effect of various interferons on the spontaneous ...international units with human leukocyte if as...

(CANCER RESEARCH 41, 294-299, January 1981]0008-5472/81 /0041-OOOOS02.00

Effect of Various Interferons on the Spontaneous Cytotoxicity Exertedby Lymphocytes from Normal and Tumor-bearing Patients1

Miguel A. Lucero, Wolf H. Fridman, Marie-Annick Provost, Claude Billarden. Pierre Pouillart, Janine Dumont,and Ernesto Falcoff

Institut Curie. Section Medicale and Section de Biologie (INSERM U 196), 26, rue d Ulm. 75231 Paris. Cedex 05, France

ABSTRACT

Natural killer cell activity, which represents the spontaneouscytotoxicity of lymphocytes toward tumor cells, has been measured in 173 tumor-bearing patients and 25 healthy volunteers;

no significant difference was found in mean natural killer cellactivity between the two groups. The parameters of interferon-

induced activation of natural killer cells were studied in orderto provide a suitable test for monitoring the effect of interferonin clinical trials.

The three interferons tested (leukocyte, lymphoblastoid, andfibroblast) were equally active in inducing spontaneous cytotoxicity of lymphocytes from all healthy individuals and tumor-

bearing patients studied. Incubation for one hr with 100 unitsof interferon was sufficient to increase spontaneous cytotoxicity activity, the maximum effect being obtained when lymphocytes were incubated with 1000 units of any of the interferonsused. This effect was blocked with the appropriate antiinter-

feron sera. The target cells for interferon seem to be positiveFey receptor lymphocytes.

INTRODUCTION

The role of the immune system in host defense againstneoplastic growth is still open to question. For years, it hasessentially been viewed in terms of T-cells2 reacting with spe

cific tumor transplantation or with tumor-associated antigens

(3). However, with time, it appeared that, while this mechanismwas certainly effective in virus-induced tumors in which T-cellsrecognize viral-coded antigens in association with histocom-patibility products (8), there was little evidence that they directlylimit the growth of chemically induced or spontaneous tumors.It has been suggested that other effector mechanisms also playa role in antitumor surveillance against these 2 types of tumors,especially NK cell cytotoxicity (17, 22).

NK cells have been found both in human (11) and in experimental (18, 20) systems. These cells, apparently devoid ofantigen specificity, kill tumor cells (11, 18, 22), transformedcells (21, 28), and under certain conditions normal cells (21).NK cells seem to be good candidates for effector cells in aprimitive defense against spontaneous tumors. Large numbersof NK cells are found in athymic nude (nu/nu) mice, and theirpresence may explain the low incidence of spontanous tumors

' This work was supported by grants 47-77-79 and 78-4 082 from Institut

National de la SantÃ©et de la Recherche MÃ©dicale,from DÃ©lÃ©gationGÃ©nÃ©raleÃ laRecherche Scientifique et Technique, and from ComitÃ©Inter-sections of Curie

Institute.' The abbreviations used are: T-cells, thymus-derived cells; NK, natural killer;

IF, interferon; RPMI 1640. Roswell Park Memorial Institute Medium 1640; FCS.fetal calf serum; EAG, Ig-G-sensitized erythrocytes; RFC. rosette-forming cells;

SC, spontaneous cytotoxicity.Received April 8. 1980; accepted October 3. 1980.

in these animals (25). A correlation has been observed betweenthe in vivo resistance of mice to the transplantable YAC lym-phoma and the in vitro level of NK cell cytotoxicity (9, 23). Asimilar correlation was found between the NK cell level and therejection of small tumor inocula (24).

IF was shown recently to enhance NK cell activity in vivo andin vitro (7, 28), and it has been suggested that such enhancement may partly account for its antitumor effects (26). Sinceseveral therapeutic assays using IF for treatment of humantumor-bearing patients are already in progress (1, 14, 16, 19,

27), it appeared of interest to study carefully the parametersfor activation of human NK cells by IF, as well as the NK cellactivity of lymphocytes from tumor-bearing patients.

The results reported here show that natural cytotoxicity ofhuman lymphocytes from normal and tumor-bearing patients is

enhanced in vitro by the same doses and kinetics by fibroblast,leukocyte, and lymphoblastoid IP's. This effect is blocked by

the relevant anti-IF sera and is directed towards Fey receptor-

positive lymphocytes. When lymphocytes from 173 patientsbearing various types of tumors were tested for the level of NKcell activity, no significant difference was found compared tolymphocytes from 25 healthy donors.

MATERIALS AND METHODS

Healthy Donors

The 25 healthy donors were laboratory workers at the InstitutCurie, Paris, France. They included 14 women and 11 men, 22to 60 years old.

Tumor-bearing Patients

The 173 patients studied before or several months aftertreatment comprised 98 women and 75 men, ages 20 to 70years. Tumor type distribution among patients was as follows:43 lymphomas; 42 malignant melanomas; 25 lung tumors; 24breast adenocarcinomas; 17 squamous cell carcinomas of theupper respiratory and digestive tract; 5 cervical carcinomas; 3ovarian carcinomas; 1 fallopian tube epithelioma; 1 vulvarepidermoid carcinoma; 2 pancreatic adenocarcinomas; 2 rectal carcinomas; 1 colon carcinoma; 1 malignant glioma; 1testicular dysembryoma; 1 leiomyosarcoma; 1 neurinosar-coma; 1 Ewing's sarcoma; 1 kidney carcinoma; 1 osteogenic

sarcoma.

Lymphocyte Preparation and Identification of LymphocyteSubpopulations

Peripheral blood was collected into a syringe containingheparin (Calciparine; Laboratories Choay, Paris, France), andlymphocytes were prepared on a Ficoll gradient (Ficoll Radio

294 CANCER RESEARCH VOL. 41

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IF and NK Cell Activity in Tumor-bearing Patients

Selectan; Sigma Chemical Co., St. Louis, Mo., and Schering,West Germany) according to the technique of Boyum (2). Thelymphocytes were resuspended in RPMI 1640 (Eurobio, Paris,France) supplemented with 20% PCS (Grand Island BiologicalCo., Glasgow, Scotland) at a final concentration of 107 cells/

ml.Spontaneous erythrocyte-forming rosettes detecting T-cells

were formed with lymphocytes and sheep RBC (Sorga, Paris,France), using a previously described technique (22).

EAG rosettes detecting Fey receptor-positive cells were per

formed according to the method of Peter et al. (22).In some experiments, lymphocyte suspensions were either

depleted or enriched in RFC or EAG-RFC by centrifugation in

Ficoll gradient after rosette formation (22). The cells recoveredat the interface were used when completely free of RFC.

Interferons

Human fibroblast IF, obtained from Hem Research, Inc.(Rockville, Md.), was prepared on HFS No. 30 normal foreskinfibroblasts. Each lyophilized vial contained 1.89 x 105 units ofpurified IF (2.37 X 106 units/mg of protein). After solubilizationin distilled water, IF samples at 105 units/ml were stored at-70Â°.

Human leukocyte IF, supplied by Dr. K. Cantell (Helsinki,Finland) (4), was obtained after treatment of human leukocyteswith Sendai virus. Each vial contained 6 x 106 units/ml (4 x106 units/mg of protein). IF samples were processed as de

scribed above.Human lymphoblastoid IF, provided by Dr. N. Finter (Well

come Research Laboratory, London, England), was preparedby infecting Namalwa lymphoid cells with Sendai virus. Eachlyophilized bottle contained 4.67 x 106 units (1.57 x 106

units/mg of protein). IF samples were processed as describedabove.

In some experiments, lymphocytes from healthy donors andcancer-bearing patients were incubated with different doses ofhuman fibroblast and leukocyte IP's (101, 102, 103, or 104

units) prior to the spontaneous cytotoxic activity assay fordifferent incubation periods (1, 4, or 24 hr). Cells (107) were

incubated in 0.9 ml of RPMI 1640 supplemented with 20%PCS and 100 fi\ of IF solution for the required time at 37Â°.At

the end of the incubation, 4 ml of medium were added, andcells were centrifuged and washed twice.

Anti-human IF Sera. The following sera were obtained fromthe National Institute of Allergy and Infectious Diseases (Be-thesda, MD.). For antiserum to human leukocyte IF, 1 ml ofserum contained 104 neutralizing units against 10 units of IF;i.e., a 10~" dilution of this serum neutralizes 10 units of human

leukocyte IF. Antiserum to human fibroblast IF was preparedagainst fibroblast IF. It contained 103 neutralizing units against

10 units of IF.In some experiments, IF was preincubated with anti-IF serum

before the NK cell activity test. For this purpose, sufficientantiserum to neutralize 100 units of IF as determined by subsequent titration (final antiserum dilution: 1/1000 for anti-leukocyte IF and 1/100 for anti-fibroblast IF) was added to the IFpreparation which was then incubated for 1 hr at 37Â°.Of this

solution, 100 /xl were then added to 900 /Â¿Iof cell suspensioncontaining 107 lymphocytes. The cells were incubated for 1 hrat 37Â°, and the cytotoxicity assay was performed. Control

tubes were prepared by incubating lymphocytes with IF andantiserum separately. In other experiments designed to studythe kinetics of IF-mediated enhancing SC, lymphocytes wereincubated with 103 units of leukocyte or fibroblast IF, and the

appropriate antisera were added at different times thereafter.Before use or after neutralization by antisera, IF titers were

determined by the cytopathogenic inhibition test (10) on GM2504 human fibroblasts, provided by Dr. A. Greene (Instituteof Medical Research, Camden, N. Y.). The vesicular stomatitisvirus was used as challenge. The IF titer was expressed ininternational units with human leukocyte IF as reference (NIH,Bethesda, Md.).

Lymphocyte Cytotoxicity Assay. Cells of the K 562 line(kindly provided by Dr. M. Fellous, HÃ´pital Saint-Louis, Paris,

France) were used as targets. They were grown in the laboratory in RPM11640 supplemented with 10% FCS and Gentalline(40 mg/liter; Unilabo, France).

The cytotoxicity assay was performed according to themethod of Cerottini and Brunner (5), 51Cr (C.E.A., Saclay,France)-labeled target (K 562) cells (10") in 100 Â¿ilof RPMI

1640 supplemented with 10% FCS were mixed with 100 fil ofincreasing numbers of lymphocytes at effector/target cell ratios varying from 1/1 to 100/1. The cell mixtures were incubated for 4 or 16 hr at 37Â°.At the end of incubation, 100 jul of

the supernatant were transferred into vials and counted in agamma counter (C.G. 4000; Intertechnique, France).

The percentage of specific 51Cr release was determined

according to the formula

E- S

T-S

where Â£is the amount of 51Cr released from the target cells in

the presence of lymphocytes, S is the spontaneous release of51Cr measured in control cultures (target cells mixed with

culture medium), and T is the maximum release when controlcultures are treated with 2 N HCI. Spontaneous release did notexceed 30%. All the cultures were set in triplicate. Variabilityfor the triplicate determination did not exceed 10%.

RESULTS

Kinetics of IF-activated SC. In a first set of experimentsdesigned to establish the kinetics of NK cell activity enhancement by IF's, lymphocytes from healthy donors were incubated

for various periods with IF and tested at different effector/target cell ratios for their SC activity on K 562 target cells.

Chart 1 shows the results obtained when 107 lymphocyteswere incubated with 100 units of leukocyte or fibroblast IF's

for 1, 4, and 24 hr. Their SC on K 562 was measured in 4- or16-hr assays (Chart 1) at an effector/target cell ratio of 100/

1.Both IF's enhanced NK cell activity in the same proportions.

The level of enhancement was similar, whether cells wereincubated with IF for 1, 4, or 24 hr. It is noteworthy thatincubation of lymphocytes for 4 hr in the absence of IF enhanced their SC. Incubation with IF produced the same effect,but the level of SC was higher than in lymphocytes incubatedfor 4 hr in IF-free medium. Finally, the effect of IF was clearlydetectable in both the 4-hr and 16-hr cytotoxic assays.

Comparative Effects of Different Doses of Leukocyte,

JANUARY 1981 295



M. A. Lucero et al.

100

75

= 50

25

4 24 I 4 ' 24

INCUBATION TIME (hr) WITH INTERFERONChart 1. Kinetics of SC activation by IP's. â€¢.controJ/A. treated with leukocyte

IF; A. treated with fibroblast IF. F dose. 100 units/ml; effector/target ratio, 10071; cytotoxicity assay: a. 4 hr; o, 16 hr

IUCE

50r

25

Q.V) control

10 1000100UNITS/ml

Chart 2. Comparison of the dose effect of different IF's. A. control, â€¢leu

kocyte IF; â€¢,fibroblast IF; D. lymphoflastoid IF. Effector/target ratio. 100/1;cytotoxicity assay, 16 hr.

Lymphoblastoid, and Fibroblast IF. To compare the effects ofvarious IF's on SC, lymphocytes from a healthy donor were

incubated with graded doses of leukocyte, lymphoblastoid, andfibroblast IF and tested for their SC on K 562 cells.

Chart 2 shows the results obtained with an effector/targetcell ratio of 100/1, for which no quantitative differences, wereobserved between the 3 IF's tested. For all 3 IF's, 100 units of

IF were sufficient to induce a significant increase in NK cellactivity.

We tested various effector/target cell ratios from 1/1 to10O/1, and the enhancing effect of IF's on NK cell activity was

highly significant for ratios of 6/1 and more (data not shown).Effect of Anti-IF Sera. To confirm that IF was indeed the

active molecule in enhancing SC, 100 units of fibroblast orleukocyte IF were incubated with the relevant anti-fibroblast oranti-leukocyte IF sera, at a dilution at which all viral activity was

neutralized (data not shown), and then tested for their effecton NK cell activity. Table 1 (Experiment 1) shows that neutralization of IF's by the relevant antisera completely abolished its

enhancing activity. In no case did the incubation of effectorcells with anti-IF sera in the absence of IF lead to a decrease

in cytotoxicity (Table 1).

In another set of experiments, anti-IF sera were used to

analyze the kinetics of IF mediation in enhancing NK activity.In the experiments shown in Chart 4, lymphocytes were incubated with 1000 units of leukocyte or fibroblast IF, and therelevant antisera were added at different times. SC was tested1 hr after incubation. When the antiserum and IF were addedsimultaneously, addition of the antiserum completely abolishedthe enhancing activity of IF in both cases, whereas addition ofanti-IF serum 30 min after IF had almost no effect. This showed

that a very brief contact between IF and lymphocytes wassufficient to induce the events leading to the expression ofcytoxicity. In no case did the incubation of effector with anti-IFsera in the absence of IF lead to a decrease in cytotoxicity(data not shown).

Characterization of the Lymphocyte Subpopulation Reacting with IF. To determine whether T-cells were necessary forIF-induced enhancement of NK cell activity, lymphocytes weredeprived of T-cells by depletion of erythrocyte RFC by centrif-

ugation in a Ficoll grandient and then incubated with 1000units of leukocyte IF.

Chart 3 shows that incubation of IF for 1 hr with a lymphocytepopulation deprived of T-cells leads to enhanced SC activity

identical to that obtained by incubating IF with an undepletedlymphocyte preparation. By contrast, depletion of Fey receptor-positive cells by Ficoll sedimentation of EAG-RFC abolished SC(Chart 3). Treatment of the Fey receptor-negative cells with IF

did not induce NK activity on K 562 cells (Chart 3).Since in humans NK cells have been shown to be contained

in the Fey receptor-positive population (22), these experiments

suggest that IF may act directly on the effector NK cells. Inanother set of experiments, where IF was incubated either withEAo-RFC-depleted cells or with unseparated lymphocytes, no

synergistic effect could be observed (data not shown), suggesting that no cell-to-cell cooperation is needed for the

expression of IF activity.

Table 1Neutralization by anti-IF sera and kinetics of the enhancing effect of IF on SC

Treatment% of specific 5'Cr release at differ

ent effector/target ratios

Type ofIFExperiment1aNo

IFNoIFLeukocyteLeukocyteNo

IFFibroblastFibroblastExperiment

2NoIFLeukocyteLeukocyteLeukocyteLeukocyteFibroblastFibroblastFibroblastFibroblastDose

(units/ml)IO210*10210"10s10'10Â»IO3103ioÂ»IO3IO3Specific

anti-IFaddedâ€”+â€”++â€”+â€”0

min30min60

minâ€”0

min30min60

min-12/15.67146.35.48.44.14.74.58.614.213.97.619.316.21425/110.811.218.411.611.819.410.613.812.422.322.626.215.333.931.233.650/116.717.535.315.615.934.213.821.314.927.73827133428.638.7100/121.718.637.236.14120.337.141.940.6

Specific antisera were incubated with or without the indicated IF for 1 hrbefore addition to the lymphocyte suspension.

'' Specific antisera were added directly to the lymphocyte suspension at the

indicated time.





Comparison of NK Cell Activity in Cancer-bearing Patients

and Healthy Donors. We measured SC toward K 562 targetcells of lymphocytes from 173 patients and 25 healthy controls.

As shown in Table 2, the overall mean of the spontaneouscytotoxicity exerted by lymphocytes from tumor-bearing pa

tients and healthy donors was similar [33.6 Â± 23.3 (S.D.)compared to 32.5 Â±14.5].

Taking into account the considerable scatter of the valuesobtained for the percentage of specific 51Cr release and in

order to enable comparison between cancer patients andhealthy donors, our cases were arbitrarily divided into 3 groups:low responders (less than 20%); medium responderÃ¡ (20 to40%); and high responders (more than 40%) at an effector/target cell ratio of 50/1. Table 1 also shows that, in eachgroup, mean SC values and the respective numbers of healthydonors and cancer patients were similar. Moreover, when SCwas compared in lymphocytes from tumor subgroups, no significant difference was found.

Reactivity to IF of Lymphocytes from Tumor-bearing Pa

tient. We have tested the in vitro enhancement of NK cellactivity of lymphocytes from 74 tumor-bearing patients. As a

general rule, incubation of their lymphocytes with IF led to anenhancement of NK cell activity comparable to the enhancement obtained for IF incubation with lymphocytes from healthydonors.

These data can be schematically summarized as following 3patterns of reactivity: in patients exerting low spontaneous NKcell activity (Chart 4a), in vitro treatment with IF brought theresponse to normal levels; in those presenting normal NK cellactivity, IF enhanced the response with dose and kinetics

UJin

uiOC

oZ

uUIaw

f

30r

20 -

10-

Chart 3. Characterization of the lymphocyte subpopulation reacting with leukocyte IF (1000 units/ml). Group 1. Total lymphocyte cell population: left bar.control: right bar, IF treated. Group 2. Lymphocyte cell population deprived of T-cells: left bar, without IF; right bar, IF treated. Group 3. Lymphocyte cellpopulations depleted in RFC-positive cells: left bar, without IF; right bar, IFtreated. Effector/target ratio. 50/1; cytotoxicity assay. 16 hr.

similar to those observed when testing healthy individuals(Chart 4b); finally, in those patients presenting spontaneoushigh NK cell activity, such as patients receiving Bacillus Cal-mette-Guerin Â¡mmunotherapy, IF could not enhance the cyto

toxicity to K 562 above the already maximum reactivity (Chart4c).

DISCUSSION

The data reported here analyze the parameters of IF-induced

enhancement of NK cell activity in humans. We showed thatthere was no detectable difference in the effects of NK cellactivity of 3 different types of IF (leukocyte, fibroblast, andlymphoblastoid). We further established that IF is the activemoiety in enhancing SC by neutralizing its effect with therelevant antisera.

Consequently, from the viewpoint of therapeutic application,all these IF's act equally well on the immunological compart

ment considered. The kinetics of the effects of IF under ourexperimental conditions shows that a short period of incubation(30 min) was sufficient to induce the events leading to enhancement of NK cell activity. These results are in agreement withdata from the mouse showing that a few min incubation ofeffector lymphocytes with IF enhanced NK cell activity (26).

To analyze the kinetics of IF effects, we also neutralized freeIF by adding the relevant antisera at different times. In contrastto a previous report (13), we did not find that antisera whenadded to effector cells in the absence of IF reduced their SCunder our experimental conditions. Production of free IF bycells being tested is therefore unlikely to play an important partof promoting natural cytotoxicity. The role of immune IF produced by the cells under test cannot be ruled out by ourexperiments since immune IF would not react with the antiseraused. Finally, the enhancement of NK cell activity was as goodin a 4-hr 51Cr release assay as in a 16-hr assay, indicating that

maturation of NK cells after their induction by IF is a short-period event which probably does not involve cell division.

In an attempt to define the cells which react with IF in thesystem considered, we showed that IF does not induce SCactivity in Fey receptor-negative T-cells (EAG-RFC-depletedpopulation), or does it render T-cells able to induce NK cells.By contrast, IF acts directly on human Fey receptor-positivecells containing mature NK cells or their immediate precursors,as suggested previously (22, 29). Finally, no synergistic effectcould be obtained when an IF-treated cell population wasadded to untreated cells, which argues against possible cell-to-cell cooperation in mediating the effects of IF.

Our observations confirm and extend previous findings (13,28) which showed that IF may enhance NK cell activity in

Table 2Comparison of spontaneous lymphocyte cytotoxicity in healthy donors and cancer-bearing patients

% of specific s'CrreleaseGroupHealthy

donorsAll cancer patients including

LymphomaMalignant melanomaBreast adenocarcinomaLung tumors<20%16.5

Â±2.2a (8)"

12.5 Â±4.7 (52)12.4 Â±2.6 (13)14.1 Â±2.5 (15)13.7 Â±3.1 (9)10.7 Â±3.8 (8)20-40%28.6

Â±6.3 (9)27.2 Â±5.6(61)25.7 Â±3.4 (15)27.7 Â±4.8(15)25.1 Â±4.1 (7)24.9 Â±3.3 (6)>40%55.3

Â±5.6 (8)61.1 Â±10.8(60)60.5 Â±6.2 (15)67.3 Â±6.5 (12)54.5 Â±5.3 (10)57.2 Â±5.7 (10)Overall

mean32.5

Â±14.5 (25)33.6 Â±25.3(173)34.3 Â±13.3 (43)31.7 Â±13.8 (42)35.6 Â±11.7 (26)33.6 Â±12.8 (24)

" Mean Â±S.D. at lymphocyte/target cell ratio of 50/1. Cytotoxicity assay 16 hr. p not significant when SC in lymphocytes from each tumor group is compared

with that of healthy donors." Numbers in parentheses, number of individuals tested.

JANUARY 1981 297



M. A. Lucero et al.

100

75

yik0

25

12 25 50 MX) 25 50 no 25 50 no

LYMPHOCYTE TO TARGET CELL RATIO

Chart 4. Effect of IP's on SC exerted by lymphocytes from tumor-bearing patients. A, lymphocytes from tumor-bearing patients with low (a), medium (6), and high

(c) spontaneous NK cell activities. O, treated with leukocyte IF; â€¢,treated with fibroblast IF. IF dose, 100 units/ml; cytotoxicity assay, 16 hr.

human lymphocytes. In the mouse, in vivo injection of IF wasshown to enhance NK cell activity (7). In humans, for monitoringof patients treated with IF in clinical trials, it seems necessaryto establish the kinetics of the in vivo effects of IF on SC. Sucha trial is currently in progress, and preliminary results indicatethat IF does indeed enhance NK cell activity in vivo (6, 15).

For the purposes of clinical trials, it appeared to be a necessary prerequisite to measure SC in tumor-bearing patients

and to compare it to a control population.We did not find any significant difference in NK cell activity

between patients and controls, which argues against the possibility of a relationship between low NK cell activity and hostdefense against cancer. NK cells from nearly all tumor-bearing

patients, except those with already high NK cell activity at thetime of our assays, can be stimulated by IF, as can be NK cellsfrom healthy individuals. It is therefore not likely that this assaycould be used to choose between IF-responsive- and IF-non-

responsive patients but rather, in the course of a clinical trial,to assess the presence of a biological effect of IF. Regardlessof its relevance to tumor evolution, enhanced NK cell activitydemonstrates an active interaction of IF and the immune systemallowing monitoring of patients. However, the important issuemay be the sensitivity of various tumor cells to destruction bythe autologous NK cells. In this respect, the results of preliminary experiments in our laboratory suggest that the sensitivityto NK cells of various tumor cells, especially lymphoma cells,differed from one individual to another. Since all tumor-bearing

patient lymphocytes were responsive to IF, it may well be thatscreening of sensitive tumors serve as a guide in applying IFtreatment.

ACKNOWLEDGMENTS

The authors are grateful to Dr. K. Cantei (Finland) for the gift of leukocyte IF.to Dr. N. Finter (Great Britain) for the gift of lymphoblastoid IF, to Dr. J. Dunnick

(NIH, Bethesda. Md.) for providing antisera against human leukocyte and fibroblast IF, and Dr. M. Fellous (France) for providing K 562 cells line.

We are also indebted to M. RiviÃ¨re and C. Kuhlen for competent technicalassistance.

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1981;41:294-299. Cancer Res Miguel A. Lucero, Wolf H. Fridman, Marie-Annick Provost, et al. PatientsExerted by Lymphocytes from Normal and Tumor-bearing Effect of Various Interferons on the Spontaneous Cytotoxicity

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