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Departments of
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Immunology and
Anatomy and Cell Biology, University of Toronto, Toronto, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain (5), which in turn signals through
the protein tyrosine kinase Syk (11). Although the NKR-P1 molecules may
recognize carbohydrate ligands on target cells (12, 13), cognate
protein ligands for these receptors remain unknown. Killer cell inhibitory receptors expressed on NK cells prevent activation and lysis of class I MHC-bearing cells (14), providing a mechanism for the "missing self" model of NK cell function (15, 16, 17). Members of the mouse Ly-49 family of receptors, which are structurally related to the NKR-P1 molecules, are included in this category (18). The ability of these receptors to inhibit NK cell function seems to be dictated by the presence in their cytoplasmic domain of a conserved immunoreceptor tyrosine-based inhibitory motif (ITIM) (single-letter amino acid code I/V/LxYxxL/V (19, 20)) (3, 21, 22). However, not all Ly-49 members are inhibitory in function, as Ly-49D and Ly-49H, which lack consensus ITIMs, have been shown to activate NK cell function (22, 23). In keeping with this, the mouse NKR-P1A and NKR-P1C molecules lack consensus ITIMs (3, 10). Alternatively, the closely related mouse NKR-P1B molecule possesses a consensus ITIM (LxYxxL) in its cytoplasmic domain (3, 10). However, due to a lack of available specific Abs, this molecule has remained relatively uncharacterized to date.
Here we show that the mouse NKR-P1B gene product serves as a ligand for the anti-NK1.1 mAb PK136, and thus represents a novel NK1.1 Ag (8, 10). Surprisingly, NKR-P1B fails to activate NK cell cytotoxicity. Moreover, in NK cells expressing both the NKR-P1B and NKR-P1C molecules, NK1.1-mediated redirected lysis of target cells is abrogated. Taken together, these data indicate that NKR-P1B functions as a killer cell inhibitory receptor (3, 14). Indeed, like other ITIM-bearing receptors expressed by NK cells, NKR-P1B binds Src homology 2 (SH2)-containing protein tyrosine phosphatase-1 (SHP-1) in a phosphorylation-dependent manner (14, 24, 25). This provides a molecular mechanism for the inhibition of NK cell cytotoxicity through this novel NK1.1 Ag and demonstrates that NKR-P1B is an inhibitory member of the CD161 family. The existence of two closely related NK1.1 Ags with opposite regulatory function suggests a role for NKR-P1 molecules, such as those of the Ly-49 family, in regulating NK cell-mediated functions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6) and Swiss.NIH (Sw) mice were obtained from the National Cancer Institute, Frederick Cancer Research and Development Center (Frederick, MD). (B6 x Sw)F1 mice were bred and maintained in our own animal facility.
Cell lines
P815 target cells were obtained from Dr. Brian Barber
(Department of Immunology, University of Toronto, Toronto, Ontario,
Canada); Jurkat cells were obtained from Dr. Neil Berinstein
(Department of Immunology, University of Toronto, Toronto, Ontario,
Canada). An Sw-derived pre-NK cell line, MNK-1, was derived in our own
laboratory. Briefly, day 15 fetal thymocytes were sorted for
NK1.1+ cells, infected overnight with a Bcl-2 retroviral
producer cell line, and transfected with replication-deficient SV40 and
human c-myc constructs by electroporation. Cells were then
maintained in complete medium (DMEM supplemented with 12% FCS, 2 mM
glutamine, 10 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml
gentamicin, 110 µg/ml sodium pyruvate, 50 µM 2-ME, and 10 mM HEPES
(pH 7.4)) plus human rIL-2 (10 U/ml) for 6 wk before adherent colonies
formed. MNK-1 cells have the following phenotype: NK1.1+.
In addition, they are FcRII/III+, CD90+,
CD117+, CD122+, Lin-,
TCR/CD3-, Ig-, and DX5- by flow
cytometric analysis; do not express Ly-49A, Ly-49C, and perforin by
RT-PCR analysis; and fail to display cytotoxic activity. These
characteristics indicate that the resulting MNK-1 cell line possesses a
pre-NK cell phenotype (26).
Flow cytometric analysis
Fetal blood cells were prepared as described previously (27). FITC-, PE-, and biotin-conjugated mAbs as well as streptavidin APCs, were obtained from PharMingen (San Diego, CA). Cell suspensions were stained in 50 µl of staining buffer (HBSS, without phenol red, plus 1% BSA and 0.05% NaN3) for 20 min on ice and washed twice before analysis. Intracellular staining was performed as directed by the supplier using the Cytofix/Cytoperm staining kit (PharMingen). Stained cells were analyzed with a FACScalibur flow cytometer using CellQuest software (Becton Dickinson, Mountain View, CA); data were live-gated by forward/side light scatter and a lack of propidium iodide uptake (except propidium iodide was omitted for intracellular stains). All plots display 10,000 events; frequencies in each quadrant are given as the percentage of the total in the upper right corner.
DNA transfections
Jurkat cells were electroporated using a BTX (San Diego, CA)
ECM600, under the following conditions: 300 V, 186 
, and 1600 µF
in 4-mm gap cuvettes. Cells were resuspended in 250 µl of RPMI 1640
with 20% FCS before electroporation and subsequently cotransfected
with 20 µg of the NKR-P1 plasmid DNA, as indicated, and with 6 µg
of the CMV-green fluorescent protein (GFP) plasmid DNA (CMV
enhancer/promoter driving the expression of enhanced GFP from the
pEGFP-1 plasmid (Clontech, Palo Alto, CA)). Transfected cells were then
cultured for an additional 6 h before analysis by flow cytometry.
RT-PCR
RNA was prepared from B6 and Sw spleen cell suspensions depleted
of CD24+ cells by Ab/complement-mediated lysis, as
described previously (28), and from the CTLL-2 (B6) and MNK-1 (Sw) cell
lines. Total RNA was isolated using the Trizol RNA isolation protocol
(Life Technologies, Gaithersburg, MD). cDNA was prepared from 1 µg of
each RNA using random hexamer primers and the cDNA Cycle kit
(Invitrogen, San Diego, CA). Subsequent PCR analysis was performed
using Expand DNA polymerase (Boehringer Mannheim, Indianapolis, IN) on
an automated GeneAmp 9600 thermocycler (Perkin-Elmer, Norwalk, CT) with
10 s of denaturation at 94°C, 30 s of annealing at 55°C,
and 2-min extensions at 72°C for 35 cycles, with a hot start at
94°C for 2 min and a final extension at 72°C for 6 min. The
gene-specific primers used for PCR were as follows: (5'
3'): NKR-P1A
(5'), GCA CAA TGG ACA CAG CAA; NKR-P1A (3'), GTA GAC ATG GCT CAG TGA
TTG; NKR-P1B (5'), CAA TGG ATT CAA CAA CAC TGG TC; NKR-P1B (3'), GGA
CAG GGG AGA GAT GGA GAT; NKR-P1C (5'), TGA AAT GGA CAC AGC AAG TAT C;
and NKR-P1C (3'), GAG TCA ACG AAT GGA AAG GAA. Inverse photo images of
ethidium bromide-stained gels are shown. PCR products were cloned using
a mammalian expression vector, a eukaryotic TOPO TA cloning kit
(Invitrogen), and were sequenced before analysis.
51Cr release Ab-induced redirected lysis (AIRL) assay
Spleen cell suspensions from B6, Sw, and (B6 x
Sw)F1 mice were depleted of CD3+,
CD4+, CD8+, and CD24+ cells by
Ab/complement-mediated lysis and subsequently sorted for
DX5+ cells using Midi-MACS (Miltenyi Biotech, Auburn, CA).
Sorted cells were grown for 1 wk in complete medium containing 100 U/ml
of human rIL-2. Effector cells were prelabeled with 10 µg/ml Ab
(NK1.1, PK136; CD16/32, 2.4G2) for 15 min in complete medium and washed
once before use. Target P815 cells were labeled with 51Cr
for 1 h and used at 2–3 x 103 cells in 100 µl
per well (V-bottom, 96-well plates). Cells were mixed at different E:T
ratios, and plates were centrifuged for 1 min at 200 x
g and placed in culture for 4 h at 37°C. After lysis,
100 µl of each culture supernatant was collected, and radioactivity
was measured in a gamma counter. Supernatant from target cells cultured
alone or target cells alone plus 1% SDS gave the spontaneous or
maximal release counts, respectively. The counts obtained from the
culture supernatant of different E:T ratios (experimental) were used to
determine the percent specific lysis (%SL), as described previously
(29). Percent specific lysis values for anti-NK1.1-mediated AIRL
were indexed relative to those for anti-FcRIII/II AIRL
(anti-CD16/32) and spontaneous lysis (no Ab) controls for each
effector subset and ratio tested, according to the following formula:
% NK1.1 cytotoxicity = ([%SL (NK1.1) - %SL (no mAb)]/[%SL
(CD16/32) - %SL (no mAb)]) x 100%. The data shown represent the
mean (± SEM) of five independent experiments performed at a 30:1 E:T
ratio; similar results were obtained with 10:1 and 3:1 E:T ratios.
Immunoprecipitation and Western blotting
A total of 50–100 x 106 MNK-1 cells were left
unstimulated or stimulated with pervanadate for 20 min, as described
previously (30), washed once in serum-free medium, and lysed for 15 min
in 0.5 ml of ice-cold lysis buffer (0.5% TNTE (0.5% Triton X-100, 150
mM NaCl, and 20 mM Tris-Cl (pH 7.4)) plus 50 mM NaF, 10 mM sodium
pyrophosphate, 1 mM sodium orthovanadate, and protease inhibitors
(pepstatin, leupeptin, aprotinin, and trypsin inhibitor)). Lysates were
centrifuged at 14,000 x g for 10 min to remove cell
debris; next, 5 µg of anti-NK1.1 (PK136) or isotype control mouse
IgG2a
(anti-trinitrophenyl, G155–178) Ab was added to
precleared lysates and incubated with gentle agitation for 1 h. A
total of 50 µl of protein G-Sepharose beads (1:5 (w/v) in 0.1%
Triton X-100 lysis buffer) were added to lysates and incubated for an
additional 1 h with gentle agitation. Beads were washed six times
with 1 ml of wash buffer (0.1% TNTE plus 1 mM sodium orthovanadate).
Beads and lysate supernatant were boiled for 2 min in protein-loading
buffer containing 2-ME, and proteins were resolved on 10% SDS-PAGE
gels and electroblotted onto membranes. Membranes were blocked with 3%
BSA in TBST (50 mM Tris-Cl (pH 8.0), 500 mM NaCl, and 0.1% Tween 20)
for 1 h, and analyzed by Western blotting using polyclonal rabbit
anti-SHP-1 Ab (Upstate Biotechnology, Lake Placid, NY),
affinity-purified rabbit anti-SHP-2 Ab (Santa Cruz Biotechnology,
Santa Cruz, CA), or rabbit anti-SH2-containing inositol phosphatase
(SHIP) Ab (a kind gift of Dr. D. J. Dumont (Department of Medical
Biophysics, University of Toronto, Toronto, Ontario, Canada) at 1:2000
dilutions in 1% BSA-TBST. All blots were visualized using 1:10,000
anti-rabbit HRP in 1% BSA-TBST and an enhanced chemiluminescence
Plus kit (Amersham, Arlington Heights, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Molecular cloning of the mouse NKR-P1C gene served to identify it
as the NK1.1 Ag (8). However, during the course of studying two
NK1.1-expressing strains of mice, we obtained evidence suggesting that
NKR-P1C may not be the only NK1.1 Ag. Specifically, we observed that
NK1.1 is differentially expressed on a subset of fetal blood
CD117+ (c-kit) progenitors for T/NK cells
derived from two NK1.1-expressing mouse strains, Sw and B6 (26, 27).
Fig. 1
shows Sw and B6 day 15 fetal blood
cells depleted for CD24high cells by Ab/complement-mediated
lysis, which enriches for CD117+ cells (27). As
demonstrated previously (27, 31), a subset of CD117low
fetal blood cells derived from both Sw and B6 mice expresses CD90
(Thy-1) (Fig. 1). However, although the majority of
CD90+/CD117low fetal blood cells from Sw mice
display the NK1.1 marker (27), the identical subset from B6 mice lacks
NK1.1-expression (Fig. 1). As both subsets of cells express high levels
of CD16/32 and were found to be functionally identical (26), this
suggested either that the developmental regulation of NKR-P1C is
strain-specific, even among NK1.1-expressing strains, or that the
anti-NK1.1 mAb PK136 recognizes a different ligand in each of the
strains. To address this issue, we analyzed the expression of NKR-P1
family members by RT-PCR.
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To investigate the expression of NKR-P1 genes in different strains
of mice, we employed RT-PCR on RNA isolated from spleen cells of adult
mice. Fig. 2 shows RT-PCR for the
expression of full-length NKR-P1A/B/C transcripts in spleen cells from
Sw, B6, and (B6 x Sw)F1 mice. Surprisingly, although
all three strains possessed similar populations of NK1.1+
splenocytes (data not shown), the Sw strain lacked transcripts for
NKR-P1C (Fig. 2). This finding suggested that the NK1.1 Ag expressed by
Sw NK cells and fetal blood cells might represent the product of a
closely related gene in the NKR-P1 family. As the NKR-P1A gene product
was shown previously not to encode an NK1.1 Ag (8), and because Sw mice
express abundant mRNA for the NKR-P1B gene (Fig. 2) (28), these data
suggested that the NKR-P1B molecule might represent a novel NK1.1 Ag in
Sw mice. Notably, NKR-P1B shares 
96% amino acid identity to NKR-P1C
in its extracellular C-type lectin domain (10).
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To test whether the NKR-P1B molecule might represent a second
NK1.1 Ag, we cloned the NKR-P1B cDNA from Sw-derived NK cells into a
mammalian expression vector for use in transfection experiments. As
controls, we also cloned the NKR-P1A and NKR-P1C cDNAs from B6-derived
NK cells (8). These constructs were transiently transfected into Jurkat
cells. To identify transfected cells, we cotransfected a plasmid
construct encoding an enhanced mammalian version of jellyfish GFP. Fig. 3a shows a representative GFP
fluorescence profile of transfected Jurkat cells (Fig. 3a,
solid dark line). Fig. 3b shows sequential extracellular vs
intracellular staining using the anti-NK1.1 mAb, PK136, after
gating on GFP+ Jurkat transfectants. We performed this dual
analysis because we were not certain whether mouse NKR-P1 molecules
would be efficiently expressed on the cell surface of transfected human
Jurkat cells. As shown previously (8), the B6-derived NKR-P1C construct
encodes an NK1.1 Ag, whereas the NKR-P1A molecule does not (Fig. 3b, NKR-P1C vs NKR-P1A). Furthermore, as predicted by our
previous data (Figs. 1
and 2) (26, 27), the Sw-derived NKR-P1B molecule
also binds the anti-NK1.1 Ab (Fig. 3b, NKR-P1B). Thus,
the mouse NKR-P1B molecule represents a second NK1.1 Ag.
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; data not
shown). Therefore, to assess the function of each NKR-P1 molecule
independently, we tested the capacity of B6, Sw, and F1 NK
cells to mediate redirected lysis using the PK136 mAb. NKR-P1B functions as an inhibitory receptor
To purify NK cells for use in functional assays, splenocytes from
each mouse strain were depleted of CD3+, CD4+,
CD8+, and CD24+ cells and subsequently sorted
for DX5+ cells using magnetic beads. These cells were then
expanded in the presence of IL-2 for 1 week in culture. As shown in
Fig. 4a, the resulting NK
cells from each of these strains expressed the NK1.1 Ag on the cell
surface. Therefore, to analyze the function of the Sw NK1.1 Ag,
Sw-derived NK cells were tested in AIRL assays using the anti-NK1.1
mAb, PK136. In AIRL assays, an activating molecule on the surface of an
NK cell augments cytotoxicity toward FcR+ target cells,
such as mouse P815 mastocytoma cells, whereas inhibitory and
nonstimulatory receptors do not significantly affect target cell lysis.
As a positive control for lysis, we performed the AIRL assay using the
anti-CD16/32 (FcRIII/II) mAb 2.4G2, which activates NK cells
(11). In addition, spontaneous cytotoxicity in the absence of
redirecting Ab was used to determine background levels of natural
killing. To simplify these data, the percent specific lysis values for
the NK1.1-mediated AIRL were indexed relative to the CD16/32-mediated
AIRL and spontaneous lysis values (a product termed the percentage of
anti-NK1.1 cytotoxicity; see Materials and Methods). As
shown in Fig. 4b, NK1.1-mediated cytotoxicity was high using
B6-derived NK cells, confirming an activating role for the B6 NKR-P1C
molecule. In contrast, NK1.1-mediated cytotoxicity was insignificant
using Sw-derived NK cells (Fig. 4b). As this suggested that
NKR-P1B might function as an inhibitory receptor, we subsequently
assessed NK1.1-mediated AIRL using (B6 x Sw)F1 NK
cells, which express both NK1.1 Ags, NKR-P1B and NKR-P1C (Figs. 2 and 4a). Fig. 4b demonstrates that the presence of
the NKR-P1B molecule on (B6 x Sw)F1 NK cells exerted
a dominant inhibitory effect over the ability of NKR-P1C to augment
AIRL cytotoxicity.
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c, the addition of both
anti-NK1.1 and anti-CD16/32 significantly reduced cytotoxicity
relative to AIRL in the presence of anti-CD16/32 alone
(p < 0.05). Notably, this effect was not seen
in assays using B6 or (B6 x Sw)F1 NK cells (data not
shown). However, NKR-P1B-mediated inhibition of CD16/32 AIRL (Fig. 4c) was not as complete as that of NKR-P1C AIRL (Fig. 4b). There may be a number of reasons for this apparent
difference. First, the anti-CD16/32 mAb is capable of binding to
the CD16/32+ P815 target cells both indirectly (by means of
AIRL) and directly (by means of Ab-dependent cellular cytotoxicity).
This may lead to a stronger association and activation of NK cells
through CD16/32 relative to other activating receptors, and might
explain why cytotoxicity values were always higher for CD16/32 than
NK1.1 (Fig. 4b). In addition, NK1.1 expression levels on a
population basis were lower and more variable on Sw NK cells than on B6
or (B6 x Sw)F1 NK cells (Fig. 4a), whereas
expression levels of CD16/32 were consistently high in all strains
tested (data not shown). In such a case, a higher number of activating
receptors in the presence of fewer inhibitory signals might lead to an
activation bias, either in individual NK cells or generally among a
population. Indeed, in contrast to the inhibition observed for Sw NK
cells in the presence of both anti-NK1.1 and anti-CD16/32, the
additional presence of the activating NKR-P1C molecule on (B6 x
Sw)F1 NK cells (Fig. 2) may explain why these cells were
not inhibited (data not shown). Notably, strong or numerous activating
signals have been shown to be capable of overriding the negative
signals induced by inhibitory NK cell receptors (32, 33). Taken
together, these data indicate that the NKR-P1B molecule functions as a
killer cell inhibitory receptor. NKR-P1B binds SHP-1 in a phosphorylation-dependent manner
A number of different inhibitory receptors in both humans (killer
cell Ig-like receptor (KIR)-2DL/3DL; CD94/NKG2A) and mice (Ly-49A/G2)
have been shown to exert their inhibitory effects on NK cell function
by interrupting the early tyrosine phosphorylation pathways responsible
for NK cell activation (33). This was first shown at the molecular
level by the demonstration that KIR cross-linking recruits SHP-1 to the
intracellular ITIM in a phosphorylation-dependent manner (19, 24, 34, 35, 36). Another structurally related tyrosine phosphatase, SHP-2, was
found to function in similar capacity to SHP-1 (36), although SHP-2 may
also be involved in transducing activating signals (37). Alternatively,
SHIP, which is involved in mediating inhibition through the FcRIIB
pathway (38), does not associate with KIRs (25, 39). Notably, the mouse
NKR-P1B molecule possesses a consensus ITIM (LxYxxL) in its
intracellular domain (3, 10). Therefore, to determine the molecular
mechanism for inhibition of cytotoxicity through the NKR-P1B molecule
(Fig. 4b), we tested whether SHP-1 is recruited to the ITIM
of NKR-P1B upon phosphorylation of the receptor. To achieve this, we
used an NK1.1+ Sw-derived pre-NK cell line, MNK-1 (see
Materials and Methods), which expresses abundant NKR-P1B
mRNA but no NKR-P1C (data not shown). Phosphorylation of the ITIM-based
tyrosine was accomplished in intact cells by the disruption of
endogenous tyrosine phosphatase activity using pervanadate stimulation
(40), as described previously for the human CD94/NKG2A (41) and murine
Ly-49A and Ly-49G2 molecules (21, 30).
As shown in Fig. 5a, Western
blot analysis for SHP-1 on anti-NK1.1 immunoprecipitates indicates
that the NKR-P1B molecule associates with SHP-1 upon pervanadate
stimulation (Fig. 5a, VO5+/IP
NK1.1+). This
association is phosphorylation-dependent (21, 30), as SHP-1 does not
coimmunoprecipitate with NKR-P1B in unstimulated cells (Fig. 5a, VO5-/IPNK1.1+). The total cell lysate
supernatant (shown on the right of Fig. 5a, S) indicates
that 0.4% of total intracellular SHP-1 coimmunoprecipitates with
the NKR-P1B molecule in response to pervanadate stimulation. The
additional bands observed in the total lysate supernatant are due to
anti-SHP-1 cross-reactivity (data not shown) (24) and are absent
from anti-NK1.1 immunoprecipitations. Importantly, the association
of NKR-P1B with SHP-1 is specific, as Western blots for SHP-2 (Fig. 5b, anti-SHP-2 blot) and SHIP (Fig. 5c,
anti-SHIP blot) revealed that these phosphatases do not
significantly coimmunoprecipitate with NKR-P1B upon pervanadate
stimulation. Moreover, coimmunoprecipitating SHP-1 was also detected in
pervanadate-stimulated Sw and (B6 x Sw)F1 NK cells,
although SHP-1 could not be detected in anti-NK1.1
immunoprecipitates from B6 NK cells or the NK1.1+ (NKR-P1C)
B6-derived cell line CTLL-2 (data not shown) (3). These data support
initial studies demonstrating that the cross-linking of NKR-P1C on
B6-derived cells leads to transient increases in the
intracellular calcium concentration and tyrosine phosphorylation of
downstream substrates (3), whereas similar responses were not observed
upon NKR-P1B cross-linking using Sw-derived cells (data not shown).
Taken together, these data indicate that the NKR-P1B molecule binds to
SHP-1 in a phosphorylation-dependent manner, confirming a functional
role for the NKR-P1B consensus intracellular ITIM.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), whereas the activating
mouse NKR-P1A and NKR-P1C molecules do not, can be explained in the
latter cases by their lack of consensus ITIMs (I/V/LxYxxL/V; Fig. 6a). Both NKR-P1A and NKR-P1C
possess hydrophilic amino acid residues (R and S, respectively) at the
-2 position relative to their YxxL motif, a position that has been
shown to be critical for SHP-1 binding (19). Like the inhibitory Ly-49
family members, NKR-P1B possesses a hydrophobic amino acid at this
position (L for NKR-P1B, V/I for Ly-49 molecules). However,
unlike the activating Ly-49 family of molecules, which possess a
phenylalanine residue in substitution of the ITIM-based tyrosine,
NKR-P1A and NKR-P1C retain the YxxL motif. This may have important
implications for the respective signaling capacities of the Ly-49 vs
the NKR-P1 families of molecules.
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a). The existence of similar residues in the
activating human CD94/NKG2C and murine Ly-49D and Ly-49H molecules has
been shown to be necessary for the association of these molecules with
the immunoreceptor tyrosine-based activation motif (ITAM)-containing
DAP-12 immunoreceptor (22, 42, 43). Furthermore, similar residues are
important for the association of the CD3 subunits with the TCR and the
CD79 subunits with the B cell receptor (42). The finding that NKR-P1C,
like CD16 (FcRIII), functionally associates with the FcR chain
(Fc
RI) (5), suggests that this residue may be important for
NKR-P1-mediated activation. Similar to DAP-12 and the CD3 subunits,
FcR possesses a negatively charged aspartic acid residue in its
transmembrane domain (42). Thus, the absence of a positively charged
amino acid in the NKR-P1B transmembrane domain supports its inability
to activate NK cell activity, as association with FcRI would be
undesirable for mediating inhibitory signals.
Taken together, the findings reported here and elsewhere (3, 5, 11)
suggest a model for the mechanism of NKR-P1 signaling (Fig. 6b). Association of the activating NKR-P1 family members
(NKR-P1A/C) with the ITAM-containing FcR chain induces tyrosine
phosphorylation of downstream substrates, including the SH2-containing
tyrosine kinase Syk, resulting in the activation of NK cell function.
Other motifs present in the intracellular domains of these receptors
may be responsible for mediating increases in intracellular calcium
concentration and phosphatidylinositol turnover. Alternatively,
phosphorylation of the cytoplasmic ITIM of inhibitory NKR-P1 molecules
(NKR-P1B) recruits SHP-1, resulting in the disruption of proximal
tyrosine phosphorylation pathways and in an inhibition of NK cell
function. The signaling molecules responsible for inducing
phosphorylation of the ITAM and ITIM motifs remain unknown, although
the presence of a conserved CxCP motif in the NKR-P1 molecules (Fig. 6a) suggests that Src family tyrosine kinases may play a
role in this event (3, 10, 44). Additional experiments are required to
elucidate the details of the molecular cascades involved in NKR-P1
signaling.
Over 20 years after its original identification (9), the mouse NK1.1 Ag
is still incompletely characterized. Our data demonstrate that the
anti-NK1.1 mAb recognizes the products of two distinct mouse genes,
NKR-P1B and NKR-P1C (Fig. 3). Moreover, these two NKR-P1 molecules,
which share a cluster of differentiation designation, CD161 (5),
possess opposite roles in the regulation of NK cell function (Fig. 4b). Until now, members of the NKR-P1 family of molecules
were thought to be responsible for transducing activating signals (2, 5). Here, we show that this is not necessarily the case, and that one
member, NKR-P1B (3, 10), acts as a inhibitory receptor.
This finding highlights the increasingly similar features that these molecules share with the structurally related mouse Ly-49 family of type II transmembrane C-type lectin receptors (1, 2, 18). However, unlike the well-characterized MHC class I ligands that regulate the specificity of the Ly-49 family of molecules (18), cognate protein ligands for the NKR-P1 molecules have yet to be identified (3, 12). If the NKR-P1 molecules do not recognize specific self molecules, such as the MHC Ags, the existence of two NKR-P1 receptors with possibly identical ligands and yet opposite regulatory functions presents a potential paradox for NK cell biology. More likely, these data suggest that the cognate ligands for the NKR-P1 molecules are similar in form and function to the MHC class I alleles specifically recognized by members of the Ly-49 family (3, 18) or to the related nonclassical MHC gene products such as human HLA-E (mouse H-2Qa) recognized by the heterodimeric CD94/NKG2 receptors (41, 43, 45, 46, 47).
In this regard, there is some emerging evidence that NK1.1 (NKR-P1C)
may be involved in allogeneic target recognition in the NK
cell-mediated F1-antiparent "hybrid resistance"
phenomenon (3, 48). The identification of a negative regulatory member
of the NKR-P1 family, NKR-P1B, now suggests a role for these
molecules in the missing self hypothesis (15, 16), in which the
perception of self vs nonself appears to rely on the presence or
absence on the target cell surface of MHC or MHC-like gene products
(17). In a manner analogous to the recent discovery of MHC class
I-related CD1 ligands recognized by the ßTCR of mouse
NK1.1+ natural T cells (4), it seems that the variety of
receptors and ligands involved in self/nonself recognition is still
expanding. Interestingly, selective expression in the Sw (this report)
and SJL/J (Richard G. Miller, unpublished observations) mouse strains
of NKR-P1B but not NKR-P1C may explain the deficiency in NK1.1
expression on the surface of the ßTCR+ natural T cells
derived from these mice; this defect has been reported to genetically
segregate within the NK gene complex independently of defects in IL-4
production and IgE secretion (49). These findings raise some important
questions as to the role of CD161 molecules in both mice and humans and
suggest a previously unrecognized level of specificity in the
regulation of the immune response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Juan Carlos Zúñiga-Pflücker, Department of Immunology, University of Toronto, 1 King’s College Circle, Toronto, Ontario, M5S 1A8, Canada. E-mail address:
3 Abbreviations used in this paper: NKR, NK cell receptor; ITIM, immunoreceptor tyrosine-based inhibitory motif; ITAM, immunoreceptor tyrosine-based activation motif; B6, C57BL/6; Sw, Swiss.NIH; AIRL, Ab-induced redirected lysis; GFP, green fluorescent protein; SH2, Src homology 2; KIR, killer cell Ig-like receptor; SHP, SH2-containing protein tyrosine phosphatase; SHIP, SH2-containing inositol phosphatase; NKR, ???.
Received for publication November 9, 1998. Accepted for publication March 10, 1999.
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is essential for activation signal through NKR-P1 (CD161) in natural killer (NK) cells and NK1.1+ T cells. J. Exp. Med. 186:1957.RIIb1 require distinct phosphatases. J. Exp. Med. 186:473.ß T lymphocyte lineage commitment. Immunity 9:187.[Medline]
RIIB1-BCR coligation. J. Immunol. 161:684.RIIB. Nature 383:263.[Medline]
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