1982;42:2480-2488. Cancer Res

Eva Lotzov, Cherylyn A. Savary, Jordan U. Gutterman, et al.

Partially Purified and Cloned Interferon-Modulation of Natural Killer Cell-mediated Cytotoxicity by

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[CANCER RESEARCH 42, 2480-2488. June 1982]0008-5472/82/0042-OOOOS02.00

Modulation of Natural Killer Cell-mediated Cytotoxicity by PartiallyPurified and Cloned Interferon-1Eva Lotzov,2 Cherylyn A. Savary,3 Jordan U. Gutterman, and Evan M. HershDepartment of Clinical Immunology and Biological Therapy, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, TheTexas Medical Center, Houston, Texas 77030

ABSTRACTWe have studied peripheral blood of 35 normal individuals,

28 solid-cancer patients, and 14 leukemic patients for naturalkiller (NK) cell cytotoxicity to K-562 and CEM tumor cells in a51Cr release cytotoxicity assay. We have found that both normalindividuals and solid-cancer patients could be grouped intohigh (NK-HR), medium (NK-MR), and low (NK-LR) NK cellresponder categories with regard to their degree of NK cellactivities. However, in general, NK-HRs were found to beprevalent in normal donor population and NK-LRs in cancerpatients. Leukemic patients always exhibited the NK-LR status.The difference between NK-HRs and NK-MRs appeared to bedue to the relative decrease in number of active NK cells. Incontrast, the NK-LR status could not be contributed to decrease of NK cells because of the dichotomy in NK cell dose-response patterns of NK-LRs versus those of NK-HRs and NK-MRs. Eighteen cancer patients undergoing interferon-a (IFN-a) therapy (3 x 106 units, i.m., daily) were also tested for NKcell activities after single or multiple IFN-a injections. Therewas a highly significant and consistent increase in NK cellcytotoxicity of patients with the NK-LR and NK-MR phenotypesfollowing one injection of IFN-a. In contrast to the NK-LR andNK-MR group of patients, most patients displaying the NK-HRphenotype failed to show any NK cell augmentation followingany number of IFN-a injections (up to 119).

We have also tested NK cell activities of cancer patientsundergoing treatment with low (3 x 106 units) and high (36 x106 and 50 x 106 units) doses of clone A of recombinant IFN-a (IFN-arA). In these studies, all patients receiving the lowerdose of IFN-arA and three of five patients receiving the higherdoses of IFN-arA displayed augmented NK cell activity 24 hrafter a single i.m. injection. Analysis of patients receiving thelow dose of IFN-arA in a single-cell assay suggested that thisagent did not modulate tumor-NK cell-binding properties butaugmented significantly NK cell cytotoxicity and killing of already-bound tumor targets.

INTRODUCTIONIFNs4 generated recently considerable interest in the scien

tific and clinical community because of their indicative antineo-plastic properties in experimental animal systems and in humans (10, 18, 41, 42, 45). In animals, these agents have beendemonstrated to delay or prevent development of chemically

1This research was supported by Grants CA 21062 and CA 14528 from theNational Cancer Institute.

2 To whom requests for reprints should be addressed.3 Recipient of Rosalie B. Hite Postdoctoral Fellowship.4 The abbreviations used are: IFN, interferoni NK, natural killer; IFN-a, inter

feron-a; IFN-arA. clone A of recombinant interferon-o; RPMI1640. Roswell ParkMemorial Institute Tissue Culture Medium 1640; T:E, target:effector; NK-HR, NK-MR, and NK-LR, high, medium, and low NK cell responder, respectively.

Received August 5, 1981; accepted March 9, 1982.

(41) and virally (42) induced tumors and to inhibit the growthof transplantable tumors (1, 11, 12, 50). In humans, IFNs havebeen shown to exert antitumor effect in cancers, such asmultiple myeloma (18, 27, 37), Hodgkin's lymphoma (2), andbreast cancer (18). However, in spite of the active research onthe mechanism of the action of IFNs, their antitumor effect isstill poorly understood. IFNs were demonstrated to inhibit tumorcell multiplication (13,14,16, 24), and thus, one of the possiblemechanisms of their antitumor action could be the direct effecton tumor cells. This, however, does not appear to be the onlymechanism by which IFNs exert antitumor activities. It has beenshown that IFN-mediated antineoplasm protection is not limitedto IFN-sensitive tumors but is exhibited also to IFN-resistanttumors (15). Since the role of the immune system in hostdefense against cancers has become increasingly evident, it isplausible that IFNs operate also via augmentation of the immune responses of the host. In fact, IFNs augment specificcell-mediated cytotoxicity (31 ) and NK cell-mediated cytotoxicity (19, 20, 22, 39, 48), the 2 functions perhaps most relevantin anticancer immunity. NK cells appear to represent goodcandidates in antitumor immunity (especially in immunosurveil-lance) because of their natural occurrence, prompt reactivityagainst various types of tumors, and high activities in congen-itally athymic mice, which, in spite of the lack of mature T-cellfunctions, do not experience a higher incidence of cancersthan their euthymic littermates (21, 29, 33, 34). The latterobservation places NK cells in a superior position to T-cells.Moreover, there is a good correlation between the growth oftumors and their metastasizing properties and levels of NK cellactivities (30, 40, 46, 47).

It is important to indicate in this context that IFNs not onlyaugment but also inhibit various immune phenomena; e.g., theysuppress lymphocyte responses to mitogens and alloantigens(32), inhibit antibody formation (4, 8, 28, 44), and depressdelayed hypersensitivity (6) and allograft reactions (38). Additionally, it has been shown that tumor cells treated with IFNs invivo or in vitro became resistant to NK cell-mediated cytotoxicity (49). Thus, the modulation of antitumor immunity by IFNsand the mechanism of such modulation has to be furtherinvestigated.

Because NK cells have been implicated in tumor immuno-surveillance and defense and IFNs have been applied as oneof the cancer therapies in our Institute, we have studied theeffect of exogenously administered IFN-a and IFN-arA on peripheral blood NK cell cytotoxicity of cancer patients. Additionally, we have studied the distribution patterns of NK cell phenotypes among normal individuals and cancer patient populations.

MATERIALS AND METHODS

Healthy Donors and Cancer Patients. Thirty-five healthy donors,

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NK Cell Cytotoxicity

employees of M. D. Anderson Hospital and Tumor Institute, were usedin these studies. They included individuals of both sexes, in the agerange of 26 to 53 years. Since no correlation between the sex or ageof normal donors and their NK cell activities was observed, the dataare not categorized according to these parameters.

Cancer patients included 28 individuals with solid tumors (10 patients with breast cancer, 6 with oat cell carcinoma, 3 with ovariancancer and multiple myeloma, 2 with Hodgkin's disease, and one each

with lymphocytic sarcoma, pharyngeal papilloma, colon, and prostatecancer) and 14 patients with leukemia (7 with acute myelogenousleukemia, 3 with chronic myelogenous leukemia, 2 with acute lymphocytic leukemia, and one each with acute myelomonocytic and acutepromyelocytic leukemia). Patients were treatment free at least 1 monthbefore the NK cell test.

Preparation of Effector Cells. Peripheral blood mononuclear cellsof normal donors and cancer patients were separated from heparinizedvenous blood by a Ficoll-Hypaque (Ficoll was from Pharmacia FineChemicals, Uppsala, Sweden; Hypaque was from Winthrop Laboratories, New York, N. Y.) gradient technique as described previously (3).The cells were then washed twice in Hanks' solution (Flow Laboratories, Inc., McLean, Va.) and diluted to required concentration in RPMI1640 (Flow Laboratories), supplemented with 10% fetal calf serum(Gibco Laboratories, Santa Clara, Calif.), 4-(2-hydroxyethyl)-1-pipera-zineethanesulfonic acid buffer (Flow Laboratories), and antibiotics (500units of penicillin and 50 ;ug of streptomycin per ml). This medium isdesignated as supplemented RPMI 1640.

Target Cells. Chronic myelogenous leukemia cell line K-562 (36)and T-lymphoblastoid cell line CEM were used as targets in all experiments. These cell lines were grown as suspension cultures at 37 ina 5% CO2 humidified atmosphere in supplemented RPMI 1640. Bothtarget cell lines were found to be Mycoplasme free.

Assays for NK Cell Cytotoxicity and Tumor Cell Binding. Thecytotoxicity assay was performed as described previously (35). Briefly,5 million target cells suspended in 0.5 ml of supplemented RPMI 1640were labeled with 5'Cr (Na/'CrO.,). Fifty i\of labeled target cells and100 /il of effector cells (T:E cell ratio, 1:50) were then incubated inquadruplicates in round-bottomed microtiter plates (Linbro Scientific,Inc., Hamden, Conn.) at 37 in 5% CO2 humidified atmosphere for 4hr. The percentage of cytotoxicity was calculated according to theformula:

[(% - %S)/(%M - %S)] x 100where %is the percentage of 5'Cr release obtained in the cultures oftarget cells and lymphocytes and %S is the percentage of spontaneousrelease, obtained from cultures containing 51Cr-labeled target cellsalone or 51Cr-labeled and unlabeled target cells, in the same ratio astarget and effector cells in experimental cultures. The latter cultureswere done in order to correct for cell densities in the cultures. Nodifference has been found between these 2 types of spontaneouscultures. %M is the maximum percentage of 51Cr release, obtained byfreezing and thawing the target cells 4 times. The spontaneous releaseranged from 5 to 9%, and the maximum release ranged from 90 to97%, for both the K-562 and CEM target cell lines. Standard error ofthe mean of replicates was 50% to both of thesetarget cell lines, at a T:E cell ratio of 1:50. Similar variabilitywas seen also with other T:E cell ratios. From these studies,however, it was not clear whether such variability in NK cellcytotoxicity was a true reflection of a difference in NK cellresponsiveness among normal donors or whether the cytotoxicity of a single donor tested on various occasions fluctuatedsimilarly. To examine this question, we have tested NK cellactivities of 13 normal donors and 2 patients (who were at thistime treatment free) repeatedly, 3 to 7 times, over a periodencompassing several months. For logistic reasons, more patients who would be untreated for a longer time period werenot available. Results of these experiments are illustrated inTable 1. It can be seen clearly that NK cell cytotoxicity to bothtarget cell lines varied to some extent within multiple tests of asingle individual; however, it was always significantly below thevariability of NK cell cytotoxicity observed among differentdonors. This was confirmed statistically by an analysis ofvariance test (see legend of Table 1). Additionally, despitecertain degrees of fluctuation in NK cell cytotoxicity of a singleindividual, the NK cell phenotype of that individual remainedinvariably unchanged. Specifically, NK-HRs retained constantly their NK-HR status, and NK-MRs and NK-LRs retainedtheir NK-MR and NK-LR status, respectively. Two cancer pa-

JUNE 1982 2481

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E. Lotzov et al.Table 1

Serial tests of NK cell cytotoxicity of a single individual% of NK cellcytotoxicity"Type

ofdonorNormal

12345678910111213Patients0

12Target

celllineK-562

CEMK-562

CEMK-562

CEMK-562

CEMK-562

CEMK-562K-562K-562

CEMK-562K-562K-562

CEMK-562

CEMK-562K-562

CEMK-5621*20.5

12.933.1

27.470.7

29.481.0

49.264.0

25.973.052.039.3

19.161.025.066.2

58.067.3

56.016.259.4

32.921.5227.3

11.029.0

29.070.5

31.679.8

52.672.0

21.354.750.030.4

21.257.421.572.8

55.048.6

60.516.358.9

30.425.1335.0

17.937.0

37.077.0

20.083.8

44.150.7

34.470.077446.1

28.759.030.879.3

53.152.0

49.823.760.3

38.329.74

534.5

42.011.623.526.0

24.036.823.862.0

34.326.082.8

92.865.649.064.5

37.380.352.476.9

41.6Mean

6 7 S.E.31.9

3.712.1 16.7 15.1 1.729.8

2.428.0 25.6 29.7 2.070.0

3.130.8 28.7 28.7 1.784.0

2.370.1 55.1 4.262.8

4.429.7 3.769.5

5.457.96.538.64.5

23.0 2.959.11.025.82.772.83.8

55.4 1.456.05.8

55.4 3.118.72.563.9

4.435.8 2.525.4

2.41 NK cell cytotoxicity was tested in a 4-hr 5lCr release cytotoxicity assay; T:E cell ratio was 1:50.

Analysis of variance indicated that the difference in NK cell variability of multiple tests of a single individualwas significantly lower than that among various individuals for both cell lines (p < 0.001). Furthermore,when evaluated by a paired t test, no significant change was found among multiple tests of single individuals.

Number of individual tests.c Two solid-cancer patients were treatment free during serial testing.

tients tested serially also exhibited relatively close values of NKcell cytotoxicity.

In contrast, a great degree of variability in NK cell cytotoxicitywas observed among different donors. In fact, when NK cellcytotoxicity of the entire normal donor population was analyzed, these could be classified on the basis of their NK cellresponsiveness to a given tumor target, as NK-LRs, NK-MRs,and NK-HRs. The NK cell cytotoxicity values for NK-LRs, NK-MRs, and NK-HRs were divided arbitrarily into groups of 45%, respectively, at a 1:50 T:E cell ratio. Theresults of such analysis and the NK cell dose-response curvesof normal donors with various NK cell phenotypes are illustratedin Chart 1. It can be seen from the chart that the NK cell dose-response curves of each class of NK cell responders, i.e., NK-HRs, NK-MRs, and NK-LRs, were significantly distinct (at allT:E cell ratios tested) from the other 2 classes (for statisticalanalysis, see legend of Chart 1). It is also evident that theslopes of the NK cell dose-response curves of the NK-HRs,and NK-MRs (to both K-562 and CEM target cells) did notdiffer significantly from each other (evaluated by analysis ofcovariance, as represented by Student's f test for comparison

of slopes), an observation suggesting that the lower NK cellresponse of the NK-MR group may be due to a relative decrease in the number of active NK cells. In contrast, the slopeof the NK cell dose-response curve of NK-LRs deviated significantly from that of NK-HRs and NK-MRs. If the low NK cellactivity of NK-LRs was due merely to reduced numbers of NKcells, lower but parallel slopes of dose-response curves wouldbe expected. Thus, it is possible that NK-LRs may be experiencing a possible aberration in NK cell activities. This is,however, only a suggestion which must be further exploredexperimentally. If NK cells indeed prove to be important constituent of defense against tumors, the disclosure of NK-LRscould be of extreme importance.

Even though the normal individuals exhibited NK-LR, NK-MR, and NK-HR phenotypes to both the K-562 and CEM tumorcell lines, the distribution of these phenotypes to the 2 tumorsdiffered. As illustrated in Table 2, only 11 % of the normal donorpopulation displayed the NK-LR status to K-562, whereas 40%exhibited the same responder status to CEM. Differences between the NK-HR phenotypes to CEM and K-562 were alsoobserved; i.e., 52% of normal donors were NK-HRs to K-562,

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E 60

1 7 11 21 017 01NUMBEROF IFN-fflINJECTIONS

7 11 21

fe

017 01 01 017 11 21NUMBEROF IFIl-OtlNJECTIONS

Chart 1. NK cell dose-response curves of normal individuals and leukemicpatients. Thirty-five normal individuals and 14 leukemic patients were tested.Points, mean percentage of NK cell cytotoxicity of NK-HRs, >45% ();NK-MRs.20 to 45% (9); NK-LRs,

E. Lotzovet al.the NK cell dose-response curves, dilution of NK cells inleukemic patients does not appear to be the explanation fortheir NK-LR status. Instead, the NK-LR status of leukemicpatients could reflect an NK cell aberrancy which underliesleukemia. Since NK cells exhibit strongest reactivity to tumorsof hemopoietic origin in vitro, it is possible that they are alsoinvolved in defense against hemopoietic cancers in vivo.

Another interesting observation originating from our studieswas that a single normal donor, as well as a cancer patient,could exhibit different NK phenotypes to the K-562 and CEMtarget cell lines; i.e., a single donor could be a NK-HR to onetarget cell line and a NK-MR or NK-LR to the other. Chart 2diagrammatically illustrates distribution of NK cell phenotypes(and their combinations) to both target cell lines among thenormal donor and cancer patient populations. Similarly to theresults obtained with NK cell reactivity to a single cell line (seeTable 2), the distribution of combined (to both cell lines) NKcell phenotypes of a single individual differed between normaldonors and cancer patients. In normal donors, only 7% ofindividuals were of the LR/LR (K-562/CEM) phenotype,whereas 17% of cancer patients were of the NK-LR phenotypeto both targets. Also, the MR/LR and LR/MR phenotypes wereprevalent in cancer patients (42%), in comparison to normalindividuals (14%). Reversely, HR/HRs and MR/HRs or HR/MRs were predominant in the normal donor population (64%)but not in the cancer patient population (34%). These observations suggest that cancer patients may be deficient in theirNK cell activities.

Effect of Exogenously Administered IFN-a on NK CellCytotoxicity of Cancer Patients. In this facet of the study, wehave investigated the effect of exogenously administered IFN-a on peripheral blood NK cell cytotoxicity of 18 cancer patientswith a variety of cancers (see "Materials and Methods"). NK

cell activities were tested before and after a single or multiplei.m. injections of 3 x 106 units of IFN-,always 24 hr aftereach injection. The NK cell activity of some patients wasfollowed through as many as 146 injections. Since no difference in NK cell reactivity of patients receiving 26 or more IFN-a injections was observed and only one patient was availableat each point, the data were pooled, and a single group,designated as >26 injections, is presented. In order to facilitatethe interpretation of the data, i.e., not to fail to detect NK cellmodulation, due to the wide range of NK cell cytotoxicity (from8 to 70%), the cancer patients were divided into NK-HR, NK-MR, and NK-LR categories, on the basis of their NK cellpretreatment levels. The NK-LR and NK-MR groups of patientswere then combined, since no difference in NK cell modulationbetween these 2 groups of cancer patients was observed.

The results of these studies are illustrated in Chart 3. Following one injection of IFN-a, there was a highly significant andconsistent increase in NK cell cytotoxicity of all patients withthe NK-MR and NK-LR phenotypes (Chart 3, A and B); theaverage cytotoxicity increased from 24% before the treatmentto greater than 52% after IFN-a injection to the K-562 cell lineand from 15 to 30% to the CEM cell line. Following multipleIFN-a injections (i.e., 7, 14, 21, and >26), the average NK cellcytotoxicity remained significantly increased but was not further potentiated above the single IFN-a injection level. However, a certain degree of variability was observed among individual patients. Specifically, 4 of 9 patients tested after multipleIFN-a injections did not exhibit any increase in cytotoxicityabove that observed at the single injection level, whereas NKcell cytotoxicity of 5 patients was potentiated further withmultiple injections (4 to K-562 and one to CEM). Two of thepatients tested after 7 injections returned to pretreatment values; however, they were again potentiated with further injec-

E 60

0 0

wnu OF im-a lucciimsChart 3. Effect of single and multiple injections of IFN-a on NK cell cytotoxicity of cancer patients. Points, responses of individual NK-LR and NK-MR patients to

K-562 (A.) and CEM (B) and of NK-HR patients to K-562 (C) at 1:50 T:E cell ratio; , average NK cell response of patients. The statistical analysis of changes inNK cell responsiveness following IFN-a treatment was evaluated by paired r test analysis. The p values for NK-LRs and NK-MRs to the K-562 tumor target cell linewere

A/K Cell Cytotoxicity

TO TARGET CELL RATIO

Chart 4. Comparison of NK cell Cytotoxicity of normal donors and cancerpatients tested simultaneously before and after single injection of IFN-a. Eightdifferent patients (,)and 24 normal donors (O) were tested in 24 differentexperiments; additionally, some of the normal donors .were tested on severaloccasions ( ), in order to demonstrate the consistency of their response, (bars,S.E. of multiple tests of a single individual). NK cell Cytotoxicity was testedagainst the K-562 target cell line (1:50 T:E cell ratio) in a 4-hr 51Cr release assay.

tions (Chart 3). That the increase in NK cell Cytotoxicity wasmediated by IFN-a and could not be contributed to the fluctuation of NK cell Cytotoxicity is indicated: (a) by changes in NKcell Cytotoxicity always in a positive direction (in contrast tonormal individuals exhibiting equal changes in NK cell cytotox-icity in both positive and negative directions), as establishedby paired f test analysis; and (b) by the differences in thedegree of change of NK cell Cytotoxicity of IFN-a-treated patients. Specifically, in NK-LR and NK-MR patients, the p valuesbetween pretreatment and posttreatment levels of Cytotoxicitywere always statistically significant (except for NK cell response of patients to CEM after 21 injections), whereas thedifferences in NK cell Cytotoxicity of normal individuals testedon various occasions were not statistically different (as determined by paired i test analysis; see legend of Chart 3 andTable 1). Additionally, as can be seen in Chart 4, when IFN-a-treated patients were tested simultaneously with normal individuals (a different normal donor was tested at each point) inan experiment, highly dichotomous patterns of NK cell activitiesbetween normal individuals and cancer patients were observed. Namely, the high (or low) levels of NK cell Cytotoxicityof IFN-a-treated patients did not coincide with high (or low) NKcell Cytotoxicity levels of normal individuals (even though eachof the normal individuals, when tested at various occasions,retained constant levels of NK cell Cytotoxicity, as indicated bythe bars). This suggests that changes in patients' NK cellCytotoxicity after IFN-a treatment could not be attributed tofluctuation in Cytotoxicity values due to the variability in the NKcell assay since, under the latter conditions, the NK cell Cytotoxicity of both patients and normal donors should fluctuate inthe same (and not in a dichotomous) direction. Further evidence eliminating fluctuation in NK cell assay as a cause ofincreased NK cell Cytotoxicity after IFN treatment will be presented in the section concerned with modulation of NK cellCytotoxicity by IFN-arA.

Although NK cell stimulation was exhibited to both target celllines, the duration of stimulation to K-562 extended beyondthat to CEM. Namely, stimulation to the K-562 cell line was stillevident after >26 injections, while that to the CEM cell line wasnot detected after 21 injections. Two of the cancer patients inthe NK-LR and NK-MR categories (lymphocytic sarcoma andchronic lymphocytic leukemia) did not show any stimulation ofNK cell Cytotoxicity by IFN-a after 21 and >26 IFN-a injections,respectively (data not shown). In contrast to the NK-LR andNK-MR group of patients, only 2 patients of those displayingthe NK-HR phenotype to K-562 showed sensitivity to NK cellaugmentation following multiple IFN-a injections (Chart 3C). Infact, some NK-HR patients exhibited decreased NK cell activities after IFN-a injections. It is important to mention in thiscontext that the NK-HR cancer patients also showed aberrantNK cell dose response when compared to NK-HR normalindividuals (data not shown); i.e., practically no increase in NKcell Cytotoxicity with increasing T:E cell ratio was observed;the Cytotoxicity reached already high levels with low T:E cellratios. It is possible that the NK-HR phenotype of cancerpatients is not their natural NK cell status but hyperactivity ofNK cells to tumors. Consequently, such hyperactive NK cellsmay not be sensitive to IFN-a stimulation.

Effect of Exogenously Administered IFN-arA on NK CellCytotoxicity of Cancer Patients. We have also tested NK cellactivities of cancer patients undergoing IFN-arA therapy. Thesepatients received a single i.m. injection of 3 x 106, 36 x 106,or 50 x 106 units of IFN-arA. In the first set of experiments,peripheral blood lymphocytes of 3 cancer patients who received 3 x 106 units of IFN-arA were tested before and 24 hrafter injection for NK cell cytoxicity in a 51Cr release Cytotoxicityassay and for NK cell-tumor cell-binding properties and -killingpotential in a single-cell assay. K-562 tumor was used astarget. As shown in Table 3, no significant changes wereobserved in tumor cell-NK cell-binding properties after a singleIFN-arA injection in any patient. This indicates that IFN-arAdoes not potentiate the binding of NK cells to tumor targets.However, there was a significant augmentation of NK cellCytotoxicity (as determined by a 51Cr release assay) and killingof already-bound tumor targets (as determined by a single-cellassay) 24 hr after IFN-arA treatment in all 3 patients.

Table 3Effect of exogenously administered IFN-arA on the peripheral blood NK cell

activity of cancer patients

Patient123Treatment0None

IFN-arANone

IFN-arANone

IFN-arA%

of tumor-bindingcells7.410.613.3

9.57.08.0%

of cytotoxictumor-binding

cells"20.9

44.4C22.1

33.3d16.3

25.7e%

of51CrCytotoxicity42.6

62.7C26.238.0o26.7

39.2e

Patients were tested for NK cell Cytotoxicity and tumor-binding propertiesbefore and 24 hr after a single i.m. injection of IFN-arA (3 x 106 units). Both a51Cr release Cytotoxicity assay and single-cell assay were used (see "Materialsand Methods").

6 K-562 tumor cell line was used as target at a T:E cell ratio of 1:12 (5'Crrelease Cytotoxicity assay) and 1:1 (single-cell assay).

c' 'e Changes in NK cell Cytotoxicity after IFN-arA treatment were analyzedstatistically by Fisher exact test. The p values were

E. Lotzov et al.Table 4

Kinetics of NK cell cytotoxicity after a single injection of high doses of IFN-arA

Experiment1234Type ofdonorPatient

1Patient2Normal

Donor1Patient

3NormalDonor2Patient

4NormalDonor3Patient

5NormalDonor 4Dose

ofIFN-arA

(unitsX106)5050505036%

of NK cellcytotoxicity3O642.712.869.814.049.89.860.314.921.34

88.6C18.30.6C

2.8d69.777.711.151.45.060.21.8C19.92435.622.6e69.837.8e43.267.3e68.022.918.5

3 K-562 tumor cell line was used as a target at a T:E cell ratio of 1:50 in a 3-hr 5'Cr release cytotoxicity assay. The differences between pretreatment andpost-IFN-arA treatment values were evaluated statistically by Fisher exact test.All differences not footnoted were not statistically significant. Mononuclear cellsof normal donors were tested simultaneously with each patient. At Time 0, freshcell suspensions were used, and refrigerated suspensions of the same normaldonor were tested again 4, 8. and 24 hr later, together with mononuclear cells ofIFN-arA-treated patients. No statistically significant differences were found between various tests of a single normal donor.

Time after single injection of IFN-arA (hr).ep< 0.001.

p < 0.01.* p < 0.02.

In the second series of experiments, NK cell cytotoxicity of4 patients who received a single injection of 50 x 106 unitsand one patient administered a single injection of 36 x 106units of IFN-arA was tested before and 4, 8, and 24 hr after thetreatment. In these experiments, the same normal donor wastested for NK cell cytotoxicity simultaneously with each patientat all time intervals in order to assess variability in NK cellassay. A fresh mononuclear cell sample of the normal donorwas tested at Time 0, and a refrigerated sample of the samenormal donor was tested again at the later time intervals. Theresults of these experiments are illustrated in Table 4. All ofthe patients tested exhibited a decrease in NK cell cytotoxicity4 and 8 hr after IFN-arA injection in comparison to theirpretreatment values (the cause of such a decrease is currentlybeing investigated). However, NK cell cytotoxicity of 3 of 4patients receiving 50 x 106 units of IFN-arA, i.e., Patients 2, 3,and 4, was significantly increased 24 hr after the injection(stimulation index was 1.8, 2.7, and 6.9, respectively). The NKcell cytotoxicity of Patient 1 who received 50 x 106 units ofIFN-arA and Patient 5 who received 36 x 106 units of IFN-arAwas not augmented in comparison to pretreatment values butwas completely restored after the initial 4- to 8-hr decline. Ascan be seen by the consistent decline in NK cell cytotoxicity at4 and 8 hr post-IFN-arA treatment and by the highly reproducible cytotoxicity values of normal individuals in each experiment, the augmentation of NK cell cytotoxicity could not beattributed to variability in NK cell assay.

These results indicate that IFN-arA also augments NK cellcytotoxicity and consequently may be a useful potentiator ofantitumor immunity. More patients are currently being studiedto determine duration of this effect, as well as the effect ofmultiple IFN-arA injections on NK cell activities.

DISCUSSION

The present investigation has shown that both normal donorsand cancer patients can be grouped into NK-HR, NK-MR, and

NK-LR categories, with regard to their degree of NK cellactivities. However, in general, NK-HRs were found to beprevalent in normal donor population, whereas NK-LRs werefound in cancer patients. The relevant question raised, but notanswered for obvious reasons by our studies, is whether thelow NK cell responsiveness of normal donors could be indicative of their susceptibility to cancers (especially leukemias,which appear to be the most sensitive NK cell targets). Thereis certainly a similarity between the NK cell dose-responsecurves of NK-LR normal donors and leukemic patients, both ofwhich significantly deviate from those of NK-HR and NK-MRnormal individuals. This suggests that NK cell activities of NK-LR normal individuals and leukemic patients may be aberrant.If NK cells indeed will prove to be an important component ofthe immunosurveillance mechanism, then such NK cell deficiency could reflect higher susceptibility of NK-LR normalindividuals to cancer. Another important question is whetherthe levels of NK cell cytotoxicity of cancer patients could beused as a prognostic factor in progression or relapse of thedisease. Our unpublished observations on the decline of NKcell cytotoxicity several weeks before relapse of leukemia couldbe indicative of possible significance of NK cells in the prognosis of this disease.

The recognition of NK cell phenotypes within the humanpopulation may also have relevance to the more precise evaluation of various biological response modifiers (and therapeuticagents in general) on NK cell modulation. The effectiveness ofthese agents may not be quite apparent when the data of theindividuals with wide range of NK cell reactivity are combined.As a matter of fact, such division of patients undergoing IFN-atherapy allowed us to observe the differential effect of IFN-o onNK cell cytotoxicity of NK-LR and NK-MR patients versus NK-HR patients. Whereas the former 2 classes of NK cell respond-ers were stimulated in their NK cell activities, only 2 of 6 of thelatter class of responders were augmented by multiple IFN-ainjections. In fact, a decrease in NK cell cytotoxicity of the NK-HRs was observed after multiple IFN-a injections. The NK cellcytotoxicity of the NK-LRs and NK-MRs was augmented already after a single IFN injection and in 44% of patients wasnot further potentiated with additional injections but remainedat the augmented levels. These data are compatible with thoseof Einhorn et al. (7) and Huddlestone e-al. (26) who alsoobserved similar augmentation of NK cell cytotoxicity after asingle IFN injection. The former group of investigators, incontrast to our studies, did not observe any dependence of NKcell augmentation on the pretreatment NK cell levels. However,the degree of NK cell cytotoxicity in the studies of these authorsnever reached the high levels of NK cell cytotoxicity displayedby NK-HR patients in our studies; they resembled rather theNK-MRs and NK-LRs. This could be contributed most likely tothe different target cells used in these 2 investigations (Changversus K-562 target cells).

There was no correlation between IFN-a-mediated NK cellaugmentation and the type of cancer, since the cytotoxicity ofpatients with various histological types of cancer was augmented with the exception of perhaps lymphoid type of tumors.One patient with lymphocytic sarcoma and one with chroniclymphocytic leukemia did not show any augmentation of theirNK cell activities after IFN-a treatment. There also was nocorrelation between NK-LR status and previous therapies. Furthermore, no relationship was found between certain types of

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NK Cell Cytotoxicity

cancer and the NK cell phenotype. No correlation was observed between IFN-a-mediated potentiation of NK cell cyto-toxicity and age, sex, leukocyte counts, proportion of lymphocytes and monocytes, or changes in clinical status of thedisease.

Preliminary results have shown that NK cell cytotoxicity ofmost cancer patients receiving IFN-arA therapy was also augmented 24 hr after the injection, as measured by 2 independentassays. Tumor cell-NK cell-binding properties were not modulated by IFN-arA in these studies, an observation suggestingthat the NK cell augmentation was not mediated via recruitmentof NK cell precursors but rather via induction of the lyticmachinery of preexisting NK cells.

It has been recognized that the NK cell assay is subject tovariability. However, the increase in NK cell cytotoxicity in ourexperiments cannot be attributed to NK cell variability forseveral reasons, as have been discussed in detail in "Results."(a) No correlation (but a dichotomy) in the levels of NK cellcytotoxicity was observed between IFN-a-treated patients andnormal donors, when these 2 groups of individuals were testedsimultaneously (see Chart 4). (b) The degree of increase afterIFN stimulation was always significantly greater than that observed in most normal individuals when tested alone serially(Table 1). (c) Augmentation has been detected by 2 independent assays (i.e., by a 51Cr release and single-cell assay; Table3). (d) When the same normal donor was tested with eachpatient in 4 different NK cell assays, the NK cell cytotoxicityvalues of a normal donor were highly consistent and reproducible, whereas those of patients were either increased (24 hrafter injection) or decreased (4 to 8 hr after injection) (Table4). This and other experimental evidence, indicated in"Results," argue strongly against the possibility that the NKcell increase after IFN treatment was due to the variability inNK cell assay.

The observation that NK cell cytotoxicity was augmentedsignificantly during IFN-a and IFN-arA therapy of cancer patients suggests that the beneficial effect of IFNs could bemediated at least partially via immune activities and that NKcells may be one of the components of such an antitumormechanism.

ACKNOWLEDGMENTSThe authors wish to acknowledge Lily K. Muesse, D. Hearn, and J. McDowell

for technical assistance.

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