Modulation of Natural Killer Cell-mediated Cytotoxicity by

Partially Purified and Cloned Interferon-

Eva Lotzov, Cherylyn A. Savary, Jordan U. Gutterman, et al.
Cancer Res 1982;42:2480-2488.

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[CANCER RESEARCH 42, 2480-2488.

0008-5472/82/0042-OOOOS02.00

June 1982]

Modulation of Natural Killer Cell-mediated Cytotoxicity by Partially

Purified and Cloned Interferon-1
Eva Lotzov,2 Cherylyn A. Savary,3 Jordan U. Gutterman, and Evan M. Hersh
Department of Clinical Immunology and Biological
Texas Medical Center, Houston, Texas 77030

Therapy, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute,

ABSTRACT
We have studied peripheral blood of 35 normal individuals,
28 solid-cancer patients, and 14 leukemic patients for natural
killer (NK) cell cytotoxicity to K-562 and CEM tumor cells in a
51Cr release cytotoxicity assay. We have found that both normal
individuals and solid-cancer patients could be grouped into
high (NK-HR), medium (NK-MR), and low (NK-LR) NK cell
responder categories with regard to their degree of NK cell
activities. However, in general, NK-HRs were found to be
prevalent in normal donor population and NK-LRs in cancer
patients. Leukemic patients always exhibited the NK-LR status.
The difference between NK-HRs and NK-MRs appeared to be
due to the relative decrease in number of active NK cells. In
contrast, the NK-LR status could not be contributed to de
crease of NK cells because of the dichotomy in NK cell doseresponse patterns of NK-LRs versus those of NK-HRs and NKMRs. Eighteen cancer patients undergoing interferon-a (IFNa) therapy (3 x 106 units, i.m., daily) were also tested for NK
cell activities after single or multiple IFN-a injections. There
was a highly significant and consistent increase in NK cell
cytotoxicity of patients with the NK-LR and NK-MR phenotypes
following one injection of IFN-a. In contrast to the NK-LR and
NK-MR group of patients, most patients displaying the NK-HR
phenotype failed to show any NK cell augmentation following
any number of IFN-a injections (up to 119).
We have also tested NK cell activities of cancer patients
undergoing treatment with low (3 x 106 units) and high (36 x
106 and 50 x 106 units) doses of clone A of recombinant IFNa (IFN-arA). In these studies, all patients receiving the lower
dose of IFN-arA and three of five patients receiving the higher
doses of IFN-arA displayed augmented NK cell activity 24 hr
after a single i.m. injection. Analysis of patients receiving the
low dose of IFN-arA in a single-cell assay suggested that this
agent did not modulate tumor-NK cell-binding properties but
augmented significantly NK cell cytotoxicity and killing of al
ready-bound tumor targets.
INTRODUCTION
IFNs4 generated recently considerable

interest in the scien

tific and clinical community because of their indicative antineoplastic properties in experimental animal systems and in hu
mans (10, 18, 41, 42, 45). In animals, these agents have been
demonstrated to delay or prevent development of chemically
1 This research was supported by Grants CA 21062 and CA 14528 from the
National 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 Park
Memorial Institute Tissue Culture Medium 1640; T:E, target:effector; NK-HR, NKMR, and NK-LR, high, medium, and low NK cell responder, respectively.
Received August 5, 1981; accepted March 9, 1982.

2480

The

(41) and virally (42) induced tumors and to inhibit the growth
of transplantable tumors (1, 11, 12, 50). In humans, IFNs have
been shown to exert antitumor effect in cancers, such as
multiple myeloma (18, 27, 37), Hodgkin's lymphoma (2), and
breast cancer (18). However, in spite of the active research on
the mechanism of the action of IFNs, their antitumor effect is
still poorly understood. IFNs were demonstrated to inhibit tumor
cell multiplication (13,14,16, 24), and thus, one of the possible
mechanisms of their antitumor action could be the direct effect
on tumor cells. This, however, does not appear to be the only
mechanism by which IFNs exert antitumor activities. It has been
shown that IFN-mediated antineoplasm protection is not limited
to IFN-sensitive tumors but is exhibited also to IFN-resistant
tumors (15). Since the role of the immune system in host
defense against cancers has become increasingly evident, it is
plausible that IFNs operate also via augmentation of the im
mune responses of the host. In fact, IFNs augment specific
cell-mediated cytotoxicity (31 ) and NK cell-mediated cytotox
icity (19, 20, 22, 39, 48), the 2 functions perhaps most relevant
in anticancer immunity. NK cells appear to represent good
candidates in antitumor immunity (especially in immunosurveillance) because of their natural occurrence, prompt reactivity
against various types of tumors, and high activities in congenitally athymic mice, which, in spite of the lack of mature T-cell
functions, do not experience a higher incidence of cancers
than their euthymic littermates (21, 29, 33, 34). The latter
observation places NK cells in a superior position to T-cells.
Moreover, there is a good correlation between the growth of
tumors and their metastasizing properties and levels of NK cell
activities (30, 40, 46, 47).
It is important to indicate in this context that IFNs not only
augment but also inhibit various immune phenomena; e.g., they
suppress lymphocyte responses to mitogens and alloantigens
(32), inhibit antibody formation (4, 8, 28, 44), and depress
delayed hypersensitivity (6) and allograft reactions (38). Addi
tionally, it has been shown that tumor cells treated with IFNs in
vivo or in vitro became resistant to NK cell-mediated cytotox
icity (49). Thus, the modulation of antitumor immunity by IFNs
and the mechanism of such modulation has to be further
investigated.
Because NK cells have been implicated in tumor immunosurveillance and defense and IFNs have been applied as one
of the cancer therapies in our Institute, we have studied the
effect of exogenously administered IFN-a and IFN-arA on pe
ripheral blood NK cell cytotoxicity of cancer patients. Addition
ally, we have studied the distribution patterns of NK cell phe
notypes among normal individuals and cancer patient popula
tions.
MATERIALS

AND

METHODS

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

CANCER

RESEARCH

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VOL. 42

NK Cell Cytotoxicity
employees of M. D. Anderson Hospital and Tumor Institute, were used
in these studies. They included individuals of both sexes, in the age
range of 26 to 53 years. Since no correlation between the sex or age
of normal donors and their NK cell activities was observed, the data
are not categorized according to these parameters.
Cancer patients included 28 individuals with solid tumors (10 pa
tients with breast cancer, 6 with oat cell carcinoma, 3 with ovarian
cancer and multiple myeloma, 2 with Hodgkin's disease, and one each

was 1 x 106 units/mg of protein. IFN-a was administered to the

patients daily in the dose of 3 x 106 units i.m. All patients were

with lymphocytic sarcoma, pharyngeal papilloma, colon, and prostate

cancer) and 14 patients with leukemia (7 with acute myelogenous
leukemia, 3 with chronic myelogenous leukemia, 2 with acute lympho
cytic leukemia, and one each with acute myelomonocytic and acute
promyelocytic leukemia). Patients were treatment free at least 1 month
before the NK cell test.
Preparation of Effector Cells. Peripheral blood mononuclear cells
of normal donors and cancer patients were separated from heparinized
venous blood by a Ficoll-Hypaque (Ficoll was from Pharmacia Fine

2 with colon cancer; and one each with prostate, pharyngeal papilloma,
lymphocytic sarcoma, chronic lymphocytic leukemia, and lymphoma.
Peripheral blood samples were obtained from each patient before IFNa therapy and then at 24 hr after 1,7, 14, 21, or >26 IFN-a injections.

Chemicals, Uppsala, Sweden; Hypaque was from Winthrop Laborato

ries, New York, N. Y.) gradient technique as described previously (3).
The cells were then washed twice in Hanks' solution (Flow Laborato
ries, Inc., McLean, Va.) and diluted to required concentration in RPMI
1640 (Flow Laboratories), supplemented with 10% fetal calf serum
(Gibco Laboratories, Santa Clara, Calif.), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (Flow Laboratories), and antibiotics (500
units of penicillin and 50 ;ug of streptomycin per ml). This medium is
designated 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 exper
iments. These cell lines were grown as suspension cultures at 37 in
a 5% CO2 humidified atmosphere in supplemented RPMI 1640. Both
target cell lines were found to be Mycoplasme free.
Assays for NK Cell Cytotoxicity and Tumor Cell Binding. The
cytotoxicity assay was performed as described previously (35). Briefly,
5 million target cells suspended in 0.5 ml of supplemented RPMI 1640
were labeled with 5'Cr (Na/'CrO.,).
Fifty i\of labeled target cells and
100 /il of effector cells (T:E cell ratio, 1:50) were then incubated in
quadruplicates in round-bottomed microtiter plates (Linbro Scientific,
Inc., Hamden, Conn.) at 37 in 5% CO2 humidified atmosphere for 4
hr. The percentage
formula:

of cytotoxicity

[(% - %S)/(%M
where %is the percentage

was calculated

according

to the

- %S)] x 100

of 5'Cr release obtained in the cultures of

target cells and lymphocytes and %S is the percentage of spontaneous

release, obtained from cultures containing 51Cr-labeled target cells
alone or 51Cr-labeled and unlabeled target cells, in the same ratio as
target and effector cells in experimental cultures. The latter cultures
were done in order to correct for cell densities in the cultures. No
difference has been found between these 2 types of spontaneous
cultures. %M is the maximum percentage of 51Cr release, obtained by
freezing and thawing the target cells 4 times. The spontaneous release
ranged from 5 to 9%, and the maximum release ranged from 90 to
97%, for both the K-562 and CEM target cell lines. Standard error of
the mean of replicates was <5%.
A single-cell assay, as described previously in detail (17, 43), was
used to evaluate changes in NK cell-tumor-binding
and -killing prop
erties after IFN treatment. The incubation time in these experiments
was 4 hr, at 37in 5% CO2 humidified atmosphere. The percentage of
tumor-binding cells was determined by counting at least 100 lympho
cytes. The percentage of cytotoxic tumor-binding cells was determined
by counting the number of dead target cells in 100 conjugates. Results
are expressed as a percentage of dead tumor target cells out of total
number of conjugates, corrected for spontaneous lysis of target cells
plated alone (43).
Interferon Treatment. Partially purified human interferon-a, pre
pared as described previously (5), was provided by Dr. K. Cantell from
the Finnish Red Cross Center. The specific activity of this preparation

JUNE 1982

treatment free at least 1 month before the first IFN-a injection, and no
other treatment was administered during the course of IFN-a therapy.
Eighteen patients, representing a heterogeneous group, mostly with
recurrent metastastic cancer, and who failed previous conventional
treatment, were included in the IFN-a study: 3 with breast cancer; 3
with ovarian cancer; 3 with multiple myeloma; 2 with Hodgkin's disease;

Three patients, i.e., 2 with multiple myeloma and one with breast
cancer, undergoing therapy with IFN-arA were tested also for their NK
cell activities before and 24 hr after i.m. administration of 3 x 106 units
of IFN-arA. Additionally, changes in NK cell cytotoxicity of 4 patients
(2 with lymphoma, one with colon, and one with ovarian cancer) treated
with 50 x 106 units of IFN-arA and one patient (breast cancer) treated
with 36 x 106 units of IFN-arA were evaluated before and at various
times after IFN-arA treatment. These patients were also treatment free
at least 1 month before IFN-arA therapy. IFN-arA was prepared as
described previously (9, 25) and was provided for clinical studies by
Roche Institute of Molecular Biology, Nutley, N. J. The specific activity
of this preparation was 2 to 4 x 10s units/mg of protein. In all
experiments, fresh blood samples were used for NK cell tests.
Statistical Analysis. Standard statistical tests were used to evaluate
the presented data. These represented analysis of variance and covariance, Student's f test, paired-sample f test, and Fisher exact test and
are specified in individual experiments.

RESULTS
Distribution of NK Cell Cytotoxicity in Normal Individuals
and Cancer Patients. It is a common observation that the
degree of peripheral blood NK cell cytotoxicity varies consid
erably among the normal donor population, even if an identical
tumor cell line is used as a target. In a study of 35 normal
individuals, we have also observed such variability in NK cell
cytotoxicity to 2 different tumor cell lines, i.e., K-562 and CEM.
NK cell cytotoxicity ranged from 11 to >50% to both of these
target cell lines, at a T:E cell ratio of 1:50. Similar variability
was seen also with other T:E cell ratios. From these studies,
however, it was not clear whether such variability in NK cell
cytotoxicity was a true reflection of a difference in NK cell
responsiveness among normal donors or whether the cytotox
icity of a single donor tested on various occasions fluctuated
similarly. To examine this question, we have tested NK cell
activities of 13 normal donors and 2 patients (who were at this
time treatment free) repeatedly, 3 to 7 times, over a period
encompassing several months. For logistic reasons, more pa
tients who would be untreated for a longer time period were
not available. Results of these experiments are illustrated in
Table 1. It can be seen clearly that NK cell cytotoxicity to both
target cell lines varied to some extent within multiple tests of a
single individual; however, it was always significantly below the
variability of NK cell cytotoxicity observed among different
donors. This was confirmed statistically by an analysis of
variance test (see legend of Table 1). Additionally, despite
certain degrees of fluctuation in NK cell cytotoxicity of a single
individual, the NK cell phenotype of that individual remained
invariably unchanged. Specifically,
NK-HRs retained con
stantly their NK-HR status, and NK-MRs and NK-LRs retained
their NK-MR and NK-LR status, respectively. Two cancer pa2481

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E. Lotzov et al.
Table 1
Serial tests of NK cell cytotoxicity
cytotoxicity"Type

donorNormal
of

cell
lineK-562

of a single individual
% of NK cell

534.5

S.E.31.9

12345678910111213Patients0
CEMK-562

42.0
12.933.1
11.029.017.937.0
23.526.0
11.6

12.1

16.7

3.7
1.729.8
15.1

CEMK-562

24.0
27.470.7
29.070.537.077.0
23.862.0
36.8

28.0

25.6

2.4
2.070.0
29.7

CEMK-562

29.481.031.679.8
20.083.826.082.8
34.3

30.8

28.7

3.1
1.784.0
28.7

CEMK-562

92.8
49.264.052.672.0
44.150.749.064.5
65.6

70.1

CEMK-562K-562K-562
25.973.052.039.3
21.354.750.030.4
34.470.077446.1
37.380.352.476.9

2.3
4.262.8
55.1
4.4
3.769.5
29.7
5.457.9

6.538.6

19.161.025.066.2
CEMK-562K-562K-562
21.257.421.572.8
28.759.030.879.3

4.5
2.959.1
23.0
1.025.8

2.772.8

CEMK-562

58.067.3
55.048.653.152.0

CEMK-562K-56256.016.259.4
60.516.358.9
49.823.760.3

3.8
.456.0
55.4 1
5.8
55.4
3.118.7

2.563.9

12Target

CEMK-5621*20.532.921.5227.3
30.425.1335.0
38.329.74
41.6Mean

4.4
2.525.4
35.8
2.4

1 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 individual
was 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 NK

cell cytotoxicity.
In contrast, a great degree of variability in NK cell cytotoxicity
was observed among different donors. In fact, when NK cell
cytotoxicity of the entire normal donor population was ana
lyzed, these could be classified on the basis of their NK cell
responsiveness to a given tumor target, as NK-LRs, NK-MRs,
and NK-HRs. The NK cell cytotoxicity values for NK-LRs, NKMRs, and NK-HRs were divided arbitrarily into groups of <20,
20 to 45, and >45%, respectively, at a 1:50 T:E cell ratio. The
results of such analysis and the NK cell dose-response curves
of normal donors with various NK cell phenotypes are illustrated
in Chart 1. It can be seen from the chart that the NK cell doseresponse curves of each class of NK cell responders, i.e., NKHRs, NK-MRs, and NK-LRs, were significantly distinct (at all
T:E cell ratios tested) from the other 2 classes (for statistical
analysis, see legend of Chart 1). It is also evident that the
slopes of the NK cell dose-response curves of the NK-HRs,
and NK-MRs (to both K-562 and CEM target cells) did not
differ significantly from each other (evaluated by analysis of
covariance, as represented by Student's f test for comparison
2482

of slopes), an observation suggesting that the lower NK cell

response of the NK-MR group may be due to a relative de
crease in the number of active NK cells. In contrast, the slope
of the NK cell dose-response curve of NK-LRs deviated sig
nificantly from that of NK-HRs and NK-MRs. If the low NK cell
activity of NK-LRs was due merely to reduced numbers of NK
cells, lower but parallel slopes of dose-response curves would
be expected. Thus, it is possible that NK-LRs may be experi
encing a possible aberration in NK cell activities. This is,
however, only a suggestion which must be further explored
experimentally. If NK cells indeed prove to be important con
stituent of defense against tumors, the disclosure of NK-LRs
could be of extreme importance.
Even though the normal individuals exhibited NK-LR, NKMR, and NK-HR phenotypes to both the K-562 and CEM tumor
cell lines, the distribution of these phenotypes to the 2 tumors
differed. As illustrated in Table 2, only 11 % of the normal donor
population displayed the NK-LR status to K-562, whereas 40%
exhibited the same responder status to CEM. Differences be
tween the NK-HR phenotypes to CEM and K-562 were also
observed; i.e., 52% of normal donors were NK-HRs to K-562,
CANCER

RESEARCH

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VOL. 42

NK Cell Cytotoxicity

to both target cell lines. This shift in the modal range of NK cell
reactivity towards the lower degree of cytotoxicity may be
indicative of impaired NK cell activities in cancer patients. More
individuals, however, must be tested before these indications
can be substantiated. These data are in agreement with the
data of Hersey ef al. (23) indicating decreased NK cell-killing

60

potential in cancer patients. Interestingly, leukemic patients

exhibited only the NK-LR phenotype, regardless of the type of
leukemia studied. This confirms our previous observations
demonstrating low NK cell responsiveness of leukemic individ
uals (35). Due to the low amount of cancer patient's peripheral
1

7 11 21
017
01
NUMBER
OF IFN-fflINJECTIONS

7 11 21

blood available for these studies, the NK cell dose-response

curves of solid-cancer patients could not be constructed. How
ever, as evident from Chart 1, the slope of the NK cell doseresponse curve of leukemic patients deviated significantly from
that of NK-MR and NK-HR normal individuals and resembled
that of NK-LR normal donors. Based on the aberrant slope of

Distribution

fe

of NK cell phenotypes

Table 2
in the normal individual and cancer patient
populations

cytotoxicityK-56216.029.659.46.933.169.49.72221.24
of NK cell
cell
pheno
type8NK-LRNK-MRNK-HRNK-LRNK-MRNK-HRNK-LR%

DonorNormal
individualsSolid-cancer

017

01
01
017
NUMBER
OF IFIl-OtlNJECTIONS

11 21

Chart 1. NK cell dose-response curves of normal individuals and leukemic

patients. 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, <20% (O); and leukemic patients ();oars, S.E.
Differences in NK cell cytotoxicity between these groups were evaluated statis
tically by Student's f test analysis. For the K-562 cell line, the p values between
NK-HRs and NK-MRs and between NK-HRs and NK-LRs were <0.001 for all
ratios tested; the p values between NK-LRs and NK-MRs were <0.02, <0.001,
and <0.05 at 1:25, 1:50, and 1:100 T:E cell ratios, respectively. For the CEM
cell line, the p values between NK-HRs and NK-MRs were <0.01 for a T:E cell
ratio of 1:12 and <0.001 for T:E cell ratios of 1:25 and 1:50, respectively; the p
values between NK-HRs and NK-LRs and between NK-LRs and NK-MRs for all
T:E cell ratios tested were <0.001.
The slopes of the dose-response curves (shown in a log-linear plot in the
chart) were determined by linear regression analysis and were found to be 35.3,
28.4. and 13.7 for NK-HRs, NK-MRs, and NK-LRs, respectively, to the K-562
cell line and 35.0, 29.6, and 14.5 for NK-HRs, NK-MRs, and NK-LRs, respec
tively, to the CEM cell line. There was no significant difference in the slope of
NK-HR and NK-MR dose-response curves, as evaluated by analysis of covariance
(using Student's t test for comparison of slopes); however, significant differences

patientsLeukemic

patientsNK
NK-LR, <20%; NK-MR, 20 to 45%; NK-HR, >45% cytotoxicity at 1:50 T:E
cell ratio. NK cell cytotoxicity was tested in a 4-hr 51Cr cytotoxicity assay.
0 Mean S.E. A total of 35 normal individuals, 28 solid-cancer patients, and
14 leukemic patients were tested.
c Numbers in parentheses, percentage of population.
d NK cell reactivity was tested in a 14-hr 51Cr cytotoxicity

assay.

were observed between the slopes of NK-HR and NK-LR dose-response curves
(p < 0.001) and between NK-MR and NK-LR curves (p < 0.02). The distribution
of NK-HR, NK-MR, and NK-LR individuals to K-562 and CEM is illustrated in
Table 2. The slope of the dose-response curve of leukemic patients (11.2) to the
K-562 cell line was found to differ significantly from that of normal NK-HR and
NK-MR (<0.001) donors but was not significantly different from the slope of NKLR normal individuals.

whereas only 29% were NK-HRs to CEM. Approximately equal

number of normal donors responded in NK-MR fashion to both
cell lines.
The distribution of NK cell phenotype to K-562 and CEM
tumors among 28 solid-cancer patients and 14 leukemic pa
tients is also shown in Table 2. Similarly to normal donors,
solid-cancer patients displayed a wide range of NK cell cyto
toxicity and could be grouped into NK-HR, NK-MR, and NK-LR
phenotype categories. Most pronounced, however, was the
observation that, in comparison to normal individuals, a higher
percentage of solid-cancer patients exhibited the NK-LR status
(27% to K-562 and 50% to CEM) and a lower percentage
exhibited the NK-HR status (33% to K-562 and 12% to CEM)
JUNE

1982

HR/HH

HR/MR
MR/HR

IB/HR

MR/MR

MR/Ifl
IB/MR

Hi/IB

NK CELL PHENOTYPES (K-562/CEM)

Chart 2. Distribution of the phenotypes of normal donors and solid-cancer

patients to K-562 and CEM tumor targets. Thirty-five normal individuals and 28
solid-cancer patients were tested and categorized as NK-HR, NK-MR, and NKLR (see Table 2). Open columns, distribution of NK phenotypes among normal
donors; shaded columns, distribution of phenotypes among cancer patients.

2483

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E. Lotzovet al.
the NK cell dose-response

curves,

dilution

of NK cells in

leukemic patients does not appear to be the explanation for

their NK-LR status. Instead, the NK-LR status of leukemic
patients could reflect an NK cell aberrancy which underlies
leukemia. Since NK cells exhibit strongest reactivity to tumors
of hemopoietic origin in vitro, it is possible that they are also
involved in defense against hemopoietic cancers in vivo.
Another interesting observation originating from our studies
was that a single normal donor, as well as a cancer patient,
could exhibit different NK phenotypes to the K-562 and CEM
target cell lines; i.e., a single donor could be a NK-HR to one
target cell line and a NK-MR or NK-LR to the other. Chart 2
diagrammatically illustrates distribution of NK cell phenotypes
(and their combinations) to both target cell lines among the
normal donor and cancer patient populations. Similarly to the
results obtained with NK cell reactivity to a single cell line (see
Table 2), the distribution of combined (to both cell lines) NK
cell phenotypes of a single individual differed between normal
donors and cancer patients. In normal donors, only 7% of
individuals were of the LR/LR (K-562/CEM)
phenotype,
whereas 17% of cancer patients were of the NK-LR phenotype
to both targets. Also, the MR/LR and LR/MR phenotypes were
prevalent in cancer patients (42%), in comparison to normal
individuals (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 obser
vations suggest that cancer patients may be deficient in their
NK cell activities.
Effect of Exogenously Administered
IFN-a on NK Cell
Cytotoxicity of Cancer Patients. In this facet of the study, we
have investigated the effect of exogenously administered IFNa on peripheral blood NK cell cytotoxicity of 18 cancer patients
with a variety of cancers (see "Materials and Methods"). NK

cell activities were tested before and after a single or multiple

i.m. injections of 3 x 106 units of IFN-,always 24 hr after
each injection. The NK cell activity of some patients was
followed through as many as 146 injections. Since no differ
ence in NK cell reactivity of patients receiving 26 or more IFNa injections was observed and only one patient was available
at each point, the data were pooled, and a single group,
designated as >26 injections, is presented. In order to facilitate
the interpretation of the data, i.e., not to fail to detect NK cell
modulation, due to the wide range of NK cell cytotoxicity (from
8 to 70%), the cancer patients were divided into NK-HR, NKMR, and NK-LR categories, on the basis of their NK cell
pretreatment levels. The NK-LR and NK-MR groups of patients
were then combined, since no difference in NK cell modulation
between these 2 groups of cancer patients was observed.
The results of these studies are illustrated in Chart 3. Follow
ing one injection of IFN-a, there was a highly significant and
consistent increase in NK cell cytotoxicity of all patients with
the NK-MR and NK-LR phenotypes (Chart 3, A and B); the
average cytotoxicity increased from 24% before the treatment
to greater than 52% after IFN-a injection to the K-562 cell line
and from 15 to 30% to the CEM cell line. Following multiple
IFN-a injections (i.e., 7, 14, 21, and >26), the average NK cell
cytotoxicity remained significantly increased but was not fur
ther potentiated above the single IFN-a injection level. How
ever, a certain degree of variability was observed among indi
vidual patients. Specifically, 4 of 9 patients tested after multiple
IFN-a injections did not exhibit any increase in cytotoxicity
above that observed at the single injection level, whereas NK
cell cytotoxicity of 5 patients was potentiated further with
multiple injections (4 to K-562 and one to CEM). Two of the
patients tested after 7 injections returned to pretreatment val
ues; however, they were again potentiated with further injec-

60

wnu OF im-a lucciims

Chart 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 in
NK 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 line
were <0.001, <0.05, <0.02, <0.02. and <0.005 for 1, 7, 14. 21, and >26 injections, respectively, and for the CEM tumor target cell line, p < 0.005, <0.05, and
<0.05 for 1, 7. and 14 injections, respectively. There was no significant change in NK cell cytotoxicity to the latter cell line after 21 injections of IFN-a. No significant
changes in NK cell cytotoxicity to the K-562 tumor target cell line were observed in NK-HR patients after IFN-a treatment.

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VOL.

42

A/K Cell Cytotoxicity

Although NK cell stimulation was exhibited to both target cell
lines, the duration of stimulation to K-562 extended beyond
that to CEM. Namely, stimulation to the K-562 cell line was still

TO TARGET CELL RATIO

Chart 4. Comparison of NK cell Cytotoxicity of normal donors and cancer

patients tested simultaneously before and after single injection of IFN-a. Eight
different patients (,)and 24 normal donors (O) were tested in 24 different
experiments; additionally, some of the normal donors .were tested on several
occasions ( ), in order to demonstrate the consistency of their response, (bars,
S.E. of multiple tests of a single individual). NK cell Cytotoxicity was tested
against 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 was

mediated by IFN-a and could not be contributed to the fluctua
tion of NK cell Cytotoxicity is indicated: (a) by changes in NK
cell Cytotoxicity always in a positive direction (in contrast to
normal individuals exhibiting equal changes in NK cell cytotoxicity in both positive and negative directions), as established
by paired f test analysis; and (b) by the differences in the
degree of change of NK cell Cytotoxicity of IFN-a-treated pa
tients. Specifically, in NK-LR and NK-MR patients, the p values
between pretreatment and posttreatment levels of Cytotoxicity
were always statistically significant (except for NK cell re
sponse of patients to CEM after 21 injections), whereas the
differences in NK cell Cytotoxicity of normal individuals tested
on various occasions were not statistically different (as deter
mined by paired i test analysis; see legend of Chart 3 and
Table 1). Additionally, as can be seen in Chart 4, when IFN-atreated patients were tested simultaneously with normal indi
viduals (a different normal donor was tested at each point) in
an experiment, highly dichotomous patterns of NK cell activities
between normal individuals and cancer patients were ob
served. Namely, the high (or low) levels of NK cell Cytotoxicity
of IFN-a-treated patients did not coincide with high (or low) NK
cell Cytotoxicity levels of normal individuals (even though each
of the normal individuals, when tested at various occasions,
retained constant levels of NK cell Cytotoxicity, as indicated by
the bars). This suggests that changes in patients' NK cell
Cytotoxicity after IFN-a treatment could not be attributed to
fluctuation in Cytotoxicity values due to the variability in the NK
cell assay since, under the latter conditions, the NK cell Cyto
toxicity of both patients and normal donors should fluctuate in
the same (and not in a dichotomous) direction. Further evi
dence eliminating fluctuation in NK cell assay as a cause of
increased NK cell Cytotoxicity after IFN treatment will be pre
sented in the section concerned with modulation of NK cell
Cytotoxicity by IFN-arA.

JUNE

1982

evident after >26 injections, while that to the CEM cell line was
not detected after 21 injections. Two of the cancer patients in
the NK-LR and NK-MR categories (lymphocytic sarcoma and
chronic lymphocytic leukemia) did not show any stimulation of
NK cell Cytotoxicity by IFN-a after 21 and >26 IFN-a injections,
respectively (data not shown). In contrast to the NK-LR and
NK-MR group of patients, only 2 patients of those displaying
the NK-HR phenotype to K-562 showed sensitivity to NK cell
augmentation following multiple IFN-a injections (Chart 3C). In
fact, some NK-HR patients exhibited decreased NK cell activ
ities after IFN-a injections. It is important to mention in this
context that the NK-HR cancer patients also showed aberrant
NK cell dose response when compared to NK-HR normal
individuals (data not shown); i.e., practically no increase in NK
cell Cytotoxicity with increasing T:E cell ratio was observed;
the Cytotoxicity reached already high levels with low T:E cell
ratios. It is possible that the NK-HR phenotype of cancer
patients is not their natural NK cell status but hyperactivity of
NK cells to tumors. Consequently, such hyperactive NK cells
may not be sensitive to IFN-a stimulation.
Effect of Exogenously Administered IFN-arA on NK Cell
Cytotoxicity of Cancer Patients. We have also tested NK cell
activities of cancer patients undergoing IFN-arA therapy. These
patients 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 re
ceived 3 x 106 units of IFN-arA were tested before and 24 hr
after injection for NK cell cytoxicity in a 51Cr release Cytotoxicity
assay and for NK cell-tumor cell-binding properties and -killing
potential in a single-cell assay. K-562 tumor was used as
target. As shown in Table 3, no significant changes were
observed in tumor cell-NK cell-binding properties after a single
IFN-arA injection in any patient. This indicates that IFN-arA
does not potentiate the binding of NK cells to tumor targets.
However, there was a significant augmentation of NK cell
Cytotoxicity (as determined by a 51Cr release assay) and killing
of already-bound tumor targets (as determined by a single-cell
assay) 24 hr after IFN-arA treatment in all 3 patients.

Effect of exogenously

Table 3
administered IFN-arA on the peripheral
activity of cancer patients

Patient123Treatment0None

of cytotoxic
of tumortumor-binding
cells"20.9
binding
cells7.410.613.3
44.4C22.1

IFN-arANone

IFN-arANone

IFN-arA%

9.57.08.0%

blood NK cell

51CrCytotoxicity42.6
of

62.7C26.238.0o26.7

33.3d16.3

25.7e%

39.2e

Patients were tested for NK cell Cytotoxicity and tumor-binding properties

before and 24 hr after a single i.m. injection of IFN-arA (3 x 106 units). Both a
51Cr release Cytotoxicity assay and single-cell assay were used (see "Materials
and Methods").
6 K-562 tumor cell line was used as target at a T:E cell ratio of 1:12 (5'Cr
release Cytotoxicity assay) and 1:1 (single-cell assay).
c' 'e Changes in NK cell Cytotoxicity after IFN-arA treatment were analyzed
statistically by Fisher exact test. The p values were <0.005C, <0.02d, and <0.058
for both the single-cell assay and 5'Cr release Cytotoxicity assay.

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E. Lotzov et al.
with regard to their degree of NK cell
in general, NK-HRs were found to be
donor population, whereas NK-LRs were
found in cancer patients. The relevant question raised, but not
Experi
(units
X106)5050505036%
answered for obvious reasons by our studies, is whether the
donorPatient
of
ment1234Type
88.6C
18.30.6C
low NK cell responsiveness of normal donors could be indica
1Patient
2.8d69.7
2Normal
tive of their susceptibility to cancers (especially leukemias,
1Patient Donor
77.711.151.45.060.21.8C19.92435.622.6e69.837.8e43.267.3e68.022.918.5
which appear to be the most sensitive NK cell targets). There
is certainly a similarity between the NK cell dose-response
3Normal
2Patient Donor
curves of NK-LR normal donors and leukemic patients, both of
which significantly deviate from those of NK-HR and NK-MR
4Normal
normal individuals. This suggests that NK cell activities of NK3Patient Donor
LR normal individuals and leukemic patients may be aberrant.
5Normal
If NK cells indeed will prove to be an important component of
Donor 4Dose
3 K-562 tumor cell line was used as a target at a T:E cell ratio of 1:50 in a 3the immunosurveillance mechanism, then such NK cell defi
hr 5'Cr release cytotoxicity assay. The differences between pretreatment and
ciency could reflect higher susceptibility of NK-LR normal
post-IFN-arA treatment values were evaluated statistically by Fisher exact test.
individuals to cancer. Another important question is whether
All differences not footnoted were not statistically significant. Mononuclear cells
the
levels of NK cell cytotoxicity of cancer patients could be
of normal donors were tested simultaneously with each patient. At Time 0, fresh
cell suspensions were used, and refrigerated suspensions of the same normal
used as a prognostic factor in progression or relapse of the
donor were tested again 4, 8. and 24 hr later, together with mononuclear cells of
disease. Our unpublished observations on the decline of NK
IFN-arA-treated patients. No statistically significant differences were found be
cell cytotoxicity several weeks before relapse of leukemia could
tween various tests of a single normal donor.
Time after single injection of IFN-arA (hr).
be indicative of possible significance of NK cells in the prog
ep< 0.001.
nosis of this disease.
p < 0.01.
* p < 0.02.
The recognition of NK cell phenotypes within the human
population may also have relevance to the more precise eval
In the second series of experiments, NK cell cytotoxicity of
uation of various biological response modifiers (and therapeutic
4 patients who received a single injection of 50 x 106 units
agents in general) on NK cell modulation. The effectiveness of
and one patient administered a single injection of 36 x 106
these agents may not be quite apparent when the data of the
units of IFN-arA was tested before and 4, 8, and 24 hr after the
individuals with wide range of NK cell reactivity are combined.
treatment. In these experiments, the same normal donor was
As a matter of fact, such division of patients undergoing IFN-a
tested for NK cell cytotoxicity simultaneously with each patient
therapy allowed us to observe the differential effect of IFN-o on
at all time intervals in order to assess variability in NK cell
NK cell cytotoxicity of NK-LR and NK-MR patients versus NKassay. A fresh mononuclear cell sample of the normal donor
HR patients. Whereas the former 2 classes of NK cell respondwas tested at Time 0, and a refrigerated sample of the same
ers were stimulated in their NK cell activities, only 2 of 6 of the
normal donor was tested again at the later time intervals. The
latter class of responders were augmented by multiple IFN-a
results of these experiments are illustrated in Table 4. All of
injections. In fact, a decrease in NK cell cytotoxicity of the NKthe patients tested exhibited a decrease in NK cell cytotoxicity
HRs was observed after multiple IFN-a injections. The NK cell
4 and 8 hr after IFN-arA injection in comparison to their
cytotoxicity of the NK-LRs and NK-MRs was augmented al
pretreatment values (the cause of such a decrease is currently
ready after a single IFN injection and in 44% of patients was
being investigated). However, NK cell cytotoxicity of 3 of 4 not further potentiated with additional injections but remained
patients receiving 50 x 106 units of IFN-arA, i.e., Patients 2, 3,
at the augmented levels. These data are compatible with those
and 4, was significantly increased 24 hr after the injection
of Einhorn et al. (7) and Huddlestone e-al. (26) who also
(stimulation index was 1.8, 2.7, and 6.9, respectively). The NK
observed similar augmentation of NK cell cytotoxicity after a
cell cytotoxicity of Patient 1 who received 50 x 106 units of
single IFN injection. The former group of investigators, in
IFN-arA and Patient 5 who received 36 x 106 units of IFN-arA
contrast to our studies, did not observe any dependence of NK
was not augmented in comparison to pretreatment values but
cell augmentation on the pretreatment NK cell levels. However,
was completely restored after the initial 4- to 8-hr decline. As
the degree of NK cell cytotoxicity in the studies of these authors
can be seen by the consistent decline in NK cell cytotoxicity at
never reached the high levels of NK cell cytotoxicity displayed
4 and 8 hr post-IFN-arA treatment and by the highly reproduc
by NK-HR patients in our studies; they resembled rather the
ible cytotoxicity values of normal individuals in each experi
NK-MRs and NK-LRs. This could be contributed most likely to
ment, the augmentation of NK cell cytotoxicity could not be the different target cells used in these 2 investigations (Chang
attributed to variability in NK cell assay.
versus K-562 target cells).
These results indicate that IFN-arA also augments NK cell
There was no correlation between IFN-a-mediated NK cell
cytotoxicity and consequently may be a useful potentiator of
augmentation and the type of cancer, since the cytotoxicity of
antitumor immunity. More patients are currently being studied
patients with various histological types of cancer was aug
to determine duration of this effect, as well as the effect of
mented with the exception of perhaps lymphoid type of tumors.
multiple IFN-arA injections on NK cell activities.
One patient with lymphocytic sarcoma and one with chronic
lymphocytic leukemia did not show any augmentation of their
DISCUSSION
NK cell activities after IFN-a treatment. There also was no
correlation between NK-LR status and previous therapies. Fur
The present investigation has shown that both normal donors
and cancer patients can be grouped into NK-HR, NK-MR, and thermore, no relationship was found between certain types of
Kinetics of NK cell cytotoxicity

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Table 4
NK-LR categories,
after a single injection of high doses of IFN-arA
activities. However,
cytotoxicity3O642.712.869.814.049.89.860.314.921.34
of NK cell
ofIFN-arA
prevalent in normal

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VOL. 42

NK Cell Cytotoxicity
cancer and the NK cell phenotype. No correlation was ob
served between IFN-a-mediated potentiation of NK cell cytotoxicity and age, sex, leukocyte counts, proportion of lympho
cytes and monocytes, or changes in clinical status of the
disease.
Preliminary results have shown that NK cell cytotoxicity of
most cancer patients receiving IFN-arA therapy was also aug
mented 24 hr after the injection, as measured by 2 independent
assays. Tumor cell-NK cell-binding properties were not modu
lated by IFN-arA in these studies, an observation suggesting
that the NK cell augmentation was not mediated via recruitment
of NK cell precursors but rather via induction of the lytic
machinery of preexisting NK cells.
It has been recognized that the NK cell assay is subject to
variability. However, the increase in NK cell cytotoxicity in our
experiments cannot be attributed to NK cell variability for
several reasons, as have been discussed in detail in "Results."
(a) No correlation (but a dichotomy) in the levels of NK cell
cytotoxicity was observed between IFN-a-treated patients and
normal donors, when these 2 groups of individuals were tested
simultaneously (see Chart 4). (b) The degree of increase after
IFN stimulation was always significantly greater than that ob
served in most normal individuals when tested alone serially
(Table 1). (c) Augmentation has been detected by 2 independ
ent assays (i.e., by a 51Cr release and single-cell assay; Table
3). (d) When the same normal donor was tested with each
patient in 4 different NK cell assays, the NK cell cytotoxicity
values of a normal donor were highly consistent and reproduc
ible, whereas those of patients were either increased (24 hr
after injection) or decreased (4 to 8 hr after injection) (Table
4). This and other experimental
evidence, indicated in
"Results," argue strongly against the possibility that the NK
cell increase after IFN treatment was due to the variability in
NK cell assay.
The observation that NK cell cytotoxicity was augmented
significantly during IFN-a and IFN-arA therapy of cancer pa
tients suggests that the beneficial effect of IFNs could be
mediated at least partially via immune activities and that NK
cells may be one of the components of such an antitumor
mechanism.
ACKNOWLEDGMENTS
The authors wish to acknowledge
for technical assistance.

Lily K. Muesse, D. Hearn, and J. McDowell

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