit for induction of
TIME
AFTER
INTERFERON
INJECTION
FIG. 2. Kinetics of interferon-induced spleen cell cytotoxicity against YAC tumor cells. Mice were injected with 8 x 104interferon units ip. Effector celktarget cell ratios: 0, lOO:l, 0,33.1; A, 11:l; n , PBSinjected control mice at 100: 1 effector:target cell ratio.
192
SENIK ET AL.
enhanced cytolytic activity was approximately lo4 interferon units in one experiment and lo3 units in another experiment (data not shown). Kinetics of induction of cytotoxicity by interferon. Mice were given a single injection of 8 x lo5 mouse interferon units, sacrificed thereafter, and the level of spleen cell cytotoxicity determined. Lytic tests were performed in the presence of anti-interferon globulin to exclude the possibility of carry-over of injected interferon into the in vitro assay (see below under section 6). As illustrated in Fig. 2, significant cytotoxicity was observed as early as 1 hr after interferon injection and had reached peak activity at 12 hr (24 hr was not tested in this experiment) followed by a progressive decline reaching background values around Day 4. A second injection of the same dose of interferon at Day 6 after primary interferon administration caused an enhancement of spleen cell cytotoxicity with similar kinetics and magnitude as those induced by the first interferon injection (data not shown). Thus no “memory” either in a positive or negative sense, was discerned using such time intervals between repeated doses of interferon. Species specificity of interferon. Interferon displays a certain species specificity. Thus, human leucocyte interferon exerts very little antiviral effect on murine cells (37). Likewise as shown in Table 1 no significant increase in spleen cell cytotoxicity was observed after injection of mice with either human leucocyte or mock mouse interferon preparations. Age of mouse. Both exogenous interferon (Fig. 3A) and NDV (Fig. 3B) enhanced spleen cell cytotoxicity in mice of different ages, but the level attained in adult mice was considerably higher than that attained in 15day-old mice. 3. Incubation of Normal Mouse Spleen Cells with Interferon against YAC Target Cells
Increases Cytotoxicitj
The rapid response to the injection of interferon in vivo (i.e., an increase in spleen cell cytotoxicity at 1 hr) suggested that interferon might enhance spleen cell cytotoxicity by a direct effect on the effector cells themselves. This possibility was examined by determining the time of contact between interferon and normal spleen TABLE
1
Comparison of Homologous and Heterologous Interferons in Induction of Mouse Spleen Cell Cytotoxicity”r* Mice injected with Mouse IF
Human IF
26.2 2 1.9* 19.3 2 1.6* 20.6 ? 2.1*
10.9 k 1.3 9.9 z!z 1.1 NS’ 9.9 + 1.6
Control preparation (mock interferon) 4.4 k 1.4 5.2 2 0.9 10.0 2 1.5
Not injected 4.0 2 0.1 6.0 k 0.3
a Mice were injected with 8 x loj mouse interferon units or 8 x lo5 human leucocyte interferon units 16 hr prior to sacrifice. h Values are percentages of specific Wr release and represent individual mice. Effector: YAC target cell ratio 100: 1. c Not significant compared to control preparation injected mice. * P < 0.001.
INTERFERON
ENHANCES
NK CELL ACTIVITY
193
60
50
40 % 2 i 30 5 d 2 20 Y ;
:w 10 0
2
6
24
2
10
AGE OF MICE (IN WEEKS)
FIG. 3. Interferon-induced spleen cell cytotoxicity as a function of age. Mice were injected 16 hr prior to test. Experiment A: n Spleen cell of mice injected with 8 x lo5 units of interferon; H spleen cells of PBS-injected mice. Experiment B: q spleen cells of mice inoculated with 10’ TCIDsO NDV; Cl spleen cells of untreated mice. Effector:target cell ratio was 100: 1, the standard error did not exceed 2. Each bar is the mean value of triplicate determinations and corresponds to an individual mouse.
cells required to trigger in vitro the cytolytic mechanism. CBA/H spleen cells were incubated for 4 hr with lo3 units/ml of interferon. Anti-interferon antibodies were added at various times during this incubation period to prevent further contact of free interferon with the spleen cells (Fig. 4). When tested on NK-sensitive YAC cells, interferon-pretreated cells displayed a cytolytic activity which was proportional to the time of contact with interferon prior to the addition of anti-interferon globulin (Fig. 4) (The capacity of the anti-interferon antibodies to neutralize the effect of interferon is evident by the fact that when added at time 0 together with interferon there was no difference in the level of cytotoxicity between these treated spleen cells and untreated control spleen cells.) It should be noted that a significant increase in cytotoxicity was evident after 10 min of contact of spleen cells with interferon. Thus an essential step in the inductive process had taken place within this brief interval. 4. Evidence
That the Interferon-Znduced
Cytotoxicity
Is Mediated
by NK Cells
In vivo induced cytotoxic cells are not T lymphocytes. NK cells are present in the spleen of nude, T-lymphocyte-deficient mice. They are not lysed after incubation with anti-0 serum and complement under conditions that cause lysis of normal T cells. Interferon and interferon inducers increased splenic cell cytotoxicity in nude mice just as they did in normal mice (36) and the cytotoxicity of spleen cells induced in normal mice by NDV or tilorone was not abrogated by incubation with anti-Thy serum and complement (Table 2). In vivo induced cytotoxic cells are nonadherent ceils devoid of surface Zg and avid Fc receptors. Fractionation of in vivo induced cytotoxic cells was undertaken
194
SENIK ET AL.
0
5 TIME
10 (min.)
30 AFTER
120
60
INTERFERON
240
ADDITION
FIG. 4. Effect of addition at varying times of anti-interferon antibodies during the in vitro incubation of spleen cells with interferon. Anti-interferon antibodies added to cultures at times indicated in abscissa. 0, Spleen cells incubated with 1Oaunits of interferon/ml. 0, Untreated spleen cells. 0, Spleen cells incubated from time 0 with interferon and anti-interferon antibodies. A, Spleen cells incubated from time 0 with anti-interferon antibodies alone. Effector target cell ratio 100: 1.
using nylon fiber columns known to remove adherent cells and B lymphocytes (32) or by addition of IgG-coated erythrocytes to remove avid Fc-receptor-positive cells (34). As shown in Table 3 the induced cytolytic cells are not adherent, do not have detectable surface immunoglobulin or avid Fc receptors. They behave as NK cells in all respects (9, 10). Cytotoxic cells induced in vitro also have characteristics of NK cells. The afore mentioned cell fractionation procedures were also carried out on in vitro TABLE
2
Induced Cells Are Resistant to Treatment with Anti-Thy Serum and Complement Control lysis’ Expt. No.” 1 2
Inducing agent* NDV Tilorone
NMS + C -3.2
t
0.8
1.0 + 0.5
Anti-Thy
Induced lysis’ + C
Not done Not done
NMS+C 19.7 + 2.9 21.4 t 2.1
Anti-Thy + C 23.2 32.2
+ 1.3 + 1.7
(1Expt. 1 Balbk and Expt. 2 CBA/H mice. b Inducing agents were used at optimal concentrations and mice were sacrificed 1 day after administration. c Target cells in Expts. 1 and 2 are YAC (= H-2” Moloney lymphoma) cells. Effector:target cell ratio 50: 1. Conditions as described under Materials and Methods. Anti-Thy serum = AKR-anti-CBA or AKR-anti-C3H sera (= anti-Thy 1.2 sera with cytotoxic titers for thymocytes in presence of guinea pig complement more than 1:lOO) used at a concentration 1:lO. NK cells in normal spleen cells are resistant to anti-Thy serum treatment using the above procedure. NMS, normal mouse serum.
INTERFERON
ENHANCES TABLE
3
In Vivo Induced Cytolytic Cells are Nonadherent Expt. No.
Pretreatment”
195
NK CELL ACTIVITY
Cells Devoid of Avid Fc Receptors
Fractionation*
Percentage specific “‘Cr release
1
NDV NDV
None Nylon fiber column
24.8 f 6.0 24.1 t 3.4
2
None None Tilorone Tilorone
None Nylon fiber column None Nylon fiber
1.2 13.3 27.4 40.3
2 rt -t c
1.2 0.9 3.1 2.8
3
Tilorone Tilorone Interferon Interferon
None Ig-anti-Ig column None FcR + cell depletion
51.3 65.7 13.0 36.0
+ r 2 ”
2.1 0.9 0.7 1.8
NDV NDV None None
None FcR + cell depletion None FcR + cell depletion
34.9 91.0 5.2 28.7
+ ? + +
2.2 5.6 1.7 3.4
4 5
a Administration of 10’ EID,, of NDV or 1 mg of tilorone 1 day before assay. b Fractionation on nylon fiber or Ig-anti-Ig columns as described in Refs. (32) and (33). Parallel experiments as to surface markers and T versus B cell responsiveness to mitogens assured the functional ability of the columns (not shown).
interferon-induced spleen cells. As shown in Table 4 these procedures failed to reduce the level of cytotoxicity of the fractionated cells. Thus, cells with NK attributes were also responsible for the in vitro interferon-induced cytotoxicity against YAC cells. TABLE
4
Cell Surface Characteristics of the Cytolytic Cells Induced by Interferon in Vitro Percentage specific 51Cr release Effector:YAC target cell ratio Method of spleen cell fractionation
Treatment
100: 1
50: 1
25: 1
Interferon” Control*
30.3 2 1.5 14.0 & 1.8
22.4 + 2.3 9.5 k 0.5
9.7 k 0.7 5.9 rt 0
Interferon Control
38.1 k 1.5 24.8 k 0.5
29.3 + 0.6 19.2 k 0.5
14.2 r 1.6 8.9 + 0.1
Nylon wool column
Interferon Control
37.9 2 1.0 24.6 2 1.8
18.8 4 0.6 15.8 ? 1.6
13.0 + 1.3 4.0 t 1.2
Anti-Ig column
Interferon Control
43.5 2 2.2 32.7 t 0.9
32.9 r 1.2 21.1 k 2.0
21.3 + 1.4 9.7 k 1.3
None Anti-Thy
1.2 + complement
D Interferon (W units) was added for 2 hr to CBA/H spleen cells. b CBA/H control spleen cells.
196
SENIK ET AL.
Spec$city oflysis. Although the fine specificity of lytic ability of NK cells is still under debate there are a number of cell lines of defined susceptibility or resistance to lysis by such cells. The in vivo interferon-induced cytotoxic cells show the same patterns of cytolysis against four well-established NK target cells as do NK cells (Table 5). Thus like NK cells, the interferon-induced cytotoxic cells are neither H-2 nor even species restricted in their lytic activity. However, they do not lyse PHA-stimulated lymphoblasts (Table 5). In summary, the in vivo or in vitro interferon-induced cytolytic spleen cells display surface features, and specific cytolytic capcity similar to those of spontaneously occurring NK cells. 5. The Increase in the Level of Spleen Cell Cytotoxicity Activity Is Not Due to a Redistribution of NK Cells with a Preferential Localization of These Cells in the Spleen The increase in the spleen cell cytolytic activity induced by interferon may result from an increase in NK cell activity or a redistribution of NK cells throughout the body of the mouse with an accumulation of these cells in the spleen. We therefore tested the level of cytolytic activity of cells from a variety of organs from normal mice injected with an interferon inducer. Figure 5 shows that after inoculation of tilorone there is a general enhancement of NK-cell activity in the spleen, lymph nodes, peripheral blood, and bone marrow. In contrast, however, thymocytes exerted no cytotoxic activity before or after inoculation of tilorone. 6. Evidence That NK-Cell Activity in Vitro Incubation Conditions
Occurs in Vivo and Is Not the Result of the
It might be suggested that the interferon induced increased spleen cell cytotoxicity does not occur in vivo but develops only during the period of incubation of spleen cells from an interferon-treated mouse with the target cells in vitro. Several lines 01 experimental data argue against this possibility. TABLE
5
Cytolytic Cells Induced by Interferon and by Interferon Inducers Manifest the Same Target Cell Specificity Pattern as NK Cells” Targets Expt. No. 1
2
Effector cells CBA/H Controls Tilorone-treated
Balbinude Controls Interferon-treated
K562
YAC
MPC 11
P815
24.1 + 0.2 43.8 k 0.6
8.4 + 0.7 20.8 k 0.6
1.6 -c 0.1 9.5 2 1.0
K.Balb
C3H line
L 1210
DBA/2 PHA blasts
23.7 2 3.7 30.2 k 0.5
10.0 + 2.6 30.5 + 1.4
1.3 + 0.2 29.6 + 5.0
-14.7 t 1.5 -17.0 + 0.7
14.2 k 0.3 26.1 k 0.2
a Effector cells from spleens of mice untreated or treated with 8 x lo5 units of interferon or 1 mg 01 tilorone 1 day earlier. Effector:target cell ratio 50: 1. Mean + SE of individual spleens. Three or four mice per group.
INTERFERON
ENHANCES
197
NK CELL ACTIVITY Thymw
spleen
50
40 1
3w ;30 0 IL 0 L m SC c s l(
~
a
b
==w a
C
b
FIG. 5. Organ distribution of tilorone induced NK-cell activity. A, Cells from CBA mice harvested 1 day after injection of 2 mg of tilorone. 0, Cells from CBA control mice. a = 100: 1; b = 50: I; c = 25: 1; effector:YAC target cell ratios.
(A) Interferon was not detected in extracts of spleen cells from interferoninjected mice. The sensitivity of the method permitted detection of small amounts of interferon, lower than the amount of interferon necessary to increase splenic cell cytotoxicity in vitro. (B) Interferon could not be detected in the culture fluid during the 4-hr incubation period, indicating that lymphoid cells did not release nor produce interferon on contact with target cells. (C) Addition of anti-interferon antibodies to mixtures of target and in vivo induced effector cells did not modify the degree of spleen cell cytotoxicity for YAC target cells. The same concentration of anti-interferon immunoglobulins could be shown to block completely the in vitro induction by IO3 units of interferon of NK cells if added together with interferon at time 0 (see Fig. 4). DISCUSSION This investigation has shown that exogenous or endogenous interferon markedly increase NK cell activity both in viva and in vitro. Thus, each of the interferon
198
SENIK
ET AL.
inducers tested in viva, (NDV, poly I:C, tilorone) caused a rapid increase in spleen cell cytotoxicity activity; while administration of anti-interferon blocked this enhanced NK-cell activity, indicating that the phenomenon was mediated by the production of endogenous interferon. Furthermore, partially purified mouse interferon type I also increased NK-cell activity both in vivo and in vitro, whereas human leucocyte interferon or control preparations failed to exert any demonstrable effect. The evidence presented under section 6 under Results shows that this increase in spleen cell cytotoxicity occurred in vivo and did not occur during the incubation of effector cells with target cells in vitro. Since inoculation of an interferon inducer enhanced NK-cell cytotoxicity in various lymphoid tissues (Fig. 5), it seems likely that the increase in spleen cell cytotoxicity is part of a generally augmented cytotoxic response rather than a localization within the spleen of NK cells from other sites. The evidence that the induced cytolytic cells are in fact NK cells and not some other cell type stems from several independent observations. Previous investigators using similar interferon inducers found that the induced cells had the characteristics of “normal” NK cells (16). In the present study we showed that the induced cytolytic cells are neither T nor B lymphocytes. Furthermore, they were nonadherent and did not have avid Fc receptors as shown by experiments utilizing nylon fiber, anti-Ig columns, or the rosetting of FcR+ cells with IgG-coated erythrocytes. These are all features characteristic of murine NK cells (9, 10). Furthermore, the analysis of target cell susceptibility using the interferon-induced cells showed a parallelism between the behavior of induced cytolytic cells and normal NK cells. These results affirm those reported recently in an in vitro human system (38,39) and in a mouse system in vivo andin vitro, by other investigators and ourselves (36, 40). What is the mechanism of this enhancement of NK activity? The increase in spleen cell cytotoxicity was manifested within 1 hr after inoculation of mice with interferon (Fig. 2). The in vitro experiments (section 3 under Results) suggest that less than 10 min of contact of interferon with spleen cells suffices to initiate an enhanced cytotoxicity (Fig. 4). Since this enhancement occurs with such rapidity, cell division is unlikely to be required. The simplest explanation would be a direct effect of interferon on NK cells. As previously shown, interferon can enhance a number of specialized cell functions (41); this includes increased cytotoxicity of sensitized spleen cells for allogeneic target cells (42), increased expression of H-2 antigens on the surface of various tumor and normal cells (43-45). Findings such as these taken together with the present data suggest that interferon treatment of NK cells also leads to a modification of the cell surface, expressed as enhanced inherent cytolytic activity, in this case assayed on tumor target cells. It has been suggested that NK-cell activity in mice can also be modulated by the activity of suppressor cells (46,47). Although our results suggest that enhancement of NK-cell activity stems from a direct effect of interferon on NK cells, we can not rule out the possibility that interferon may also inhibit the activity of cells which have been reported to suppress NK-cell activity. Such an effect would also result in an increase in NK-cell activity. If, as has been suggested (12, 13), NK ceils are important in the natural resistance to tumors, it may be that some part of the antitumor effects of interferon and interferon inducers (23,48) is mediated by the enhancement of NK-cell activity. In
INTERFERON
ENHANCES
NK CELL ACTIVITY
199
our experiments, a second injection of interferon or interferon inducer 7 days after a primary injection (i.e., at a time when NK cell activity had returned to normal) induced a “normal” increase in NK-cell activity as judged by the kinetics and extent of the response. It would seem worthwhile to determine whether it is feasible to develop and maintain constant high levels of NK-cell activity in tumor susceptible mouse strains by repeated injection of interferon, or interferon inducers, as a defense against neoplastic growth. ACKNOWLEDGMENTS We are grateful to Dr. Wolf H. Fridman for many helpful discussions and to Mrs. Marie-The&e Bandu, Mrs. Josiane Buywid, and Mrs. Francoise Zambetti for skillful technical assistance. We are also grateful to Dr. Maurice Landy for reading the article and for his helpful suggestions. This work was aided by grants from I.N.S.E.R.M. (ATP 78-91, 44.77.79/13/16), D.R.E.T. (77/203), and Swedish Cancer Society, NIH Contract NOl-CB-64033.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Rygaard, J., and Povbsen, C. C., Transplant. Rev. 28, 43, 1976. Warner, N. L., Woodruff, M. F. A., and Burton, R. C., Int. J. Cancer 20, 146, 1977. Greenberg, A. H., and Greene, M., Nature (London) 264, 356, 1976. Kiessling, R. E., Klein, E., and Wigzell, H., Eur. J. Immunol. 5, 112, 1975. Herberman, R. B., Nunn, M. E., and Laurin, D. H., Int. J. Cancer 16, 216, 1975. Peter, H. H., Pavie-Fischer, J., Fridman, W. H., Aubert, C., Cesarini, J. P., Roubin, R., and Kourilsky, F. M., J. Immunol. 115, 539, 1975. West, W. H., Cannon, G. B., Kay, H. D., Bonnard, G. D., and Herberman, R. B.,J. Immunol. 118, 355, 1977. Oehler, J. R., Lindsay, L. R., Nunn, M. E., and Herberman, R. B., Int. J. Cancer 21,204, 1978. Kiessling, R. E., Klein, E., Pross, H., and Wigzell, H., Eur. J. Immunol. 5, 117, 1975. Herberman, R. B., Nunn, M. E., Holden, T. H., and Laurin, D. H., Int. J. Cancer 16, 230, 1975. Haller, O., Kiessling, R., Orn, A., and Wigzell, H., J. Exp. Med. 145, 1411, 1977. Petranyi, G. G., Kiessling, R., Povey, S., Klein, G., Heizenberg, L., and Wigzell, H., Immunogenetics 3, 15, 1976. Haller, O., Hansson, M., Kiessling, R., and Wigzell, H., Nature (London) 270, 609, 1977. Herberman, R. B., Nunn, M. E., Holden, H. T., Staal, S., and Djeu, J. Y., Int. J. Cancer 19, 555, 1977. Welsch, R. M., and Zinkernagel, R. M., Nature {London) 268, 646, 1977. Oehler, J. R., Lindsag, L. R., Nunn, M. E., Holden, H. T., and Herberman, R. B., Int. J. Cancer 21, 210,
1978.
17. Issacs, A., and Lindenmann, J., Proc. Roy. Sot. Ser.B 147, 258, 1957. 18. Field, A. K., Tytell, A. A., Lampson, G. P., and Hilleman, M. R. Proc. Nat. Acad. Sci. USA 58, 1004, 1967. 19. Kirchner, H., Hirt, H. M., Becker, H., and Munk, K., Cell. Immunol. 31, 172, 1977. 20. Trinchieri, G., Santoli, D., and Knowles, B. B., Nature (London) 270, 611, 1977. 21. Gresser, I., Bourali, C., Levy, J. P., Fontaine-Brouty-Boy&, D., and Thomas, M. T., Proc. Nat. Acad. Sci. USA 63, 51, 1969. 22. Gresser, I., and Bourali, C., Nature (London) 223, 844, 1969. 23. Gresser, I., In “Advances in Cancer Research” (G. Klein and S. Weinhouse, eds.), pp. 97-141. Academic Press, New York/London, 1972. 24. Levy, H. B., Law, L. W., and Rabson, A. S., Proc. Nat. Acad. Sri. USA 62, 357, 1969. 25. Gresser, I., Brouty-BoyC, D., Thomas, M. T., Macieira-Coelho, A., Proc. Nat. Acad. Sci. USA 66, 1052, 1970. 26. Gresser, I., Maury, C., and Brouty-Boy& D., Nature (London) 239, 167, 1972. 27. Tovey, M. G., Begon-Lours, J., and Gresser, I., Proc. Sot. Exp. Biol. Med. 146, 809, 1974. 28. Gresser, I., Bandu, M. T., Brouty-Boy& D., J. Nat. Cancer Inst. 52, 553, 1974. 29. Gresser, I., Fontaine-Brouty-Boy& D., Bourali, C., and Thomas, M. T., Proc. Sot. Exp. Biol. Med. 130, 236, 1969.
200
SENIK ET AL.
30. Gresser, I., Tovey, M. G., Bandu, M. T., Maury, C., and Brouty-Boy& D.,J. Exp. Med. 144, 1305, 1976. 31. Gresser, I., Maury, C., Bandu, M. T., Tovey, M. G., and Maunoury, M. T.,Znr. J. Cancer 21,72, 1978. 32. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3, 645, 1973. 33. Wigzell, H., Stand. J. Immunol. Suppl. 5, 23, 1976. 34. Fridman, W. H., and Golstein, P., Cell. Immunol. 11, 442, 1974. 35. Cerottini, J. C., and Brunner, K. T., In “In Vitro Methods in Cell Mediated Immunity” (B. R. Bloom and P. R. Glade, Eds.), p. 369. Academic Press, New York, 1971. 36. Gidlund, M., Orn, A., Wigzell, H., Senik, A., and Gresser, I., Nature (London) 273, 759, 1978. 37. Levy-Koenig, R. E., Golgher, R. R., and Paucker, K., J. Immunol. 104, 791, 1970. 38. Trinchieri, G., Santoli, D., Dee, R. R., and Knowles, B. B., J. Exp. Med. 147, 1299, 1978. 39. Trinchieri, G., and Santoli, D., J. Exp. Med. 147, 1314, 1978. 40. Djeu, J. Y., Heinbaugh, J. A., Holden, H. T., and Herberman, R. B., J. Immunol. 122, 17.5, 1979. 41. Gresser, I., Cell. fmmunol. 34, 406, 1977. 42. Lindahl, P., Leary, P., and Gresser, I., Proc. Nut. Acad. Sci. USA 69, 721, 1972. 43. Lindahl, P., Leary, P., and Gresser, I., Proc. Nut. Acad. Sci. USA 70, 2785, 1973. 44. Lindahl, P., Leary, P., and Gresser, I., Eur. J. lmmunol. 4, 779, 1974. 45. Vignaux, F., and Gresser, I., Proc. Sot. Exp. Biol. Med. 157, 456, 1978. 46. Hochman, P. S., Cudkowicz, G., and Dausset, J., J. Nat. Cancer Inst. 61, 265, 1978. 47. Cudkowicz, G., and Hochman, P. S., “Developmental Immunobiology” (G. W. Siskind, Ed.). Grune & Stratton, New York, 1978. 48. Gresser, I.,ln “Cancer-A Comprehensive Treatise” (F. Becker, Ed.), pp. 521-571. Plenum, New York. 1977.
IMMUNOLOGY
Enhancement ANNA
SENIK*,
44, 186-200 (1979)
by Interferon ION
GRESSERt,
ANDERS *Lahorutoire d’lmmunologie Scientifiques sur le Cancer, Uppsulu University,
of Natural
MAuRY,t
CHANTAL
ORN,S
AND
Killer Cell Activity
HANS
MAGNUS
in Mice
GIDLUND,$
WIGZELLS
Celluluire el TLuboratoire d’oncologie Virule, Institut de Recherches B.P. 8, 94800 Villejuif, France, and $Department of Immunology, Biomedical Center, Box 582, S-751, 23 Uppsala, Sweden Received
September
25, 1978
Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for 5’chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as IO3to IO4units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic (nuinu) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro. Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity ofthese cells.
INTRODUCTION It is still a matter of some debate whether the mammalian host possesses an immune surveillance system against neoplastic growth. Until recently this concept had been viewed primarily in terms of thymus (T)-dependent cells as the effecters of cell-mediated anti-tumor responses, especially in virus-induced tumors. However, with time only little evidence was obtained to support the notion that such cells are important in limiting the growth of chemically or spontaneously occurring tumors not expressing surface viral antigens. Moreover, recent studies have demonstrated that athymic (nulnu) nude mice display a very low incidence of spontaneous tumors (1); that they resist some lymphoid tumor grafts better than heterozygote (au/+) 186 OOOS-8749/79/050186-15$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
INTERFERON
ENHANCES
NK
CELL
ACTIVITY
187
mice (2); and that mice having undergone thymectomy, lethal irradiation, and bone-marrow reconstitution resist small tumor inocula, as well, if not better, than untreated mice (3). Findings such as these point toward the operation of an antitumor surveillance mechanism mediated by non-T-dependant noninduced killer” (NK) cells may well represent the principal cytotoxic cells. “Natural cellular component of this defense. Discovered in the mouse (4,5), and in man (6,7) then in the rat (8), NK cells were able to lysein vitro avariety of tumor cells. Studies in mice (9, 10) have characterized these cells as small lymphocytes devoid of adherent properties and lacking the cell surface markers of mature T and B lymphocytes. Able to differentiate into cytotoxic cells from bone-marrow precursors without thymic influence (1 l), NK cells are under polygenic control involving participation of genes of the major histocompatibility complex H-2 (4, 12). Inbred strains of mice have thus been classified as “low” or “high” on the basis of their NK cell activity in vitro. A correlation has been found between the in vivo resistance to the YAC transplantable lymphoma and the level of natural cytotoxicity displayed in vitro against this tumor (12, 13). Although little is known about the mechanism which induces NK-cell cytotoxicity, the following results are germane to the present study. (i) Consistant elevation in levels of NK cell activity have been induced by injecting mice with Corynebacterium parvum, tumor cells, or viruses (14, 15). (ii) Increase of NK-cell-mediated cytotoxicity has been observed in rats injected with double-stranded synthetic polynucleotides (16). It is noteworthy that while viruses (17) and double-stranded polynucleotides (18) are potent interferon inducers, interferon production can also be induced by C. parvum in murine spleen cell cultures (19) and it has recently been shown that certain tumor cell lines on interaction with human lymphocytes are also able to induce in vitro high levels of interferon (20). Furthermore, both interferon and interferon inducers (21-24) exert marked antitumor effects in mice. This effect probably results from direct inhibition of tumor cell multiplication by interferon (23, 25), but the in vivo growth of tumor cells resistant to interferon can also be inhibited in interferon-treated mice (26). It therefore seemed likely that interferon was activating or enhancing some host mechanism involved in tumor cell rejection. Taking into account (i) the enhancement of NK-cell activity by interferon inducers, (ii) the antitumor effect displayed in vivo by interferon and interferon inducers, we have examined the hypothesis that interferon was the responsible factor of enhancement of NK-cell activity. Experiments designed to test this hypothesis show that interferon itself is in fact a potent inducer in vivo and in vitro of splenic cell cytotoxicity for a variety of syngeneic, allogeneic, and xenogeneic target cells when assayed in vitro. The distinctive characteristics of these cytotoxic cells are those associated with NK cells. MATERIALS
AND METHODS
Mice
Fifteen-day- and 2-month-old Balb/c mice were obtained from a specific pathogen-free colony at the Institut du Cancer (Villejuif). Athymic nude Balb/c (H-2d), C3H/He (H-2k), and C57B1/6 (H-2b) mice were obtained from the Bomholgkd laboratories (Denmark) and from the breeding center of the C.N.R.S.
188
SENIK
at Orleans (France). (Uppsala, Sweden).
CBA/H
ET AL.
mice came from the department
of Immunology
Target Cells Characteristics
Cell K-Balb (H-2d) C3H/He (H-2k) L1210 (H-2d) LSTRA (H-2d) YAC (H-2”) MPC 11 (H-2d) P815 (H-2d) K562
Type culture
Kirsten-Moloney sarcoma virus (MSV) transformed nonproducer cell line (established by Dr. S. A. Aaronson) Embryo fibroblast cell line (established by Dr. C. A. Reznikoff) Methylcholanthrene-induced lymphoma
Monolayer
Moloney
virus-induced
lymphoma
Suspension
Moloney
virus-induced
lymphoma
Suspension
Monolayer Suspension
Balb/c myeloma
Suspension
DBA/2 mastocytoma
Suspension
Human
Suspension
myeloid
leukemia
Cells were cultivated in RPM1 1640 medium (Eurobio) containing 10% fetal calf serum (Eurobio), 100 IU/ml penicillin, and 100 pglml streptomycin. Phytohemagglutinin (PHA)-induced lymphoblasts were obtained by cultivating 25 x lo6 mouse spleen cells for 72 hr in Corning 25cm2 flasks containing 20 ml of culture medium supplemented with 5 x 10e5 M 2M-E and 1 puglml PHA (Wellcome Laboratories, England). Preparations
Used to Enhance NK Cell Activity
Interferon. Mouse interferon was prepared from Swiss mouse C-243 cells induced with Newcastle Disease virus (NDV), concentrated, partially purified, as previously described (27). In the control preparation (mock interferon) NDV was either omitted or added for 1 hr immediately before collection of the culture supernatant. All interferon and control preparations were dialyzed further for 24 hr at 4°C against 0.01 M perchloric acid before testing for toxicity on a line of L1210 cells resistant to interferon (28). The specific activity of such mouse C-243 cell interferon preparations was in the range of 2.8 x lo6 to 1.7 x IO’ reference unitslmg of protein. Partially purified human interferon (specific activity 3.5 x IO5 units/mg of protein) prepared in leukocyte suspensions inoculated with SendaI virus was a gift of Dr. K. Cantell. Mouse interferon units referred to in this report equal 4 mouse interferon reference units. Mice were injected intraperitoneally (ip) with 0.2 ml of interferon or control preparation. Interferon inducers. Polyinosinic-polycytidylic acid (poly 1:C) was obtained from C. F. Boehringer and Sohne GMBH (Mannheim, Germany). Mice were injected intravenously (iv) with 100 pg of poly 1:C. Tilorone was provided by Dr.
INTERFERON
ENHANCES
NK
CELL
ACTIVITY
189
G. D. Mayer of Merrell-National Laboratory, Cincinnati, Ohio, and was administrated per OS at a dose of 4 mg/mouse. Newcastle Disease virus (NDV) was propagated in the allantoic cavity of IO-day-old embryonated eggs; lo7 chick embryo fibroblast infectious dose,, in 0.2 ml was injected iv. Assay
of Interferon
Interferon preparations were assayed on mouse L cells in 0.2-ml volumes of Falcon microtest II plates, challenged with 100 TCID,, of vesicular stomatitis virus as previously described (29). The same techniques were used to determine serum or spleen interferon levels or the amount of interferon present in the nutrient medium from cultures of lymphocytes and target cells. Sheep Anti-mouse
Interferon
Globulin
The preparation, partial purification and assay of sheep anti-mouse interferon globulin and normal sheep globulin have been previously described (30). Anti-interferon globulin was diluted 1:3 in phosphate-buffered saline to give a neutralizing titer of 6 x 1O-5 against 8 units of mouse interferon induced by Newcastle Disease virus in mouse C-243 cells. The neutralizing titer of this globulin against serum interferons induced in mice by poly 1:C and tilorone was of similar magnitude (3 1). Mice were injected iv with 0.1 ml of anti-interferon or normal serum globulin (i.e., approximately 2 mg of sheep globulin/mouse). Spleen Cell Fractionation
Procedures
To remove adherent cells and B lymphocytes. Spleen cell suspensions were passed through nylon wool columns according to the method of Julius et al. (32). The cell yield after column passage was approximately 30% and the filtered spleen cells contained 4% Ig-bearing cells, as determined by indirect immunofluorescence using fluorescein-labeled anti-mouse Ig antibodies, and 80% T cells as determined by susceptibility to anti-Thy 1.2 serum and complement. Spleen cells were also fractionated by passage through an anti-mouse Ig column as described by Wigzell et al. (33). In this technique cells are passed through a column of Degalan beads coated with mouse Ig (obtained by 40% ammonium sulfate precipitation) and an excess of rabbit anti-mouse Ig serum. Such anti-Ig column-passed cell populations are known to be depleted of adherent cells, of surface Ig-positive cells and of avid Fc-receptor-bearing cells. To remove T lymphocytes. The spleen cell population was incubated with anti-Thy 1.2 serum and complement. This serum was prepared in AKR mice by eight weekly ip injections of 10’ C3H/He thymocytes, and was absorbed twice on AKR thymus and spleen cells (1 x lo8 cells for 1 ml serum), the first absorption being performed at +4”C and the second at 37°C. When tested on Balbic thymocytes the anti-Thy 1.2 serum had a 50% cytotoxic titer of 1:200. It was used at a final dilution of 1:30 for 10’ spleen cells in 1 ml. Depletion of Fc-receptor-bearing cells. Rosettes were formed between Fc-receptor-bearing spleen cells (FcR+ cells) using sheep red blood cells (SRBC) coated with rabbit anti-Forssman IgG as previously described (34). These rosettes were removed by velocity sedimentation on Ficoll: 20-30 x IO6 spleen cells in 2 ml
190
SENIK
ET AL.
culture medium were layered on 2 ml Ficoll-Urovison (density 1.077) (lymphopreparation Eurobio) in 12 x 75mm plastic tubes. These tubes were centrifuged for 15 min at 1500 rpm, and the cells lying at the interface were recovered, washed twice, and again tested for their rosetting capacity. These steps were performed at +4”C. After these procedures, the remaining 10% of cells did not form rosettes with antibody-coated erythrocytes. In Vitro Treatment
of Spleen Cells with Interferon
Spleen cells (15 x 106) suspended in 1 ml of culture medium were incubated for 2 or 3 hr at 37°C with IO3 units of interferon. After two washings these cells were tested for cytotoxicity on YAC target cells. Cytotoxic
Assay
This assay was based on the technique described by Cerottini and Brunner (35). Target cells (10’) were suspended in serum-free medium and labeled with 100 &i of 51Cr for 1 hr, washed twice, and then adjusted to a concentration of 2 x lo5 cells/ml in culture medium. Target cells (0.1 ml) were dispensed into wells of 6-mm flat-bottomed Falcon TC 3040 microtest plates and mixed with 0.1 ml of spleen cell suspensions obtained from normal or treated mice at an effector target cell ratio varying from 100: 1 to 3: 1 in a total volume of 0.2 ml. Monolayers of K-Balb and C3H were trypsinized and seeded into microtest plates at concentrations of 5 x lo4 cells per well. Sixteen hours later they were labeled with 2 PCi of 51Crfor 2 hr and washed three times before addition of 0.2 ml effector cell suspensions. Prior to addition of effector cells to target cells, anti-mouse interferon globulin was added (titer 5 x 10-3) to the effector cell suspension and left in the culture wells. All these cultures were incubated for 4 hr at 37°C in an humidified atmosphere containing 5% CO, in air. At the end of this incubation period, 0.1 ml of the supernate was transferred into tubes and counted in a gamma counter (SAIP, Malakoff, France). Percentage specific 51Cr release was calculated according to the formula: E-S T-S
x 100,
where E is the 51Cr released from the target cells in the presence of spleen cells, S is the spontaneous release of 51Cr, and T is the maximum release of 51Cr upon addition of 2 N HCl. All determinations were made in triplicate. YAC tumor cells have been previously described as extremely susceptible target cells for natural spleen cell cytotoxicity (4). They were therefore used in most of our assays to examine the effect of interferon on spleen cell cytotoxicity. RESULTS 1. Administration of Interferon Inducers to Mice Causes an Interferon-Mediated Increase in Spleen Cell Cytotoxicity against NK Sensitive Tumor Cells
As previously shown, injection of mice with the interferon inducers, NDV, and tilorone markedly increased spleen cell cytotoxicity for NK-sensitive tumor target cells (36). This effect was abrogated by simultaneous injection of mice with potent sheep anti-mouse interferon globulin, suggesting that the increase in NK cell
INTERFERON
ENHANCES
NK CELL ACTIVITY
191
FIG. 1. Interferon dose-dependent increase of spleen ceII cytotoxicity against YAC tumor cells. Interferon units were injected ip 16 hr before assay. Effector cell:target cell ratios: 0, 1OO:l; 0, 33:l; A, 11:l.
activity was mediated by the production of endogenous interferon. More recently experiments have shown that the increased spleen cell cytotoxicity in mice inoculated with poly 1:C was also inhibited by injection of anti-interferon globulin. Thus, at a 100: 1 spleen cell:target cell ratio, the specific percentage lysis for control mice was -5.4%, for polyI:C-injected mice 17.6%, and for poly I:C-injected mice treated with anti-interferon globulin - 3.2%. 2. Administration of Mouse Interferon to Mice Increases Spleen Cell Cytotoxicity against NK-Sensitive Tumor Cells Znfluence of dose of interferon. Mice were injected with varying amounts of mouse interferon and their spleen cell cytotoxicity was determined 16 hr later. As illustrated in Fig. 1, a marked increase in spleen cell cytotoxic activity was observed in mice injected with lo4 to lo6 interferon units. The lower limit for induction of
TIME
AFTER
INTERFERON
INJECTION
FIG. 2. Kinetics of interferon-induced spleen cell cytotoxicity against YAC tumor cells. Mice were injected with 8 x 104interferon units ip. Effector celktarget cell ratios: 0, lOO:l, 0,33.1; A, 11:l; n , PBSinjected control mice at 100: 1 effector:target cell ratio.
192
SENIK ET AL.
enhanced cytolytic activity was approximately lo4 interferon units in one experiment and lo3 units in another experiment (data not shown). Kinetics of induction of cytotoxicity by interferon. Mice were given a single injection of 8 x lo5 mouse interferon units, sacrificed thereafter, and the level of spleen cell cytotoxicity determined. Lytic tests were performed in the presence of anti-interferon globulin to exclude the possibility of carry-over of injected interferon into the in vitro assay (see below under section 6). As illustrated in Fig. 2, significant cytotoxicity was observed as early as 1 hr after interferon injection and had reached peak activity at 12 hr (24 hr was not tested in this experiment) followed by a progressive decline reaching background values around Day 4. A second injection of the same dose of interferon at Day 6 after primary interferon administration caused an enhancement of spleen cell cytotoxicity with similar kinetics and magnitude as those induced by the first interferon injection (data not shown). Thus no “memory” either in a positive or negative sense, was discerned using such time intervals between repeated doses of interferon. Species specificity of interferon. Interferon displays a certain species specificity. Thus, human leucocyte interferon exerts very little antiviral effect on murine cells (37). Likewise as shown in Table 1 no significant increase in spleen cell cytotoxicity was observed after injection of mice with either human leucocyte or mock mouse interferon preparations. Age of mouse. Both exogenous interferon (Fig. 3A) and NDV (Fig. 3B) enhanced spleen cell cytotoxicity in mice of different ages, but the level attained in adult mice was considerably higher than that attained in 15day-old mice. 3. Incubation of Normal Mouse Spleen Cells with Interferon against YAC Target Cells
Increases Cytotoxicitj
The rapid response to the injection of interferon in vivo (i.e., an increase in spleen cell cytotoxicity at 1 hr) suggested that interferon might enhance spleen cell cytotoxicity by a direct effect on the effector cells themselves. This possibility was examined by determining the time of contact between interferon and normal spleen TABLE
1
Comparison of Homologous and Heterologous Interferons in Induction of Mouse Spleen Cell Cytotoxicity”r* Mice injected with Mouse IF
Human IF
26.2 2 1.9* 19.3 2 1.6* 20.6 ? 2.1*
10.9 k 1.3 9.9 z!z 1.1 NS’ 9.9 + 1.6
Control preparation (mock interferon) 4.4 k 1.4 5.2 2 0.9 10.0 2 1.5
Not injected 4.0 2 0.1 6.0 k 0.3
a Mice were injected with 8 x loj mouse interferon units or 8 x lo5 human leucocyte interferon units 16 hr prior to sacrifice. h Values are percentages of specific Wr release and represent individual mice. Effector: YAC target cell ratio 100: 1. c Not significant compared to control preparation injected mice. * P < 0.001.
INTERFERON
ENHANCES
NK CELL ACTIVITY
193
60
50
40 % 2 i 30 5 d 2 20 Y ;
:w 10 0
2
6
24
2
10
AGE OF MICE (IN WEEKS)
FIG. 3. Interferon-induced spleen cell cytotoxicity as a function of age. Mice were injected 16 hr prior to test. Experiment A: n Spleen cell of mice injected with 8 x lo5 units of interferon; H spleen cells of PBS-injected mice. Experiment B: q spleen cells of mice inoculated with 10’ TCIDsO NDV; Cl spleen cells of untreated mice. Effector:target cell ratio was 100: 1, the standard error did not exceed 2. Each bar is the mean value of triplicate determinations and corresponds to an individual mouse.
cells required to trigger in vitro the cytolytic mechanism. CBA/H spleen cells were incubated for 4 hr with lo3 units/ml of interferon. Anti-interferon antibodies were added at various times during this incubation period to prevent further contact of free interferon with the spleen cells (Fig. 4). When tested on NK-sensitive YAC cells, interferon-pretreated cells displayed a cytolytic activity which was proportional to the time of contact with interferon prior to the addition of anti-interferon globulin (Fig. 4) (The capacity of the anti-interferon antibodies to neutralize the effect of interferon is evident by the fact that when added at time 0 together with interferon there was no difference in the level of cytotoxicity between these treated spleen cells and untreated control spleen cells.) It should be noted that a significant increase in cytotoxicity was evident after 10 min of contact of spleen cells with interferon. Thus an essential step in the inductive process had taken place within this brief interval. 4. Evidence
That the Interferon-Znduced
Cytotoxicity
Is Mediated
by NK Cells
In vivo induced cytotoxic cells are not T lymphocytes. NK cells are present in the spleen of nude, T-lymphocyte-deficient mice. They are not lysed after incubation with anti-0 serum and complement under conditions that cause lysis of normal T cells. Interferon and interferon inducers increased splenic cell cytotoxicity in nude mice just as they did in normal mice (36) and the cytotoxicity of spleen cells induced in normal mice by NDV or tilorone was not abrogated by incubation with anti-Thy serum and complement (Table 2). In vivo induced cytotoxic cells are nonadherent ceils devoid of surface Zg and avid Fc receptors. Fractionation of in vivo induced cytotoxic cells was undertaken
194
SENIK ET AL.
0
5 TIME
10 (min.)
30 AFTER
120
60
INTERFERON
240
ADDITION
FIG. 4. Effect of addition at varying times of anti-interferon antibodies during the in vitro incubation of spleen cells with interferon. Anti-interferon antibodies added to cultures at times indicated in abscissa. 0, Spleen cells incubated with 1Oaunits of interferon/ml. 0, Untreated spleen cells. 0, Spleen cells incubated from time 0 with interferon and anti-interferon antibodies. A, Spleen cells incubated from time 0 with anti-interferon antibodies alone. Effector target cell ratio 100: 1.
using nylon fiber columns known to remove adherent cells and B lymphocytes (32) or by addition of IgG-coated erythrocytes to remove avid Fc-receptor-positive cells (34). As shown in Table 3 the induced cytolytic cells are not adherent, do not have detectable surface immunoglobulin or avid Fc receptors. They behave as NK cells in all respects (9, 10). Cytotoxic cells induced in vitro also have characteristics of NK cells. The afore mentioned cell fractionation procedures were also carried out on in vitro TABLE
2
Induced Cells Are Resistant to Treatment with Anti-Thy Serum and Complement Control lysis’ Expt. No.” 1 2
Inducing agent* NDV Tilorone
NMS + C -3.2
t
0.8
1.0 + 0.5
Anti-Thy
Induced lysis’ + C
Not done Not done
NMS+C 19.7 + 2.9 21.4 t 2.1
Anti-Thy + C 23.2 32.2
+ 1.3 + 1.7
(1Expt. 1 Balbk and Expt. 2 CBA/H mice. b Inducing agents were used at optimal concentrations and mice were sacrificed 1 day after administration. c Target cells in Expts. 1 and 2 are YAC (= H-2” Moloney lymphoma) cells. Effector:target cell ratio 50: 1. Conditions as described under Materials and Methods. Anti-Thy serum = AKR-anti-CBA or AKR-anti-C3H sera (= anti-Thy 1.2 sera with cytotoxic titers for thymocytes in presence of guinea pig complement more than 1:lOO) used at a concentration 1:lO. NK cells in normal spleen cells are resistant to anti-Thy serum treatment using the above procedure. NMS, normal mouse serum.
INTERFERON
ENHANCES TABLE
3
In Vivo Induced Cytolytic Cells are Nonadherent Expt. No.
Pretreatment”
195
NK CELL ACTIVITY
Cells Devoid of Avid Fc Receptors
Fractionation*
Percentage specific “‘Cr release
1
NDV NDV
None Nylon fiber column
24.8 f 6.0 24.1 t 3.4
2
None None Tilorone Tilorone
None Nylon fiber column None Nylon fiber
1.2 13.3 27.4 40.3
2 rt -t c
1.2 0.9 3.1 2.8
3
Tilorone Tilorone Interferon Interferon
None Ig-anti-Ig column None FcR + cell depletion
51.3 65.7 13.0 36.0
+ r 2 ”
2.1 0.9 0.7 1.8
NDV NDV None None
None FcR + cell depletion None FcR + cell depletion
34.9 91.0 5.2 28.7
+ ? + +
2.2 5.6 1.7 3.4
4 5
a Administration of 10’ EID,, of NDV or 1 mg of tilorone 1 day before assay. b Fractionation on nylon fiber or Ig-anti-Ig columns as described in Refs. (32) and (33). Parallel experiments as to surface markers and T versus B cell responsiveness to mitogens assured the functional ability of the columns (not shown).
interferon-induced spleen cells. As shown in Table 4 these procedures failed to reduce the level of cytotoxicity of the fractionated cells. Thus, cells with NK attributes were also responsible for the in vitro interferon-induced cytotoxicity against YAC cells. TABLE
4
Cell Surface Characteristics of the Cytolytic Cells Induced by Interferon in Vitro Percentage specific 51Cr release Effector:YAC target cell ratio Method of spleen cell fractionation
Treatment
100: 1
50: 1
25: 1
Interferon” Control*
30.3 2 1.5 14.0 & 1.8
22.4 + 2.3 9.5 k 0.5
9.7 k 0.7 5.9 rt 0
Interferon Control
38.1 k 1.5 24.8 k 0.5
29.3 + 0.6 19.2 k 0.5
14.2 r 1.6 8.9 + 0.1
Nylon wool column
Interferon Control
37.9 2 1.0 24.6 2 1.8
18.8 4 0.6 15.8 ? 1.6
13.0 + 1.3 4.0 t 1.2
Anti-Ig column
Interferon Control
43.5 2 2.2 32.7 t 0.9
32.9 r 1.2 21.1 k 2.0
21.3 + 1.4 9.7 k 1.3
None Anti-Thy
1.2 + complement
D Interferon (W units) was added for 2 hr to CBA/H spleen cells. b CBA/H control spleen cells.
196
SENIK ET AL.
Spec$city oflysis. Although the fine specificity of lytic ability of NK cells is still under debate there are a number of cell lines of defined susceptibility or resistance to lysis by such cells. The in vivo interferon-induced cytotoxic cells show the same patterns of cytolysis against four well-established NK target cells as do NK cells (Table 5). Thus like NK cells, the interferon-induced cytotoxic cells are neither H-2 nor even species restricted in their lytic activity. However, they do not lyse PHA-stimulated lymphoblasts (Table 5). In summary, the in vivo or in vitro interferon-induced cytolytic spleen cells display surface features, and specific cytolytic capcity similar to those of spontaneously occurring NK cells. 5. The Increase in the Level of Spleen Cell Cytotoxicity Activity Is Not Due to a Redistribution of NK Cells with a Preferential Localization of These Cells in the Spleen The increase in the spleen cell cytolytic activity induced by interferon may result from an increase in NK cell activity or a redistribution of NK cells throughout the body of the mouse with an accumulation of these cells in the spleen. We therefore tested the level of cytolytic activity of cells from a variety of organs from normal mice injected with an interferon inducer. Figure 5 shows that after inoculation of tilorone there is a general enhancement of NK-cell activity in the spleen, lymph nodes, peripheral blood, and bone marrow. In contrast, however, thymocytes exerted no cytotoxic activity before or after inoculation of tilorone. 6. Evidence That NK-Cell Activity in Vitro Incubation Conditions
Occurs in Vivo and Is Not the Result of the
It might be suggested that the interferon induced increased spleen cell cytotoxicity does not occur in vivo but develops only during the period of incubation of spleen cells from an interferon-treated mouse with the target cells in vitro. Several lines 01 experimental data argue against this possibility. TABLE
5
Cytolytic Cells Induced by Interferon and by Interferon Inducers Manifest the Same Target Cell Specificity Pattern as NK Cells” Targets Expt. No. 1
2
Effector cells CBA/H Controls Tilorone-treated
Balbinude Controls Interferon-treated
K562
YAC
MPC 11
P815
24.1 + 0.2 43.8 k 0.6
8.4 + 0.7 20.8 k 0.6
1.6 -c 0.1 9.5 2 1.0
K.Balb
C3H line
L 1210
DBA/2 PHA blasts
23.7 2 3.7 30.2 k 0.5
10.0 + 2.6 30.5 + 1.4
1.3 + 0.2 29.6 + 5.0
-14.7 t 1.5 -17.0 + 0.7
14.2 k 0.3 26.1 k 0.2
a Effector cells from spleens of mice untreated or treated with 8 x lo5 units of interferon or 1 mg 01 tilorone 1 day earlier. Effector:target cell ratio 50: 1. Mean + SE of individual spleens. Three or four mice per group.
INTERFERON
ENHANCES
197
NK CELL ACTIVITY Thymw
spleen
50
40 1
3w ;30 0 IL 0 L m SC c s l(
~
a
b
==w a
C
b
FIG. 5. Organ distribution of tilorone induced NK-cell activity. A, Cells from CBA mice harvested 1 day after injection of 2 mg of tilorone. 0, Cells from CBA control mice. a = 100: 1; b = 50: I; c = 25: 1; effector:YAC target cell ratios.
(A) Interferon was not detected in extracts of spleen cells from interferoninjected mice. The sensitivity of the method permitted detection of small amounts of interferon, lower than the amount of interferon necessary to increase splenic cell cytotoxicity in vitro. (B) Interferon could not be detected in the culture fluid during the 4-hr incubation period, indicating that lymphoid cells did not release nor produce interferon on contact with target cells. (C) Addition of anti-interferon antibodies to mixtures of target and in vivo induced effector cells did not modify the degree of spleen cell cytotoxicity for YAC target cells. The same concentration of anti-interferon immunoglobulins could be shown to block completely the in vitro induction by IO3 units of interferon of NK cells if added together with interferon at time 0 (see Fig. 4). DISCUSSION This investigation has shown that exogenous or endogenous interferon markedly increase NK cell activity both in viva and in vitro. Thus, each of the interferon
198
SENIK
ET AL.
inducers tested in viva, (NDV, poly I:C, tilorone) caused a rapid increase in spleen cell cytotoxicity activity; while administration of anti-interferon blocked this enhanced NK-cell activity, indicating that the phenomenon was mediated by the production of endogenous interferon. Furthermore, partially purified mouse interferon type I also increased NK-cell activity both in vivo and in vitro, whereas human leucocyte interferon or control preparations failed to exert any demonstrable effect. The evidence presented under section 6 under Results shows that this increase in spleen cell cytotoxicity occurred in vivo and did not occur during the incubation of effector cells with target cells in vitro. Since inoculation of an interferon inducer enhanced NK-cell cytotoxicity in various lymphoid tissues (Fig. 5), it seems likely that the increase in spleen cell cytotoxicity is part of a generally augmented cytotoxic response rather than a localization within the spleen of NK cells from other sites. The evidence that the induced cytolytic cells are in fact NK cells and not some other cell type stems from several independent observations. Previous investigators using similar interferon inducers found that the induced cells had the characteristics of “normal” NK cells (16). In the present study we showed that the induced cytolytic cells are neither T nor B lymphocytes. Furthermore, they were nonadherent and did not have avid Fc receptors as shown by experiments utilizing nylon fiber, anti-Ig columns, or the rosetting of FcR+ cells with IgG-coated erythrocytes. These are all features characteristic of murine NK cells (9, 10). Furthermore, the analysis of target cell susceptibility using the interferon-induced cells showed a parallelism between the behavior of induced cytolytic cells and normal NK cells. These results affirm those reported recently in an in vitro human system (38,39) and in a mouse system in vivo andin vitro, by other investigators and ourselves (36, 40). What is the mechanism of this enhancement of NK activity? The increase in spleen cell cytotoxicity was manifested within 1 hr after inoculation of mice with interferon (Fig. 2). The in vitro experiments (section 3 under Results) suggest that less than 10 min of contact of interferon with spleen cells suffices to initiate an enhanced cytotoxicity (Fig. 4). Since this enhancement occurs with such rapidity, cell division is unlikely to be required. The simplest explanation would be a direct effect of interferon on NK cells. As previously shown, interferon can enhance a number of specialized cell functions (41); this includes increased cytotoxicity of sensitized spleen cells for allogeneic target cells (42), increased expression of H-2 antigens on the surface of various tumor and normal cells (43-45). Findings such as these taken together with the present data suggest that interferon treatment of NK cells also leads to a modification of the cell surface, expressed as enhanced inherent cytolytic activity, in this case assayed on tumor target cells. It has been suggested that NK-cell activity in mice can also be modulated by the activity of suppressor cells (46,47). Although our results suggest that enhancement of NK-cell activity stems from a direct effect of interferon on NK cells, we can not rule out the possibility that interferon may also inhibit the activity of cells which have been reported to suppress NK-cell activity. Such an effect would also result in an increase in NK-cell activity. If, as has been suggested (12, 13), NK ceils are important in the natural resistance to tumors, it may be that some part of the antitumor effects of interferon and interferon inducers (23,48) is mediated by the enhancement of NK-cell activity. In
INTERFERON
ENHANCES
NK CELL ACTIVITY
199
our experiments, a second injection of interferon or interferon inducer 7 days after a primary injection (i.e., at a time when NK cell activity had returned to normal) induced a “normal” increase in NK-cell activity as judged by the kinetics and extent of the response. It would seem worthwhile to determine whether it is feasible to develop and maintain constant high levels of NK-cell activity in tumor susceptible mouse strains by repeated injection of interferon, or interferon inducers, as a defense against neoplastic growth. ACKNOWLEDGMENTS We are grateful to Dr. Wolf H. Fridman for many helpful discussions and to Mrs. Marie-The&e Bandu, Mrs. Josiane Buywid, and Mrs. Francoise Zambetti for skillful technical assistance. We are also grateful to Dr. Maurice Landy for reading the article and for his helpful suggestions. This work was aided by grants from I.N.S.E.R.M. (ATP 78-91, 44.77.79/13/16), D.R.E.T. (77/203), and Swedish Cancer Society, NIH Contract NOl-CB-64033.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Rygaard, J., and Povbsen, C. C., Transplant. Rev. 28, 43, 1976. Warner, N. L., Woodruff, M. F. A., and Burton, R. C., Int. J. Cancer 20, 146, 1977. Greenberg, A. H., and Greene, M., Nature (London) 264, 356, 1976. Kiessling, R. E., Klein, E., and Wigzell, H., Eur. J. Immunol. 5, 112, 1975. Herberman, R. B., Nunn, M. E., and Laurin, D. H., Int. J. Cancer 16, 216, 1975. Peter, H. H., Pavie-Fischer, J., Fridman, W. H., Aubert, C., Cesarini, J. P., Roubin, R., and Kourilsky, F. M., J. Immunol. 115, 539, 1975. West, W. H., Cannon, G. B., Kay, H. D., Bonnard, G. D., and Herberman, R. B.,J. Immunol. 118, 355, 1977. Oehler, J. R., Lindsay, L. R., Nunn, M. E., and Herberman, R. B., Int. J. Cancer 21,204, 1978. Kiessling, R. E., Klein, E., Pross, H., and Wigzell, H., Eur. J. Immunol. 5, 117, 1975. Herberman, R. B., Nunn, M. E., Holden, T. H., and Laurin, D. H., Int. J. Cancer 16, 230, 1975. Haller, O., Kiessling, R., Orn, A., and Wigzell, H., J. Exp. Med. 145, 1411, 1977. Petranyi, G. G., Kiessling, R., Povey, S., Klein, G., Heizenberg, L., and Wigzell, H., Immunogenetics 3, 15, 1976. Haller, O., Hansson, M., Kiessling, R., and Wigzell, H., Nature (London) 270, 609, 1977. Herberman, R. B., Nunn, M. E., Holden, H. T., Staal, S., and Djeu, J. Y., Int. J. Cancer 19, 555, 1977. Welsch, R. M., and Zinkernagel, R. M., Nature {London) 268, 646, 1977. Oehler, J. R., Lindsag, L. R., Nunn, M. E., Holden, H. T., and Herberman, R. B., Int. J. Cancer 21, 210,
1978.
17. Issacs, A., and Lindenmann, J., Proc. Roy. Sot. Ser.B 147, 258, 1957. 18. Field, A. K., Tytell, A. A., Lampson, G. P., and Hilleman, M. R. Proc. Nat. Acad. Sci. USA 58, 1004, 1967. 19. Kirchner, H., Hirt, H. M., Becker, H., and Munk, K., Cell. Immunol. 31, 172, 1977. 20. Trinchieri, G., Santoli, D., and Knowles, B. B., Nature (London) 270, 611, 1977. 21. Gresser, I., Bourali, C., Levy, J. P., Fontaine-Brouty-Boy&, D., and Thomas, M. T., Proc. Nat. Acad. Sci. USA 63, 51, 1969. 22. Gresser, I., and Bourali, C., Nature (London) 223, 844, 1969. 23. Gresser, I., In “Advances in Cancer Research” (G. Klein and S. Weinhouse, eds.), pp. 97-141. Academic Press, New York/London, 1972. 24. Levy, H. B., Law, L. W., and Rabson, A. S., Proc. Nat. Acad. Sri. USA 62, 357, 1969. 25. Gresser, I., Brouty-BoyC, D., Thomas, M. T., Macieira-Coelho, A., Proc. Nat. Acad. Sci. USA 66, 1052, 1970. 26. Gresser, I., Maury, C., and Brouty-Boy& D., Nature (London) 239, 167, 1972. 27. Tovey, M. G., Begon-Lours, J., and Gresser, I., Proc. Sot. Exp. Biol. Med. 146, 809, 1974. 28. Gresser, I., Bandu, M. T., Brouty-Boy& D., J. Nat. Cancer Inst. 52, 553, 1974. 29. Gresser, I., Fontaine-Brouty-Boy& D., Bourali, C., and Thomas, M. T., Proc. Sot. Exp. Biol. Med. 130, 236, 1969.
200
SENIK ET AL.
30. Gresser, I., Tovey, M. G., Bandu, M. T., Maury, C., and Brouty-Boy& D.,J. Exp. Med. 144, 1305, 1976. 31. Gresser, I., Maury, C., Bandu, M. T., Tovey, M. G., and Maunoury, M. T.,Znr. J. Cancer 21,72, 1978. 32. Julius, M. H., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3, 645, 1973. 33. Wigzell, H., Stand. J. Immunol. Suppl. 5, 23, 1976. 34. Fridman, W. H., and Golstein, P., Cell. Immunol. 11, 442, 1974. 35. Cerottini, J. C., and Brunner, K. T., In “In Vitro Methods in Cell Mediated Immunity” (B. R. Bloom and P. R. Glade, Eds.), p. 369. Academic Press, New York, 1971. 36. Gidlund, M., Orn, A., Wigzell, H., Senik, A., and Gresser, I., Nature (London) 273, 759, 1978. 37. Levy-Koenig, R. E., Golgher, R. R., and Paucker, K., J. Immunol. 104, 791, 1970. 38. Trinchieri, G., Santoli, D., Dee, R. R., and Knowles, B. B., J. Exp. Med. 147, 1299, 1978. 39. Trinchieri, G., and Santoli, D., J. Exp. Med. 147, 1314, 1978. 40. Djeu, J. Y., Heinbaugh, J. A., Holden, H. T., and Herberman, R. B., J. Immunol. 122, 17.5, 1979. 41. Gresser, I., Cell. fmmunol. 34, 406, 1977. 42. Lindahl, P., Leary, P., and Gresser, I., Proc. Nut. Acad. Sci. USA 69, 721, 1972. 43. Lindahl, P., Leary, P., and Gresser, I., Proc. Nut. Acad. Sci. USA 70, 2785, 1973. 44. Lindahl, P., Leary, P., and Gresser, I., Eur. J. lmmunol. 4, 779, 1974. 45. Vignaux, F., and Gresser, I., Proc. Sot. Exp. Biol. Med. 157, 456, 1978. 46. Hochman, P. S., Cudkowicz, G., and Dausset, J., J. Nat. Cancer Inst. 61, 265, 1978. 47. Cudkowicz, G., and Hochman, P. S., “Developmental Immunobiology” (G. W. Siskind, Ed.). Grune & Stratton, New York, 1978. 48. Gresser, I.,ln “Cancer-A Comprehensive Treatise” (F. Becker, Ed.), pp. 521-571. Plenum, New York. 1977.
