Mechanisms of Ageing and Development
112 (1999) 197 – 215
www.elsevier.com/locate/mechagedev

Genetic differences in the age-associated
decrease in inducibility of natural killer cells by
interferon-a/b
Artur Plett, Donna M. Murasko *
Department of Microbiology and Immunology, MCP Hahnemann Uni6ersity School of Medicine,
2900 Queen Lane, Philadelphia, PA 19129, USA
Received 8 June 1999; accepted 1 October 1999

Abstract
Natural killer (NK) cells, which are important in viral infections and anti-tumor activity,
show reduced cytotoxicity in aged mice. The mechanism(s) for this age-related decline in NK
activity has not been clearly established. We assessed changes in NK cytotoxicity in
splenocytes and peripheral blood mononuclear cells after interferon (IFN)-a/b stimulation in
adult (6 months) and aged (22–26 months) C57Bl/6, Balb/c, and (Balb/c×C57Bl/6)F1 mice.
Aged C57Bl/6 and Balb/c mice had a signiﬁcantly reduced IFN-a/b-stimulated NK cytotoxicity compared to adult mice. In contrast, adult and aged F1 mice showed similar NK
cytotoxicity after IFN-a/b induction. The decreased ability of NK cells of aged mice to
respond to induction by IFN-a/b was not due to a requirement for an increased amount of
IFN or for a longer period of treatment with IFN. Further, this decreased response did not
appear to be the result of suppressive activity of adherent cells or T cells. While the
percentage of NK cells (NK1.1 +) was similar in adult and aged mice, the (CD8 + NK1.1+
) subset of NK cells was signiﬁcantly increased in aged mice. Importantly, the percentage of
CD8+ NK1.1+ cells was inversely related to the cytotoxicity observed after IFN-a/b
treatment. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
Keywords: NK cells; Aging; Interferon

* Corresponding author. Tel.: +1-215-991-8357; fax: +1-215-848-2271.
E-mail address: murasko@mcphu.edu (D.M. Murasko)
0047-6374/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 4 7 - 6 3 7 4 ( 9 9 ) 0 0 0 9 1 - 3

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1. Introduction
Natural killer (NK) cells, which are an important component of the innate
immune system, play a critical role in defense against viral infections and cancer.
They are large granular lymphocytes (LGL) that are cytotoxic without the need for
prior activation, display no immunologic memory, and are not restricted by the
major histocompatibility complex (MHC). In addition, cytokines, such as interleukin (IL)-2, IL-12, and interferons (IFN) type I (IFN-a/b) and II (IFN-g), can
enhance NK cell activity (Reiter, 1993; Kutza and Murasko, 1994).
Although initially deﬁned for its antiviral activity (Dianzani and Antonelli, 1989),
IFN-a/b is now known as a pleotrophic cytokine that has a major impact on the
proliferation of a wide range of cells, including tumor cells (Fleischmann and
Fleischmann, 1992). In addition, IFN-a/b can modify various aspects of the
immune response, including increasing expression of MHC Class I antigens (Minato et al., 1980), modifying recirculation of lymphocytes (Korngold et al., 1983;
Mann et al., 1989), and augmenting cytotoxic responses of both NK cells and
CD8 + T cells (Minato et al., 1980). IFN-a/b has been used in anti-cancer therapy
because of both its anti-proliferative and immunomodulatory activities. As an
anti-cancer agent, IFN-a/b has been shown to protect from tumor development and
metastatic dissemination in animals (Markovic and Murasko, 1990), as well as to be
effective in some human cancers, including head and neck carcinoma and
lymphomas (Tyring, 1992).
Since the incidence of cancers increases with age, the effectiveness of IFN-a/b as
an anti-cancer therapy in the elderly needs to be carefully evaluated. Using a
murine melanoma model in which we have shown that IFN can prevent metastatic
dissemination and mortality in 50% of young mice (Markovic and Murasko, 1990),
we have obtained preliminary data demonstrating that IFN has no effect on tumor
dissemination in aged mice. The effectiveness of IFN-a/b treatment in this model in
young mice is dependent on enhancement of NK activity (Markovic and Murasko,
1991). Thus it is possible that the lack of anti-tumor activity of IFN-a/b in aged
mice is due to the inability of IFN-a/b to augment NK activity in aged mice.
Age-related alterations in immune function have been described in both human
and in animals. Although decreased T cell proliferative responses to both mitogenic
and antigenic stimulation have been the most consistent and profound changes that
occur with age (Murasko and Goonewardene, 1990; Miller, 1996), decreased
cytotoxic T cell function (Effros and Walford, 1983) and impaired antibody
responses have also been reported. However, the effects of age on NK activity have
been explored less extensively. Although initial reports in Balb/c mice suggested
that basal NK activity decreased signiﬁcantly with age (Kiessling et al., 1975),
additional studies indicated that basal NK activity actually peaks at about 2–3
months of age, decreases by 6 months of age and then remains fairly constant from
6 months through death (Albright and Albright, 1983; Provinciali et al., 1989). NK
activity can be enhanced after treatment with either IL-2 or IFN-a/b in 6 month,
but not 24 month, old Balb/c mice (Provinciali et al., 1989). These limited data
suggest that aging has a preferential, deleterious effect on inducible, but not basal,
NK cell activity in the murine model.

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Since the melanoma model utilized by our laboratory was originally established
in C57Bl/6 (B6) mice, the current study addressed whether IFN-a/b can augment
NK activity in adult and aged B6 and (Balb/cByJNia × C57Bl/6JNia)F1 (CB6F1)
mice. The age-related requirements for enhancement of NK by IFN-a/b in both
amount and length of treatment were evaluated, as well as the possible suppressive
roles of T cells or adherent cells. Finally, the possibility that there is an age-associated decrease in the total number of NK (NK1.1+ ) cells, was explored.

2. Materials and methods

2.1. Animals
Speciﬁc pathogen-free C57Bl/6JNia (B6), Balb/cNia (Balb/c) and (Balb/cByJNia× C57Bl/6JNia)F1 (CB6F1) mice were obtained from the NIA colony maintained by Charles River (Kingston, NY) at 2 months (young), 6–8 months (adult)
and 22 – 26 months (aged) of age. All mice were maintained in AAALAC certiﬁed
barrier facilities at MCP Hahnemann University. All mice were given food and
water ad libitum. Experiments were conducted after mice had acclimated to our
facilities for at least 2 weeks. Mice that demonstrated enlarged spleens or tumors
were eliminated from the study.

2.2. Cell isolation and preparation
Peripheral blood was obtained from the retro-orbital sinus and collected into
heparanized blood collection tubes (Fisher Scientiﬁc, Pittsburgh, PA). Spleens were
removed, placed in RPMI-1640 (Biowhittaker, Walkersville, MD), and homogenized to obtain single cell suspensions. Mononuclear cells were isolated as described
previously (Kutza and Murasko, 1994). Brieﬂy, spleen cell homogenates or diluted
whole blood was layered onto ﬁcoll hypaque-1083 (Sigma, St. Louis, MO) to isolate
mononuclear cells by density gradient centrifugation (2500 rpm for 20 min at RT).
Cells were washed twice in RPMI-1640 (1000) rpm 10 min at RT) and then
resuspended at 1×106 cells/ml in complete media containing RPMI-1640, 10% fetal
bovine serum (FBS) (BioWhittaker, Walkersville, MD) and 1% glutamine
(BioWhittaker).

2.3. NK cytotoxicity assay
A standard 4 h51Cr-release assay with YAC-1 cells as targets to assess NK cell
activity was utilized (Albright and Albright, 1983). Brieﬂy, 1 × 106 YAC-1 cells
were incubated with 200 mCi Na 51CrO4 (ICN, Costa Mesa, CA) for 2 h at 37°C.
During this incubation, cells were mixed every 20 min by gentle tapping to ensure
maximal uptake of Na51 CrO4. The cells were then washed twice with RPMI-1640,
resuspended in complete media, and then rotated for 1 h at RT. After the ﬁnal
wash, YAC-1 cells were resuspended at 1× 104 cells/ml in complete media and

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plated in round bottom 96 well microtiter plates (ICN, Costa Mesa, CA). Effector
cell preparations from spleen or blood were then added to wells at effector to target
(E:T) ratios of 100, 50, or 25:1. All samples were assayed in triplicate. Target cells
were incubated with either media alone or 5% Triton X-100 to quantitate spontaneous and maximum release, respectively. After a 4 h incubation at 37°C, supernatants were harvested using the Skatron harvesting system (Skatron, Sterling, VA)
and radioactivity in supernatants was quantitated using a gamma counter (Packard,
Sterling, VA). Percent cytotoxicity was calculated as follows:
% Cytotoxicity =

[CPM(sample)− CPM(spontaneous release)]
[CPM(maximum release)− CPM(spontaneous release)]
×100

Spontaneous release was always less than 10% of maximal release. Maximal release
was always between 1200 and 6000 cpm.

2.4. Interferon treatment
2.4.1. In 6itro
Effector cells (2× 106cells/ml) were incubated with varying doses of IFN-a/b
(ranging from 1× 103 – 1 × 105 units per 106 cells) for 1–8 h at 37°C in 5% CO2.
Preliminary experiments (see Section 3) established that maximal cytotoxicity
occurred after a 4 h incubation with IFN-a/b (Western Biomedical and Diagnostics,
San Diego, CA); therefore, this exposure time was used in all subsequent experiments. After IFN treatment, microtiter plates were centrifuged to pellet the cells
and the media containing IFN-a/b was discarded. Cells were then plated at the
appropriate E:T ratios and NK cytotoxicity was assayed as described above.
2.4.2. In 6i6o
IFN-a/b (ranging from 2×103 –2× 105 units/ml) was diluted in pyrogen-free
saline. Mice were given a 0.5 ml intraperitoneal inoculation. Spleens were harvested
24 h later and NK cytotoxicity was evaluated as described above.
Results are presented as percent cytotoxicity or as fold induction. Fold induction
is calculated according to the following formula: (% IFN-a/b induced cytotoxicity
} % basal cytotoxicity), where a fold induction of one equals no induction over
basal.
2.5. Assessment of surface phenotype
Splenocytes were washed with phosphate buffered saline (PBS) and resuspended
in staining buffer (1% FBS, 0.1% sodium azide-PBS) at 5× 106 cells/ml. Cells were
then incubated with mouse-anti-NK1.1 antibody conjugated to phycoerythrin (PE),
and rat-anti-CD8 antibody conjugated to allophycocyanin (APC) (Pharmingen, San
Diego, CA) for 30 min at 4°C in the dark. Cells were then washed with staining
buffer and ﬁxed with 1% paraformaldehyde. 10 000–30 000 lymphocyte gated
events were acquired for each sample on a FACScan or FACSCalibur ﬂow

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cytometer using LYSYS II software and analyzed using CellQuest software (Becton
Dickinson, Mountain View, CA).

2.6. Depletion of cells
2.6.1. Separation of adherent and non-adherent cells
Adherent cells were isolated using methods that have been described previously
(Brunda et al., 1983). Brieﬂy, pooled mononuclear splenocytes from adult (6–8
months) or aged (22 – 26 month) mice were incubated for 2 h at 37°C in 5% CO2 in
96 well ﬂat bottom microtiter plates (Falcon, Lincoln Park, NJ). Non-adherent cells
were then carefully removed to minimize dislodging of the adherent cells. Both
non-adherent and adherent cells were washed three times with complete media.
Non-adherent splenocytes from adult mice were added to adherent cells from either
adult or aged mice or were incubated alone as non-adherent controls. These same
combinations were used for non-adherent splenocytes of aged mice. All treatment
groups were then stimulated with IFN-a/b as described above. After a 4 h
incubation with IFN-a/b, non-adherent cells were transferred to round bottom 96
well microtiter plates and mixed with 51Cr-labeled YAC-1 targets to assess cytotoxic
activity.
2.6.2. T cell depletions
T cells were depleted by negative selection using immunomagnetic beads from
DYNAL Laboratories (Lake Success, NY), with minor modiﬁcations of the
manufacturer’s instructions. Brieﬂy, 2×107 cells were incubated with puriﬁed rat
anti-mouse CD4 antibody, rat-anti-mouse CD8 antibody (Pharmingen, San Diego,
CA), or both for 30 min on ice. The cells were washed with RPMI-1640 and
resuspended in 1% cold bovine serum albumin (BSA) in RPMI-1640 containing
4 × 107 DYNAL magnetic beads coated with goat-anti-rat-IgG antibodies. The
tubes were resuspended by gentle tapping every minute for 30 min, after which they
were placed onto a magnet and the supernatant aspirated. To ensure that NK cell
numbers were constant despite the enrichment of NK cells in T cell depleted
samples, control and depleted samples were resuspended to the same volume. Cells
were then plated in 96 well round-bottom microtiter plates and assayed for NK
activity as described above. These samples were also analyzed by ﬂow cytometry to
determine the effectiveness of depletion. Staining showed that \ 95% of CD8 and
\85% of CD4 cells were removed by this depletion protocol.
2.7. Statistics
Differences between basal and IFN-a/b-induced cytotoxicity in young, adult and
aged animals were evaluated using a Students’ t-test. The signiﬁcance of differences
in the kinetics and dose response experiments was determined using ANOVA
followed by a Bonferroni multiple-comparison post-hoc test. Statistics were done
using the SPSS statistics package (SPSS, Chicago, IL).

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3. Results

3.1. Age-associated changes in induction of NK acti6ity in B6 mice: effects of
IFN-h/i concentration
Previous reports indicated that while maximal NK activity is induced after a 4 h
stimulation with IFN-a/b in vitro in both young and aged Balb/c mice, the level of
activity in aged mice was lower than that of young mice (Provinciali et al., 1989).
We questioned whether B6 mice also demonstrated an age-associated change in
IFN-induced NK activity similar to that observed in Balb/c mice. To examine this
question, IFN-induced NK activity was measured in 2- (young), 6- (adult), and
24-month old (aged) B6 mice. As shown in Fig. 1, IFN-a/b-induced NK cytotoxicity was signiﬁcantly higher than basal NK activity (PB 0.001) in B6 mice of all
three ages. However, several points concerning this induction became apparent: (1)
Both young and adult mice showed a 3- to 3.5-fold increase in NK activity, whereas
aged mice showed only a 1.5- to 2-fold increase. (2) The level of NK activity after
stimulation with all doses of IFN-a/b was signiﬁcantly higher in both 2- and
6-month old mice compared to 24-month old mice. (3) Basal NK activity of
2-month old mice was signiﬁcantly higher than basal NK activity of both 6- and
24-month old B6 mice; basal NK activities of 6- and 24-month old mice were
comparable. Since 2-month old mice had higher basal NK activity, which could

Fig. 1. NK activity in young, adult and aged B6 mice after in vitro IFN-a/b treatment. Splenocytes from
three animals in each age group were stimulated with IFN-a/b for 4 h at the doses indicated and assayed
separately for in vitro cytotoxicity. The ﬁgure shows representative data from one of three experiments
at E:T = 100:1. Bars reﬂect mean + SD. IFN-a/b-induced activity was signiﬁcantly increased over basal
6
activity with all IFN concentrations (P B0.001; c). IFN-a/b-induced cytotoxicity of both the 2 and 6
month groups were signiﬁcantly higher (P B0.05; *) than the 24 month group at every dose of IFN-a/b
tested. Basal cytotoxicity of 2 month animals was signiﬁcantly higher (P = 0.002; $) than basal
cytotoxicity of both 6 and 24 month old mice.

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Fig. 2. NK activity in adult and aged B6 mice after in vivo stimulation with IFN-a/b and Poly I:C.
Adult (6 month) and aged (24 month) B6 mice (3 – 4 per age group) were injected with 0.5 ml saline
containing 100 mg of Poly I:C or the doses of IFN-a/b indicated. In vitro NK activity was assayed 24
h later. Bars represent mean percent cytotoxicity 9SEM (E:T= 100:1). NK cytotoxicity of adult mice
was signiﬁcantly enhanced relative to basal levels (PB 0.02; *) and to cytotoxicity of aged mice
(P B0.01; c ), after treatment with Poly I:C or 105 Units of IFN-a/b. Cytotoxicity in aged animals was
not signiﬁcantly increased after treatment with Poly I:C or any of the IFN-a/b doses. Basal (saline
treated) cytotoxicity was not signiﬁcantly different between adult and aged animals.

account for differences in the absolute level of NK activity after treatment with
IFN, all subsequent comparisons were made between 6–8 month (adult) and
22– 26 month (aged) animals. Collectively, these data suggest that the defect in
induction of NK activity in aged B6 mice cannot be overcome by treatment with
higher doses of IFN.
To determine whether the effects on NK activity after treatment with IFN-a/b
in vitro reﬂect the in vivo circumstance, mice were given intraperitoneal inoculations of either IFN-a/b, (103 – 105 U) or 100 mg Poly I:C. Splenocytes were
harvested and NK cytotoxic activity was assessed 24 h after treatment. As seen
in Fig. 2, although treatment with 105 units of IFN-a/b increased NK activity
signiﬁcantly in 6-month old mice, this amount of IFN-a/b did not alter NK
activity in 24-month old B6 mice. Further, treatment with Poly I:C, which induces the production of endogenous IFN-a/b, signiﬁcantly increased NK activity
of adult, but not aged, B6 mice. The inability of IFN-a/b or Poly I:C to
stimulate NK activity suggests that aged mice do not respond to IFN given
exogenously or produced endogenously. These results conﬁrm previous studies in
Balb/c mice (Provinciali et al., 1989) showing that NK activity in aged mice
cannot be augmented signiﬁcantly by treatment with exogenous IFN-a/b.

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3.2. Aged-associated changes in induction of NK acti6ity by IFN-h/i in B6 mice:
kinetics
The above data clearly demonstrated that the augmentation of NK activity by
IFN-a/b was diminished in aged B6 mice and could not be restored by higher
concentrations of IFN-a/b. However, it was still possible that this decrease in
inducible NK activity of aged mice could either be enhanced or restored after a
longer exposure to IFN-a/b in vitro. To test this possibility, splenocytes from adult
(6 months) and aged (24 months) B6 mice were incubated with 103 units of IFN-a/b
per 106 cells for 1, 2, 4, or 8 h. As seen in Fig. 3, NK activity of adult mice
increased signiﬁcantly after 1 h of treatment with IFN-a/b, and after an 8-h
exposure to IFN-a/b was enhanced ﬁve-fold. In contrast, NK activity of aged mice
did not increase signiﬁcantly over basal activity until 4 h of treatment with
IFN-a/b. However, even at its peak, inducible NK activity of aged mice was still
only 2.5-fold higher than basal NK activity. Fig. 3 also shows that NK activity was
signiﬁcantly higher in adult compared to aged mice after IFN-a/b treatment at all
time points, although basal NK activity did not differ signiﬁcantly between adult
and aged animals (3 and 4%, respectively). Collectively, these two sets of experiments clearly indicate that the limited induction of NK cytotoxicity by IFN-a/b in
aged B6 mice, relative to the induction achieved in adult B6 mice, cannot be
attributed to differences in the dose of, or time of exposure to, IFN-a/b.
Fig. 3 suggests that inducible NK activity did not plateau in either adult or aged
mice even after treatment with IFN-a/b for 8 h. Despite this, longer time periods of

Fig. 3. In vitro kinetics of IFN-a/b stimulated NK activity in adult and aged B6 mice. Splenocytes (three
to four mice per age group) were stimulated with IFN-a/b at 103U/106 cells for the time indicated and
assayed individually. Results show the mean fold induction 9 SEM relative to basal cytotoxicity. Level
of induction in adult (6 month) animals is signiﬁcantly higher than induction in aged (24 month) mice
(P B 0.01; *) at every time-point. Signiﬁcant (PB 0.05; c ) increase in activity relative to basal activity
was apparent by 1 h of stimulation in adult mice, but not until 4 h in aged mice. Data represent one of
two experiments.

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Fig. 4. NK activity of adult and aged CB6F1 and Balb/c mice. Splenocytes from 5 – 6 animals of each
age and each strain were assayed separately after 4 h stimulation with IFN-a/b (103U/106 cells). Bars
indicate mean cytotoxicity (E:T = 100:1)9SEM. IFN-a/b-induced cytotoxicity was signiﬁcantly increased
relative to basal cytotoxicity for CB6F1 and Balb/c mice, in both adult (PB 0.001; $) and aged
(P B 0.04; c ) groups. In Balb/c mice, IFN-a/b-induced cytotoxicity was signiﬁcantly lower in aged
compared to adult mice (PB 0.02; *). Levels of cytotoxicity after IFN-a/b treatment were comparable in
adult and aged CB6F1 mice. Basal cytotoxicity was not signiﬁcantly different between adult and aged
animals of either strain.

IFN-a/b treatment were not explored for several reasons. First, a preliminary
experiment indicated that NK activity did not increase signiﬁcantly when splenocytes from young or aged mice were treated with IFN-a/b from 8–12 h (data not
shown). Second, NK activity increased, but not signiﬁcantly, from 4–8 h in both
young and aged mice, suggesting a plateau in augmentation. Most importantly,
lymphokine activated killer (LAK) cell activity is also induced after treatment with
IFN-a/b for 12 h. Thus, at later time points, the activities of NK and LAK cells
overlap, making it difﬁcult to clearly delineate the responding cell. In all subsequent
studies, splenocytes were treated with 103 units of IFN-a/b per 106 cells for 4 h
because these conditions resulted in a signiﬁcant level of difference between young
and aged animals. In addition, our results could be easily compared with previous
reports in Balb/c mice that measured NK activity at the time point.

3.3. Age-associated changes in NK acti6ity after IFN-h/i stimulation: genetics
The above results clearly demonstrate that NK activity is lower after treatment
with IFN-a/b in aged compared to adult B6 mice. Since we have shown that the
anti-metastatic effects of IFN-a/b treatment are maximal in our melanoma model
in young CB6F1 mice (unpublished data), we next questioned whether CB6F1 mice
would demonstrate age-related changes in inducible NK activity similar to that seen
in B6 mice. Thus, we compared induction of NK activity by IFN-a/b in adult and
aged CB6F1 mice. Fig. 4 shows that the induction of NK cytotoxicity by IFN-a/b
in aged CB6F1 mice was comparable to that achieved in adult mice.

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The differential effects of age on IFN-induced NK activity in B6 compared to
CB6F1 mice were surprising. Previous studies had shown that the other parental
strain of the CB6F1 mice, the Balb/c strain, also demonstrates age-associated
decreases in NK activity after treatment with IFN-a/b (Provinciali et al., 1989).
Although it was possible that different age-associated defects in induction of NK
activity were operative in B6 and Balb/c mice and the results of the CB6F1 mice
represent genetic complementation, it was imperative to determine whether the
Balb/c parent of the CB6F1 mice used in the present studies exhibited the age-associated defect in inducible NK activity that had been reported previously. Fig. 4
shows that although IFN-a/b enhanced NK activity in both adult and aged Balb/c
mice, the level of induction was signiﬁcantly lower in aged compared to adult
Balb/c mice. These data strongly suggest that the similar augmentation of NK cell
activity by IFN-a/b in aged and adult CB6F1 mice cannot be directly attributed to
a contribution of either parental strain, since NK cells from aged animals in both
Balb/c and B6 strains show a reduced induction by IFN-a/b.

3.4. Age-associated changes in IFN-induced NK acti6ity: organ distribution
It has been reported that IFN-a/b-induced NK activity does not decrease with
age in humans (Kutza et al., 1995), which is in direct contrast to our ﬁndings with
aged B6 and Balb/c mice. These differences could reﬂect a species difference in the
pattern of decline in IFN-a/b-inducible NK activity. Alternatively, the differences
could be organ speciﬁc since NK activity was assessed in peripheral blood mononuclear cells (PBMC) in humans, but in splenocytes in our studies. To address this
possibility, IFN-a/b-induced NK activity of PBMC from adult and aged B6 and
CB6F1 mice was assessed. As shown in Fig. 5, PBMC from both adult and aged
CB6F1 mice, and from adult B6 mice, demonstrated enhanced NK activity after
IFN-a/b treatment, reﬂecting about a 2.5-fold increase in all three groups. However, NK activity only increased 1.5-fold relative to basal activity after IFN-a/b
treatment in aged B6 mice. This induced level of NK activity in PBMC of aged B6
mice was signiﬁcantly lower than adult B6 mice (P= 0.03). These data clearly
indicate that the same pattern of inducibility by IFN-a/b was observed in PBMC
and splenocytes from both B6 and CB6F1 mice. These data suggest that differences
in the ability of IFN-a/b to stimulate NK activity in mice and humans may reﬂect
not only variation between species, but also variation among strains within the
same species.

3.5. Age-associated changes in induction of NK cell acti6ity by IFN-h/i:
mechanism
3.5.1. Percentage of NK (NK1.1+) cells
It is possible that a decreased percentage of NK cells may account for reduced
inducibility of NK activity by IFN-a/b in aged B6 mice. To address this possibility,
splenocytes from adult and aged B6 mice were stained with the NK-speciﬁc
monoclonal antibody, NK1.1. Representative staining patterns for adult and aged

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207

B6 mice are shown in Fig. 6. The pattern of staining (Fig. 6, panels A and B) and
the percentage of NK1.1 cells (Table 1) in spleen and PBMC were similar in adult
and aged B6 mice. In addition, there was no correlation between the percentage of
NK1.1 + cells and the level of cytotoxicity after IFN-a/b treatment (R= 0.09,
r 2 =0.04, P =0.14) for 24 adult and 24 aged mice. The similar percentages of NK
cells in adult and aged animals suggest that the difference in induction of NK
activity by IFN-a/b is not simply due to a reduced percentage of NK1.1 cells in the
mononuclear cell population of aged mice.

3.5.2. Percentage of NK1.1+ CD8+ cells
Although the percentage of NK1.1+ cells was not signiﬁcantly different between
adult and aged B6 mice, the percentage NK1.1+ CD8+ cells within the population of NK1.1 + cells was signiﬁcantly increased in aged compared to adult B6
mice (Fig. 6, panels C and D). Table 1 shows that the percentage of NK1.1+
CD8+ cells was two-fold higher in aged B6 mice. Importantly, the percentage of
NK1.1 +CD8 + cells was negatively correlated with cytotoxicity after IFN-a/b
treatment (R = − 0.86, r 2 =0.75, P= 0.02; n= 6 adult and 6 aged mice). The
correlation is even observed when results from multiple experiments are considered
(R= − 0.48, r 2 = − 0.21, P =0.005,n = 29 adult and 29 aged), which suggests a
robust correlation considering the high interassay variability of the 51Cr release
assay. These results suggest that the increased percentage of NK cells expressing the
CD8 marker may be at least partially responsible for the reduced induction of NK
activity by IFN-a/b in aged B6 mice.

Fig. 5. NK activity in PBMC of adult and aged CB6F1 and B6 mice after IFN-a/b treatment. PBMCs
from six to eight animals of each age group in each strain were assayed separately for cytotoxicity after
a 4 h stimulation with IFN-a/b (103U/106 cells). Bars show the mean fold induction 9 SEM relative to
basal levels of cytotoxicity. While the fold increase in CB6F1 mice is not signiﬁcantly different between
adult and aged, fold induction in PBMC of aged B6 animals is signiﬁcantly lower compared to adult
mice (PB 0.03; *).

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Fig. 6. Phenotypic proﬁle of B6 splenocytes. Splenocytes were stained with a PE-conjugated anti-NK1.1
antibody and/or APC-conjugated anti-CD8 antibody. 10 000 – 30 000 lymphocyte-gated events were
acquired. Panels A and B show a representative staining proﬁle for NK1.1 alone in adult and aged mice,
respectively. Panels C and D show a representative proﬁle for NK1.1 and CD8 two-color staining in
adult and aged mice respectively. Quadrants were set based on isotype controls.

3.5.3. Suppression of NK cell cytotoxicity by adherent cells
Several reports in mice have indicated that splenic and peritoneal adherent cells
suppress both basal and IFN-induced NK activity (Brunda et al., 1983; Irimajiri et
al., 1985; Riccardi et al., 1986). To determine if adherent cells were responsible for
the limited effect of IFN-a/b on NK activity in aged mice, adherent and non-adherent splenocytes from adult and aged B6 mice were separated prior to IFN-a/b
treatment. Cells were then combined as follows: non-adherent cells from adult mice
were added to adherent cells from adult or aged mice, and similarly, non-adherent
cells from aged mice were added to adherent cells from aged or adult mice.
Non-adherent cells alone or non-adherent cells mixed with adherent cells were
stimulated with 103 units of IFN-a/b for 4 h. As shown in Fig. 7, IFN-a/b-induced
cytotoxicity in adult animals showed higher cytotoxicity in the absence of adherent

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209

Table 1
Percentages of NK1.1 cells in spleen and peripheral blood of adult and aged B6 micea,b
Spleenc

Peripheral bloodd

%NK1.1e
Adult
Aged

%NK1.1+CD8+f

%NK1.1

%NK1.1+CD8+

4.229 0.35
4.589 0.56

12.39 9 1.62
29.349 3.21g

5.65 90.27
6.61 91.12

ND
ND

a

Mononuclear cells of spleen or peripheral blood (5×105)were stained with NK1.1-PE and CD8APC, followed by acquisition of 10 000 to 30 000 lymhocyt-gated events.
b
There was no signiﬁcant different in percent of NK1.1+ cells between adult and aged mice in either
spleen or peripheral blood (P=0.2)
c
Represents the mean of 33 adult and 33 aged B6 mice.
d
Represents the mean of 5 adult and 5 aged mice.
e
Percent of total lymphocytes.
f
Percent of total NK1.1+ cells.
g
Percent of NK1.1+CD8+ cells was signiﬁcantly elevated in aged compard to adult mice (PB
0.0001).

cells, although this increase was not signiﬁcant. In contrast, removal of non-adherent cells had no effect on inducible NK activity in aged mice. Further, IFN-a/b
induction of NK cytotoxicity in non-adherent cells of adult and aged mice was
comparable in the presence of either aged or adult adherent cells. This suggests that
whatever suppressive effect adherent cells may have, is not different between
adherent cells from adult or aged animals.

Fig. 7. Effect of adherent cells on IFN-a/b-induced NK activity in adult and aged B6 mice. Pooled
splenocytes from two mice of the same age were assessed for NK activity with and without adherent
cells. Solid bars indicate mean IFN induced cytotoxicity 9 SD of triplicate samples. Basal cytotoxicity of
unseparated splenocytes of adult and aged groups (hatched bars), were not signiﬁcantly different.
IFN-a/b-induced cytotoxicity of adult mice was signiﬁcantly higher in all three treatment groups
compared to aged mice treated comparably (PB 0.01; *). Figure shows a representative experiment in B6
mice, two additional experiments were done in both B6 and Balb/c mice, with similar results.

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Fig. 8. Effect of T cell depletion on IFN-a/b-induced NK activity in adult and aged B6 mice. Spleen cells
were treated with magnetic beads alone (CTRL), or speciﬁcally depleted of T cell subsets. ‘ − 4’ indicates
depletion of CD4 + cells, ‘ −8’ indicates depletion of CD8 + cells, and ‘ −4/8’ indicates depletion of
both CD4 + and CD8 + cells. All groups were stimulated with IFN-a/b (103U/106 cells) for 4 h. Bars
show the mean percent cytotoxicity9 SEM of two identical experiments done with pools of three adult
and three aged B6 mice in each age group. Depletion does not induce any statistically signiﬁcant changes
in basal or IFN-induced cytotoxicity in adult or aged groups.

3.5.4. Suppression of NK cell cytotoxicity by T cells
T cells have been shown to modify NK cell responses in vivo and in vitro after
IFN-a/b stimulation in young mice (Markovic and Murasko, 1991, 1993). To
determine if T cells inﬂuence the age-associated decrease in IFN-a/b-induced NK
activity, CD4 and CD8 cells were depleted separately and together before or after
IFN-a/b stimulation of splenocytes. As seen in Fig. 8, depletion of CD4 and/or
CD8 cells before IFN-a/b stimulation did not affect basal or IFN-a/b-induced
cytotoxicity in aged animals. Although in adult animals there is a small trend for
increased basal and IFN-a/b-stimulated cytotoxicity after CD8 cell depletion, the
increase is not statistically signiﬁcant. Depleting T cells after IFN-a/b stimulation
had no effect on either basal or induced NK activity in adult or aged mice (data not
shown). These data indicate that neither CD8 nor CD4 cells directly suppress the in
vitro induction of NK cells by IFN-a/b in aged mice.
4. Discussion
Since there is an increase in the incidence of cancer in the elderly, the validation
of the effectiveness of various cancer therapies must be extended to this population.
One treatment modality has used IFN-a/b. This cytokine has been successful as an
immunotherapeutic agent for some human cancers (Tyring, 1992), as well as in
animal models (Markovic and Murasko, 1991). It is clear that at least some of this
anti-tumor activity of IFN-a/b dependent on enhancement of NK cytotoxicity.
Based on these observations, the ability of IFN-a/b to enhance NK cytotoxicity in
aged and adult mice was evaluated in this study.

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211

The results presented have not only conﬁrmed previous data, but also have
extended the information currently available on age-associated changes in IFN-a/binducible NK activity in mice. Our data conﬁrmed previous results from Balb/c
mice indicating that the decrease in basal NK activity is not an aging phenomenon
per se, but rather a phenomenon of maturation (Provinciali et al., 1989). The most
dramatic decrease in basal NK activity occurs between 2–6 months of age, with the
level of basal NK activity remaining fairly constant from 6 months through the rest
of the lifespan. Therefore, any useful assessment of age-associated modiﬁcation in
NK activity in mice must compare animals at least 6 months old to mice at or
about their median lifespan. One study reported that enhancement of NK cytotoxicity after IFN-a/b treatment showed a gradual decrease with increasing age, with
6 month old Balb/c mice maintaining a greater IFN-a/b-inducibility of NK activity
compared to 24-month old mice (Provinciali et al., 1989). The current results extend
the previous ﬁndings to B6 mice, indicating that while basal NK activity is stable
from 6 through 28 months of age, there is an age-associated decrease in the ability
of NK cells of aged mice to be augmented by IFN-a/b.
Since both B6 and Balb/c mice demonstrate an age-associated decrease in
inducibility of NK cells after stimulation with IFN-a/b, it was assumed that this
decrease would also be seen in their F1 progeny. In contrast to our prediction,
CB6F1 mice showed no age-associated decrease in induction of NK cytotoxicity by
IFN-a/b. Although this may be a random genetic difference, it is possible that this
represents a complementation in different defects in the Balb/c and B6 parents.
Since the CB6F1 mouse does not show the decline, it may hold genetic clues to the
maintenance of an augmentable NK response in aged animals. This would not only
help elucidate NK cell function in aging mice, but could also be useful in
investigations of IFN therapy in elderly humans. Although most elderly do not
show a decline in induction of NK activity by cytokines with increasing age, it has
been reported that there is a subset of individuals who demonstrate a decreased
response of NK cells after IFN induction (Mysliwska et al., 1992). Since the human
population is very heterogeneous, it is possible that most individuals represent the
complementation of the appropriate genes to maintain NK activity. The subset of
individuals who show decreased inducibility may reﬂect homozygosity at the genes
that are responsible for the decreased response to IFN of inbred mouse strains. It
is important to note, however, that our results demonstrate that the differences seen
between mice and humans is not due to the source of cells that are being utilized,
since NK activity of PBMC reﬂects the NK activity of splenocytes. While one
previous report showed that basal NK activity of PBMC did not decrease with age
in CBA/J and C3H/HeN mice (Lanza and Djeu, 1982), it did not assess inducibility
of NK activity in PBMC. Our results demonstrate that B6 mice show an age-related
decline in inducibility of NK activity in PBMC that is comparable to the decrease
seen in splenocytes, while the CB6F1 mouse shows comparable augmentation of
NK activity by IFN-a/b in both peripheral blood and spleen throughout the ages
examined.
The mechanism of decreased inducibility of NK activity with increasing age was
explored. The microenvironment of the NK cell could inﬂuence the level of

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cytotoxicity achieved in aged mice. A suppressive interaction with monocytes is one
possibility, since suppression of basal NK activity by adherent cells has been
reported in aged humans and mice (Brunda et al., 1983; Irimajiri et al., 1985;
Mysliwska et al., 1992; Riccardi et al., 1986). In addition, the ability of splenic
adherent cells to suppress basal NK cytotoxicity was observed to be more pronounced in aged (24 months) compared to young (2 months) C3H/HeN mice
(Riccardi et al., 1986). Our results, however, show no effect of adherent cells on
IFN-a/b-induced NK activity in either adult or aged mice, suggesting that after the
maturational change in NK has happened, the adherent cells from adult or aged
mice do not have different suppressive abilities on NK cytotoxicity.
Although there is limited information on the ability of T cells to suppress NK
cytotoxicity in aged mice, there are several studies suggesting that T cells can
regulate NK activity. One study reported that CD8 cells of some humans suppress
basal NK activity in vitro (Garcia-Penarrubia et al., 1989). In addition, several
˜
reports have indicated that anesthesia inhibits IFN-a/b induction of NK cytotoxicity in mice (Markovic et al., 1993) and humans (Kutza et al., 1997). In the mouse
model, depletion of CD8 cells after anesthesia, but prior to in vitro stimulation with
IFN-a/b, was able to restore anesthesia-mediated inhibition of NK cell activity
(Markovic and Murasko, 1993). Our mouse model of immunotherapy using B16
melanoma cells also demonstrates regulation by T cells. The effectiveness of
IFN-a/b in this model is dependent on IFN-a/b augmentation of NK activity, since
depletion of NK cells completely abrogates any protection. However, depletion of
only CD4 cells also abrogates protection, while depletion of both CD4 and CD8
cells results in protection comparable to that seen in intact animals. One interpretation of these results is that in this melanoma model CD8 cells inhibit IFN induction
of NK activity, while CD4 cells counterbalance this CD8 suppression (Markovic
and Murasko, 1991). In contrast, our current results suggest that the age-associated
decrease in IFN-a/b-inducibility of NK cells in vitro is not affected by the presence
of T cells.
Innate changes to NK cells could also be responsible for the decrease response of
NK to IFN-a/b. Our data clearly demonstrate that the deﬁciency is not related to
a requirement of the NK cells of aged mice for more IFN-a/b or for an extended
incubation with IFN-a/b to allow the activation to occur. The possibility that NK
cells of aged mice demonstrate a decreased number or afﬁnity of receptors for
IFN-a/b is currently being explored.
Another simple explanation could be that aged mice have a reduced number of
NK cells compared to adult mice. While one report showed that the percentage of
splenic NK cells (determined by reactivity with anti-asialo GM1 antibody) was
reduced in young (2-month old) compared to aged (10–13 month old) DBA mice
(Dussault and Miller, 1994), another study using a more speciﬁc indicator of NK
cells (reactivity with anti-NK1.1 antibody) found that the percentage of splenic NK
cells was not different between young (3–4 month) and aged (22–26 month) B6
mice (Mikael et al., 1994). Evaluation of the percentage of NK1.1+ cells in our
studies also indicated that a decrease in NK cell number was not responsible for the
age-associated decrease in NK activity. However, upon further evaluation, we

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213

found that aged mice demonstrate an increase in the percent of NK cells that
express the T cell marker, CD8. Aged mice had a two-fold higher percentage of
CD8 +NK1.1 + cells than adult mice, representing about a third of all NK cells
in aged mice. Importantly, the level of cytotoxicity observed after induction with
IFN-a/b was inversely related to the percentage of NK1.1 + CD8+ cells.
Presently, the functions and activity of CD8+ NK1.1+ cells have not been
extensively explored. They are thought to be LAK cell precursors in mice (Lee et
al., 1996). In humans, an equivalent population that is CD8+ CD57+ is increased in disease conditions such as rheumatoid arthritis and are proposed to
have a role in autoimmunity (Imberti et al., 1997). Interestingly, the CD8+
CD57 + cell population increases in elderly people (Miyaji et al., 1997), indicating some similarity in the generation of these cells in mice and humans with
aging. Since removal of CD8+ cells, which would include the CD8+ NK cells,
did not affect cytotoxicity in the depletion studies (Fig. 8), it is possible that
CD8 + NK1.1 + cells may not be cytotoxically active in the short-term NK
assay. Techniques are being developed to isolate this small percentage of splenic
lymphocytes to address this question more directly. It will be interesting to see
whether NK cytotoxic activity is limited to the NK1.1+ CD8− population, and
whether IFN-a/b will have differential effects on the CD8+ and CD8− NK
subpopulations.
There are other mechanisms in the activation pathway of NK cell activity that
must be evaluated for their contribution in the age-associated decrease of IFN-a/
b-induced NK cytotoxicity. Reduced cytotoxicity by NK cells of aged mice could
reﬂect defective binding of IFN-a/b or impaired signal transduction after interaction with the cytokine. Alternatively, NK cells of aged mice might exhibit a
reduced binding to the target cells; this has been reported aged DBA/2 mice
(Dussault and Miller, 1994). Additionally, decreased production and/or release of
cytotoxic effectors such as granzymes, perforins, and Fas ligand could result in
decreased cytotoxicity. Further evaluation of these mechanisms in adult and aged
mice in relation to IFN-a/b-induced NK cytotoxicity will provide important
clues to the age-related decrease in NK activity in B6 mice. The mechanisms in
B6 animals could then be evaluated in CB6F1 mice to identify possible genetic
differences linked to the defect and possible application in treatment of elderly
humans.

Acknowledgements

This manuscript was supported by NIH Grant AG13542. We would like
to thank Dr Elizabeth Gardner for her assistant with both the interpretation
of the ﬂow cytometry data and with editorial revisions of this manuscript, and Ms Christine Kinsinger for assistance in preparation of this
manuscript.

214

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