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Production and Cytotoxicity of IL-2-Activated Murine NK Cells Are Differentially Regulated by MHC Class I Molecules1
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Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, Canada;
Unité de Biologie Moléculaire du Gène, Institut National de la Santé et de la Recherche Médicale Unit 277, Institute Pasteur, Paris, France; and
Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. MHC class I
molecules on target cells regulate NK cytotoxicity. However, little is
known about the regulation of IFN- production by NK cells. We
examined the production of IFN- in individual murine NK cells
stimulated with tumor cell lines by flow cytometric analysis of
intracellular IFN-. Among several tumor lines tested, the rat
basophilic leukemia line RBL-1 induced particularly high level of
IFN- production in IL-2-activated NK cells, whereas other lines,
including the prototypic NK target YAC-1, induced very low or no
IFN- production. Transfection of murine classical MHC class I
molecules into RBL-1 cells substantially inhibited IFN- production.
This inhibition of IFN- production by MHC class I was independent of
Ly-49 or CD94/NKG2A expression on NK cells. These results indicate that
some target cells directly stimulate IL-2-activated NK cells and induce
IFN- production, but the requirements for the induction of IFN-
production seem different from those for NK cytotoxicity. Furthermore,
similar to NK cytotoxicity, induction of IFN- production is
inhibited by MHC class I on stimulating cells. However, the MHC class
I-specific receptors inhibiting IFN- production are different from
those for NK cytotoxicity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is
thought to be particularly important as it activates macrophages to
kill phagocytosed pathogens and to secrete various cytokines. IFN-
also promotes Th1 immune responses (2). Therefore, IFN-
production by NK cells seems to be one of the important links between
innate and adaptive immune responses. NK cytotoxicity is regulated by a balance between receptor-induced stimulatory and inhibitory signals (3). Whereas the identities of stimulatory NK receptors remain unclear, recent studies have identified multiple NK inhibitory receptors that recognize class I MHC on target cells. In mice, these inhibitory receptors belong to two families of C-type lectins, namely the Ly-49 family (4) and CD94/NKG2 heterodimers (5, 6). The former recognizes specific classical class I MHC, whereas the latter recognize the nonclassical MHC class I Qa-1b (5). It is thought that NK cells are activated by a wide range of target cells but are maintained to be self-tolerant due to the inhibitory receptors that recognize self-MHC on normal cells (7, 8).
NK cells can be activated to produce cytokines by monocyte-derived
cytokines. Although IL-12, IL-15, or IL-18 alone does not induce
significant cytokine production in resting human NK cells, combinations
of two of these cytokines are potent inducers of IFN- production
(9). IFN- production by NK cells can also be induced by
some tumor cells (10, 11). However, the regulatory
mechanisms controlling NK cell IFN- production are largely
unknown.
In this study, we examined the production of IFN- by IL-2-activated
NK cells stimulated with tumor cell lines. Our results show that
activation of IFN- production and cytotoxicity are differentially
regulated in NK cells. Furthermore, classical MHC class I molecules on
stimulating cells inhibit IFN- production. However, Ly-49 or
CD94/NKG2A does not seem to be responsible for the inhibition,
suggesting that murine NK cells express novel inhibitory receptors for
MHC class I that inhibit IFN- production.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Tumor cell lines were obtained from American Type Culture Collection (Manassas, VA). The rat basophilic leukemia RBL-1 lines transfected with mouse MHC class I have been described (12).
Abs and flow cytometry
The hybridomas PK136 (anti-NK1.1), 20-8-4S (anti-Db, Kb, Kd), 28-8-6S (anti-Db), 3-83P (anti-Kk), and 34-5-8S (anti-Dd) were obtained from the American Type Culture Collection (Manassas, VA). The mAbs YE1/48 (anti-Ly-49A) and 5E6 (anti-Ly-49C and -I) have been described (13). For the staining of RBL-1 cells transfected with murine MHC class I, cells were first incubated with anti-MHC class I mAbs at 4°C for 30 min. After two washes, the cells were incubated with appropriate FITC-conjugated secondary Abs (Jackson ImmunoResearch, West Grove, PA) for 30 min at 4°C and analyzed by FACSCalibur (Becton Dickinson, San Jose, CA).
Generation of IL-2-activated NK cells
Adherent IL-2-activated NK cells were generated as described (14). In brief, spleen cells were passed through a nylon wool column to remove B cells. The nylon wool-nonadherent cells were incubated on tissue culture plates for 1 h to remove adherent cells. Nonadherent cells were cultured at a density of 106 cells/ml in RPMI 1640 medium supplemented with 10% FCS, 5 x 10-5 M 2-ME, and 1000 U/ml murine IL-2. After 3 days of culture, nonadherent cells were removed, and the adherent cells were then cultured in the presence of IL-2 for an additional 4 days.
Cytotoxicity assay and isolation of Ly-49A+ NK cells
For specific lysis of RBL-1 target cells, which were poorly labeled with 51Cr, we used nonradioactive cytotoxicity assay kit (CytoTox 96, Promega, Madison, WI) in which lactate dehydrogenase released from lysed target cells was quantitatively measured according to the manufacturer’s protocols. For other target cells, specific lysis was determined by using a standard 4-h 51Cr release assay as previously described (14). Both assays well correlated with each other when YAC-1 cells were used as target cells. For the isolation of Ly-49A+ NK cells, IL-2-activated NK cells were enriched by using the miniMACS (Miltenyi Biotec, Auburn, CA) immunomagnetic-based separation method. In this method, cells were first incubated with 1 µg/ml of biotinylated YE1/48 mAb followed by streptavidin microbeads. The labeled cells were then passed through the RS+ separation column. Ly-49A+ NK cells retained in the column were removed by several rinses with saline. The purified NK cells were subsequently cultured overnight in 1000 U/ml of IL-2 before used in cytotoxicity assays. For mAb blocking assay, effector cells were preincubated for 30 min at room temperature with 50 µg/ml mAb before the addition of target cells.
Detection of intracellular IFN-
NK cells (2 x 105) and stimulator
cells were washed and mixed at a ratio of 2:1 in 200 µl of RPMI 1640
+ 10% FCS in sterile 96-well plates, centrifuged at 1000 rpm for 1
min, and incubated at 37°C for 11 to 12 h. Brefeldin A (10
µg/ml, Sigma, St. Louis, MO) was added during the final 6 h to
inhibit cytokine secretion. Intracellular IFN- was determined by
flow cytometric analysis as described by Prussin et al.
(15) with slight modifications. Briefly, cells were washed
and incubated for 30 min with PK136-FITC on ice in the dark. After
washing, cells were fixed with 4% paraformaldehyde for 5 min at room
temperature. On the following day, cells were treated for 30 min with
cell permeation buffer (0.1% saponin, 1 mM
CaCl2, 1 mM MgSO4, 0.05%
NaN3, 1% BSA, 10 mM HEPES), and then incubated
for 30 min with PE-labeled IFN--specific mAb (PharMingen, San
Diego, CA) or PE-labeled rat IgG1 (PharMingen) in the permeation buffer
(2.5 µg/ml). Cells were washed with the permeation buffer,
resuspended in PBS, and analyzed by FACSCalibur. Tumor cells were gated
out based on their high forward scatter. In some experiments, NK cells
and RBL-1 cells were incubated in 24-well flat-bottom plates with
transwell inserts with 3-µm pore size membranes (Becton Dickinson).
NK cells (1 x 106) were incubated in the
lower wells, whereas RBL-1 cells (5 x 105)
were incubated in the upper wells in 1 ml media. In control wells, NK
cells were incubated without RBL-1 cells or were incubated together
with RBL-1 cells in the lower wells.
Qa-1b tetramer
The Qa-1b tetramer has been described (16). Briefly, a tetrameric complex of biotinylated Qa-1b, human ß2-microglobulin, and peptide (AMAPRTLL) was formed using Streptavidin Red 670 (Life Technologies, Gaithersburg, MD) at a 4:1 molar ratio. For cell staining, Qa-1b tetramer was used at a total protein concentration of 7 µg/ml in PBS, and cells were incubated for 30 min on ice.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production with tumor cell lines
Murine NK cells were stimulated with various NK-sensitive tumor
cell lines, and intracellular IFN- content in individual NK cells
(NK1.1+) was determined by two-color flow
cytometry. When freshly isolated NK cells were used, the tumor cell
lines tested in this study did not induce a significant amount of
IFN- production (data not shown). However, adherent IL-2-activated
NK cells that were cultured for 7 days in the presence of IL-2 produced
variable levels of IFN- on incubation with tumor cells (Fig. 1
). The intracellular IFN- accumulated
relatively slowly as compared with NK cytotoxicity and peaked at
12 h of incubation with tumor cells (Fig. 2A). This was dependent on the
stimulation with tumor cells, in that IL-2-activated NK cells incubated
with media alone did not produce IFN- (Fig. 2B). Although
most cell lines tested in this study were sensitive to NK cell
cytotoxicity, the ability to stimulate IFN- production was quite
variable. The percentages of NK1.1+ cells
positively stained with anti-IFN- mAb as well as the intensities
of the staining varied significantly depending on the cell lines used
as stimulators (Table I). The prototypic
NK target YAC-1 was highly sensitive to NK cytotoxicity, but
stimulation of IFN- production was almost negligible. Among the cell
lines tested, the rat basophilic leukemia line RBL-1 was the most
effective stimulator of IFN- production. More than 70% of
IL-2-activated NK cells produced IFN- in response to this cell line.
Another rat cell line, YB2/0, was a much poorer stimulator of IFN-
production. The induction of IFN- production by RBL-1 did not seem
to be due to production of soluble factors by RBL-1, because culture
supernatant of RBL-1 cells had no effect (Fig. 2B). We also
tested whether direct cell-cell contact between NK and RBL-1 cells is
required for the stimulation of IFN- production using transwells.
When NK cells were separated from RBL-1 cells by a porous membrane,
they did not produce IFN-, whereas NK cell incubated in close
contact with RBL-1 cells in the same wells produced significant amount
of IFN- (Fig. 2C). Therefore, direct cell-cell contact
between NK and RBL-1 cells is required for the induction of IFN-
production.
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production by MHC class I
To investigate whether expression of MHC class I on stimulator
cells affects NK cell IFN- production, RBL-1 cells transfected with
murine MHC class I were used as stimulators. The transfected RBL-1
cells expressed comparable levels of surface MHC class I molecules
(Fig. 3). When incubated with MHC class I
-transfected RBL-1, IFN- production by NK cells was significantly
lower than the controls in which NK cells were incubated with either
the parental or the vector-transfected RBL-1 cells (Fig. 4). Db and
Kb, which are self-MHC for C57BL/6 NK cells, as
well as foreign MHC class I Dd and
Kk significantly inhibited IFN-
production.
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production
To determine whether the inhibition of IFN-
production by MHC class I observed above is mediated by the binding of
MHC class I to the Ly-49 family of NK-inhibitory receptors, we first
examined the effect of MHC class I molecules on the cytotoxicity of
Ly-49-positive NK cells. As shown in Fig. 5, Dd expressed on
RBL-1 cells efficiently inhibited the cytotoxicity of Ly-49A-positive
NK cells, and this inhibition was reversed by anti-Ly-49A Ab. This
result showed that MHC class I molecules on the transfected RBL-1 cells
are properly expressed and sufficient to inhibit NK cytotoxicity in a
Ly-49-dependent manner. To determine whether the Ly-49 family of
inhibitory receptors on NK cells is also responsible for the inhibition
of IFN- production by class I MHC on the transfected RBL-1 cells, we
performed multicolor flow cytometric analysis to simultaneously detect
cytoplasmic IFN- and cell surface Ly-49 on
NK1.1+ cells. The YE1/48 mAb detects Ly-49A that
recognizes Dd (13), whereas the 5E6
mAb reacts with Ly-49C that recognizes all the class I MHC tested in
this study as well as Ly-49I (17). Interestingly,
production of IFN- in both YE1/48+ and
YE1/48- NK subsets was significantly reduced by
Dd expression on the stimulator RBL-1 cells (Fig. 6A). Dd
also inhibited IFN- production in 5E6+ and
5E6- NK cells. For all MHC class I molecules
tested in this study, no significant difference in the degree of
inhibition was observed between Ly-49+ and
Ly-49- population (Fig. 6B).
Furthermore, preincubation of NK cells with a mixture of anti-Ly-49
mAbs, namely, YE1/48 (anti-Ly-49A), 5E6 (anti-Ly-49C and -I),
and 4D11 (anti-Ly-49G), had no significant effect on IFN-
production (data not shown). These results indicate that Ly-49 is not
responsible for the inhibition of IFN- production by MHC class I. To
determine whether CD94/NKG2A is involved in the inhibition of IFN-
production, Qa-1b tetramer that binds to
CD94/NKG2A (5, 16) was used to identify
CD94/NKG2A+ NK cells. Flow cytometric analysis of
NK cells stimulated with Kb- or
Db-transfected RBL-1 showed that IFN-
production in NK cells stained with Qa-1b
tetramer as well as those not stained by Qa-1b
were equally inhibited by these MHC class I molecules (Fig. 7). Therefore, CD94/NKG2 does not seem to
be responsible for the inhibition of IFN- production by MHC
class I.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 2) MHC class I
molecules on tumor cells inhibit this activation of IFN- production,
and 3) inhibitory receptors on NK cells recognizing classical MHC class
I responsible for this inhibition seem different from known murine NK
receptors for MHC class I. The results suggest that murine NK cells
express novel MHC class I-specific inhibitory receptors that regulate
IFN- production. They further suggest that activation signals
required for NK cytotoxicity and IFN- production may be different.
Many tumor cell lines, including the standard NK target YAC-1, are
highly sensitive to NK cytotoxicity, and yet they are very poor
inducers of IFN- production. NK cytotoxicity and IFN- production
also differ in their time courses. The former is rapidly triggered on
contact with susceptible target cells (18) and can be
detected within 3 h, whereas IFN- production is a slower
process and takes 12 h incubation to reach the maximum level (Fig. 2
A). This difference is likely due to the fact that NK
cytotoxicity is mediated by exocytosis of preformed perforin and
granzymes on activation of NK cells by targets, whereas IFN-
production involves de novo gene transcription (19, 20, 21).
The differences also suggest that ligands on tumor cells triggering NK
cytotoxicity do not seem to be able to provide activation signals
required for the induction of IFN- production. It is also possible
that additional stimulatory signals may be required for IFN-
production. The differential regulation of NK cytotoxicity and cytokine
production has also been suggested by previous studies. Some human NK
cell clones killed K562 and 721.221 targets equally well, but they
secreted more IFN- in response to K562 than to 721.221 cells
(10).
The possible difference in stimulatory signals for NK cytotoxicity and
IFN- production may explain why Ly-49 or CD94/NKG2A does not seem to
provide inhibitory signals for IFN- production. It has been
established that Ly-49A recognizes Dd (22, 23) whereas Ly-49C recognizes a wide range of classical MHC
class I molecules (13). Consistent with these findings, we
have previously shown that MHC class I-transfected RBL-1 cells bind to
Ly-49-transfected COS cells (12). Furthermore, we showed
in this study that Ly-49 on IL-2-activated NK cells are able to
recognize murine MHC class I molecules on the transfected RBL-1 cells
and provide inhibitory signals for NK cytotoxicity. Although the same
MHC class I molecules on RBL-1 also inhibited IFN- production of NK
cells, this inhibition was Ly-49 independent. Two-color flow cytometric
analysis showed that expression of Dd on RBL-1
inhibits IFN- production in not only Ly-49A+
but also Ly-49A- NK cell subsets. Similarly,
there was no correlation between inhibition of IFN- production by
any of the MHC class I molecules tested and the expression of Ly-49C/I
on NK cells detected by the 5E6 mAb. Furthermore, anti-Ly-49 mAb
had no effects on the inhibition of IFN- production by MHC class I
(data not shown), whereas the same Ab reversed the inhibition of NK
cytotoxicity by MHC class I on target cells. These results indicate
that Ly-49 is not responsible for the inhibition of IFN- production.
It is also unlikely that CD94/NKG2A is responsible for the inhibition.
CD94/NKG2A recognizes the nonclassical MHC class I
Qa-1b (5) that presents peptides
derived from the leader peptides of certain classical MHC class I
molecules (24, 25). It is possible that transfection of
murine classical MHC class I into RBL-1 cells may enhance expression of
the rat Qa-1b homologue. However, transfection of
different MHC class I, regardless of whether they contain the Qa-1
determinant modifier motif (25) in the leader sequence,
inhibited IFN- production. Moreover, the inhibition of IFN-
production did not correlate with the expression of CD94/NKG2 on NK
cells, as determined by the binding of tetrameric
Qa-1b.
Taken together, these results strongly suggest that murine NK cells
express novel MHC class I-specific receptors that inhibit IFN-
production, the identity of which is currently unknown. It is also not
known whether a single receptor recognizes different MHC class I
molecules or another family of receptors, each recognizing specific MHC
class I, mediates the inhibition of IFN- production. In humans,
killer cell-inhibitory receptors that belong to the Ig superfamily have
been reported to inhibit both cytotoxicity and cytokine production
(10). In addition, another type of MHC class I-specific
inhibitory receptors belonging to the Ig superfamily, leukocyte
immunoglobulin-like receptor (26), has been identified on
human lymphocytes. However, murine homologues of killer cell-inhibitory
receptors and leukocyte immunoglobulin-like receptor have not been
identified. Another receptor, termed gp49, that also belongs to the Ig
superfamily has been identified on murine NK cells and mast cells
(27, 28), but the ligands for gp49 are not known. It
remains to be determined whether any of these receptors are involved in
the inhibition of IFN- production by MHC class I.
Direct cell-mediated cytotoxicity and cytokine production are thought
to be two important functions of NK cells. Although recent studies have
revealed the mechanisms by which NK cytotoxicity is regulated by MHC
class I on target cells, little is known about the regulation of
IFN- production by NK cells. Our study has shown, for the first
time, that MHC class I molecules on tumor cells inhibit NK cell IFN-
production by a mechanism distinct from that responsible for the
inhibition of NK cytotoxicity.
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Acknowledgments |
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Footnotes |
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2 Current address: Immuno-Designed Molecules (IDM), 172 rue de Charonne, 75011 Paris, France.
3 Address correspondence and reprint requests to Dr. Fumio Takei, Terry Fox Laboratory, British Columbia Cancer Research Center, 601 West 10th Avenue, Vancouver, British Columbia, V5Z 1L3 Canada. E-mail address:
Received for publication July 30, 1999. Accepted for publication October 6, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA synthesis. J. Immunol. 137:3836.[Abstract]
and its receptor. Annu. Rev. Immunol. 11:571.[Medline]
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