Download Enhancement of natural killer (NK) cell cytotoxicity by fever

Enhancement of natural killer (NK) cell cytotoxicity by feverrange thermal stress is dependent on NKG2D function and
is associated with plasma membrane NKG2D clustering
and increased expression of MICA on target cells
Julie R. Ostberg,1,2 Baris E. Dayanc,1 Min Yuan, Ezogelin Oflazoglu, and Elizabeth A. Repasky3
Department of Immunology, Roswell Park Cancer Institute, Buffalo, New York, USA
Abstract: Circulating NK cells normally experience temperature gradients as they move about the
body, but the onset of inflammation can expose
them and their targets to febrile temperatures for
several hours. We found that exposure of human
peripheral blood NK cells and target cells to feverrange temperatures significantly enhances lysis of
Colo205 target cells. A similar effect was not observed when NK cell lines or IL-2-activated peripheral blood NK cells were used as effectors, indicating that thermal sensitivity of effectors is maturation or activation state-dependent. Use of blocking
antibodies revealed that this effect is also dependent on the function of the activating receptor
NKG2D and its ligand MHC class I-related chain A
(MICA). On NK cells, it was observed that thermal
exposure does not affect the total level of NKG2D
surface expression, but does result in its distinct
clustering, identical to that which occurs following
IL-2-induced activation. On tumor target cells, a
similar, mild temperature elevation results in transcriptional up-regulation of MICA in a manner that
correlates with increased sensitivity to cytolysis.
Overall, these data reveal that NK cells possess
thermally responsive regulatory elements, which
facilitate their ability to capitalize on reciprocal,
stress-induced changes simultaneously occurring
on target cells during inflammation and fever. J.
Leukoc. Biol. 82: 1322–1331; 2007.
Key Words: inflammation
䡠 innate immunity 䡠 IL-2
䡠
febrile temperatures
䡠
hyperthermia
activating versus inhibitory signals through the ligation of
specific receptors. Activation is also associated with “clustering” of the activating receptors in lipid raft regions of the
plasma membrane [3]. Specifically, experimental evidence
shows that the early and what is sometimes called the “nonspecific phase” of NK cell activation are regulated by the
proinflammatory milieu of the microenvironment [4, 5]. Certain
cytokines, including IL-2, IL-7, IL-12, IL-15, and Type I IFNs
[4, 6 –9], are known to activate NK cells rapidly, and TNF-␣
has been found specifically to be involved in the recruitment of
NK cells to inflammatory sites [10, 11]. It is at this point, after
a nonspecific activation has resulted from cytokine inflammatory mediators, that NK cell-mediated killing must also be
triggered by the dominance of activation receptor complexes
over inhibitory receptor complexes [12–14].
It is important that some of the same proinflammatory cytokines (e.g., TNF-␣, IFN-␣/␤) involved in the early events
leading to activation of NK cells are also pyrogenic, causing an
increase in body temperature or fever through several overlapping physiological, behavioral, and neurological changes (reviewed in refs. [15, 16]); this increase in body temperature is
considered one of the cardinal features of inflammation and
takes place in the same general time-frame in which NK cells
are initiating their activation and functional activity, as both
events are triggered by the same proinflammatory signals.
However, despite the temporal linkage between cytokinedriven NK cell activation and the pyrogenicity of these same
cytokines, a separate thermal component in the regulation of
NK cell activation has not been fully characterized. Is it
possible that at least some of the physiological effects of local
inflammatory cytokines on NK cell function are mediated indirectly through a thermal shift? If so, how do cells “recognize”
thermal signals in the activation pathway leading to cytotoxicity, and how is specificity for appropriate targets maintained
INTRODUCTION
NK cells provide essential, innate host protection against viral
infections and tumor formation. Not only do these cells kill
target cells directly, but they also have the ability to manipulate downstream, long-term, adaptive immune responses [1, 2].
Recent work has revealed remarkable complexity in the regulation of NK cell activation and cytotoxic potential. Indeed, NK
cell activation and killing only occur after exposure to inflammatory cytokines and after achieving the correct balance of
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Journal of Leukocyte Biology Volume 82, November 2007
1
These authors contributed equally to this work.
Current address: Division of Cancer Immunotherapeutics and Tumor Immunology, City of Hope National Medical Center, Beckman Research Institute,
Duarte, CA, USA.
3
Correspondence: Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA. E-mail:
[email protected]
Received November 24, 2006; revised July 6, 2007; accepted July 25, 2007.
doi: 10.1189/jlb.1106699
2
0741-5400/07/0082-1322 © Society for Leukocyte Biology
when millions of cells well beyond the initial inflammatory site
are affected by an increase in body temperature? When others
examined the effects of thermal stress on human NK cell
cytotoxic activity, use of temperatures significantly above fever-range (i.e., ⬎42°C or 107°F) was found to generally inhibit
NK cytotoxicity [17–20], and more mild elevations in temperature (i.e., more similar to fever-range) have been associated
with enhanced NK cytotoxic activity [21–23]. Indeed, earlier
studies by our group [24] using various murine tumor models
suggested that the ability of a mild, fever-range, whole body
hyperthermia (39.5– 40°C for 6 – 8 h) to inhibit tumor growth in
vivo was a result of the action of NK cells based on use of
function-blocking antibodies. However, in a physiological situation, elevated temperatures would affect both effectors and
targets. To date, the requirement for heating the NK effector
cells and/or targets has never been examined, nor has the
mechanisms by which temperature affects NK cell activation
been defined. Thus, to begin to address these issues, we have
examined the effects of temperature on effector cells and target
cells using freshly isolated human peripheral blood NK cells or
NK cell lines and human Colo205 tumor cells.
We report here that cytotoxic activity of human peripheral
blood NK cells (but not long-term cultured cell lines, nor cells
activated by IL-2) is optimally enhanced when NK effectors
and tumor target cells are exposed to mild hyperthermic conditions. We also describe the mechanistic linkage of this effect
to the involvement of the activating receptor NKG2D on NK
cells and its MHC class I-related chain A (MICA) ligand on
tumor target cells. These data strongly suggest that peripheral
blood NK cells are responsive to the evolutionarily conserved
temperature gradients associated with fever/inflammation and
may have evolved to take advantage of simultaneous, reciprocal, stress-induced alterations on the surface of target cells
exposed to the same temperature gradient in the body.
MATERIALS AND METHODS
Cell lines and human NK cell isolation
Human NK cells were isolated from healthy donor peripheral blood by density
gradient centrifugation with Histopaque-1077 (Sigma-Aldrich Inc., St. Louis,
MO, USA) and purified by negative magnetic separation using a NK cell
isolation kit (Miltenyi Biotech, Auburn, CA, USA), according to the manufacturer’s protocol. Cell purity was found to be ⬎95%. The human colon adenocarcinoma cell lines Colo205, HCT116, and HT29 [American Type Culture
Collection (ATCC), Manassas, VA, USA] were propagated in RPMI-1640
medium with 2 mM L-glutamine and 10% FBS. The human NKL cell line, a
generous gift from Dr. Jerome Ritz (Dana Farber Cancer Institute, Boston, MA,
USA), was propagated in RPMI 1640 with L-glutamine, sodium pyruvate, 15%
FBS, and 100 IU/mL human recombinant IL-2 (Sigma-Aldrich Inc.). The
human NK cell line NK92MI, a malignant, non-Hodgkin’s lymphoma, transfected with human IL-2 cDNA (ATCC), was propagated in MEM-␣ modified
with ribonucleosides, deoxyribonucleosides, and 2 mM L-glutamine, supplemented with 0.2 mM inositol, 0.1 mM 2-ME, 0.02 M folic acid, 12.5% horse
serum, and 12.5% FBS. Viability of all cells in culture, even IL-2-starved NKL
cells, was ⬎90% for each experiment.
Cytotoxicity assays
Cytotoxicity assays were performed using the CytoTox 96 nonradioactive
cytotoxicity assay (Promega Corp, Madison WI, USA), according to the manufacturer’s protocol. In some cytotoxicity assays, cells were incubated with 1
␮g/mL anti-NKG2D or 5 ␮g/mL anti-MICA/B F(ab)2, or blocking antibodies,
or 6 ␮g/mL isotype control, IgG (R&D Systems, Minneapolis, MN, USA) for 15
min before mixing with target cells. In other cytotoxicity assays, cells were
incubated with 5 mM methyl-␤ cyclodextrin (M␤CD) or DMSO vehicle control
during the last 30 min of a 6-h culture at 37°C or 39.5°C and then washed to
remove drug prior to coculture of cells in a 6-h cytotoxicity assay. The
maximum and spontaneous release was determined by incubating the target
cells and effector cells with 0.5% Triton X-100 or medium alone, respectively.
In all experiments, spontaneous release of effectors and targets was ⬍15% of
total release. Percent cytotoxicity was determined using the following equation:
100 ⫻ (experimental release–spontaneous release)/(maximum release–spontaneous release).
mAbs and flow cytometry
For flow cytometric staining, 106 cells were washed with PBS before being
incubated with anti-NKG2D (R&D Systems), anti-MICA (Immatics Biotechnologies, Tubingen, Germany), PE-conjugated anti-HLA ABC (BD Biosciences
PharMingen, San Diego CA, USA), or isotype control antibodies for 30 min on
ice. Cells incubated with nonconjugated primary antibodies were then washed
and stained with PE-conjugated goat anti-mouse secondary antibody (BD
Biosciences PharMingen) for 20 min on ice. Cells were then fixed with freshly
prepared 2% paraformaldehyde in PBS and analyzed within 12 h with a
FACScan (BD Biosciences, San Jose, CA, USA) and WinMDI 2.8 software.
Immunofluorescent staining for NKG2D
localization
Freshly purified human NK cells were centrifuged at 300 g for 5 min, washed
with cold PBS, and fixed with 2% paraformaldehyde in PBS for 15 min at 4°C
prior to incubation with 10 ␮g/mL mouse anti-human NKG2D mAb (BD
Biosciences PharMingen) for 1 h at 4°C. Cells were then washed with cold
PBS, incubated with 10 ␮g/mL FITC-conjugated goat anti-mouse polyclonal
antibody (BD Biosciences PharMingen) for 1 h at 4°C, washed with cold PBS,
and resuspended in Vectashield mounting media with 4⬘,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame CA, USA) for analysis by confocal
microscopy (Leica Microsystems AG, Wetzlar, Germany).
RNA preparation, RT-PCR, and real-time
quantitative PCR (qPCR) procedure
RNA was extracted from 107 cells using the RNeasy mini kit (Qiagen Inc.,
Valencia, CA, USA) and quantified by measuring absorbance at 260 and 280
nm using the GeneQuant RNA/DNA calculator (Pharmacia/Pfizer, New York,
NY, USA). Extracted, total RNA (1 ␮g) was mixed with 0.5 ␮g oligo-dT 12–18
primers (Molecular Probes Inc., Invitrogen, Carlsbad, CA, USA), 10 mM
deoxy-(d)NTP mix, and water, incubated at 65°C for 10 min, chilled on ice, and
centrifuged briefly. The mixture was combined with 5⫻ first-strand buffer,
DTT, RNase inhibitor, and SuperScript II RT (Molecular Probes Inc., Invitrogen), incubated at 42°C for 50 min and at 70°C for 15 min. The resulting
cDNA was then combined with PCR buffer (10 mmol/L Tris-HCl, 50 mmol/L
KCL), 1.5 mmol/L MgCl2, 2.5 mmol/L each dNTP and 2.5U Taq polymerase
(Molecular Probes Inc., Invitrogen), as well as 10 pmol each MICA sense and
antisense primers. Amplification included 30 cycles of denaturation (95°C for
1 min), annealing (56°C for 1 min), and extension (90 s at 72°C). GAPDH
message was amplified and used as an internal control. PCR products were
analyzed with 1.5% agarose gel electrophoresis, ethidium bromide staining,
and the BioDoc-It transilluminator system (UVP, Upland, CA, USA). Relative
quantification was performed using the comparative threshold (CT) method. For
qPCR experiments, SYBR Green I dye (Molecular Probes Inc., Invitrogen) was
used according to the manufacturer’s protocols with MICA reverse (5⬘GCAGGGAATTGAATCCCAGCT-3⬘) and forward (5⬘-ACACCCAGCAGTGGGGGGAT-3⬘) or GAPDH reverse (5⬘-TCCACCACCCTGTTGCTGTA-3⬘)
and forward (5⬘-ACCACAGTCCATGCCATCAC-3⬘) primers and 96-well qRTPCR plates (Axygen Scientific Inc. Union City, CA, USA). For real-time
quantitative detection, an ABI Prism 7700 sequence detection system (Applied
Biosystems, Foster City, CA, USA) was used, and fold change in relative MICA
signal was determined using the 2–⌬⌬Ct formula against the GAPDH signal.
Ostberg et al. Enhanced NK cytotoxicity by mild thermal stress
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ELISA for soluble MICA (sMICA)
Culture supernatants were harvested at the indicated times and analyzed for
sMICA levels using a human MICA DuoSet ELISA development kit (R&D
Systems) as per the manufacturer’s instructions.
RESULTS
Enhancement of NK cell cytotoxic activity by
mild thermal stress
Here, we report on the existence of a thermally responsive
component in the regulation of NK cell function, one that
appears to significantly enhance the cytotoxic activity of peripheral blood-derived, human NK cells against tumor targets.
In a series of experiments, freshly isolated human NK cells
from the peripheral blood of normal donors were cocultured
with human colon tumor target cells (Colo205) at 33°C, which
reflects skin temperature at the body’s extremities, the normothermic temperature of 37°C, or physiologically relevant, febrile temperatures of 38°C (lower febrile range) or 39.5°C
(upper febrile range) during a 6-h cytotoxicity assay. Each of
these was then also compared with coculture of cells at 42°C
for 1 h, which reflects a classical heat shock condition, followed by incubation at 37°C for the remaining 5 h of a 6-h
assay. Although some experiments indicated an enhancement
of cytotoxicity at 38°C using this time-frame (data not shown),
a consistently significant alteration in cytotoxic activity compared with the 37°C cultures was observed when the cells were
cultured at 39.5°C for 6 h (Fig. 1A). If these cells were heated
at 39.5°C for 6 h and then allowed to recover for 24 h at 37°C
Fig. 1. Temperatures influence the cytotoxic activity of peripheral blood-derived
human NK cells. Freshly purified human NK cells were incubated for 6 h with
Colo205 human colon adenocarcinoma cells (A–C) or autologous PBMCs (D) at
33°C (e), 37°C (䡬), 38°C (f), or 39.5°C (Œ) or at 42°C for the 1st hour before
continuing the culture at 37°C (●). (A–C) Data acquired with NK cells from three
different human donors. Target cell killing was assayed with a lactate dehydrogenase (LDH) release assay. All data are expressed as average percent specific
cytotoxicity ⫾ SE of triplicate wells and are representative of three independent
experiments. *, P ⬍ 0.05, when comparing 39.5°C with 37°C control values at
each E:T ratio using an unpaired Student’s t-test.
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Fig. 2. Activation with IL-2 correlates with inability of mild thermal stress to
enhance cytotoxic activity of human NK cell lines and purified human NK cells.
Human NK cell lines NKL (A) and NK92MI (B) or peripheral blood-derived
human NK cells activated with IL-2 (100 IU/ml) for 2 days (C) were incubated for
6 h with Colo205 human colon adenocarcinoma cells at 37°C (䡬) or 39.5°C (Œ).
NKL cells were also starved of IL-2 for 2 h prior to coincubation with Colo205
targets (D). Target cell killing was assayed with a LDH release assay. All data are
expressed as average percent specific cytotoxicity ⫾ SE of triplicate wells and are
representative of three independent experiments. *, P ⬍ 0.05, when comparing
control and heated values at each E:T ratio using an unpaired Student’s t-test.
before coculture in a 6-h cytotoxicity assay, a complete loss of
the enhanced cytotoxic activity was observed (data not shown).
Examination of the time-frame required at 39.5°C to get an
enhanced cytotoxic effect revealed that 4 h of coculture was not
always enough to see statistically significant enhancement of
cytotoxic potential, and 8 h of coculture did not appear to be
any better than 6 h (data not shown). Indeed, despite interpersonal variation among donors, statistically significant increases
in cytotoxic efficiency were consistently observed with NK
cells collected from the peripheral blood of various healthy
donors and incubated with targets for 6 h at 39.5°C (Fig. 1, B
and C).
This thermal enhancement of NK cell cytotoxic activity was
highly specific and dependent on the presence of an appropriate activating NK target. Thermal enhancement of cytotoxicity
was not observed when coheated, autologous PBMCs were used
as targets (Fig. 1D), indicating that there is no nonspecific
induction of NK cytolytic activity by heat; i.e., heat does not
appear to release lytic molecules nonspecifically, which should
kill all cells in the environment. Instead, target cells bearing
appropriate ligands must be present. Moreover, when human
NK cell lines such as NKL (Fig. 2A) and NK92MI (Fig. 2B)
were used as effectors with tumor targets, they displayed no
enhancement of cytotoxicity with mild thermal stress. Conversely, if the NKL cells were starved of IL-2 immediately prior
to coculture with the Colo205 targets, thermal enhancement of
cytotoxic activity was then observed (Fig. 2D). As these cell
lines might be considered more highly activated than peripheral blood-derived NK cells, in part through their constitutive
culture with IL-2 and as evidenced by their higher percent
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cytotoxicity values when cultured at 37°C (compare Fig. 1,
A–C, with Fig. 2, A or B), we also examined the effects of mild
thermal stress on human peripheral blood NK cells, which had
been cultured with IL-2. These “preactivated” NK cells (from
a human donor, which had displayed thermal enhancement of
cytotoxicity previously in the absence of IL-2) now revealed an
inability to be thermally stimulated (Fig. 2C). Thus, overall, it
appears that the cytotoxic activity of freshly derived human
peripheral blood-derived NK cells, but not NK cell lines or
IL-2-activated human NK cells, is sensitive to this mild, physiologically relevant elevation in temperature. However, as
shown here, even when using freshly isolated, peripheral
blood-derived NK cells, thermal enhancement is not seen,
unless there is an appropriate target (i.e., tumor cells but not
autologous PBMC).
Thermal enhancement of NK cytotoxicity requires
thermal stimulation of NK cells and tumor targets
To determine whether NK cells, tumor cells, or both were responsive to these temperature changes, we next preincubated the NK
effectors and tumor cells separately at 37°C or 39.5°C for 6 h
before placing them together in a 6-h cytotoxicity assay at 37°C.
Although incubation of just the target cells at elevated temperatures appeared to enhance cytotoxicity slightly more than incubation of just the NK cells at elevated temperatures (Fig. 3A), it was
observed that the most consistent and significant enhancement of
cytotoxicity occurred when NK cells and Colo205 tumor cells
underwent preheat treatment. This suggests that both the effectors
and targets can be affected by changes in their thermal microenvironment in a manner that increases the overall cytotoxic response of NK cells against tumor cell targets.
Dependence of NK cytotoxicity on NKG2D and
its ligand MICA
NKG2D is one of the major activating receptors on NK cells,
which mediate cytotoxic activity by recognizing defined ligands, often overexpressed on stressed or transformed cells
[25]. To test the hypothesis that the function of this receptor
was necessary for thermal enhancement of cytotoxicity, we
used a blocking antibody against NKG2D (Fig. 3B). Addition of
this function-blocking antibody not only significantly inhibited
the cytotoxicity observed when cells were incubated at normo-
thermic temperatures (as expected), but thermally enhanced
cytotoxicity was also lost completely. This suggests that the
cellular pathway by which NK cells respond to thermal signals
in terms of enhanced cytotoxicity ultimately still depends on
NKG2D receptor function. These data also support the conclusion drawn from data shown earlier that there is no nonspecific
release of lytic molecules by heat without engagement of appropriate targets.
Similar to heat shock protein genes, the 5⬘ end flanking
regions of the NKG2D ligands MICA and MICB, in particular,
are known to harbor putative heat shock response elements,
which permit their up-regulation upon heat shock at 42°C [26].
Thus, this potential regulation of MICA by heat shock led us to
investigate the dependence of the thermally induced cytotoxicity of Colo205 cells on this ligand. Using a blocking antibody
against MICA, there was only a partial (although significant)
loss of cytotoxic activity observed with the heated cells (data
not shown). However, this result could be misleading because
of the potential for whole IgG molecules against MICA to
mediate FcR-associated, antibody-dependent cell-mediated
cytotoxicity by the NK cells. Thus, blocking experiments were
also carried out with F(ab)2 molecules against MICA (Fig. 3C).
This resulted in complete inhibition of the thermal effect on
cytotoxicity and suggests that MICA is the predominant
NKG2D ligand by which mild thermal stress mediates the
enhancement of NK cell cytotoxic activity.
Regulation of NKG2D membrane localization but
not total surface expression by mild
thermal stress
As blocking antibodies against NKG2D were found to inhibit
basically all thermally induced, NK-mediated, cytotoxic activity, we suspected that the cellular mechanism by which thermal signals were mediated ultimately depended on this receptor. Thus, in our search for an underlying mechanism for the
thermal enhancement of NK cytotoxicity, we began by examining the effects of mild thermal stress on this activating
receptor. As NK cells have been found to display increased
expression of NKG2D when they are treated in vitro with the
stimulatory agent IL-15 [27], we first examined the effects of
mild thermal stress on NKG2D expression levels on NK cells.
Flow cytometric analysis revealed no effect of culture at 39.5°C
Fig. 3. Enhanced cytotoxicity requires
treatment of both human NK cells and
Colo205 targets with elevated temperatures
and involves NKG2D and its ligand MICA.
(A) Cells were precultured separately at
37°C or 39.5°C for 6 h prior to coincubation
at 37°C during the cytotoxicity assay. NK
cells and target cells precultured at 37°C
(䡬); NK cells cultured at 39.5°C and targets at 37°C (䉬); target cells cultured at
39.5°C and NK cells at 37°C (f); NK cells
and target cells precultured at 39.5°C (Œ). Data are expressed as average percent specific cytotoxicity ⫾ SE of triplicate wells. A representative of three different
experiments, each performed using NK cells from three different donors, is shown. *, P ⬍ 0.05, using Student’s t-test to compare NK cells and target cells
precultured at 37°C (䡬) versus NK cells and target cells precultured at 39.5°C (Œ) at each E:T ratio. (B and C) Cells were incubated for 6 h at 37°C (open bars)
or 39.5°C (solid bars) prior to addition of isotype control IgG or anti-NKG2D-blocking antibody (B) or anti-MICA-blocking F(ab)2 fragments (C) at 37°C and
performance of cytotoxicity assays. Data are expressed as average percent-specific cytotoxicity ⫾ SE of triplicate wells at a 10:1 E:T ratio. Representative data of
four experiments are shown. †, P ⬍ 0.05, comparing heated to nonheated cultures under each condition using a Student’s t-test. *, P ⬍ 0.05, comparing IgG to
blocking mAb or F(ab)2 conditions within the heated or nonheated cultures using a Student’s t-test.
Ostberg et al. Enhanced NK cytotoxicity by mild thermal stress
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Fig. 4. NKG2D surface localization, but
not overall expression levels, are altered by
mild thermal stress. (A) Freshly purified
human NK cells were cultured for 6 h at
37°C (solid line) or 39.5°C (dashed line)
and then stained with anti-NKG2D or isotype control antibody (filled histogram). (B)
Freshly purified human NK cells were cultured for 6 h at 37°C (C), 39.5°C (HT), or
37°C with 10 ␮g/mL cross-linking antiNKG2D antibody (Ab) for 15 min before
being fixed, stained for NKG2D, and visualized by confocal microscropy. These were
compared with human NK cells, which had
been activated with 100 IU/mL IL-2 for 2
days (IL2), as well as NKL cells, which are
propagated in 100 IU/mL IL-2 (NKL). Representative cells with nonclustered NKG2D
after culture at 37°C, with clustered
NKG2D after culture at 39.5°C, or with
clustered NKG2D after culture with crosslinking antibodies are depicted. (C) Upon
visualizing 50 or more cells in each group,
average percentages of cells with surface
clustering of NKG2D were determined in n ⫽ 3 experiments ⫾ SE. (D) Percentages of cells with clustered surface NKG2D when heated cells were allowed to recover
at 37°C for 12 (⫹12 h) and 24 (⫹24 h) h were also determined as described for C. *, P ⬍ 0.05, when compared with control values using a Student’s t-test.
on overall NKG2D surface expression on human NK cells (Fig.
4A). However, previous studies in our laboratory have shown
that exposure of cells to mild thermal stress can alter the
physical positioning of several different membrane-associated
molecules in a manner associated with heightened activation
potential. These include alterations in the organization of the
spectrin-based cytoskeleton and several PKC isoforms as well
as the formation of uropods [28, 29]. As redistribution or
clustering of activating receptors at the site of target recognition is known to occur when NK cells form conjugates with
target cells is known to occur during IL-2-enhanced NK cell
cytotoxicity [3], we next examined the ability of mild thermal
stress to affect NKG2D localization on the NK cell surface (Fig.
4, B and C). It is notable that a significant increase in cells with
aggregates of clustered NKG2D on the cell surface was observed after NK cells were exposed to mild thermal stimulation
compared with those which were incubated at normothermic
temperatures. Although addition of anti-NKG2D cross-linking
antibody resulted in a larger number of cells with clustered
NKG2D, the significant increase in the number of cells with an
identical pattern of clustered NKG2D at 39.5°C as that seen
with IL-2 treatment strongly supports the hypothesis that membrane localization of NKG2D is highly sensitive to mild temperature shifts in a manner that enhances cytotoxic ability.
Furthermore, a reversal of the thermally induced NKG2D
clustering was seen within 24 h after the heated, purified
human NK cells were returned to 37°C (Fig. 4D). This loss of
the clustered phenotype correlated directly with a reversal of
the enhanced cytotoxic activity observed when these cells,
which had been allowed to recover for 24 h at 37°C, were then
cultured with tumor cell targets (data not shown). The same
analysis of NKG2D positioning on IL-2-treated peripheral bloodderived human NK cells and the NK cell line NKL revealed that
even at normothermic temperatures, there was a higher percent of
cells with NKG2D clustering, similar to that seen in NKG2D
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Journal of Leukocyte Biology Volume 82, November 2007
cross-linked cells; yet there was no further increase seen after
mild thermal stress (Fig. 4C and data not shown).
As clustering of NKG2D at the cell surface is known to be
related to the association of this receptor with lipid raft domains in the plasma membrane [3], we next examined the
ability of M␤CD to affect thermally induced cytotoxicity.
M␤CD is known to cause cholesterol to effuse from the membrane and thereby disrupt lipid raft organization, and this
reagent has been used by others to determine the dependence
of an event upon the integrity of lipid rafts [30]. Thus, M␤CD
was added to the last 30 min of the 6-h culture of cells at
39.5°C versus 37°C, and then the drug was washed away before
targets and effectors were cocultured at 37°C for the cytotoxicity assay. Cell survival was not affected, but surface, heatinduced protein clustering was eliminated by this M␤CD treatment (data not shown). Further, we found that the thermal
enhancement of cytotoxicity was totally eliminated by M␤CD
treatment (Fig. 5). These data suggest that normal lipid raft
structure and organization are required for the thermally induced effects on NK-mediated cytotoxic activity, and that the
ability of the activating receptor NKG2D to cluster at the
membrane surface is at least part of the mechanism by which
NK cells respond to fever-like thermal stress.
Regulation of MICA expression by mild thermal
stress
Based on the anti-MICA F(ab)2-blocking studies described
above, as well as the potential regulation of MICA by heat
shock, we also investigated the ability of a physiologically
relevant, mild thermal stress of 39.5°C to affect MICA expression in Colo205 cells. RT-PCR analysis revealed significantly
enhanced MICA mRNA expression in the tumor cells within
3 h of incubation at 39.5°C (Fig. 6). This enhancement of the
MICA mRNA levels appeared to plateau between 4 h and 8 h
of culture at this fever-range temperature (data not shown).
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Fig. 5. Thermal enhancement of cytotoxicity is lost with M␤CD treatment.
Colo205 cells were incubated for 6 h at 37°C (open bars) or 39.5°C (solid bars)
with a final concentration of 5 mM. M␤CD was added during the last 30 min
prior to washing the cells and performance of cytotoxicity assays. Data are
expressed as average percent-specific cytotoxicity ⫾ SE of triplicate wells at a
20:1 E:T ratio. Representative data of four experiments are shown. †, P ⬍
0.05, comparing heated to nonheated cultures under each condition using a
Student’s t-test; *, P ⬍ 0.05, comparing controls to M␤CD treatment within the
heated or nonheated cultures using a Student’s t-test.
Flow cytometric analysis then revealed enhanced surface
MICA expression on Colo205 cells cultured at 39.5°C for 6 h
compared with those cultured at 37°C (Fig. 7A). Thermally
enhanced surface expression of this NKG2D ligand was also
found with the colon tumor line HCT116 (Fig. 7B) but not
HT29 colon tumor cells, possibly as these cells had higher
constitutive MICA expression with which to begin (Fig. 7C).
Fig. 7. Elevated temperatures can enhance MICA but not MHC class I
surface expression. Human colon tumor cell lines Colo205 (A), HCT116 (B).
and HT29 (C), and human PBMC (D) or freshly purified human NK cells (E)
were cultured for 6 h at 37°C (gray line) or 39.5°C (thick, black line) and then
stained with anti-MICA (left) or anti-MHC class I (HLA-ABC, right) or isotype
control antibody (thin, black line).
Fig. 6. Elevated temperatures enhance MICA mRNA levels in Colo205 cells.
(A) Total RNA was extracted from Colo205 cells after incubation at 37°C for
6 h or cultured at 39.5°C for 1, 3, and 6 h, and MICA and GAPDH message
was detected with RT-PCR. (B) MICA message is quantified with real-time
qRT-PCR. Bars represent fold induction in MICA message over 37°C control ⫾ SE of triplicate samples. All data are representative of three independent
experiments. *, P ⬍ 0.05, when compared with 37°C controls using a Student’s
t-test.
Importantly that these MICA expression patterns correlated
with the thermal sensitivity of these cell lines to NK-mediated
killing, as the NK cytotoxicity of HCT116 was enhanced significantly by mild thermal stress [and was blocked by antiMICA F(ab)2 molecules], but that of HT29 was not (data not
shown). Enhanced MICA surface expression was not observed
with total human PBMCs under the same conditions (Fig. 7D),
as may have been expected by the cytotoxicity results described for Figure 1D above. Detectable MICA expression was
also not induced on purified human NK cells (Fig. 7E). Overall,
MHC class I expression on the tumor cells, PBMC, or NK cells
was also not affected by 6 h culture at 39.5°C (Fig. 7), sug-
Ostberg et al. Enhanced NK cytotoxicity by mild thermal stress
1327
Fig. 8. sMICA is not induced by mild thermal stress. Using human colon
tumor cell line HCT116 (5⫻104/mL) as a positive control, culture supernatants
from Colo205 cells (5⫻104/mL), freshly purified human NK cells (106/mL), or
a coculture of NK and Colo205 cells were collected after 6 h at 37°C (C) or
39.5°C (HT) or after heated cells were allowed to recover for 6 (⫹6 h) or 12
(⫹12 h) h at 37°C. Data are expressed as average concentration of sMICA ⫾
SE of two to three different experiments. sMICA was not detected in all but the
HCT116 supernatants; *, supernatants not tested.
gesting that mild thermal stress has no obvious effect on levels
of these molecules, which are known to act as inhibitory
ligands for NK cytotoxicity. As soluble MIC (sMIC) proteins
are known to be released by some human tumors in a manner,
which appears to help them escape immune recognition [31,
32], we also examined the ability of mild thermal stress to
affect levels of sMICA in the supernatants of our cultures (Fig.
8). It is interesting that mild thermal stress did not induce
detectable levels of sMICA when NK cells or Colo205 cells
were cultured alone, nor when they were cultured together.
HCT116 cells did produce sMICA, as reported previously [33],
but levels did not appear to be enhanced significantly after 6 h
of culture at 39.5°C. Together, these data suggest that mild
thermal stress preferentially up-regulates the surface expression of the NKG2D ligand MICA on certain colon tumor cells
in a transcriptionally regulated manner. The ability of mild
thermal stress to up-regulate the expression of MICA thus also
appears to play an important role in determining the sensitivity
of the target cell to NK-mediated cytotoxicity.
DISCUSSION
Circulating human immune effector cells normally briefly experience mild temperature gradients as they move through
cooler regions of the skin and airway mucosal surfaces (29 –
33°C) to deeper, warmer sites maintained at core temperature
(⬃37°C). The onset of fever following infection would result in
a steeper thermal gradient experienced by immune effector
cells (and their potential targets), as core temperature in humans typically increases by 1–3°C [34]. The studies reported
here reveal the potential of a physiologically relevant thermal
shift to enhance the lytic ability of NK cells against similarly
heat-stressed target cells. The fact that this enhancement occurs in the febrile range and not at higher “heat shock”
temperatures strongly suggests an evolutionarily conserved,
physiologically relevant response to inflammatory conditions
(including a universally recognized thermal component), which
1328
Journal of Leukocyte Biology Volume 82, November 2007
occur following exposure to antigens. This ability of mild
thermal stress to regulate freshly derived blood NK cells but
not NK cell lines, which are constitutively cultured with the
activating cytokine IL-2, nor freshly derived NK cells treated
with IL-2, has revealed for the first time a selective thermal
sensitivity in NK cells, that is apparently dependent on the
activation status of these effector cells. Furthermore, we have
observed that a fever-range, mild thermal stress can enhance
NK cell cytotoxic activity through membrane-associated effects
on both NK cell effectors and human colon tumor targets; and
we have identified the activating receptor NKG2D and its
ligand MICA as primary targets in the thermal regulation of
human NK cytotoxicity.
These studies extend previous work by our group [35–39],
suggesting 1) the potential of thermal shifts to act as a “danger
signal” similar to the effects of other known danger signals [40]
and 2) the ability of whole body hyperthermia to inhibit tumor
growth in vivo using murine tumor models, an effect which
appears to be dependent, at least in part, on the increased
infiltration and function of NK cells [24]. Unlike previously
identified types of danger signals, which are often molecules
released from dying cells and thus, may act predominantly in
a local manner (reviewed in ref. [41]), body temperature can
affect a large number of effector cells at the same time,
including those that are not near the inflammatory milieu or
have not come into contact with any form of soluble mediator or
antigen- or ligand-expressing target. Thus, there is potential for
thermal stress to lower rapidly and systemically, the activation
threshold of distant immune cells well in advance of their
encounter with antigen or other soluble mediators of danger.
However, if this notion is correct, and regional or systemic
increase in temperature has the potential of affecting millions
of NK cells at the same time, there must be mechanisms in
place to prevent nonspecific killing of self or inappropriate
target cells. We observed here several clues indicating the
existence of such a regulatory mechanism in control of thermal
signaling. We found that thermally enhanced, NK cell-mediated cytotoxicity is highly specific: no killing of autologous
PBMC targets occurred upon heating, revealing the absence of
heat-induced release of lytic substances in the absence of
appropriate activation signals. Indeed, heated NK cells must
still be exposed to an appropriate target and experience normal
activation signals (coming from that target) for mild thermal
stress to have an effect. Antibodies blocking NKG2D eliminated killing of targets completely, including any enhancement
by heat. Further, significant up-regulation of MICA also helped
to enhance and guide the thermally enhanced lysis of tumor
targets, thus minimizing the potential for autoimmune activity.
Previous in vitro studies have revealed that activation of NK
cells is associated with clustering of receptors [3]. Although
activation-induced clustering is traditionally thought to occur
only at the contact site with a target, we propose that a
morphologically similar, thermally induced receptor clustering
all over the cell surface, as observed here, could still be at least
part of the underlying basis by which thermal signals increase
the lytic potential of NK cells. Although much work remains to
identify the biophysical process by which membrane-associated molecules rearrange in response to temperature shifts, our
data about the lack of detectible surface or sMICA on NK cells
http://www.jleukbio.org
indicate that thermally induced NKG2D clustering is not a
result of sMICA or surface-expressed MICA on these freshly
purified NK cells. Furthermore, the importance of thermally
induced clustering of activating receptors is also supported by
our observations of an activation state-dependent thermal sensitivity. There is a complete lack of fever-range thermal sensitivity/regulation in long-term NK cell lines, which are cultured in IL-2 and in purified human peripheral blood NK cells
that have been preactivated with IL-2. However, long-term,
cultured NK cell lines (which are known to express functional
NKG2D molecules [42, 43]) and IL-2-activated NK cells were
revealed here to constitutively display a higher percent of cells
with similarly clustered NKG2D receptors. Thus, it is possible
that in this circumstance, receptor clustering cannot be enhanced further with mild thermal stress, which may help to
explain their thermal insensitivity. That enhanced cytotoxic
activity of freshly purified NK cells is lost when cells are
recultured at 37°C, or the cells are incubated with M␤CD,
which disrupts lipid rafts, further supports the hypothesis that
thermally induced NKG2D clustering within lipid rafts is associated directly with thermal enhancement of NK cell cytotoxicity.
Up-regulation of the NKG2D ligand MICA has been reported previously to occur upon more severe stresses such as
heat shock (⬎42°C) [26], bacterial or viral infection [42, 44,
45], DNA damage [46], oxidative stress [47], and treatment
with retinoic acid [48] or the histone deacetylase inhibitor
sodium valproate [49]. The data presented here add mild
thermal stress, easily achievable under physiological conditions, to this list of conditions which can up-regulate MICA.
Furthermore, although there is fever-range enhancement of
surface MICA, there was no observable change in MHC class
I expression or sMICA, both of which could inhibit NK cytotoxic activity, suggesting that NK cells can capitalize on this
thermally induced shift in the balance of signals, resulting in
increased lytic ability. Although association of higher MICA
expression with higher NK-mediated cytotoxicity is not surprising, given previous data about the effects of various stressors on MICA expression [26, 42, 44 – 49], our data examining
the effects of heating target cells alone (Fig. 3) suggest that
such up-regulation of MICA by mild thermal stress is not
sufficient to induce statistically significant enhancement of
NK-mediated cytotoxicity. However, the fact that febrile temperatures are sufficient to increase MICA expression suggests
the intriguing possibility that NK cells have learned to take
advantage of this stress-induced effect on target cells by a
reciprocal response: clustering of NKG2D receptors, which
may strengthen NK cell interactions with the MICA ligand on
target cells. Indeed, Figure 3A suggests that NK cells and
target cells must each respond to the change in their thermal
microenvironment. This is supported by the observation that
although the NK cells may be induced to alter surface NKG2D
localization with mild thermal stress, a lack of thermally induced enhancement of MICA/ligand expression on the target
cell (as seen with HT29 cells) results in an absence of a
statistically significant or discernable increase in overall cytotoxicity. Examining the effects of mild thermal stress on a
broader range of tumor cell types, and determining whether
fever-range temperatures enhance MICA surface expression on
tumor cells and NKG2D organization on NK cells in vivo will
be critical future studies. Whether there is also a thermal effect
on the expression or function of inhibitory receptor molecules
(i.e., killer inhibitory receptors) on NK cells and their corresponding specific ligands on target cells also needs to be
examined further.
The enhancement of NK cytotoxic activity at the mild hyperthermic temperature ranges shown here contrasts with previous studies, which used temperatures significantly above
fever range (⬎42°C or 107°F), where NK cytotoxicity was
inhibited significantly [17–20]. Indeed, our data using Colo205
cells (in Fig. 1) also show that coincubation at 42°C did not
enhance cytotoxicity. This might indicate that heat shock conditions inhibit NK cell function, despite the known ability of
heat shock to up-regulate MICA on tumor cells. However, our
data support previous reports that indicate enhancement of
human NK cytotoxic activity at lower, more physiologically
relevant temperatures [21–23]. Our data also extend these
observations significantly by indicating that the enhanced cytotoxicity requires heating of the target cells and is not a result
of increased secretion of lytic molecules, thus providing mechanistic evidence regarding the role of NKG2D and its ligand
MICA in the thermal enhancement of NK-mediated cytotoxicity.
The remarkable evolutionary conservation of the febrile
response [50] suggests that there is a beneficial and specific
role for temperature shifts in the inflammatory process (reviewed in ref. [51]. Multiple studies suggest that application of
a mild hyperthermia can enhance several endpoints of the
immune system selectively in a manner which can help control
infections [52–54] and the growth of tumors in murine models
[24, 55]. Overall, we believe the data presented here further
support the hypothesis [39] that elevations in the thermal
microenvironment might serve as a danger signal, directly or
indirectly (via effects on targets themselves) alerting NK cells
to cellular insult and facilitating their more rapid (yet specific)
lytic potential through receptor clustering and perhaps additional downstream signaling events. A systemic elevation in
temperature would have the potential of alerting and/or increasing the immune-activating potential of cells over a widespread region of the body not in immediate contact with other
danger-signal molecules in a local inflammatory situation.
These data may also help to identify anti-tumor activity of
heat-exposed NK cells as one of the factors providing significantly improved tumor control, as revealed in several recent
Phase III trials using hyperthermia [56]. Although further
study is required to dissect the precise mechanisms by which
NK cells and other immune effector cells are able to perceive
and respond to an altered physical parameter of their microenvironment (i.e., temperature), the observations described
here could help in the development of new treatments, in which
heat is strategically delivered in combination with immunotherapy to enhance NK cell-mediated cytotoxicity.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health P01
CA94045, R01 CA71599, and R21 CA098852 and the Ro-
Ostberg et al. Enhanced NK cytotoxicity by mild thermal stress
1329
swell Park Cancer Institute core grant CA16056. B. E. D. was
supported by the Komen Foundation DISS0402487 and a
Fulbright predoctoral grant. The authors thank Bonnie Hylander and Sarah Hejaily for their review of the manuscript and
Jeanne Prendergast, Diane Thompson, and Christopher Gregorie for their expert laboratory assistance.
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