By
From the * Department of Medicine, University of California, San Francisco, California 94143; the
Veterans Administration Medical Center, San Francisco, California 94121; The Jackson Laboratory,
Bar Harbor, Maine 04609; and the § Department of Microbiology and Immunology, University of
California, San Francisco, California 94143
The lytic activity of natural killer (NK) cells is inhibited by the expression of class I major histocompatibility complex (MHC) antigens on target cells. In murine NK cells, Ly-49A mediates inhibition of cytotoxicity in response to the class I MHC antigen H-2Dd. In this report, we studied the function of mouse Ly-49A in both the rat NK cell tumor line, RNK-16, transfected with Ly-49A cDNA, and in primary NK cells. We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation. We also show that Ly-49A directly associates with the cytoplasmic tyrosine phosphatase SHP-1, and that Ly-49A function is impaired in NK cells from SHP-1 mutant viable motheaten mice and from SHP-1-deficient motheaten mice. Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor. These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.
NK cells and some T cells express a variety of type II
transmembrane receptors characterized by extracellular C-type lectin domains (1). In mice, these proteins include the members of the Ly-49 family (2), which recognize MHC class I molecules on target cells (6), and
the NKR-P1 family, whose physiologic ligands have yet to
be determined (11). While the NKR-P1 molecules activate NK cell cytotoxicity (14), at least three members
of the Ly-49 family inhibit NK cell function (6).
Susceptible targets stimulate phosphoinositide turnover,
calcium mobilization, and the induction of protein tyrosine
phosphorylation in NK cells. These signals have been associated with activation of NK cell cytotoxic responses (17).
The NKR-P1 lectin-like receptor also transduces these activating signals in NK cells (14). In contrast, mouse Ly49A inhibits NK cell cytotoxicity upon ligation by the target cell MHC class I molecules H-2Dd or H-2Dk (6, 9, 10).
The mechanisms by which Ly-49A interrupts NK cell activation are poorly understood, but important clues can be
derived from structural motifs within the Ly-49A molecule.
The Ly-49A cytoplasmic domain contains the amino
acid sequence VxYxxV, which constitutes a proposed binding motif for the cytoplasmic tyrosine phosphatase, SHP-1
(21, 22). SHP-1 is an SH2 domain containing tyrosine phosphatase, expressed in hematopoietic cells that can inhibit specific cellular functions. SHP-1 negatively modulates signaling through the erythropoietin receptor (23, 24), and it
inhibits activation of B cells through its association with
Fc Cells.
RNK-16, a spontaneous NK cell leukemia from F344
rats, was the gift of C. Reynolds (National Cancer Institute, Frederick, MD) and was adapted for in vitro growth in RPMI-1640
supplemented with 10% heat-inactivated FCS, 25 µM 2-ME, 2 mM
L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin
(complete RPMI) (31). Tumor target cell lines cultured in complete RPMI included YAC-1 (mouse lymphoma, H-2a), P388D1
(mouse macrophage, H-2d), and C1498 (mouse monocyte, H-2b)
from the American Type Culture Collection (Rockville, MD).
D12 (C1498 transfected with H-2Dd, C1498.Dd), a gift from W. Yokoyama, was described previously (6). B-16S, a mouse melanoma line (H-2b), was a gift from K. Kärre (Karolinska Institute,
Stockholm, Sweden).
Mice.
Viable motheaten mice C57BL/6 (mev/mev) mice were
obtained from the Jackson Laboratory at 6 wk of age along with
littermate heterozygotes (+/mev) and age-matched wild-type
C57BL/6 (+/+) mice. Motheaten C57BL/6 (me/me) mice and
littermate heterozygotes (+/me) were killed at 4-5 wk of age at
the Jackson Laboratory, and isolated spleen cells were obtained.
Antibodies and Flow Cytometry.
mAbs to mouse Ly-49A (A1,
mouse IgG2a), NK1.1 (PK136, mouse IgG2a), gp42 (3G7, mouse
IgG2a), and phosphotyrosine (APT) (4G10, mouse IgG2b) were
produced from their respective hybridoma lines. Antibodies were
partially purified from ascites by ammonium sulfate precipitation.
Tissue culture supernatant of anti-mouse Fc receptor antibody
(2.4G2, rat IgG2b) was a gift from P. Linnemeyer (University of
California at San Francisco, CA). Fluorescein conjugation of protein A-purified mAb utilized standard methods (32). F(ab Cytotoxicity Assays.
Specific lysis of NK targets was determined by using a standard 4-h 51Cr-release assay as previously described (16). In brief, target cells were harvested and labeled for 1 h
at 37°C with 200 µCi of sodium 51Cr (Amersham, Arlington
Heights, IL) in complete RPMI. Labeled target cells were washed
and resuspended at 105 cells/ml, and 0.1 ml of this cell suspension
was added to each well of 96-well plates containing 0.1 ml of effector cells at the indicated effector to target ratios. Plates were
incubated at 37°C for 4 h, then centrifuged for 5 min. 100 µl of
supernatant was counted in a gamma counter. All assays were performed in triplicate. For antibody inhibition studies, effector cells
were preincubated for 15 min at room temperature with F(ab IL-2-activated NK Cells.
IL-2-activated NK cells (LAK) were
prepared from fresh splenocytes as previously described (33).
Spleen cells harvested from me/me and +/me mice were transported from Jackson Laboratories in complete RPMI, after red
cell lysis, and received within 24 h of death. Spleen cells were
then passaged through nylon wool and placed in culture with IL-2,
as with fresh splenocytes (33). Ly-49A positive and negative IL-2-
activated NK cells were isolated as previously described (6). In
brief, day 6 IL-2-activated NK cells were panned with the anti-
Ly-49A mAb, A1. The purity of the Ly-49 cDNA Constructs and Transfections.
The construct for expression of Ly-49A in RNK-16 cells was prepared by subcloning
the Ly-49A cDNA into the EcoRI site of the expression vector
BSR Measurement of Inositol-Trisphosphate.
To measure changes in
water-soluble inositol trisphosphates (InsP3), cells were washed
into inositol-free medium containing [3H]myoinositol (20 µCi/ml,
80-120 Ci/mmol, Amersham, Arlington Heights, IL) at a concentration of 5 × 106 cells/ml. After incubation at 37°C for 3 h,
cells were washed in complete RPMI. Then 5 × 106 labeled effector cells were stimulated with 107 target cells in a total volume
of 1 ml. Cells were immediately centrifuged at 50 g for 10 s and
incubated at 37°C for the appropriate interval. [3H]InsP3 was extracted and resolved by ion exchange chromatography on Dowex
AG-50, 1X-8 (Bio-Rad, Hercules, CA) as previously described (12).
Target Stimulation of [32P]-phosphate-labeled Effector Cells.
For
32P-metabolic labeling, effectors were incubated with 1 mCi/ml
[32P]orthophosphate in phosphate-free RPMI supplemented with
10% dialyzed FCS, at 2 × 106 cells/ml for 90 min at 37°C. After
labeling, cells were washed once in phosphate-free RPMI and
used immediately for stimulation. In each sample, 107 labeled effector cells were stimulated with 107 unlabeled target cells in a total
volume of 1 ml complete phosphate-free RPMI. Cell suspensions
were immediately centrifuged for 10 s at 50 g, then incubated at
37°C for the indicated time, after which cells were rapidly centrifuged at 500 g and cell pellets were resuspended in cold HNTG
lysis buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM EDTA,
1 mM MgCl2, 10% glycerol, 1 mM sodium orthovanadate, and protease inhibitors) with 1% Triton X-100. Lysates were precleared with protein A-Sepharose beads (Pharmacia, Piscataway, NJ) that had been previously coated with 1 ml 2.4G2 supernatant, for 2 h
at 4°C, then immunoprecipitated with 30 µl of protein A beads coated with 5 mg APT mAb (4G10) overnight at 4°C. After
washing with cold HNTG buffer with 1% Triton X-100, immunoprecipitates were resolved by 8% SDS-PAGE under reducing
conditions. Gels were stained, fixed, and dried, and labeled proteins were resolved by autoradiography.
Immunoprecipitations and Western Blotting.
Where indicated, transfected and wild-type RNK-16 cells (1.5 × 107 cells/ sample)
were incubated for 5 min at 37°C in complete RPMI (6 × 105
cells/ml) with 0.03% H2O2 and 100 µM sodium orthovanadate (pervanadate), which pharmacologically increases protein tyrosine phosphorylation by inhibiting phosphatase activity (34). Cell pellets were
lysed at 4°C in complete HNTG lysis buffer containing 1% Triton X-100. Lysates were precleared for 2 h at 4°C on Protein
A-Sepharose beads that had been loaded with 10 µg control mAb
(mouse IgG2a myeloma protein, Cappel, Malvern, PA). Lysates
were then immunoprecipitated overnight at 4°C on 30 µl protein
A-Sepharose beads loaded with anti-Ly-49A (A1) or isotypematched control mAb (anti-NK1.1). Precipitates were washed
four times with complete HNTG buffer containing 0.1% Triton
X-100. Precipitated samples were resolved by 8% SDS-PAGE under nonreducing conditions and transferred to PVDF membranes (Immobilon-P, Millipore, Marlborough, MA). After blocking with TBS-T (10 mM Tris, pH 8, 150 mM NaCl, 0.05% Tween-20)
and 3% milk, the membranes were incubated with 0.5 µg/ml of
anti-SHP-1 polyclonal rabbit antibody (UBI, Lake Placid, NY) in
TBS-Tween with 3% milk for 1 h at room temperature. After
extensive washing in TBS-T, blots were developed using 125I-protein A (Amersham, Arlington Heights, IL) followed by autoradiography.
Cold-target Inhibition Studies.
Cytotoxicity assays to measure
cold-target inhibition were performed at an E/T ratio of 10:1.
105 cold targets were added to effectors at the same time as 104 labeled targets. Effectors were preincubated with F(ab To examine the intracellular signaling pathways that mediate the inhibitory function of mouse Ly-49A, we transfected mouse Ly-49A into RNK-16, a rat cell line with
phenotypic and functional characteristics of rat NK cells
(31). Nine RNK-mLy-49A clones expressing Ly-49A at
different levels were obtained, three of which are represented in Fig. 1. Clones 2, 8, and 9 are representative of
clones with low, medium, or high expression of Ly-49A,
respectively (Fig. 1, B, C, D). Wild-type RNK-16 effector
cells lysed P388D1 (H-2Dd) tumor cells (Fig. 1 E), but
RNK-16 cells transfected with Ly-49A (RNK-mLy-49A
cells) demonstrated reduced lysis of P388D1 (F, G, H). Inhibition of lysis of P388D1 cells was proportional to the level of Ly-49A expression on the RNK-16 transfectants.
RNK-mLy-49A.2 had low expression and demonstrated only
minimal inhibition of lysis (Fig. 1 F), whereas RNK-mLy49A.8 and RNK-mLy-49A.9, with progressively higher levels of expression, demonstrated marked inhibition of lysis (G
and H). Inhibition of lysis in Ly-49A transfectants was reversible by the addition of anti-Ly-49A F(ab
Using the RNK-mLy-49A.9 cell line,
we examined the inositol phosphate response following
stimulation by YAC-1 (sensitive) and P388D1 (resistant)
targets. Wild-type RNK-16, but not RNK-mLy-49A.9 cells, responded to the H-2Dd target P388D1 with a rapid
increase in InsP3 at 2 min, as shown in Fig. 2. YAC-1, which
is susceptible to lysis by both wild-type RNK-16 and
RNK-mLy-49A.9, stimulates an InsP3 response in both cell
types. The lack of phosphoinositide turnover in RNKmLy-49A.9 in response to H-2Dd target cells indicates that
Ly-49A interrupts proximal signaling events in RNK-16.
To examine the
effect of Ly-49A-mediated inhibition on protein tyrosine
phosphorylation, [32P]orthophosphate-labeled RNK-16 and
RNK-mLy-49A.9 cells were stimulated with YAC-1 (H-2a)
and P388D1 (H-2Dd) target cells. Because Ly-49A interrupts InsP3 turnover, an early signaling event, we examined
the effect of target stimulation at 30 s, 1 min, and 5 min
time points. Lysates from cells stimulated for these time intervals were immunoprecipitated with APT (4G10) and resolved by SDS-PAGE (Fig. 3). RNK-16 cells stimulated
with P388D1 (H-2Dd) showed a rapid increase in protein
tyrosine phosphorylation at 30 s to 1 min, which diminished toward basal levels at 5 min (Fig. 3, left). In contrast,
RNK-mLy-49A.9 cells stimulated with P388D1 failed to
show an increase in protein tyrosine phosphorylation at 30 s
to 1 min, but showed a minimal increase at 5 min (Fig. 3,
center). Nonetheless, RNK-mLy-49A.9 cells demonstrated
rapid protein tyrosine phosphorylation in response to YAC-1
after 30 s, demonstrating that this signaling pathway was intact (Fig. 3, right). Wild-type RNK-16 showed a similar response to YAC-1 target cells (data not shown). Thus, susceptible, but not resistant, targets induce very brisk increases
in protein tyrosine phosphorylation, and mouse Ly-49A
specifically interrupts rapid tyrosine phosphorylation in response to P388D1 (H-2Dd) targets.
In other
lymphoid cells, SHP-1 has been shown to interrupt early
tyrosine phosphorylation events. The mouse Ly-49A cytoplasmic domain includes the proposed SHP-1 binding motif VxYxxV (21, 22). These structural features of Ly-49A,
and the results obtained in Fig. 3, suggested that Ly-49A-
dependent inhibition of NK cell function might be mediated by SHP-1. Therefore, we first investigated the binding
of SHP-1 to Ly-49A in RNK-16 cells by performing immunoprecipitation experiments. RNK-16 and RNK-mLy49A.9 cells were stimulated with pervanadate, a phosphatase inhibitor that pharmacologically increases protein
tyrosine phosphorylation (34). Lysates from stimulated and
unstimulated cells were precipitated with anti-Ly-49A, and precipitates were examined for the presence of Ly-49A-associated SHP-1 by Western blot analysis. As shown in Fig. 4,
SHP-1 was present only in anti-Ly-49A immunoprecipitates from pervanadate-stimulated RNK-mLy-49A.9 cells.
SHP-1 was not detected in anti-Ly-49A immunoprecipitates from unstimulated RNK-mLy-49A.9 cells or in isotype-matched control mAb immunoprecipitates from unstimulated or stimulated RNK-mLy-49A.9 cells. SHP-1
was not detected in anti-Ly-49A or control mAb immunoprecipitates from RNK-16 cells, regardless of stimulation.
To examine the role of SHP-1
in mouse Ly-49A-mediated inhibition, we isolated Ly49A+ and Ly-49A We isolated Ly-49A+ and Ly-49A In each of four experiments, with homozygous mev/mev
mice the function of Ly-49A was impaired in that C1498.Dd
targets were lysed by Ly-49A+ effectors, albeit less effectively than by Ly-49A
The partial function of Ly-49A in mev/mev mice could
reflect the residual activity of SHP-1 in these mice. To examine the effect of the complete absence of SHP-1, we
next examined Ly-49A function in IL-2-activated NK cells
isolated from me/me mice. Using spleen cells harvested from
me/me mice (sacrificed just before their natural demise), +/me
littermate heterozygote controls, and wild-type +/+ mice,
we isolated Ly-49A+ and Ly-49A
As shown in Fig. 7, Ly-49A function was significantly
impaired in NK cells isolated from me/me mice, but, as in
mev/mev mice, it was not completely absent. Ly-49A+ cells
from wild-type +/+ or from heterozygous +/me were
unable to lyse C1498.Dd targets (A and B), but Ly-49A+
cells from me/me mice were able to lyse these targets (C),
albeit less effectively than Ly-49A
To examine further the role of SHP-1 in the
function of Ly-49A, we mutated the tyrosine residue
within the proposed SHP-1 binding motif in the Ly-49A
cytoplasmic domain. RNK-16 cells transfected with this
tyrosine mutant, (RNK-mLy-49A/Y8F) stained with anti- Ly-49A mAb at levels similar to those seen in RNK-mLy49A.9 (Fig. 8, D, F, H). Despite this level of Ly-49A expression, RNK-mLy-49A/Y8F clones 1 and 4, derived
from separate transfections, were not inhibited in their capacity to lyse P388D1 (H-2Dd) cells (Fig. 8, E and G). Consistent with our earlier results, RNK-mLy-49A.9 cells could
not lyse P388D1 (Fig. 8 C), whereas wild-type RNK-16
lysed P388D1 efficiently (A). Notably, neither anti-Ly-49A
nor control mAb (anti-NK1.1) had any effect on lysis of
P388D1 by RNK-mLy-49A/Y8F (Fig. 8, E and G), but
anti-Ly-49A reversed the inhibition of P388D1 lysis by
RNK-mLy-49A.9 (C). RNK-mLy-49A/Y8F clones 1 and
4 effectively lysed YAC-1 targets (data not shown). These
data show that the tyrosine within the proposed immunoreceptor tyrosine-based inhibitory motif (ITIM) is required for the inhibitory effect of Ly-49A on RNK-16 cytotoxicity. These experiments complement the studies of
motheaten mice, demonstrating a functional role for SHP-1
in the inhibitory activity of Ly-49A.
Initial reports demonstrated that ligation of H-2Dd
targets by the Ly-49A receptor resulted in inhibition of natural killing, of antibody-dependent cellular cytoxicity (ADCC),
and of lectin-induced cytotoxicity by NK cells (6, 38). These
studies, performed on bulk populations of Ly-49A+ NK
cells, suggested that Ly-49A might transduce signals that globally inhibit NK cell function. Using our RNK-Ly-49A
transfectants, we were able to examine mouse Ly-49A function in a clonal cell population. To examine the effect of inhibitory H-2Dd targets on the lysis of labeled bystander
non-H-2Dd targets, we performed cold-target competition
experiments with wild-type RNK-16 or RNK-mLy-49A.9
as effectors, and 51Cr-labeled B-16S (H-2b) melanoma target cells. The effect of unlabeled H-2Dd or non-H-2Dd
targets on lysis of B-16S was determined for each effector
(Fig. 9). As expected, unlabeled B-16S (H-2b) were effective cold-target competitors for the lysis of 51Cr-labeled
B-16S by both RNK-16 and by RNK-mLy-49A.9. Unlabeled P388D1 (H-2Dd) targets, which are sensitive to lysis
by RNK-16, effectively inhibited B-16S lysis by RNK-16.
In contrast, P388D1, which was not killed by RNK-mLy49A.9, was relatively ineffective as a cold-target inhibitor of
B-16S lysis by RNK-mLy-49A.9. In the presence of F(ab
Thus, the expression of H-2Dd on bystander targets does
not globally inhibit the Ly-49A+ effector cell response towards a non-H-2Dd cell. Rather, because H-2Dd cells are
not susceptible to NK cell lysis by Ly-49A+ effectors, they
compete less well than susceptible (B-16S) cold targets for
the lysis of labeled B-16S. These data indicate that there is
no bystander inhibition through Ly-49A. They suggest that
inhibitory effects mediated through this receptor are spatially oriented toward ligand-bearing H-2Dd targets on the
NK cell membrane, and this does not affect killing of susceptible targets recognized by the same NK cell. Alternatively, the duration of Ly-49A-mediated inhibition could
be brief, and temporally limited to periods of NK cell contact with H-2Dd targets. Temporal restriction would allow
the subsequent lysis of susceptible H-2b targets by the same
NK cell.
In an attempt to elucidate the pathways through which
Ly-49 molecules inhibit natural killing, we examined mouse
Ly-49A, which prevents NK cell lysis of targets expressing
H-2Dd or H-2Dk (6). To study Ly-49A function in a uniform clonal NK cell population, we transfected the rat NK
cell line RNK-16 with the mouse Ly-49A cDNA. Ly-49A
was functional in these RNK-16 transfectants, specifically
inhibiting lysis of the H-2Dd target P388D1.
Because activation of cytotoxicity is associated with phosphoinositide turnover and an increase in protein tyrosine
phosphorylation in NK cells, we examined the effect of
Ly-49A ligation on these early signaling events during
target-induced NK cell activation. Rapid rises in InsP3
were stimulated in wild-type RNK-16 cells in response to
P388D1 (H-2Dd) targets, but the expression of Ly-49A on
RNK-16 cells prevented this response. Ligation of Ly-49A
also inhibited target cell-induced protein tyrosine phosphorylation in response to P388D1 targets. In contrast,
YAC-1 targets stimulated a prompt increase in protein tyrosine phosphorylation in wild-type RNK-16 cells and in
RNK-mLy-49A.9 cells.
The ability of mouse Ly-49A to inhibit the generation of
InsP3 is similar to the effect previously shown in human
NK cells, where inhibition of inositol phosphate turnover
parallels the inhibition of NK cell cytotoxicity by class I
molecules (39). Although no crosshybridizing human Ly-49
homologues have yet been identified, human NK cells express a different family of KIRs (40). The structure of KIRs,
which are type I immunoglobulin-like receptors, is not related to that of Ly-49A. However, like Ly-49A, the KIRs
inhibit NK cell cytotoxicity upon ligation by specific MHC
class I antigens on target cells (41).
Initial reports of human NK cells indicated that protein
tyrosine phosphorylation was not inhibited by the expression of MHC class I on targets (29, 39). One study examined target-induced phosphorylation in NK cells stimulated
for 1, 5, and 30 min, and another study examined NK cells
stimulated for 5 min (29, 39). In these human studies, there
were no appreciable differences in levels of phosphorylation when NK cells were stimulated with susceptible or resistant targets. In our RNK-mLy-49A.9 transfectant, we
also could detect no significant differences in protein tyrosine phosphorylation at 5 min (Fig. 3), or at 15 and 30 min (data not shown) after target cell stimulation with either sensitive or resistant targets. However, we were able to
demonstrate clearly that rapid (30-60 s) protein tyrosine
phosphorylation is markedly reduced when Ly-49A mediates inhibition of cytotoxicity. The inhibitory effects of Ly49A on phosphoinositide turnover and on tyrosine phosphorylation indicate that Ly-49A interrupts signaling events early in target cell-induced activation of NK cells.
A possible mechanism for the inhibitory activity of Ly49A was suggested by the observation that several other inhibitory receptors bind to SHP-1, a cytoplasmic tyrosine
phosphatase (47). In other hematopoetic cells, SHP-1
has been implicated in the downregulation of signals for
cellular activation. Thus, SHP-1 inhibits activation through
the erythropoietin receptor, and erythropoietin receptors mutated at the SHP-1 binding site exhibit prolonged activation in response to ligand (23, 24). In B cells, activation
through the immunoglobulin receptor is inhibited by coligation with Fc Because of this inhibitory effect, the binding domains for
SHP-1 on immune receptors have been termed ITIMs.
As originally proposed, the ITIM motif was described as
S/TxxYxxL (25), but the KIR motifs are V/IxYxxL, and
phosphopeptides containing this motif have been shown to
bind to SHP-1 (21, 22, 29). Similar motifs, constrained by
the sequence VxYxxV/L are found in Ly-49A, in the inhibitory receptors Ly-49C and Ly-49G2, and in the functionally uncharacterized murine molecules Ly-49B, E, F,
G1, and G3. Interestingly, mouse Ly- 49D, which has been
implicated in NK cell activation (rather than inhibition),
lacks an ITIM motif as does the functionally uncharacterized
molecule Ly-49H (2, 5, 51). Previous studies by Olcese et al.
(22) showed that a synthetic, tyrosine-phosphorylated, 13amino acid peptide derived from the mouse Ly-49A cytoplasmic domain binds SHP-1 in vitro. In the present experiments, we have demonstrated that intact mouse Ly-49A
also binds to SHP-1 in pharmacologically stimulated RNK16 transfectants.
In mouse NK cells, we examined the functional importance of the Ly-49A/SHP-1 association. To clarify the role
of SHP-1 in NK cell inhibition through Ly-49A, we examined NK cells from viable motheaten (mev/mev) and motheaten (me/me) mice. Consistent with previous reports, the
lysis of YAC-1 targets by mev/mev or me/me NK cells was
somewhat diminished compared with lysis by wild-type,
heterozygous (+/mev or +/me) NK cells (data not shown)
(52). However, lysis of C1498 target cells by mev/mev or
me/me NK cells was essentially normal. When compared
with C1498 (H-2b), lysis of D12 (C1498.Dd) was almost
completely inhibited by Ly-49A in NK cells from wildtype or from heterozygous (+/mev or +/me) mice. By contrast, Ly-49A-mediated inhibition of C1498.Dd lysis in
both mev/mev and me/me NK cells was significantly impaired. We initially thought that incomplete impairment of
Ly-49A function in mev/mev mice might be due to the incomplete loss of SHP-1 activity in these mice. However,
Ly-49A also retained partial activity in NK cells from me/me
mice, which are completely deficient in SHP-1. This implies that other cytoplasmic mediators must also be capable of facilitating inhibition by Ly-49A. One candidate mediator is the structurally related tyrosine phosphatase SHP-2,
which, like SHP-1, has been shown to bind to a tyrosinephosphorylated peptide containing the ITIM of Ly-49A
(22). SHP-2 has recently been shown to associate with the
CTLA-4 receptor on T cells, suggesting a role for SHP-2
in the down regulation of T cell activation (53). In support
of a role for SHP-2, we have recently been able to demonstrate an association between intact Ly-49A and SHP-2 in anti-Ly-49A immunoprecipitates from pervanadate-stimulated RNK-mLy-49A cells (data not shown).
Site-directed mutational analysis of the Ly-49A molecule
confirmed the functional significance of the Ly-49A/SHP-1
interaction. Disruption of the putative ITIM motif in Ly49A by mutating the tyrosine to phenylalanine at residue 8 (Y8F), completely eliminated Ly-49A-mediated inhibition of
cytotoxicity against H-2Dd target cells by RNK-16 transfectants.
Our studies demonstrate that Ly-49A interrupts proximal signaling events during natural killing, and they provide evidence that this inhibitory function is largely mediated through the SHP-1 phosphatase. The mechanisms by
which SHP-1 inhibits cytotoxicity have not been fully elucidated. Our cold-target inhibition studies suggest that the
Ly-49A-mediated inhibitory effect is localized within the
NK cell, because targets expressing H-2Dd are protected
from lysis but fail to inhibit NK cell cytotoxicity against
non-H-2Dd targets. Thus, Ly-49A does not transduce global inhibitory signals to NK cells. Rather, it appears that
Ly-49A may locally interrupt activating signals transduced
by other receptors during natural killing.
In summary, we have demonstrated that the ligation of
Ly-49A by its MHC class I ligand, H-2Dd, interrupts early
signaling events stimulated during target-induced activation
of NK cells, including phosphoinositide turnover and protein tyrosine phosphorylation. We have also shown the direct association of the cytoplasmic tyrosine phosphatase SHP-1
with Ly-49A. Our studies indicate a requirement for SHP-1
for the optimal function of Ly-49A, as we found that Ly49A function is markedly impaired in SHP-1 mutant mev/mev
and me/me mice. The finding of partial Ly-49A function
despite the complete absence of SHP-1 in me/me mice indicates the possible involvement of other cytoplasmic mediators, such as SHP-2. In addition, we have shown that
the tyrosine within the proposed SHP-1 binding motif in
Ly-49A is strictly required for the inhibitory effects of this
receptor on cytotoxicity. Finally, we have demonstrated that inhibition through Ly-49A is target specific. Although
a number of candidate activating receptors on NK cells
have been described, including the lectin-like NKR-P1
molecule (54), the specific receptors that bind tumors and
activate NK cell cytotoxicity have not yet been identified.
Our studies indicate that the inhibitory Ly-49A receptor
interrupts target-induced activation signals through its recruitment of inhibitory mediators to the effector/target interface in a spatially or temporally restricted manner.
RIIB1 (25). In human NK cells, SHP-1 has been
implicated in inhibition of cytotoxicity through its association with the killer inhibitory receptors (KIRs)1, members of
the Ig family that bind to human MHC class I molecules (21, 29, 30). The presence of a proposed SHP-1 binding
motif in the cytoplasmic domain of Ly-49A suggests that
this murine receptor may also functionally associate with
SHP-1. A tyrosine-phosphorylated synthetic tridecapeptide
derived from the cytoplasmic domain of Ly-49A has recently been shown to bind to SHP-1 and to the related phosphatase SHP-2, but the functional relevance of these
findings to intact Ly-49A has not yet been examined (22).
In this report, we demonstrate that ligation of Ly-49A interrupts early signals for NK cell activation, inhibiting tyrosine phosphorylation and polyphosphoinositide turnover.
We also demonstrate that intact Ly-49A directly associates
with SHP-1. Moreover, we show that the full inhibitory
effect of Ly-49A in NK cells requires intact SHP-1 function as well as the tyrosine residue within the proposed SHP-1 binding site of Ly-49A.
)2 fragments were generated by pepsin digestion, and undigested mAb was absorbed over protein A-Sepharose columns as described
(32). Purity of F(ab
)2 fragments was verified by SDS-PAGE and
silver staining. For fluorescence analysis, mAbs were used at a
concentration of 1 µg/106 cells. Staining of IL-2-activated NK cells
was performed using directly conjugated mAb in the presence of
blocking antibodies to prevent binding to Fc receptors (1 µg/106
cells mouse IgG2a myeloma antibody [Cappel, Malvern, PA] in
0.1 ml 2.4G2 supernatant). Routine analysis was performed by using a FACScan®.
)2
at a concentration of 50 µg/106 effectors or with intact antibody
at a concentration of 20 µg/106 effectors before the addition of
targets.
NK cell population
was ensured by treatment with anti-Ly-49A and rabbit anti-mouse
Ig (Cappel, Malvern, PA), followed by rabbit complement (Cedarlane, Westbury, NY) for 1 h at 37°C. Ly-49
and Ly-49+ cell
populations were then cultured overnight in complete RPMI supplemented with 1,000 U/ml human IL-2 (National Cancer
Institute, Frederick, MD). Cells were washed extensively with
BSS with 3% FCS on day 7, replated and used for assays on day 9. This resulted in populations of NK cells that were >95% pure as
assessed by their expression of NK1.1.
EN (A. Shaw and M. Olszowy, Washington University, St.
Louis, MO). The Ly-49A/Y8F mutation was generated using sitedirected mutagenesis by PCR with the oligonucleotide 5
-ATATATGAATTCTCGAGATGAGT GAGCAGGAGGTCACTTTTTCAATGGTGAG-3
, which was cloned into the EcoRI site
of BSR
EN. Constructs were confirmed by sequencing in both directions before transfection. Transfections were performed using cesium-purified plasmids or plasmids purified over two sequential Qiagen maxiprep tips according to the instructions of the manufacturer (Qiagen, Chadworth, CA). RNK-16 cells in exponential growth were transfected with 20 µg of ScaI-linearized plasmid
DNA using a BTX-600 Electro Cell manipulator. Electroporation was performed using 3 × 106 cells/ml in 2-mm cuvettes in a
total volume of 400 µl of complete RPMI, at 115 V, 850 µF,
129 ohms. Cuvettes were incubated on ice for 15 min after electroporation. Cells were cultured overnight, then plated in 96-well
plates at a density of 104 cells/well in complete RPMI containing
1 mg/ml active G418 (Boehringer, Indianapolis, IN). G418-resistant cells grew out in 10-14 days in 5-10% of the wells. Transfected cells were maintained in 1 mg/ml active G418, but were
grown in complete RPMI without G418 for at least 2 d before
use in functional assays.
)2 fragments at 25 µg/106 cells for 15 min at room temperature before addition
of targets when indicated. Results are expressed as percent inhibition = (1
[percent cytotoxicity with cold target/percent cytotoxicity without cold target]) × 100.
Mouse Ly-49A Inhibits Killing of H-2d Targets by RNK-16.
)2 fragments, while control F(ab
)2 fragments (anti-NK1.1) had no effect.
Addition of intact antibody had the same effect as F(ab
)2
fragments (data not shown). YAC-1 targets have previously
been shown to be susceptible to lysis by mouse Ly-49A+
NK cells (6). YAC-1 target cells were lysed equally well by wild-type RNK-16 cells and by RNK-16 cells expressing
Ly-49A at various levels, and lysis of YAC-1 targets was not
altered by the presence of antibodies to Ly-49A (Fig. 1, I-L).
These studies demonstrate that expression of Ly-49A renders RNK-16 cells ineffective in the killing of H-2Dd targets. The data indicate that the clonal RNK-mLy-49A
transfectants behave similarly to freshly isolated murine Ly49A+ NK cells, and that they are a valid model in which to
study Ly-49A function.
Fig. 1.
Inhibition of lysis of P388D1 (H-2Dd) targets correlates with
the level of expression of mouse Ly-49A on RNK-16 cells. The various
levels of Ly-49A expression are shown in FACS® histograms (A-D). Cells
were incubated with anti-Ly-49A (solid line) with FITC-goat anti-mouse
Ab (FITC-GAM) or FITC-GAM alone (dotted line). Standard 4-h cytotoxicity assays were performed with either P388D1 cells (E-H) or YAC-1
targets (I-L). Effectors were wild-type RNK-16 cells (closed symbols) or
RNK-16 transfected with Ly-49A (open symbols). Effector cells used were
RNK-16 (A, E, and I) and clones of RNK-16 expressing Ly-49A: low
expression, RNK-mLy-49A.2 (B, F, J); intermediate expression, RNKmLy-49A.8 (C, G, K); and high expression, RNK-mLy-49A.9 (D, H, L).
Assays were carried out in the absence of antibody (squares), or in the presence of anti-Ly-49A F(ab)2 (diamonds) and control F(ab
)2 (antiNK1.1) (circles). The dotted line in F-H is the killing curve for wild-type
RNK-16 without mAb (from E for comparison).
[View Larger Version of this Image (34K GIF file)]
Fig. 2.
Ly-49A inhibits phosphoinositide turnover in response to
H-2Dd target cells. RNK-mLy-49A.9 cells fail to generate InsP3 upon stimulation with P388D1 targets. [3H]myoinositol-labeled RNK-16 and
RNK-mLy-49A.9 effectors (5 × 106 cells) were stimulated with 107 targets in a total volume of 1 ml at 37°C. Soluble InsP3 was resolved by ion
exchange chromatography. A brisk rise in InsP3 was seen in RNK-16
cells (left) in response to either YAC-1 (open squares) or P388D1 (H-2Dd)
(closed circles). Phosphoinositide turnover in RNK-mLy-49A.9 (right) was
stimulated by YAC-1, but not by P388D1.
[View Larger Version of this Image (12K GIF file)]
Fig. 3.
Ly-49A inhibits an
early rise in tyrosine phosphorylation induced by target cell
stimulation. Tyrosine phosphorylation of proteins in RNK-16
cells stimulated with P388D1
cells is shown on the left side of
the figure. RNK-mLy-49A.9
cells stimulated with P388D1
cells are in the center, and
RNK-mLy-49A.9 cells stimulated with YAC-1 cells are on
the right. Target cell stimulation
time points were 0, 0.5, 1.0, and
5.0 min. RNK-16 and RNKmLy-49A.9 effector cells were
metabolically labeled with 32Porthophosphate. After washing,
107 labeled effector cells and 107
unlabeled target cells were stimulated in a total volume of 1 ml
complete phosphate-free RPMI
with a brief 50 g contact spin, followed by incubation at 37°C
for the indicated time. Cells
were then immediately lysed in
cold HNTG buffer with 1% Triton X-100. Clarifed precleared
cell lysates were immunoprecipitated with APT (4G10),
washed, and resolved by reducing 8% SDS-PAGE. Gels were
dried and developed by autoradiography.
[View Larger Version of this Image (65K GIF file)]
Fig. 4.
The tyrosine phosphatase SHP-1 associates with
mouse Ly-49A in pervanadatestimulated RNK-16 cells. 1.5 × 107 unstimulated or pervanadatestimulated RNK-16 cells or
RNK-mLy-49A.9 cells were incubated in complete RPMI for 5 min at 37°C, washed, and lysed
in cold HNTG lysis buffer containing 1% Triton X-100. Clarified
precleared lysates were immunoprecipitated with anti-Ly-49A (A1, lanes 5-8) or isotypematched control mAb (NK1.1,
PK136, lanes 1-4), washed, and
resolved by 8% SDS-PAGE under nonreducing conditions. Proteins were transferred to PVDF
membranes, immunoblotted with anti-SHP-1 antiserum, and developed with 125I-protein A followed by autoradiography.
[View Larger Version of this Image (29K GIF file)]
IL-2-activated NK cells from SHP-1
mutant mice. These included viable motheaten (mev/mev)
mice, which have an incomplete defect in SHP-1, and the
completely SHP-1-deficient motheaten (me/me) mice. We
first examined NK cells from SHP-1 mutant viable motheaten (mev/mev) mice because they survive to 8-9 wk of
age, when NK cell development is complete. These mice
contain a point mutation in the SHP-1 gene that destroys a
donor splice site. This mutation results in aberrant splicing,
creating either an in-frame insertion or deletion in the catalytic domain of the SHP-1 phosphatase (35). Although
SHP-1 activity is reduced, some residual SHP-1 function remains and mev/mev mice have an attenuated motheaten
phenotype.
NK cells from homozygous mev/mev, heterozygous +/mev, and wild-type
C57BL/6 +/+ mice. Using these effectors, we tested Ly49A function in cytotoxicity assays against C1498 (H-2b)
cells and D12 (C1498.Dd) cells as targets. Ly-49A+ cells
from all mice expressed Ly-49A at similar levels and >95% of all cells were positive for NK1.1 and negative for CD3
by FACS® (data not shown).
effectors. However, Ly-49A remained partially effective in cells from mev/mev mice in that
addition of anti-Ly-49A still increased lysis of the C1498.Dd
targets. Fig. 5 shows a representative experiment in which
Ly-49A+ cells from wild-type C57BL/6 +/+ or from heterozygous +/mev mice were unable to lyse C1498.Dd targets (A and B), but Ly-49A+ cells from homozygous mice
were able to lyse these targets (C). Ly-49A
cells from all
mice were able to lyse C1498.Dd equally well (Fig. 5, D-F).
Addition of anti-Ly-49A mAb reversed the Ly-49A-mediated inhibition to levels similar to those of Ly-49A
cells
from all mice, whereas isotype-matched control anti-gp42 mAb had no effect. Ly-49A+ and Ly-49A
cells from all
mice were able to lyse the H-2b target C1498 and addition
of mAb anti-Ly-49A or anti-gp42 had no effect (data not
shown). These findings indicate that the function of Ly49A is partially impaired in IL-2-activated NK cells isolated from homozygous mev/mev mice.
Fig. 5.
Ly-49A function is impaired in mev/mev LAK cells. 9-d Ly-49A+
and Ly-49A LAK cells were tested in 4-h cytotoxicity assays against D12 (C1498.Dd) targets against Ly-49A+ effector cells (A-C) or Ly-49A
effector cells (D-F) from +/+ (A and D), +/mev (B and E), or mev/mev mice
(C and F). Assays were done in the absence of antibody (open squares), or
in the presence of anti-Ly-49A (A1, closed diamonds), or isotype-matched
control antibody (anti-gp42, 3G7, open circles).
[View Larger Version of this Image (24K GIF file)]
cells. As shown in Fig. 6
(B, D, F), Ly-49A expression was equivalent on cells isolated
from all mice. Ly-49A
cells (Fig. 6, C and E) contained
<5% Ly-49A+ cells, whereas the +/+ Ly-49A
population (A) contained ~10% Ly-49A+ cells. >95% of all cells
were positive for NK1.1 and negative for CD3 by FACS®
(data not shown).
Fig. 6.
Ly-49A expression on Ly-49A+ and Ly-49A LAK cells isolated from +/+, +/me, me/me mice. 6-d LAK cells were separated into
Ly-49A+ and Ly-49A
populations by panning with anti-Ly-49A Ab.
Ly-49A
cells were additionally treated with rabbit anti-mouse Ab and complement depletion. FACS® analysis was performed on day 9 LAK cells
using FITC-anti-Ly-49A (A1). Staining was performed in the presence of
unlabeled blocking antibodies (IgG2a mouse myeloma protein, 1 µg/106
cells in 0.1 ml 2.4G2 supernatant). Ly-49A expression is shown in FACS®
histograms (A-F). Dotted lines represent cells incubated with saline; solid
lines represent FITC-anti-Ly-49A staining.
[View Larger Version of this Image (26K GIF file)]
cells. Ly-49A
cells
from all mice were able to lyse C1498.Dd targets equally
well (Fig. 7, D-F). Addition of anti-Ly-49A mAb reversed
Ly-49A-mediated inhibition of Ly-49A+ cells from all mice
(Fig. 7, A-C), whereas control mAb had no effect. Ly-49A+
and Ly-49A
cells from all mice were able to lyse the H-2b
target C1498 (Fig. 7, G-L), and addition of anti-Ly-49A
or control mAb had no effect. These findings indicate that
Ly-49A is functionally impaired in NK cells isolated from
homozygous me/me mice. However, even in the complete
absence of SHP-1, Ly-49A had some remaining inhibitory
activity.
Fig. 7.
Ly-49A function is impaired in me/me LAK cells. 9-d Ly-49A+
and Ly-49A LAK cells were tested in 4-h cytotoxicity assays against D12 (C1498.Dd) and C1498 (H-2b) targets. D12 targets (A-F) and C1498 targets (G-L) were tested against Ly-49A+ effector cells (A-C and G-I) or
Ly-49A
effector cells (D-F and J-L) from +/+ (A, D, G, J), +/me (B, E, H, K), or me/me mice (C, F, I, L). Assays were done in the absence of
antibody (open squares), or in the presence of anti-Ly-49A (A1, closed diamonds), or isotype-matched control antibody (anti-gp42, 3G7, open circles).
[View Larger Version of this Image (32K GIF file)]
Fig. 8.
Lysis of P388D1 (H-2Dd) cells is not altered in RNK-mLy49A/Y8F transfectants. Ly-49A expression on RNK transfectants was assessed by staining cells with either saline (dotted line) or FITC-anti-Ly-49A (solid line). FACS® histograms show Ly-49A expression in wild-type
RNK-16 (B), RNK-mLy-49A.9 (D), RNK-mLy-49A/Y8F.1 (F) and
RNK-mLy-49A/Y8F.4 (H). Standard 4-h cytotoxicity assays were performed using P388D1 (H-2Dd) as targets. Effector cells were wild-type
RNK-16 (A), RNK-mLy-49A.9 (C), RNK-mLy-49A/Y8F.1 (E), or
RNK-mLy-49A/Y8F.4 (G). Effectors were preincubated with either media alone (open squares), anti-Ly-49A (closed circles), or isotype-matched
control antibody (anti-NK1.1, PK136, closed triangles), before addition of
targets.
[View Larger Version of this Image (26K GIF file)]
)2
anti-Ly-49A, P388D1 became sensitive to lysis by RNKmLy-49A.9. This allowed cold P388D1 cells to compete
effectively with labeled B-16S for the lytic machinery of
RNK-mLy-49A.9. Control F(ab
)2 anti-NK1.1 fragments
had no effect on cold-target competition.
Fig. 9.
Cold-target competition of unlabeled P388D1 and B-16S in
the lysis of labeled B-16S targets. 4-h cytotoxicity assays were performed
with RNK-16 (left) or RNK-mLy-49A.9 cells (right) as effectors. B-16S
(H-2b) target cells were labeled with 51Cr and tested at an effector to labeled target ratio of 10:1. Cold targets were either unlabeled B-16S cells
(H-2b) or P388D1 cells (H-2Dd). Cold targets were added in 10-fold excess to labeled targets. 105 cold targets and 104 labeled targets were added
at the same time to 105 effectors in a total volume of 0.2 ml. Effectors
were preincubated with no antibody, F(ab)2 anti-Ly-49A or F(ab
)2 antiNK1.1. Results are expressed as percent inhibition = (1
[percent cytotoxicity with cold target/percent cytotoxicity without cold target]) × 100.
[View Larger Version of this Image (43K GIF file)]
RIIB1 and this effect correlates with SHP-1
binding to Fc
RIIB1 (25). SHP-1 binding has also been
correlated with inhibition of human NK cell function (21,
29, 30). The KIRs associate with SHP-1 in human NK
cells (21, 29, 30), and expression of a dominant negative
mutant SHP-1 molecule in a human NK clone inhibits KIR function (21). Thus, in NK cells and in other immune cells, SHP-1 has been functionally implicated in the inhibition of
cellular activation signals.
Address correspondence to Dr. Mary Nakamura, Immunology/Arthritis Section 111-R, Veterans Administration Medical Center, 4150 Clement Street, San Francisco, California 94121.
Received for publication 17 October 1996
1Abbreviations used in this paper: APT, phosphotyrosine; InsP3, inositol triphosphates; ITIM, immunoreceptor tyrosine-based inhibitory motif; KIRs, killer inhibitory receptors; LAK, IL-2-activated NK cells.We thank F.M. Karlhofer and W. Yokoyama for providing important reagents that enabled these studies, M. Olszowy and A. Shaw for the BSR vector and invaluable help with transfection of RNK-16, S. Christianson for preparing me/me splenocytes, D.A. Lacy for assistance with the viable motheaten studies, E. Gum for
sequencing, and T.P. Quinn for helpful discussions.
This work was supported by the Veterans Administration and National Institutes of Health grant RO1 CA69299 (W.E. Seaman). J.C. Ryan is the recipient of National Institutes of Health grant R29 CA60944 and is supported by the International Human Frontiers in Science program. M.C. Nakamura is supported by National Institutes of Health grant K11 AR01927, the Rosalind Russell Arthritis Foundation, and Multipurpose Arthritis Center grant P60AR20684. L.D. Shultz is the recipient of National Institutes of Health grant RO1 CA20408.
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