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Enhanced co-culture and enrichment of human natural killer cells for the selective clearance of senescent cells

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Abstract

In the context of aging and age-associated diseases, Natural Killer (NK) cells have been revealed as a key cell type responsible for the immune clearance of senescent cells. Subsequently, NK cell-based therapies have emerged as promising alternatives to drug-based therapeutic interventions for the prevention and treatment of age-related disease and debility. Given the promise of NK cell-mediated immunotherapies as a safe and effective treatment strategy, we outline an improved method by which primary NK cells can be efficiently enriched from human peripheral blood across multiple donors (ages 20-42 years old), with a practical protocol that reliably enhances both CD56dim and CD56bright NK cells by 15-fold and 3-fold, respectively. Importantly, we show that our co-culture protocol can be used as an easily adaptable tool to assess highly efficient and selective killing of senescent cells by primary NK cells enriched via our method using longer co-culture durations and a low target to effector ratio, which may be more physiological than has been achieved in previous literature.
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INTRODUCTION
Advanced age is the greatest risk factor for most
chronic diseases, with over 90% of adults aged 65 or
older experiencing at least one chronic disease such as
cancer, diabetes and cardiovascular disease [1].
Progressively increasing numbers of these adults are
suffering from multimorbid age-related conditions [2].
Many aging phenotypes and pathologies, including
diverse age-associated diseases and disorders, are
causally linked to the accumulation of senescent cell
burden with increasing age [3, 4]. Senescent cells are
characterized by an irreversible cell-cycle arrest of
proliferation-competent cells, along with morphological
and metabolic changes, altered gene expression,
chromatin reorganization, and a complex pro-
inflammatory senescence-associated secretory
phenotype (SASP), which contributes to chronic
inflammation and damage to surrounding cells and
tissues [4, 5]. Senescent cells are thought to be cleared
by the immune system, but increasing age or disease
severity allows senescent cells to escape the process of
immunosurveillance and accumulate in older
individuals [6]. The removal of senescent cells via
drugs that selectively kill senescent cells (“senolytic”
drugs) or genetic manipulation in transgenic mouse
models can prevent or delay tissue dysfunction, improve
age-related pathologies, and extend health span,
suggesting that a reduced senescent cell burden in aging
adults merits further study as a therapeutic target for the
treatment and prevention of disease of aging [7, 8].
However, exploiting the ability of the innate immune
system to surveille senescent cells has emerged as an
alternative approach for their elimination [9]. Several
lines of evidence show that Natural Killer (NK) cells
play a vital role in the targeted elimination of senescent
cells [911]. In fact, regulation of senescence burden by
NK cells is not only considered essential for tissue
homeostasis [12, 13], but also has been shown to be
www.aging-us.com AGING 2022, Vol. 14, Advance
Research Paper
Enhanced co-culture and enrichment of human natural killer cells for
the selective clearance of senescent cells
Kristie Kim1, Tesfahun Dessale Admasu1, Alexandra Stolzing1,2, Amit Sharma1
1SENS Research Foundation, Mountain View, CA 94041, USA
2Loughborough University, Centre for Biological Engineering, Wolfson School of Electrical, Material and
Manufacturing Engineering, Loughborough, UK
Correspondence to: Amit Sharma; email: amit.sharma@sens.org
Keywords: aging, senescence, natural killer cells, NKCC, immune surveillance
Received: September 24, 2021 Accepted: February 22, 2022 Published: March 4, 2022
Copyright: © 2022 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution
License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
author and source are credited.
ABSTRACT
In the context of aging and age-associated diseases, Natural Killer (NK) cells have been revealed as a key cell
type responsible for the immune clearance of senescent cells. Subsequently, NK cell-based therapies have
emerged as promising alternatives to drug-based therapeutic interventions for the prevention and treatment of
age-related disease and debility. Given the promise of NK cell-mediated immunotherapies as a safe and
effective treatment strategy, we outline an improved method by which primary NK cells can be efficiently
enriched from human peripheral blood across multiple donors (ages 20-42 years old), with a practical protocol
that reliably enhances both CD56dim and CD56bright NK cells by 15-fold and 3-fold, respectively. Importantly, we
show that our co-culture protocol can be used as an easily adaptable tool to assess highly efficient and selective
killing of senescent cells by primary NK cells enriched via our method using longer co-culture durations and a
low target to effector ratio, which may be more physiological than has been achieved in previous literature.
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important for the regulation of pathological states such
as tumor growth [11, 14]. Moreover, impairment of NK
cell function has been shown to accelerate aging in
perforin-deficient mice due to the accumulation of
senescent cells [15].
Although senescent cells are known to secrete
chemokines as a part of the SASP to attract NK cells,
they have also evolved strategies to evade clearance by
NK cells [16]. For instance, senescent fibroblasts can
escape immune surveillance by increasing expression of
HLA-E, which upon interaction with NKG2A inhibits
NK cell cytotoxicity [17]. Furthermore, senescent cells
can shed MICA and MICB (ligands for the NK cell-
activating receptor NKG2D), preventing the binding of
NK cells to their targets [18, 19]. Thus, whether through
NK cell-based adoptive cell therapies or removal of NK
cell inhibitory ligands, there is tremendous promise in
modulating NK cells as an intervention against age-
related diseases [9].
However, an impediment to the development of NK cell-
based senotherapeutic strategies is that although several
publications have demonstrated the immune surveillance
potential of NK cells towards senescent cells, the co-
culture strategies employed do not necessarily reflect
physiological conditions. For instance, many studies
demonstrating immune surveillance of senescent cells by
NK cells used very high target to effector (T:E) ratios. It
has been shown that most NK cell killing occurs through
serial killing whereby a single NK cell can kill up to 10
targets [20]. Furthermore, a single IL-2-activated NK cell
releases about a tenth of its total lytic granule reserve
after 16 hours of co-culture [21, 22]. However, human
YT cells (human NK cell line) have been used with T:E
ratios as high as 1:20 to achieve modest cytotoxicity
towards senescent IMR-90 cells [11, 23, 24]. Lannello et
al. reportedly used T:E ratios as high as 1:81 [25].
Interestingly, although Pereira et al. developed an
autologous co-culture system with skin-derived primary
human fibroblasts and NK cells, cytotoxicity towards
senescent fibroblasts was low (10-20%) even with a high
T:E ratio of 1:20 [17, 24, 25]. In addition, in most of
these studies, co-culture experiments were performed for
relatively short (2-6 hours) durations [11, 17, 2325].
Finally, Interleukin-2 (IL-2) is a cytokine widely known
to induce proliferation and activation of resting NK cells
and is vital for their cytotoxic function both in cell culture
and in vivo [26]. However, the concentration of IL-2 used
to demonstrate the ability of NK cells to kill senescent
cells varies substantially [18, 21, 27, 28].
In the present study, we demonstrate an easily adaptable
and more physiological co-culture system in which we
use freshly isolated peripheral NK cells cultured for 3
days with a relatively low concentration (100 IU/ml) of
human recombinant Interleukin-2 (rIL-2). We have
shown that even with different target cell types, NK cell
donors, and methods of senescence induction, our co-
culture method is robust and reliable, consistently
achieving two- to three-fold higher cytotoxicity of NK
cells towards senescent cells compared to non-senescent
cells at a co-culture duration of 16 hours and low 1:1 T:E
ratio. Our protocols for NK cell isolation and enrichment
and co-culture may more fully capture the interaction
between senescent cells and NK cells than has been
previously achieved. Importantly, this simple, robust
protocol may serve as a platform for the development of
novel NK cell-based senescent cell ablation strategies.
RESULTS
Genotoxic stress-induced model of senescence
Human fetal lung fibroblasts (IMR-90) cells were used
as a cell culture model of cellular senescence. Robust
induction of SA-β-gal activity was observed in 87% of
doxorubicin-treated (300 nM) cells, nine days after
treatment whereas 1% of non-senescent control cells
showed SA-β-gal activity (Figure 1A, 1B). In addition
to doxorubicin treatment, additional models of
senescence induction such as irradiation (20 Gy) and
treatment with the mitochondria damaging agent,
etoposide (20 µM, 48 h) were tested. A statistically
significant percentage of cells with increased SA-β-gal
activity was observed in cells exposed to x-rays or
etoposide (87% and 81%, respectively) nine days after
treatment compared to non-senescent control cells (1%)
(Supplementary Figure 1B, 1C).
The increase in SA-β-gal activity of doxorubicin-treated
fibroblasts correlated with persistent DNA damage, as
measured by immunofluorescence staining for γ-H2AX
(Figure 1C). The percentage of senescent cells with >2 γ-
H2AX foci per cell was eight-fold higher compared to
that of non-senescent cells (Figure 1D). As expected,
mRNA levels of cell cycle checkpoint markers p16INK4A
and p21CIP1 were significantly elevated in senescent
compared to non-senescent cells (Figure 1E). Loss of high
mobility group box 1 (HMGB1), a highly conserved
nuclear protein from senescent cells, is often used as a
biomarker of senescence [29]. The loss of HMGB1 from
the nucleus of our senescent fibroblasts was confirmed in
doxorubicin-treated cells (Supplementary Figure 1A).
Further, loss of Lamin B1 expression, another hallmark
of senescence [30], was also observed in senescent
compared to non-senescent cells (Figure 1F). Finally,
increased mRNA levels of classical SASP factors IL-6,
IL-8 and IL-1α in senescent cells was confirmed by qRT-
PCR [31, 32] (Figure 1G). Collectively, these findings
validated robust induction of a senescence phenotype in
our cell culture model.
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Figure 1. Senescent human fibroblasts express markers of senescence. IMR-90 fibroblasts were induced to senesce by doxorubicin
(300 nM, 24 h) and SA-β-Gal staining was performed on day 9 after doxorubicin treatment. (A) Representative images of SA-β-Gal stained
senescent (S) and non-senescent (NS) cells. (B) Quantification of SA-β-gal-positive cells in NS and S IMR-90 cells. Four fields were quantified
per well (n=6) with a total of 7713 and 2666 cells counted for NS and S cells, respectively. (C) Immunofluorescence was performed to detect
γ-H2AX in NS and S IMR-90 cells, 10 days after exposure to vehicle or doxorubicin, respectively. Representative images of NS and S cells
stained for γ-H2AX (green) and Hoechst (blue). (D) The percentage of cells with 3 or more γ-H2AX foci/cell (γ-H2AX+ cells) was scored from a
total of 780 NS and 387 S cells. The results are presented as mean % of cells with 3 or more foci/cell. (EG) mRNA levels of cell cycle
regulators p16 and p21, Lamin B1, and various SASP factors, IL-6, IL-8, and IL- assessed through Quantitative Realtime PCR in NS and S IMR-
90 cells (n=3). All results are presented as a mean and error bars represent ±SEM. Statistical analysis performed using unpaired t test. *p <
0.05, **p < 0.01, and ***p < 0.001.
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Isolation and enrichment of NK cells
Freshly drawn whole blood (40 ml) was treated with
RosetteSep human NK cell cocktail (Stem Cell
Technologies, USA), which removes unwanted cells
with Tetrameric Antibody Complexes that binds to
white blood cells (except NK cells) and crosslinks
them to red blood cells (RBCs). Following density
gradient centrifugation, the NK cell population at the
interface between the plasma and buoyant density
medium was isolated. Freshly isolated NK cells
were then incubated in RPMI media supplemented
with 20% FBS and 100 IU/ml rIL-2 for 72 hours for
enrichment (Figure 2A).
The relative expression of CD56 and absence of CD3
are routinely used to identify human NK cells [33].
Low (CD56dim) and high (CD56bright) CD56 expression
levels define major subsets of NK cells [33]. CD56bright
CD16- cells are considered immature NK cells that
secrete interferon-γ (IFNγ), whereas CD56dim CD16+
NK cells are responsible for cytotoxicity [34]. Upon
physical interaction with target cells, cytotoxic NK
cells release perforin, granzymes (serine proteases),
and other cytotoxic granules that kill their targets [35].
FACS analysis was performed to characterize NK cells
freshly isolated from donors between the ages of 20-42
years. Results revealed a classic distribution of
CD56bright CD16- and CD56dim CD16+ NK cells, as
evidenced by CD16 versus CD56 flow cytometry
analysis in the scatter plots of fresh human Peripheral
blood mononuclear cells (PBMCs) exemplified by one
donor (after gating out CD3+ T cells as shown in
Figure 2B-i and Supplementary Figure 2B). Specificity
of antibodies was confirmed by Fluorescence minus
one (FMO) control experiments with NK cell
populations (Supplementary Figure 2C-i, 2C-ii).
Interestingly, statistically significant enrichment of
both sub-populations of NK cells was observed after
culturing cells in media with rIL-2 for three days
(Figure 2B-ii). This increase in the numbers of NK cell
subtypes was consistent in NK cells isolated and
enriched from multiple donors, with a fifteen-fold
increase in the numbers of CD56dim CD16+ NK cells
and approximately three-fold increase in the numbers
of CD56bright CD16- NK cells (Figure 2C-i, 2C-ii).
NK cell-mediated killing of senescent cells
Senescent cells are known to secrete a variety of factors
as part of the SASP that can attract NK cells and either
activate or inhibit cytotoxic function [9, 14, 36]. To test
whether senescent cells in our senescence model
produced increased levels of these factors, we measured
mRNA expression of multiple cytokines. A robust and
statistically significant increase in the mRNA levels of
CCL5, CXCL9 and CXCL11, which are known
chemoattractants for NK cells, was observed in senescent
cells compared to non-senescent controls (Figure 3A). A
modest increase in mRNA expression of CCL2,
decreased expression of CXCL12, and non-significant
difference in Chemerin compared to non-senescent cells
was also observed (Supplementary Figure 2A).
Although the role of NK cells in targeting senescent
cells has been previously demonstrated, the protocols
used are quite variable and employ conditions that may
not be as physiologically representative. Hence the
cytotoxic potential of NK cells isolated and enriched
with our protocol for the selective killing of senescent
cells was determined by lactate dehydrogenase (LDH)
release. Even at a T:E ratio of only 1:1, NK cells
selectively eliminated senescent fibroblasts at a
significantly higher (43%) level compared to non-
senescent control cells (15%) after 16 hours of co-
culture. In addition, twice (83%) as many senescent
fibroblasts were killed by NK cells when the T:E ratio
was doubled to 1:2 compared to 41% in NS cells, and
further increasing the T:E ratio to 1:3 killed nearly all
senescent fibroblasts (93%) compared to 51% in NS
cells. However, at higher T:E ratios of 1:2 and 1:3,
substantial killing of non-senescent cells was also
observed (Figure 3B and Supplementary Figure 3A).
Recent publications have reported substantial variability
among senescent cells based on mode of senescence
induction and cell type [37]. Thus, whether NK cells
enriched by our protocol can eliminate senescent cells
induced to senesce by different stressors was
investigated. NK cells killed 43-50% of senescent cells
whether senescence was induced by doxorubicin (Figure
3C-i), irradiation (Figure 3C-ii) or etoposide (Figure 3C-
iii) at statistically higher levels compared to cytotoxicity
towards non-senescent cells (15%). In addition, the
senescence phenotype may also vary depending on cell
type. Thus the efficacy of enriched NK cells in targeting
senescent endothelial cells was also investigated [38].
Enriched and activated NK cells were observed to be
effective at killing senescent endothelial cells at a three-
fold higher rate than NS endothelial cells (Figure 3C-iv)
in a statistically significant manner.
Freezing primary NK cells often results in a decline
in cytotoxicity [39]. However, our results showed that
NK cells isolated and enriched with the protocol
described above remained significantly more cytotoxic
towards senescent cells compared to non-senescent
cells following cryopreservation at a rate comparable to
freshly isolated NK cells, as measured by LDH release
after 16 hours of co-culture (Supplementary Figure 3B).
Interestingly, when co-culture duration was extended
to four days, the vast majority (85-90%) of NS
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Figure 2. Isolation and enrichment strategy of primary NK cells from human PBMC. (A) Experimental design of the NK cell
enrichment strategy. PBMCs were collected from multiple donors (ages 20-42 years old), and NK cells were isolated and enriched. (B) Flow
cytometry analysis of CD56 and CD16 expression in NK cells before and after enrichment for a representative donor. (B-i) Before enrichment,
1.64% of NK cells (0.18% of PBMCs) were CD56Bright CD16- and 46.34% of NK cells (2.8% of PBMCs) were CD56Dim CD16+ NK cells. (B-ii) After
enrichment, 2.29% of NK cells (0.47% of PBMCs) were CD56Bright CD16- and 66.18% of NK cells (12.6% of PBMCs) were CD56Dim CD16+ NK cells.
(C-i) Average percentage of CD56Dim CD16+ NK cell population in PBMCs from five donors. (C-ii) Average percentage of CD56Bright CD16- NK cell
population in PBMCs from five donors. Donor sex and age are indicated in the figure. Statistical analysis performed using paired t test.
*p < 0.05, **p < 0.01, and ***p < 0.001.
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Figure 3. Activated primary NK cells selectively eliminate
senescent cells. (A) Quantitative Realtime PCR was performed
to detect the mRNA levels of CCL5, CXCL9, and CXCL11 in non-
senescent and senescent IMR-90 fibroblasts. The results are
presented as mean fold change in NS compared to S samples
from two independent experiments performed in triplicate, and
error bars represent ±SEM. Statistical analysis performed using
unpaired t test. *p < 0.05, **p < 0.01, and ***p < 0.001. (B) NS or
S IMR-90 fibroblasts were co-incubated with NK cells for 16 h at
T:E ratios of 1:1, 1:2 and 1:3 and cytotoxicity was evaluated by
LDH release. The graphs show the mean and S.E. of % LDH
release. NS or S (C-i) doxorubicin-treated (n=6), (C-ii) irradiated
(n=3) or (C-iii) etoposide-treated (n=3) IMR-90 fibroblasts or
(C-iv) doxorubicin-treated endothelial cells (n=2) were overlayed
with NK cells for 16 hours at T:E ratio of 1:1, and cytotoxicity was
evaluated by LDH release. The results are plotted as mean %
cytotoxicity for NS and S cells with each experiment performed
in at least triplicate. The graphs show mean % LDH release. (D)
NK cells isolated and enriched from three different individuals
were co-cultured with NS or S IMR-90 cells at T:E ratio of 1:1 and
cytotoxicity was evaluated by LDH release after 16 hours of co-
culture. Experiments were performed in triplicate and the
results are plotted as mean % cytotoxicity for NS and S. Donor
sex and age are indicated in the figure. Statistical analysis
performed using unpaired t test. *p < 0.05, **p < 0.01, and
***p < 0.001.
cells survived, and only 10% and 30% of senescent cells
were viable following co-culture with fresh or revived
NK cells, respectively, as determined by Calcein AM
(Supplementary Figure 3C).
Results from four days of co-culture were confirmed,
demonstrating that only 6% and 17% of senescent cells
survived following co-culture with NK cells even when
senescence was induced by irradiation (Supplementary
Figure 3D-i) or etoposide (Supplementary Figure 3D-
ii), respectively, whereas 85-100% of non-senescent
cells survived following co-culture.
Finally, to test whether the cytotoxicity of primary NK
cells prepared using our protocol was influenced by
donor variability, senescent or non-senescent IMR-90
cells were co-cultured with NK cells isolated from
multiple donors. A significantly higher cytotoxicity
towards senescent compared to non-senescent cells
was observed, as measured by LDH assay for each
donor (Figure 3D). Furthermore, although the levels of
Granzyme B released by activated NK cells correlated
with overall cytotoxicity efficiency of individual NK
cell donors, NK cells from each donor released similar
amounts of Granzyme B upon co-culture with either
senescent or non-senescent cells (Supplementary
Figure 3E).
DISCUSSION
The involvement of senescent cells in aging and
diseases of aging is well documented and has generated
tremendous excitement amongst geroscientists as a
potential therapeutic target for various age-related
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diseases. Several laboratories are attempting to develop
therapeutic interventions to eliminate senescent cells
using senolytic drug therapies. Although promising in
animal models, unintended outcomes from senolytic
interventions may be a potential concern [4042]. A
better understanding of the immune surveillance of
senescent cells, and especially the key role of NK cells,
offers a potential avenue to novel immunotherapies that
can target senescent cells and expand healthspan.
NK cells are known to be one of the main effectors
responsible for the immune surveillance of senescent
cells. Hence, utilization of engineered NK cells as novel
senotheraputics has emerged as an alternative to drug-
based senolytic approaches. We investigated the
viability of such an approach by demonstrating that our
protocol is efficient in the isolation and enrichment of
NK cells from PBMCs. Furthermore, this method can
be used to assess the selective killing of senescent cells
by NK cells in a practical and highly reproducible
manner. Since senescence phenotype varies depending
on the mode of senescence induction or cell type, we
further showed that NK cells isolated and enriched with
our protocol were also effective in killing senescent
cells independent of cell origin or mode of senescence
induction by doxorubicin, ionizing radiation or
etoposide treatment.
To improve the efficiency of NK cell isolation, we
isolated NK cells from PBMCs before expanding them
in cell culture. The relative expression of CD56 and
CD16 are commonly used to identify human NK cells
[33]. CD56dim (CD16+) cells are the predominant NK
cell population in circulation, whereas CD56bright (that
are CD16-) cells are considered a less mature stage of
NK cells [33, 43, 44]. Cytotoxic function is thought to
be performed by CD56dim NK cells, while CD56bright
NK cells are potent producers of cytokines such as IFN-
γ and TNF-α [45, 46]. However, CD56bright NK cells are
known to acquire cytotoxic function in the presence of
cytokines such as IL-2 [47]. Consistent with previous
reports, we observed a higher proportion of CD56dim
NK cells in freshly isolated PBMCs. Following
isolation and enrichment, we observed expansion of
both CD56dim and CD56bright NK cell numbers by 15-
and 3-fold, respectively, expressed as the mean of all
five subjects at a relatively low rIL-2 concentration. IL-
2 is considered essential for the proliferation and
function of NK cells. Although other cytokines such as
IL-4, IL-7, and IL-12 have been used for NK cell
enrichment, they have been reported to be overall less
potent [48]. Of note, cryopreserved NK cells that have
been untouched by cytokines in cell culture have been
shown to have phenotype and cytotoxicity that
resembles those of fresh cells; whether this is true for
IL-2 activated and expanded NK cells has not been
established [39]. Interestingly, our data reveal that
cryopreservation of IL-2 activated NK cells isolated and
enriched via our method have comparable cytotoxicity
to freshly isolated NK cells.
Our results show that NK cells kill 40-50% of senescent
cells after 16 h at a T:E ratio of 1:1, which is more
efficient and under more physiological conditions than
has been previously reported. For instance, others have
reported T:E ratios as high as 1:81 and co-culture
durations as short as 2 h, which may not reflect
physiological conditions [17, 18, 49]. A longer co-
culture can also be useful for capturing the mechanism
of Natural Killer Cell Cytotoxicity (NKCC). For
example, in addition to releasing lytic granules to kill
their targets within a few hours after the start of co-
culture, NK cells are known to use additional
mechanisms that require longer exposure to their
targets, such as receptor-mediated apoptosis by
expressing TRAIL or Fas ligand [50]. Thus, our
improved co-culture strategy that employs more
physiological T:E ratios (1:1) and longer co-culture
durations (16 h or 4 d) likely captures broader and
important NK-senescent cell interactions that may be
more informative for studying the immune surveillance
of senescent cells.
To test the robustness of our NK cell enrichment
protocol, we isolated NK cells from five healthy
individuals between the ages of 20-42 years. Our results
show two- to three-fold higher cytotoxicity toward
senescent cells when compared to non-senescent
controls. Interestingly, our data suggest that NK cells
from younger males generally exhibited higher
cytotoxicity than females and older donors. This
observation warrants further investigation with a larger
sample size. Several studies have characterized changes
in the numbers of circulating NK cells as well as the
distribution of NK cell subsets with increasing age
[51, 52]. Additionally, an age-related increase has been
observed in the numbers of dysfunctional or exhausted
NK cells, which can be identified by decreased NK
effector functions, reduced IFN-γ secretion, and
lower perforin and granzyme expression [53]. These
observations suggest a potential decline in NKCC with
age. Whether this general age-related decline in NKCC
contributes to the age-dependent increase in senescence
burden in older adults should be investigated.
A greater understanding of the mechanisms by which
NK cells interact with senescent cells is needed to
identify novel interventions for improving immune
surveillance by NK cells. Senescent cells are known to
secrete CCL2, CCL5, CXCL9 and CXCL11 along with
other SASP factors [5457]. To confirm that our
senescent cells secreted elevated levels of cytokines and
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chemoattractants, we tested gene expression levels of
CCL2, CCL5, CXCL9 and CXCL11. We observed
substantially higher expression of these cytokines from
senescent compared to non-senescent cells, which is
important for the context of future in vivo studies as
these chemokines are known to be main chemo-
attractants for NK cells [5860]. However, we did not
observe an increase in the expression of CXCL12 or
chemerin, which were reported to be critical
chemotactic factors for NK cell recruitment, by
senescent fibroblasts. Chemerin/RARRES2 is
commonly downregulated across several tumor types
and often employed by tumor cells to escape immune
clearance by tumor-infiltrating effector leukocytes [61].
Our data suggest that senescent cells may also employ
similar strategies in vivo to escape immune clearance.
Since ectopic expression of chemerin in the tumor
microenvironment (TME) results in increased
recruitment of NK cells [62], a similar strategy could be
used for improving immune surveillance of senescent
cells by NK cells.
In summary, we have developed a robust, easily
adaptable cell co-culture model that involves isolation
of human NK cells from peripheral blood, followed by
enrichment in a relatively low IL-2 concentration. Our
method for co-culturing NK cells with senescent cells
may more fully capture the highly specific cytotoxicity
towards senescent human fibroblasts and endothelial
cells under more physiological conditions. We achieved
significant cytotoxicity of NK cells towards senescent
cells independent of donor variability using our
enrichment strategy. Moreover, the specificity of
primary NK cells towards senescent cells was further
confirmed by a 4-day co-culture in which virtually all
senescent cells were killed whereas viability of non-
senescent cells with NK cell effectors was visually
indistinguishable from negative control cells untouched
by effector cells. A deeper understanding of the
interplay between senescent cells and NK cells is
essential for the development of better therapeutic
interventions, especially novel immunotherapies, for the
treatment and prevention of age-associated diseases.
The co-culture strategy presented here may serve as an
important platform for the development of effective
immunotherapies in the future that may involve the
alteration and ultimately improvement of NK cell
cytotoxicity towards senescent cells.
MATERIALS AND METHODS
Cell culture
Human primary NK cells were maintained at 37° C in
humidified air containing 5% CO2, and complete media
containing RPMI-1640 medium (ATCC, USA; Cat#30-
2001) supplemented with 20% Fetal Bovine Serum
(FBS) (Millipore Sigma, USA; Cat# F4135), 1X
PenicillinStreptomycin (Corning; Cat# 30-001-CI),
and 100 IU/ml human rIL-2 (recombinant Interleukin-2)
(TECIN teceleukin; Bulk Ro 23-6019). IMR-90 primary
lung fibroblasts (ATCC, USA: Cat# CCL-186) were
used at population doubling level (PDL) 30-47 and were
cultured in an atmosphere with 5% CO2 and 3% O2 with
complete media containing Dulbecco’s Modified
Eagle’s Medium (DMEM) (Corning; Cat# 10-013-CV)
supplemented with 10% Fetal Bovine Serum (FBS)
(Millipore Sigma, US; Cat# F4135) and 1X Penicillin
Streptomycin (Corning; Cat# 30-001-CI). Cumulative
PDL was calculated using the following equation:
log H log S
PDL log 2
=
where H is the number of cells at harvest and S is the
number of cells seeded. Primary human arterial
endothelial cells purchased from Coriell Institute for
medical research (AG10770) were maintained in promo
cell basal medium MV2 (PromoCell; Cat# C-22221)
supplemented with Growth Medium MV 2 Supplement
Pack (PromoCell; Cat# C-39221) and assayed within 10
or less passages. Endothelial cells were maintained at
37° C in humidified air containing 5% CO2.
Senescence induction
Senescence was induced in IMR-90 fibroblasts as
described before with some modifications [63]. Cells
were treated with 300 nM doxorubicin hydrochloride
(Millipore Sigma, USA; Cat# 504042) in DMEM
complete media for 24 h and maintained in culture as
described for 10 days (d). Human endothelial cells were
treated with 250 nM doxorubicin in promo cell basal
medium MV2 supplemented with Growth Medium MV
2 Supplement Pack for 24 h and maintained in culture
as described [63]. For cells induced to senesce by
irradiation, IMR-90 cells were treated with ionizing
radiation (20 Gy X-ray). For cells induced to senesce by
etoposide, fibroblasts were treated with 20 µM of
etoposide (Millipore Sigma, USA; Cat# E1383) for 48 h
and maintained in culture as described before.
Senescence-associated ß-galactosidase staining
Senescence in IMR-90 cells was determined by
senescence-associated ß-galactosidase (SA-ß-gal)
activity, as reported [32], using the Senescence
Detection Kit (BioVision; Cat# K320), following the
manufacturer’s instructions. Fibroblasts were plated (5
x 104 per well) 1 day before treatment in a 6-well cell
culture plate (Greiner Bio-One; Cat# 657160) with 2.5
ml of DMEM complete media per well. Non-senescent
www.aging-us.com 9 AGING
cells were plated (1 x 105) also in 6-well cell culture
plates 1 d before staining. Staining was performed 9 d
after doxorubicin treatment. During staining, cells were
incubated for 48 h at 37° C in the absence of CO2, then
visualized by bright-field microscopy and imaged.
Percentage of SA-ß-gal-positive cells was counted
manually.
Real-time quantitative PCR
Non-senescent IMR-90 fibroblasts were plated (1 x 106)
in a T-75 cell culture flask (Cellstar; Cat# 658170) with
10 ml of DMEM complete media 2 d before cell pellet
harvest. For senescent cells, fibroblasts were plated (1 x
106) 1 d before doxorubicin treatment also in a T-75 cell
culture flask, with 10 ml of DMEM complete media.
Senescent cells were pelleted 9 d after doxorubicin
treatment. All cell pellets were stored at -80° C before
RNA isolation. Total RNA was isolated from cell
pellets using Quick-RNA MiniPrep (Zymo Research;
Cat# R1055), following the manufacturer’s protocol. 1
µg of total RNA per sample was reverse transcribed
using PrimeScript RT Master Mix (Takara; Cat#
RR036B), and cDNA was analyzed by real-time qPCR
using TaqMan Fast Advanced Master Mix (Applied
Biosystems; Cat# 4444557) (StepOnePlus™ Real-Time
PCR System). Gene expression analyses were
performed with Applied Biosystems TaqMan Gene
Expression single-tube assays. All reactions were
performed in triplicate, and relative expression levels of
each gene were normalized to actin. The relative
expression of mRNA was determined using the
comparative threshold (Ct) method by normalizing
target cDNA Ct values to that of actin.
Immunofluorescence (IF)
IMR-90 fibroblasts were plated for senescence
induction (2 x 104/well) 1 d before doxorubicin
treatment in a black 96-well plate with square wells and
a flat, clear bottom (Ibidi; Cat# 89626), with 250
µl/well of DMEM complete media. Non-senescent cells
were plated (2 x 10^5/well) 2 d before staining. All
immunostaining was performed 8 d after doxorubicin
treatment. Cells were fixed with 200 µl/well of 4%
paraformaldehyde in 1X PBS (Thermo Scientific; Cat#
AAJ19943K2) for 15 min at room temperature,
carefully rinsed with 1X PBS (Corning; Cat# 21-031-
CV), then permeabilized with 300 µl per well of 0.5%
Triton X-100 for 10 min at room temperature. Cells
were rinsed once with 1X PBS and incubated with 250
µl per well of anti-γH2AX [p Ser139] (Novus
Biologicals; Cat# NB100-74435) and anti-HMGB1
(abcam; Cat# ab18256) antibodies diluted in 5% BSA
(Research Products International; Cat# A30075) in 1X
PBS overnight at 4° C. Subsequently, cells were washed
5 times with 1X PBS and incubated with 250 µl/well of
both Alexa Fluor 488 goat anti-mouse antibody
(Invitrogen; Cat# A11029) and Alexa Fluor 546 goat
anti-rabbit antibody (Invitrogen; Cat# A11010) along
with Hoechst 33342, trihydrochloride, trihydrate
(Invitrogen; Cat# H3570) in 5% BSA for 20 min at
room temperature in the dark. Cells were washed 5
times with 1X PBS, then 200 µl of 1X PBS was added
to each well before images were acquired with
Molecular Devices Image Express Micro (Molecular
Devices, San Jose, CA, USA). Seven images were
acquired per well, with five wells captured per
condition (NS or S). Cells with >2 γ-H2AX foci per
nucleus were defined as senescent (S).
NK cell isolation and enrichment
Blood samples were obtained from healthy donors (n =
5, age range 20-42) in heparin coated vacutainers. All
subjects provided informed written consent. Inclusion
criteria for healthy individuals included those who did
not take medication that could impact immunity (e.g.,
corticosteroids) and had no clinical indication of
immunodeficiency. NK cells were isolated from freshly
drawn human peripheral blood with RosetteSep Human
NK Cell Enrichment Cocktail (Stemcell Technology,
USA; Cat# 15065) 3 d before co-culture with target
cells. Rosette antibody cocktail was added to each blood
sample (30 µl/ml) to negatively select unwanted cells
with Tetrameric Antibody Complexes that crosslink
non-NK cells in human whole blood to red blood cells
(RBCs). Blood samples were then incubated at room
temperature for 40 min, diluted with 1X PBS (Corning;
Cat# 21-031-CV), and combined with Lymphocyte
Separation Medium (Corning; Cat# 25-072-CI)
followed by density gradient centrifugation, according
to the manufacturer’s instructions. Once isolated, NK
cells were enriched and activated for 3 days in RPMI
complete media with 100 IU/ml human rIL-2
(recombinant Interleukin-2) (R and D Systems; Cat#
8879-IL-010) before co-culturing with senescent or
non-senescent IMR-90 fibroblasts.
NK cell characterization
Peripheral blood mononuclear cells (PBMCs) or enriched
NK cells were resuspended to a concentration of 1 x 106
cells/ml in FACS buffer (1% BSA in PBS + 0.01%
sodium azide) and aliquoted. Cells were washed 2 times
with 1X PBS and resuspended in FACS buffer. Cells
were then incubated with APC-conjugated anti-human
CD3 antibody (Miltenyi Biotec; Cat# 130-113-135), PE-
conjugated anti-human CD16 antibody (Miltenyi Biotec;
Cat# 130-113-393) and FITC-conjugated anti-human
CD56 antibody (Miltenyi Biotec; Cat# 130-114-549) for
30 min at room temperature in the dark. Then, cells were
www.aging-us.com 10 AGING
washed 3 times with FACS buffer and resuspended in
200 μl of FACS buffer. Finally, 500,000 events were
collected by flow cytometer (DB Accuri C6). Cell
viability was determined by PI staining and live cells
were gated for downstream analysis. Lymphocytes were
gated based on forward and side scatter followed by
gating to CD3- cells as NK cells. Finally, percentages of
CD56dim CD16+ and CD56bright CD16- NK cells were
determined across multiple donors. The data were
acquired for at least 100,000 cells/sample. Data were
analyzed using Flowlogic software (Miltenyi Biotech,
Germany).
Method for co-culture of NK effector cells with
IMR-90 target cells
IMR-90 fibroblasts were seeded (2 x 104/well) onto 24-
well cell culture plates (Greiner Bio-One; Cat# 662160)
with a 1 ml volume of DMEM complete media 10 d
before co-culture. Senescence was induced 9 d before
co-culture by treatment with 300 nM of doxorubicin for
24 h. Media was replaced with fresh DMEM complete
media every 2-3 d. 3 d before co-culture with NK cells,
non-senescent IMR-90 fibroblasts were seeded (1 x
10^4 per well) onto 24-well cell culture plates (Greiner
Bio-One; Cat# 662160) with 1 ml DMEM complete
media. 24 h before co-culture, each well was replaced
with 250 µl/well of DMEM complete media to allow for
SASP accumulation from senescent cells. Additional
wells containing no target cells were included for media
only and NK cell spontaneous lactate dehydrogenase
(LDH) release controls. At the start of co-culture, NK
cells were added in 250µl/well of RPMI complete
media at a target to effector (T:E) ratio of 1:1 for a total
of volume of 500 µl per well. Co-cultures were
incubated at 37° C in humidified air containing 5%
CO2 for 16 h. Bright-field images were taken after
16 h of co-culture for qualitative analysis of NK cell
cytotoxicity.
Cytotoxicity quantification
Cytotoxicity was assessed after 16 h of co-culture
incubation by quantifying LDH release levels in target
cells using the CytoTox 96 Non-Radioactive Cyto-
toxicity Assay (Promega, Madison, WI, USA), which
quantitatively measures LDH released upon cell lysis.
Baseline expression of LDH in culture medium alone,
medium with target cells only, and medium with NK
cells only were used as negative controls. Fibroblasts
treated with 0.2% Triton X-100 served as positive
controls. Absorbance was recorded at 490 nm using
SpectraMax i3 Multi-Mode Microplate Reader
(Molecular Devices, San Jose, CA, USA). Average
absorbance values for the culture medium background
were subtracted from absorbance values for experimental
and target cell spontaneous LDH release. The data are
presented as an index calculated as:
% 1 00
=
Experimental Effector
Spontaneous
TargetSpontaneous
Cytotoxicity Positivecontrol
TargetSpontaneous
Granzyme B release assay
Secretion of granzyme B was determined in the
supernatant of NK/IMR90-cell co-cultures after 16 h. Co-
culture plates were subject to centrifugation at 400xg for
5 min. Supernatant was collected and used to measure
human granzyme B by enzyme-linked immunosorbent
assay (ELISA) kit (R and D Systems; DY2906-05),
according to the manufacturer’s instructions.
Cell viability assay with Calcein AM
Fibroblasts were carefully rinsed 4 times with 1X PBS
(1 ml/well) to wash away effector (NK cells) and dead
detached cells. Target cells were stained with 1 µM of
Calcein AM (diluted from a 4 mM stock solution in
dimethyl sulfoxide (DMSO), Invitrogen; Cat# C3099)
in no-serum DMEM for 30 min in a 37° C incubator at a
volume of 250 µl/well. Following incubation, fluo-
rescence values were recorded at 530 nm using
SpectraMax i3 Multi-Mode Microplate Reader
(Molecular Devices, San Jose, CA, USA).
AUTHOR CONTRIBUTIONS
KK designed and performed experiments, analyzed
data, and wrote the manuscript. TDA analyzed results
and helped edit the manuscript. AS helped edit the
manuscript. AS designed experiments, supervised
research, and helped to prepare the manuscript.
ACKNOWLEDGMENTS
We thank postbaccalaureate fellow Ms. Gina Zhu for
assisting with some initial experiments with endothelial
cells.
CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest.
FUNDING
This work was funded by grants from National
Academy of Medicine’s (NAM) Healthy Longevity
www.aging-us.com 11 AGING
Catalyst Award # 2000011734. The study’s funders had
no role in the study design, data collection, analysis,
interpretation and report writing. The corresponding
author had full access to all the data and had final
responsibility for the decision to submit for publication.
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www.aging-us.com 15 AGING
SUPPLEMENTARY MATERIALS
Supplementary Figures
Supplementary Figure 1. Senescent IMR-90 cells express markers of senescence. Related to Figure 1. (A) Representative
immunofluorescence images of NS and S IMR-90 cells stained for HMGB1 (red). Nuclei were stained with Hoechst (blue). (B) Representative
images of SA-β-Gal-stained senescent (S IR, S Etoposide) and non-senescent (NS) IMR-90 cells. SA-β-Gal staining was performed on day 9
after treatment with irradiation (20 Gy) or Etoposide (20 µM, 48 h). (C) Quantification of SA-β-gal-positive IMR-90 cells in NS, S IR and S
Etoposide. Four fields were quantified per well (n=3) with a total of 3483, 1079 and 777 cells counted for NS, S IR and S Etoposide,
respectively.
www.aging-us.com 16 AGING
Supplementary Figure 2. Cytokine expression of non-senescent versus senescent IMR-90 cells and NK cell enrichment. (A)
qRT-PCR of CCL2, CXCL12 and Chemerin mRNA in non-senescent and senescent IMR-90 fibroblasts. The results are presented as mean fold
change in NS compared to S of two independent experiments performed in triplicate, and error bars represent ±SEM. Statistical analysis
performed using unpaired t test. *p < 0.05, **p < 0.01, and ***p < 0.001. (B) Flow cytometry analysis of CD56 and CD3 expressing cells before
(left) and after (right) enrichment. Rosette antibody-based isolation resulted in a pure population (99.07%) of NK cells. Red is CD56bright and
green is CD56dim NK cells. (C-i) Fluorescence Minus One (FMO) control for CD56 from a representative donor. NK cell populations were
stained with anti-CD16 antibody. (C-ii) FMO control for CD16. NK cell populations were stained with anti-CD56 antibody.
www.aging-us.com 17 AGING
Supplementary Figure 3. Activated primary NK cells selectively eliminate senescent cells. Related to Figure 3. (A) Representative
light microscopy images showing NK cell cytotoxicity towards NS versus S IMR-90 cells after 16 h of co-culture. (B) Cytotoxicity of NK cells that
had been freshly isolated 3 d before co-culture (“fresh”) versus NK cells that had been revived from storage in liquid nitrogen 3 d before co-
culture (“frozen”) after 16 h of co-culture. Cytotoxicity was evaluated by LDH release. (C) Quantitative analysis of IMR-90 target cell viability
after 4 d of co-culture with frozen or fresh NK cells, as measured by Calcein AM. (D-i) Percentage of live cells after 4 d of co-culture with NS cells
or fibroblasts induced to senesce by irradiation (20 Gy) or (D-ii) etoposide (20 µM, 48 h). (E) Granzyme B production of NK cells from 5 different
donors in response to 16 h of incubation with NS or S IMR-90 cells. Donor sex and age are indicated in the figure.
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