Ly-49s3 Is a Promiscuous Activating Rat NK
Cell Receptor for Nonclassical MHC Class
I-Encoded Target Ligands
This information is current as
of June 15, 2017.
Christian Naper, Shigenari Hayashi, Lise Kveberg, Eréne C.
Niemi, Lewis L. Lanier, John T. Vaage and James C. Ryan
J Immunol 2002; 169:22-30; ;
doi: 10.4049/jimmunol.169.1.22
http://www.jimmunol.org/content/169/1/22
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Copyright © 2002 by The American Association of
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References
The Journal of Immunology
Ly-49s3 Is a Promiscuous Activating Rat NK Cell Receptor for
Nonclassical MHC Class I-Encoded Target Ligands1
Christian Naper,*† Shigenari Hayashi,* Lise Kveberg,† Eréne C. Niemi,* Lewis L. Lanier,
John T. Vaage,2,3†§ and James C. Ryan2*
‡
N
atural killer cells play an important role in immune defense against intracellular infections (1). Infected cells
are eliminated by NK cells without prior sensitization.
NK cells also rapidly reject allogeneic bone marrow grafts and
lymphoblasts, presumably using complex arrays of activating and
inhibitory receptors that recognize MHC class I (MHC-I)4 molecules on target cells (2–5). In rodent NK cells, inhibitory Ly-49
receptors were the first MHC-I-binding receptors to be characterized (6). In the presence of self MHC-I ligands, target cells are
protected from NK cytolysis, consistent with the missing self hypothesis proposed by Kärre and colleagues (7), i.e., that targets
cells are killed if they fail to express a full array of self MHC-I
ligands. As was predicted by early studies of the rapid rejection of
MHC-disparate lymphocytes in rats termed allogeneic lymphocyte
cytotoxicity (ALC) (8 –10), NK cells also express stimulatory receptors for target MHC-I ligands (2, 11–14). These activating
MHC receptors stimulate NK cell effector functions through their
*Veterans Affairs Medical Center, Northern California Institute for Research and
Education, and University of California, San Francisco, CA 94121; †Department of
Anatomy, University of Oslo, Oslo, Norway; ‡Department of Microbiology and
Immunology and Cancer Research Institute, University of California, San Francisco,
CA 94143; and §Institute of Immunology, Rikshospitalet, University Hospital, Oslo,
Norway
Received for publication February 5, 2002. Accepted for publication April 22, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
J.C.R., S.H., and E.C.N. were supported by National Institutes of Health RO1 AI
44126 and by the U.S. Veterans Administration. L.L.L. was supported by National
Institutes of Health RO1 CA 89294. J.T.V., C.N., and L.K. were supported by the
Norwegian Cancer Society and the Research Council of Norway. C.N. is a research
fellow of the Norwegian Cancer Society.
2
J.T.V. and J.C.R. contributed equally to this work.
3
Address correspondence and reprint requests to Dr. John T. Vaage, Institute of
Immunology, Rikshospitalet, N-0027 Oslo, Norway. E-mail address: j.t.vaage@
basalmed.uio.no
4
Abbreviations used in this paper: MHC-I, MHC class I; ALC, allogeneic lymphocyte cytotoxicity; CHO, Chinese hamster ovary; ITIM, immunoreceptor tyrosinebased inhibitory motif; SIRP, signal regulatory protein.
Copyright © 2002 by The American Association of Immunologists, Inc.
association with a membrane adapter protein whose cytoplasmic
domain contains immunoreceptor tyrosine-based activation motifs.
Following receptor ligation, the immunoreceptor tyrosine-based
activation motifs in these adapter molecules are tyrosine phosphorylated and recruit stimulatory tyrosine kinases such as Syk and
ZAP-70, through which NK cells are triggered. One such adapter
molecule is DAP12, which couples to structurally diverse NK
receptors including activating members of the lectin (Ly-49,
CD94/NKG2) superfamily (15, 16) and of the Ig (killer cell Ig-like
receptor) superfamily (reviewed in Ref. 11). DAP12-associated
members of the Ly-49, CD94/NKG2, and killer cell Ig-like receptor families have been implicated in the specific recognition of
individual MHC-encoded target structures by NK cells.
In vivo studies in mice have shown that the overriding effects of
inhibitory receptors largely overshadow activation of NK killing
by MHC-binding receptors (17). In contrast, ALC studies in rats
provided ample evidence for stimulatory NK allorecognition both
in vivo and in vitro (8, 9), and suggested that rat NK cells express
a more potent array of activating receptors directed against several
distinct target cell allo-MHC determinants encoded within the nonclassical class Ib RT1-C/E/M region (5, 10, 18, 19). We speculated
that, like MHC-binding NK receptors in mice and in humans, these
rat NK receptors might be functionally associated with DAP12.
Using a previously described expression cloning system that identifies cell surface receptors by their ability to associate with
DAP12 (20, 21), in this study we describe a novel stimulatory rat
Ly-49 receptor (Ly-49s3) that activates the natural killing of allogeneic lymphocytes. Unlike mouse-activating Ly-49 molecules,
which in general have narrow specificities for individual MHCencoded alleles, rat Ly-49s3 recognizes a broad array of MHCencoded target determinants in rat strains of the c, av1, lv1, and
n haplotypes. Using intra-MHC recombinants, we have localized putative Ly-49s3 target ligands in the n and av1 haplotypes near to or within the nonclassical MHC-I region, RT1-C/
E/M. Our results suggest that killing of allogeneic lymphocytes
is determined in part by an activating Ly-49 receptor that is
0022-1767/02/$02.00
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Previous studies of the rapid rejection of MHC-disparate lymphocytes in rats, named allogeneic lymphocyte cytotoxicity, have
indicated that rat NK cells express activating receptors for nonclassical MHC class I allodeterminants from the RT1-C/E/M region.
Using an expression cloning system that identifies activating receptors associated with the transmembrane adapter molecule
DAP12, we have cloned a novel rat Ly-49 receptor that we have termed Ly-49 stimulatory receptor 3 (Ly-49s3). A newly generated
anti-Ly-49s3 Ab, mAb DAR13, identified subpopulations of resting and IL-2-activated NK cells, but not T or B lymphocytes.
Depletion of Ly-49s3-expressing NK cells drastically reduced alloreactivity in vitro, indicating that this subpopulation is responsible for a major part of the observed NK alloreactivity. DAR13-mediated blockade of Ly-49s3 inhibited killing of MHC-congenic
target cells from the av1, n, lv1, and c haplotypes, but not from the u or b haplotypes. A putative ligand was mapped to the
nonclassical MHC class I region (RT1-C/E/M) using intra-MHC recombinant strains. Relative numbers of Ly-49s3ⴙ NK cells were
reduced, and surface levels of Ly-49s3 were lower, in MHC congenic strains expressing the putative Ly-49s3 ligand(s). In conclusion, we have identified a novel Ly-49 receptor that triggers rat NK cell-mediated responses. The Journal of Immunology, 2002,
169: 22–30.
The Journal of Immunology
23
broadly reactive with polymorphic class Ib ligands from several
different MHC haplotypes.
Materials and Methods
Animals
Four- to 6-wk-old female BALB/c mice and BALB/c nu/nu mice were
from Simonsen (Gilroy, CA) and were reared under conventional conditions in accordance with institutional guidelines. The rat strains and their
MHC haplotypes used in these studies are listed in Table I. Breeding pairs
from PVG (RT1c or rat MHC haplotype c; RT1-Ac-B/Dc-C/E/Mc or c-c-c),
PVG.1U (u or u-u-u), PVG.1AV1 (av1 or a-a-av1), PVG.R23 (u-a-av1),
and PVG.R8 (a-u-u) rats were obtained from Harlan U.K. Limited (Bicester, U.K.), whereas PVG.1LV1 (lv1) and PVG.1N (n or n-n-n) were from
G. W. Butcher (The Babraham Institute, Cambridge, U.K.). These rat
strains were reared under conventional conditions in Oslo and screened for
common rat pathogens. Buffalo (b or b-b-b) rats were purchased from
Harlan (Horst, The Netherlands). BN.1B (b-b-n) were obtained from H. J.
Hedrich (Medizinische Hochschule, Hannover, Germany). The animals
were housed in compliance with guidelines set by the Experimental Animal
Board under the Ministry of Agriculture of Norway.
For single-color analysis, 50 ␮l cells (0.2– 0.5 ⫻ 107 cells/ml) were incubated with primary Ab for 30 min on ice. After three washes, labeled cells
were incubated with F(ab⬘) 2 of FITC-conjugated goat anti-mouse Ig (ICN/
Cappel, Aurora, OH). For three-color flow cytometry, mononuclear splenocytes from 2- to 4-mo-old male rats were separated by centrifugation on
Lymphoprep and depleted of Ig⫹ cells with sheep anti-rat Ig-coated M450
magnetic Dynabeads (Dynal, Oslo, Norway). Primary labeling was with a
combination of FITC-conjugated anti-NKR-P1 (mAb 3.2.3; from J.C. Hiserodt, Pittsburgh, PA; FITC conjugated according to standard methods),
PE-conjugated anti-CD3 (mAb G4.18; from BD PharMingen, San Diego,
CA), and biotinylated DAR13 (anti-Ly-49s3, see below; mouse IgG1; biotinylated according to standard procedures), followed by R-PE-indodicarbocyanine-conjugated streptavidin (DAKO, Glostrup, Denmark). The cells
were analyzed on a FACScan (BD Biosciences, Mountain View, CA).
Generation of effector cells and cytotoxicity assays
IL-2-activated NK cells were generated from mononuclear splenocytes that
were depleted of T cells with mAb anti-CD3 G4.18 and rabbit serum as a
source of complement, then isolated by positive selection of NKR-P1⫹
cells using magnetic Dynabeads, as previously described (18). Selected
CD3⫺NKR-P1⫹ cells were then cultured in RPMI 1640 supplemented with
10% FCS, 2 mM glutamine, 1 mM Na pyruvate, 5 ⫻ 10⫺5 M 2-ME,
antibiotics, and rat rIL-2, which was obtained from the dialyzed cell culture
supernatant of a Chinese hamster ovary (CHO) cell line stably transfected
with a rat IL-2 expression construct (22). Ly-49s3-negative cells were obtained from the IL-2 NK cultures by negative selection with rat anti-mouse
IgG1-coated M450 magnetic Dynabeads (Dynal) preincubated with
DAR13 mAb (mouse IgG1) ascites. Cellular depletion was done immediately before the cytotoxicity assays. The resultant Ly-49s3⫺ NK populations were routinely contaminated with less than 1% Ly-49s3⫹ cells, as
determined by flow cytometry (data not shown).
The generation of Con A-activated lymphoblast target cells and 4-h 51Cr
release assay was performed as previously described (10). The P815 mouse
mastocytoma line used as target cells in redirected killing assays was from
Expression cloning of Ly-49s3 cDNA
The cDNA library used to clone Ly-49s3 was generated from an NK cell
subset (KLRH1⫹) from PVG rats, and has been described previously (23).
In short, template mRNA from IL-2-activated KLRH1⫹ NK cells was used
for the production of adapted cDNA, which was unidirectionally ligated
into the EcoRI (5⬘) and XhoI (3⬘) sites of the expression vector pMET7
(DNAX, Palo Alto, CA). The ligation product was transformed into
DH10B ElectroMAX Escherichia coli (Life Technologies, Gaithersburg,
MD), titered, and amplified on Luria-Bertrani ampicillin agar plates. Library DNA (complexity 550,000) was purified using Qiagen Tip-500 columns (Qiagen, Chadworth, CA).
A total of 20 ␮g cDNA library was transiently transfected into a 50%
confluent monolayer of 293T cells stably transfected with mouse DAP12 as
a fusion protein tagged with the FLAG epitope (293T-DAP12/FLAG) (20)
in a 175-cm2 flask using lipofectamine and serum-free OPTI-MEM, according to the manufacturer’s instructions (Life Technologies). At 48 h,
transfected cells were incubated for 30 min with anti-FLAG mAb M2 (0.5
␮g/106 cells; mouse IgG1; Sigma-Aldrich, St. Louis, MO) in FACS buffer
(PBS with 10 mM EDTA, 3% FBS). After washing, cells were incubated
for 30 min with FITC-conjugated goat anti-mouse Ig (ICN/Cappel), and
then washed extensively. The brightest 0.2% of FLAG⫹-transfected cells
were sorted by flow cytometry and lysed for 30 min at room temperature
in Hirt solution (10 mM EDTA, 0.6% SDS). After addition of NaCl to 1 M,
the nuclei and proteins were precipitated at 4°C overnight and removed.
The episomal pMET7 sublibrary was purified and precipitated from the
Hirt supernatant, then transformed by bacterial electroporation into DH10B
ElectroMAX E. coli, followed by amplification on Luria-Bertrani ampicillin plates and Qiagen plasmid purification. The resultant plasmid sublibrary
was transfected into 293T-DAP12/FLAG cells for two additional rounds of
sorting and plasmid recovery. Individual bacterial colonies were thereafter
transfected in pools, and finally, as individual clones. Clones that stained
positive with the anti-FLAG mAb M2 by flow cytometry were sequenced
and analyzed using the Wisconsin Genetics Computer Group Program
package (Madison, WI).
Stable transfection of cells
The stable Ly-49s3 transfectant of the RNK-16 rat NK cell line (RNK16.Ly-49s3) was generated, as previously described (24), using the Ly49s3 cDNA subcloned into the EMCV-SR␣ expression vector, followed by
electroporation and selection in RPMI, 10% FCS, L-glutamine, penicillin/
streptomycin, and 2-ME supplemented with 1 mg/ml G418 (complete
G418 RPMI). The wild-type RNK-16 cell line was from C. Reynolds (National Cancer Institute, Frederick, MD). Using identical settings, 3 ⫻ 106
CHO cells (ATCC) were electroporated in the presence of 3 ␮g pMX-neomouse-DAP12/FLAG and 15 ␮g pMET7-Ly-49s3. After selection in complete G418 RPMI, stable Ly-49s3 and DAP12/FLAG cotransfectants of
CHO (CHO.Ly-49s3) were identified by the cell surface expression of the
Table I. MHC constitution of the rat strains used
RT1 Regions
Strain
RT1 (rat MHC)
Haplotype
A
B/D
C
PVG
PVG.1LVl
PVG.1AV1
PVG.1U
PVG.R8
PVG.R23
PVG.1N
Buffalo
BN.1B
c
lvl
av1
u
r8
r23
n
b
r37
c
l
a
u
a
u
n
b
b
c
l
a
u
u
a
n
b
b
c
lv1
av1
u
u
av1
n
b
n
a
Recombination point.
2a
2
2
Non-MHC
Background
(c-c-c)
(l-l-lv1)
(a-a-av1)
(u-u-u)
(a-u-u)
(u-a-av1)
(n-n-n)
(b-b-b)
(b-b-n)
PVG
PVG
PVG
PVG
PVG
PVG
PVG
Buffalo
BN
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Flow cytometry
the American Type Culture Collection (ATCC, Manassas, VA). In mAb
blocking or redirected killing experiments, 3–5 ␮g purified DAR13 mAb,
or isotype-matched control mAbs Wes42 (anti-human signal regulatory
protein (SIRP), a gift from M. C. Nakamura and W. E. Seaman, University
of California, San Francisco, CA) or TIB96 (anti-mouse IgD b allotype
from ATCC) were added to effectors 20 min before the addition of target
cells. Spontaneous release was usually between 5 and 15% of the total cpm
in the cells. The results are presented as median values from triplicates for
each E:T cell ratio.
24
Ly-49s3, PROMISCUOUS ACTIVATING MHC RECEPTOR ON RAT NK CELLS
FLAG epitope, as determined by flow cytometry. CHO cells stably cotransfected with human SIRP␤ and DAP12/FLAG were a gift from M. C.
Nakamura and W. E. Seaman (San Francisco, CA).
Production of the anti-Ly-49s3 mAb DAR13
BALB/c mice were immunized four times 2 wk apart with i.p. injections of
5 ⫻ 107 CHO.Ly-49s3 cells. Mice were boosted i.p. 3 wk after the last
immunization, and splenocytes were harvested 3 days thereafter. A total of
5 ⫻ 107 splenocytes was fused with 107 SP2/0 mouse myeloma cells
(ATCC) using Hybrimax PEG/DMSO solution (Sigma-Aldrich) and
IMDM according to standard methods. After selection in complete hypoxanthine/aminopterin/thymidine medium (IMDM-Iscove’s medium, 20%
FBS, pyruvate, penicillin/streptomycin, 2-ME, vitamins, nonessential
amino acids, 1⫻ hypoxanthine/aminopterin/thymidine, 10% hybridoma
cloning factor (Igen, Gaithersburg, MD)), hybridoma supernatants were
screened by flow cytometry for the presence of Abs against 293T-DAP12/
FLAG cells transiently transfected with Ly-49s3 cDNA. Following subcloning, the specificity of the resultant anti-Ly-49s3 DAR13 mAb was
confirmed against primary PVG rat NK cells and against control transfectants. For large-scale mAb production, 107 DAR13 hybridoma cells were
injected i.p. into pristane-primed BALB/c nu/nu mice. Malignant ascites
was harvested and purified by solvent-accessible surface precipitation, followed by dialysis against PBS.
For cell surface labeling, 4 ⫻ 107 IL-2-activated NK cells from PVG rats
were washed in PBS and resuspended in 30 ␮l lactoperoxidase solution (1
mg/ml in PBS), 30 ␮l glucose oxidase solution (150 ␮g/ml in PBS), and 30
␮l glucose solution (50 mg/ml) in the presence of 0.5 mCi Na125I (Amersham Pharmacia, Arlington Heights, IL). After incubation for 20 min at
room temperature, cells were washed extensively with cold PBS and subjected to lysis in Triton X-100 buffer (150 mM NaCl, 20 mM HEPES pH
7.4, 1% Triton X-100, 1 mM EDTA, 10% glycerol, aprotinin, leupeptin,
and PMSF). Clarified lysates were subjected to overnight immunoprecipitation on protein A-Sepharose beads (Amersham Pharmacia) coated with
rabbit anti-mouse Ig (ICN/Cappel) and loaded with DAR13 mAb, or with
isotype-matched control mAb Wes42 (anti-human SIRP). After resolution
on 8% SDS-PAGE, the gel was dried and subjected to autoradiography.
To demonstrate the association of Ly-49s3 with DAP12, 4 ⫻ 107 wildtype RNK-16 cells and an equivalent number of RNK-16.Ly-49s3 stable
transfectants were stimulated with pervanadate, a tyrosine phosphatase inhibitor. After washing in cold TBS (20 mM Tris, pH 7.4, 150 mM NaCl),
cells were lysed for 2 h at 4°C in digitonin lysis buffer (20 mM triethanolamine, pH 7.8, 150 mM NaCl, 1 mM MgSO4, 2.5 mM CaCl2, 1 mM
sodium orthovanadate, 1% digitonin (Calbiochem, San Diego, CA), aprotinin, leupeptin, and PMSF). Clarified lysates were subjected to a 4-h immunoprecipitation on protein A-Sepharose beads coated with rabbit antimouse Ig and either DAR13 mAb or control mAb Wes42. After extensive
washing in 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
(CHAPS) wash buffer (10 mM CHAPS, 150 mM NaCl, 20 mM Tris, pH
7.8, 1 mM sodium orthovanadate, aprotinin/leupeptin/PMSF), immunoprecipitates were resolved on 15% SDS-PAGE under reducing conditions.
Following semidry transfer onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Marlborough, MA), membranes were blocked in
TBS with 0.1% Tween 20 and 3% BSA and incubated with mAb antiphosphotyrosine (4G10; Upstate Biotechnology, Saranac Lake, NY) or
with a previously described rabbit anti-mouse DAP12 antiserum (generated
in rabbits by immunization with a synthetic peptide corresponding to the
cytoplasmic domain of mouse DAP12 (21)). After extensive washing, antiphosphotyrosine blots were developed using alkaline phosphatase-conjugated anti-mouse IgG2b and colorimetric substrates (nitroblue tetrazolium
and 5-bromo-4-chloro-3-indolyl phosphate; Roche, Indianapolis, IN), according to the manufacturer’s instructions. Anti-DAP12 blots were developed using HRP-conjugated donkey anti-rabbit Ab and Supersignal chemiluminescent substrate (Pierce, Rockford, IL) before exposure to film.
Results
Generation of the anti-Ly-49s3 mAb DAR13
To generate a mAb against the Ly-49s3 receptor, we cotransfected
CHO cells with Ly-49s3 and a mouseDAP12/FLAG construct.
These CHO.Ly-49s3 transfectants apparently express the Ly-49s3mouseDAP12/FLAG complex on their surface, as judged by flow
cytometry using the anti-FLAG mAb M2 (Fig. 1D). Following
immunization of BALB/c mice and fusion with SP2/0 myeloma
targets, we isolated the DAR13 mAb that specifically stained the
CHO.Ly-49s3 transfectants, but did not stain control CHO cells or
CHO cells cotransfected with DAP12/FLAG and another DAP12associated receptor, human SIRP␤ (CHO.huSIRP␤). As shown in
Fig. 1D, while both CHO.huSIRP␤ and CHO.Ly-49s3 transfectants react with anti-FLAG mAb M2, only CHO.Ly-49s3 stains
with the DAR13 mAb, while an isotype-matched control mAb
Wes42, which recognizes human SIRP, fails to bind CHO.Ly-49s3
(but does bind CHO.huSIRP␤ cells). These data demonstrate that
DAR13 mAb reacts with Ly-49s3 and not with the FLAG epitope
or with DAP12 alone. It cannot be excluded that the DAR13 mAb
reacts with other uncharacterized rat Ly-49 receptors that might be
highly homologous with Ly-49s3 in the extracellular region. But if
so, the distinct functional and phenotypic results obtained for
DAR13 in different MHC haplotypes (see below) suggest that
these Ly-49 receptors most likely also have overlapping functions
and specificities for MHC-related ligands.
Complementation expression cloning of the Ly-49s3 cDNA
A major NK cell subset in PVG rats expresses Ly-49s3
Previous experiments from our laboratory (5, 10, 18) and from
others (19) showed that rat NK cells most likely express activating
receptors directed against target Ags encoded within the nonclassical rat MHC class Ib region RT1-C/E/M. Since structurally divergent MHC-binding NK receptors in mice and humans have
been found to associate with the signaling adapter DAP12 (11), we
speculated that rat NK receptors for RT1-C/E/M-encoded ligands
As shown in Fig. 2A, the DAR13 mAb defines a major NK subset
in PVG rats. A total of 64% of IL-2-activated PVG NK cells react
with DAR13 mAb and hence are Ly-49s3⫹, while only 24% label
with the STOK2 mAb (26), which we have shown to react with the
inhibitory Ly-49 receptor Ly-49i2 (44). Relative numbers of Ly49s3⫹ NK cells are somewhat increased as a result of IL-2 culture,
as can be deduced from the results described below for fresh PVG
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Cell surface labeling, immunoprecipitation, and Western blotting
might also be functionally associated with DAP12. In an attempt to
identify novel DAP12-associated receptors in rat NK cells, we
used a complementation cloning approach, as previously described
(20, 21). The negatively charged aspartic acid residue in the transmembrane domain of DAP12 precludes its expression on the cell
surface of 293T cells unless coupled to an associating receptor.
Thus, cDNAs for DAP12-associated receptors can be identified in
293T cells stably transfected with DAP12, by their ability to induce cell surface expression of DAP12. We used this expression
requirement of DAP12 to screen a cDNA library derived from a
highly alloreactive NK cell subset (KLRH1⫹) from the PVG rat
strain (23). By transiently transfecting the cDNA library into 293T
cells stably expressing FLAG-tagged mouse DAP12 (293TDAP12/FLAG cells), we were able to isolate several clones that
induced DAP12/FLAG expression on the cell surface. Sequence
analysis of one clone, which induced surface expression of DAP12
at levels similar to mouse Ly-49H (Fig. 1A), revealed it to be a
novel member of the lectin-like Ly-49 (KLRA) family with structural features of an activating receptor. As this was the third reported rat Ly-49 molecule with stimulatory structural features, we
have named it Ly-49 stimulatory receptor 3, or Ly-49s3 (Fig. 1B).
Like the stimulatory mouse Ly-49D and -H receptors, rat Ly-49s3
does not possess a cytoplasmic immunoreceptor tyrosine-based
inhibitory motif (ITIM), yet it contains a transmembrane basic
residue (R) that is most likely involved in the recruitment of
DAP12. Among previously published rat Ly-49 molecules, the
extracellular domain of Ly-49s3 shares 53–56% amino acid
identity with the corresponding domains of the F344 strain receptors Ly-49i1, -s1, and -s2 (formerly Ly-49.9, .29, and .12,
respectively; Fig. 1, B and C (25).
The Journal of Immunology
NK cells. By contrast, the majority of T and B cells are Ly-49s3
negative; thus, only ⬃2% of freshly isolated cervical lymph node
cells and ⬍1% of thymocytes stain with DAR13 mAb (Fig. 2, B
and C).
We have previously shown that the inhibitory Ly-49i2 receptor
is expressed by small subsets of NKR-P1⫹ T cells in addition to
NK cells (26), but this is apparently not the case for the stimulatory
Ly-49s3 receptor. Triple labeling studies of rat splenocytes with
DAR13 mAb in combination with anti-CD3 and anti-NKR-P1
showed that ⬃30% of the CD3⫺NKR-P1⫹ NK cells were Ly49s3⫹. In contrast, less than 1% of the CD3⫹NKR-P1⫹ T cell
population was Ly-49s3 bright positive. It could not be excluded
that some NKR-P1⫹ T cells express Ly-49s3 at very low levels, as
FIGURE 2. A major NK cell subpopulation in PVG rats expresses the
Ly-49s3 receptor, as judged by staining with mAb DAR13. A, DAR13
mAb labeling of IL-2-activated PVG NK cells, in comparison with STOK2
mAb reacting with the Ly-49i2 receptor. B and C, Ly-49s3-specific fluorescence of cervical lymph node (LN) cells and thymocytes, respectively;
D, reactivity with a subpopulation of freshly isolated CD3⫺NKR-P1⫹
splenic NK cells (but not with CD3⫹NKR-P1⫹ or CD3⫹NKR-P1⫺ T lymphocytes) is demonstrated.
there was a shoulder on the negative peak on the indicated fluorescence histogram, but if so, their expression levels were much
lower than for NK cells (Fig. 2D). Conventional NKR-P1⫺ T cells,
in contrast, were clearly Ly-49s3 negative (Fig. 2D).
Ly-49s3 stimulates redirected lysis in RNK-16 cells
To aid in functional studies, we transfected the rat NK-like cell line
RNK-16 with the Ly-49s3 cDNA and examined the receptor-specific effects in redirected cytotoxicity assays against FcR⫹ target
cells. Transfected RNK-16 cells were screened by flow cytometry
for clones stably expressing Ly-49s3, as depicted for one clone
(RNK-16.Ly-49s3) in Fig. 3A. Neither wild-type RNK-16 cells nor
the RNK-16.Ly-49s3 transfectants killed the FcR⫹ mouse mastocytoma target P815. However, the RNK-16.Ly-49s3 line exhibited
brisk redirected lysis of P815 in the presence of stimulating quantities of DAR13 mAb (Fig. 3B). Addition of mAb DAR13 had no
effect on P815 killing by wild-type RNK-16, as compared with the
isotype control mAb Wes42, which had no appreciable effect on
P815 killing by either NK effector cell (Fig. 3B and data not
shown).
The homodimeric Ly-49s3 receptor associates with rat DAP12
Immunoprecipitation of Ly-49s3 from lysates of 125I-surface-labeled IL-2-activated rat NK cells revealed that Ly-49s3 migrates
as a 80- to 100-kDa glycoprotein under nonreducing conditions
and as a 45- to 50-kDa glycoprotein under reducing conditions,
confirming that, like other Ly-49 molecules, Ly-49s3 is a disulfide-linked homodimer on NK cells (Fig. 4A).
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FIGURE 1. Isolation of Ly-49s3 cDNA and generation of the anti-Ly49s3 mAb DAR13. A, The rat Ly-49 stimulatory receptor 3 (Ly-49s3) was
cloned by its ability to induce surface expression of a mouse DAP12/FLAG
fusion protein in 293T cells. Induction was comparable with that of the
activating mouse Ly-49H receptor (MLY-49H). Mock-transfected 293T
cells are shown as a control (CTR.). B, Deduced amino acid sequence of
the Ly-49s3 receptor, in comparison with the Ly-49 inhibitory receptor 1
(Ly-49i1; previously denoted Ly-49.9), Ly-49s1 (previously Ly-49.29),
and Ly-49s2 (previously Ly-49.12). The position of the transmembrane
basic residue (R) in Ly-49s1, Ly-49s2, and Ly-49s3 is indicated with an
asterisk. A putative ITIM motif in Ly-49i1 is marked with a stippled line,
and the predicted transmembrane region is underlined. C, Dendrogram of
the different rat Ly-49 receptors based on total amino acid similarity. D,
Staining of CHO cells stably cotransfected with Ly-49s3 and a mouse
DAP12/FLAG construct (CHO.Ly-49s3) with mAb DAR13 mAb (a) and
anti-FLAG mAb M2 (b), but not with the isotype-matched anti-SIRP mAb
Wes42 (c). By contrast, mAb DAR13 (a) did not stain CHO cells stably
cotransfected with human SIRP␤ and DAP12/FLAG (CHO.huSIRP␤),
while anti-FLAG (b) and anti-SIRP (c) mAbs stained positively. Sequence
data are available from GenBank/EMBL/DDJB under accession no.
AY102036.
25
26
Ly-49s3, PROMISCUOUS ACTIVATING MHC RECEPTOR ON RAT NK CELLS
⫹
FIGURE 3. The Ly-49s3 receptor mediates redirected lysis of FcR
P815 tumor cells. A, Expression of Ly-49s3 by the NK-like RNK-16 cell
line stably transfected with the Ly-49s3 cDNA (RNK-16.Ly-49s3). Flow
cytometry using the DAR13 mAb (open curve) and the isotype-matched
mAb Wes42 as a control (Ctr.; shaded curve) is demonstrated. B, Cytotoxicity of the Ly-49s3⫹ RNK-16.Ly-49s3 cell line against P815 tumor
target cells in the presence of 3 ␮g/well DAR13 mAb (f) or the Wes42
mAb control (F).
NK reactivity against MHC-mismatched lymphoblast targets is
highly enriched in the Ly-49s3⫹ NK subset
We have shown previously that NK alloreactivity is markedly enriched in the Ly-49i2⫹ subset (26, 27), but enrichment was even
more pronounced in the Ly-49s3⫹ NK subset. Bulk cultures of
FIGURE 4. The Ly-49s3 receptor is a DAP12-associated homodimeric transmembrane protein. A, Immunoprecipitation from Triton X-100 lysates from 125I
surface-iodinated IL-2-activated PVG NK cells using
the DAR13 mAb and the isotype-matched control
Wes42 mAb. Migration of precipitates on 8% SDSPAGE is shown under nonreducing (NR) and reducing
(Red.) conditions. B, Coprecipitation of the Ly-49s3
receptor with endogenous DAP12 in the rat NK-like
line RNK-16 NK stably transfected with Ly-49s3
cDNA (RNK-16.Ly-49s3). RNK-16 wild-type cells
(RNK-16 wt) are shown as a control. Immunoprecipitations were with DAR13 mAb and the isotypematched control Wes42 mAb, as indicated. After resolution on reducing 15% SDS-PAGE, the Western blot
was developed with the anti-phosphotyrosine 4G10
mAb, showing coprecipitation of a 12- to 14-kDa tyrosine-phosphorylated protein with Ly-49s3. This Ly49s3-associated protein likely is DAP12, as shown in a
parallel Western blot developed with a rabbit antiDAP12 antiserum (C).
IL-2-activated NK cells were depleted of Ly-49s3⫹ cells using
DAR13 mAb and magnetic Dynabeads, and alloreactivity of undepleted and Ly-49s3-negative NK cells was compared against a
panel of MHC-mismatched Con A blast targets that were all on the
PVG strain background. As shown in Fig. 5, undepleted NK cells
from PVG.1U (RT1u haplotype or u) rats displayed high cytolytic
activity against MHC-mismatched n, av1, lv1, and c haplotype
blasts, while syngeneic (u) control targets were spared. Removal of
the Ly-49s3⫹ NK cell subset reduced killing against all targets
near to that seen for syngeneic control targets. Similar results were
obtained when using NK cells from additional MHC-congenic
strains such as PVG (c) and PVG.1AV1 (av1) (data not shown).
Based on these findings, we speculated that the Ly-49s3 receptor
is an important triggering MHC alloreceptor for rat NK cells.
Ly-49s3 is a broadly reactive receptor for MHC-encoded target
determinants in the n, av1, lv1, and c haplotypes; mapping of a
putative Ly-49s3 ligand to the MHC class Ib RT1-C/E/M region
Receptor-blocking experiments using DAR13 mAb and alloreactive NK effector cells revealed that Ly-49s3 exhibits an apparent
broad specificity for MHC-encoded ligands. Addition of purified
DAR13 to the cytotoxic assays markedly inhibited killing of
MHC-mismatched n, av1, and lv1 haplotype targets by IL-2-activated PVG (c haplotype) NK cells (Fig. 6A). The blocking effect
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The Ly-49s3 receptor was initially identified by its ability to
associate with mouse DAP12 on the surface of 293T cells. Coprecipitation with Ly-49s3 of a surface-labeled protein that migrated
with a molecular mass of ⬍17 kDa under reducing conditions (Fig.
4A) suggested that Ly-49s3 was constitutively associated with this
adapter molecule in IL-2-activated rat NK cells. To confirm that
the Ly-49s3 receptor associates with rat DAP12, we immunoprecipitated Ly-49s3 from digitonin lysates of pervanadate-stimulated
RNK-16.Ly-49s3 transfectants and from untransfected RNK-16
wild-type cells. After resolution of immunoprecipitates on 15%
reducing SDS-PAGE, anti-phosphotyrosine Western blotting revealed that Ly-49s3 specifically coprecipitates with a 12- to 14kDa tyrosine-phosphorylated protein (Fig. 4B), which most likely
represents DAP12 as judged by anti-DAP12 Western blot analysis
(Fig. 4C). Thus, Ly-49s3 associates with rat DAP12 in RNK-16
transfectants.
FIGURE 5. The Ly-49s3⫹ NK subset is strongly alloreactive. In vitro
depletion of Ly-49s3⫹ NK cells with mAb DAR13 from a culture of IL2-activated NK cells from the PVG.1U strain (u MHC haplotype) led to a
strongly reduced cytolysis of lymphoblast targets expressing the n, av1,
lv1, and c MHC haplotypes. Levels of killing of syngeneic (u haplotype)
targets (Ctr.) were not affected. Shown are cytolytic activities of depleted
(Ly-49s3⫺, f) and undepleted (CTR., F) NK cells.
The Journal of Immunology
was especially pronounced toward haplotype n allogeneic targets
with a reduction of cytotoxicity almost to syngeneic baseline levels, suggesting that the Ly-49s3 receptor is essential for the killing
of n haplotype allotargets by PVG NK cells. Addition of DAR13
mAb had no effect on killing of allogeneic blasts from u haplotype
rats. These data suggested that the n, av1, and lv1, but not the u
MHC haplotypes encode target determinants for the Ly-49s3 receptor. Haplotype c blasts were predictably not killed by syngeneic
PVG NK effectors, and Ab blockade experiments using this E:T
cell combination were uninformative. Targets from c haplotype
rats share a functionally defined activating NK allodeterminant,
ALC-2, with blasts from av1 and lv1 haplotype rats (28). To check
for additional ligands in c haplotype targets, we used NK cells
from allogeneic PVG.1U (u) rats as effectors. DAR13 mAb inhibited killing of c as well as of av1 haplotype allogeneic targets by
PVG.1U effectors, confirming the existence of a Ly-49s3 target
structure in the c haplotype (Fig. 6B).
Using lymphoblast targets from intra-MHC recombinant strains,
the Ly-49s3 target structure(s) was mapped to the class Ib region
27
of the rat MHC (RT1-C/E/M), and not to the class Ia (RT1-A) or
class II (RT1-B/D) regions. This MHC class Ib region has been
shown previously to contain several functionally defined stimulatory NK allodeterminants (10). DAR13 mAb-mediated blocking of
Ly-49s3 on PVG NK cells led to reduced killing of RT1-Au-B/DaC/E/Mav1 (or u-a-av1) targets from PVG.R23 rats, but not of a-u-u
lymphoblasts from PVG.R8 rats, while the isotype-matched control mAb TIB-96 had no appreciable effect on the killing of either
target. These data showed that the av1-encoded Ly-49s3 target
determinant was located in either the class II (RT1-B/D) or the
class Ib (RT1-C/E/M) region. Direct RT1-C/E/M mapping was obtained using targets derived from the BN.1B r37 intra-MHC recombinant (b-b-n) rat. Similar to u-u-u targets (Fig. 6A), DAR13
mAb did not affect PVG NK cell lysis of b-b-b targets, but markedly inhibited killing of b-b-n recombinant targets (Fig. 6D). These
experiments localized the prominent Ly-49s3 ligand in the n haplotype to the nonclassical MHC-I region, RT1-C/E/M, and it seems
reasonable to assume that the putative Ly-49s3 ligands in the other
MHC haplotypes may also be encoded in this region.
Like the inhibitory rat Ly-49i2 receptor (27) and inhibitory Ly-49
receptors in the mouse (29, 30), expression levels of Ly-49s3 on
the surface of individual NK cells were decreased in rats bearing
its putative MHC ligand(s), as judged by three-color flow cytometric studies of ex vivo isolated spleen NK cells from a panel of
MHC-congenic rats on the PVG strain background. These studies
showed that Ly-49s3 expression levels on CD3⫺NKR-P1⫹ NK
cells were 6 –10 times higher in Ly-49s3 ligand-negative PVG.1U
rats (u-u-u) when compared with levels seen in ligand-positive
PVG.1N rats (n-n-n) (Fig. 7A). The markedly reduced Ly-49s3
expression in n-n-n haplotype NK cells correlates with the deduced
importance of this receptor in inducing NK-mediated lysis of n-n-n
allotargets (see above), and suggests that this haplotype encodes a
dominant (or multiple) ligand(s) for the Ly-49s3 receptor. Intermediate expression levels were observed for NK cells from ligandbearing PVG.1AV1 (a-a-av1), PVG.1LV1 (l-l-lv1), and PVG (cc-c) rats, which displayed staining intensities that were approximately
half that seen in NK cells from ligand-deficient PVG.1U (u-u-u) rats
(Fig. 7A and data not shown). As noted above, these three haplotypes
share a common operationally defined NK allodeterminant, ALC-2,
which has been previously mapped to the MHC class Ib region, RT1C/E/M (28). Similarly, the locus in the a-a-av1 haplotype that leads to
FIGURE 6. Ly-49s3 is a receptor for target MHC ligands from the n,
av1, lv1, and c rat MHC (RT1) haplotypes, but not from the u or b haplotypes: mapping of target ligands to the nonclassical class I region RT1C/E/M. Blocking of NK cytolytic activities against different Con A blast
targets by the addition of DAR13 mAb to the cytotoxic cultures. The MHC
constitutions of the target cells are indicated above each graph, specified
for the RT1-A (classical class I), -B/D (class II), and -C/E/M (nonclassical
class I) regions, respectively. Lymphoblast targets were from PVG.1N
(RT1-An-B/Dn-C/E/Mn or n-n-n), PVG.1AV1 (a-a-av1), PVG.1LV1 (l-llv1), PVG.1U (u-u-u), PVG (c-c-c), PVG. R8 (a-u-u), PVG. R23 (u-a-av1),
Buffalo (b-b-b), and BN.1B (b-b-n). IL-2-activated NK cell effectors were
from either PVG (A, C, and D) or PVG.1U rats (B). Cytolysis is shown in
the presence of blocking quantities (5 ␮g/well) of purified DAR13 mAb
(f) or of the isotype-matched control mAb TIB-96 (F).
FIGURE 7. In vivo reduction of the NK cell surface level of Ly-49s3 in
MHC congenic strains expressing putative Ly-49s3 ligand. Freshly isolated
CD3⫺NKR-P1⫹ splenic NK cells from the PVG.1N (RT1-An-B/Dn-C/E/Mn
or n-n-n), PVG.1AV1 (a-a-av1), and PVG.1U (u-u-u) rat strains (A), or
from PVG.R23 (u-a-av1) and PVG.R8 (a-u-u) intra-MHC recombinant rats
(B) were analyzed by three-color flow cytometry for reactivity with mAb
DAR13.
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Ly-49s3 expression is reduced in the presence of its putative
MHC ligand(s)
28
Ly-49s3, PROMISCUOUS ACTIVATING MHC RECEPTOR ON RAT NK CELLS
Discussion
Although NK cells were first identified by their ability to recognize
and kill targets in an MHC-unrestricted manner, early experiments
in rodents have shown that the MHC haplotype of a target can
affect its susceptibility to NK lysis. In mice, the lectin-like Ly-49
molecules are the predominant MHC-binding receptors on NK
cells, and the same is likely the case in rats (25, 27). Inhibitory
Ly-49 receptors have cytoplasmic ITIM motifs that recruit inhibitory tyrosine phosphatases such as SHP-1, whereas activating receptors lack ITIMs, but have basic transmembrane residues
through which they recruit the activating transmembrane adapter
protein DAP12. In the current study, we have used a complementation expression cloning technique to isolate a novel member of
the DAP12-associated rat Ly-49 family, Ly-49s3, which stimulates
the selective NK cell killing of targets expressing MHC-encoded
molecules in lv1, av1, c, and n haplotype rats. Fine mapping of the
Ly-49s3 target structures in the n and av1 haplotypes localized
putative Ly-49s3 ligands telomeric to the class Ia MHC region
RT1-A, and within or near to the class Ib region RT1-C/E/M.
These data extend and support previous functional and genetic
studies implicating Ly-49-encoded receptors in the killing of
MHC-disparate lymphoblast targets by highly alloreactive rat NK
cells (25–27). In these and related studies, allospecific inhibitory
functions in rat NK cells are in general restricted by classical RT1-
Table II. Proportion of Ly-49s3⫹ NK cells in reduced in strains
expressing MHC ligand(s) for the Ly-49s3 receptora
Strain
RT1 (MHC)
Haplotype
% of Ly-49s3⫹ NK Cells
nb
PVG.1N
PVG
PVG.1LV1
PVG.1AV1
PVG.1U
n
c
lv1
av1
u
23 (22–28)c
29 (27–31)
32 (28–34)
28 (27–29)
38 (35–41)d
4
7
6
4
6
a
Mononuclear spleen cells, depleted of Ig⫹ cells, from a panel of MHC congenic
strains were analyzed by three-color flow cytometry for the percentage of Ly-49s3⫹
cells among CD3⫺NKR-P1⫹ NK cells, as judged by DAR13 staining. Values represent medians and observed ranges.
b
Individual animals analyzed.
c
Statistically different from PVG.1U ( p ⬍ 0.001), PVG.1LV1 ( p ⬍ 0.01), PVG,
and PVG.1AV1 (both p ⬍ 0.05) with a two-sample t test (two sided).
d
Statistically different from PVG.1N, PVG.1LV1 (both p ⬍ 0.001), PVG, and
PVG.1AV1 (both p ⬍ 0.0001) with a two-sample t test (two sided).
A-encoded ligands (27, 28), while all currently known alloactivating NK functions are restricted by class Ib molecules encoded
within the RT1-C/E/M region (5, 10, 31). Although the exact number of MHC class I genes and gene fragments encoded from RT1C/E/M has yet to be fully determined, it is estimated that this
region contains ⬃45 MHC class I-like genes in the n haplotype
(32, 33). Similar to the H2-D/Q/T/M region of the mouse MHC
complex, the telomeric RT1-C/E/M region contains four distinct
clusters of class I genes that are separated by sets of framework
genes conserved between the two species. The last three clusters
contain genes that resemble the H2-T and H2-M genes, while the
most centromeric of these four clusters harbors genes that are more
similar to classical RT1-A genes than to H2-D and -Q. Cell surface
density of the RT1-C/E/M-encoded molecules is low, being ⬍10%
the density of the classical RT1-A molecules, and little is known
about their tissue distribution and regulation. Only one example of
T cell restriction by an RT1-C/E/M-encoded molecule has been
described (34). The RT1-C/E/M molecules have therefore been
regarded as nonclassical MHC-I molecules, and their physiologic
functions remained largely obscure. However, previously published functional and genetic data have shown that target ligands
that activate alloreactive NK cells reside within the most centromeric class Ib cluster (the RT1-C/E/M cluster), which harbors between 10 and 15 functional class I genes, including alleles of the
RT1-C, RT1-E, and RT1-U loci (32, 35), notably the RT1.Cl molecule, which is deleted in the lm1 MHC mutant rat strain, and
RT1.Eu (5, 10, 19). It can only be speculated whether Ly-49s3 is
cross-reactive with several distinct RT1-C/E-encoded ligands, being the products of one or more loci within this MHC-I cluster, or
whether Ly-49s3 reacts with a ligand that is shared between several unrelated MHC haplotypes.
Rat Ly-49s3 shares common structural and functional features
with the activating Ly-49 receptors in mice (12–14). Notably, like
the stimulatory mouse Ly-49D, -H, -P, and -W receptors, rat Ly49s3 is a lectin-like dimer with a putative DAP12-docking transmembrane residue. In addition, like activating mouse Ly-49 receptors, rat Ly-49s3 appears to bind MHC-related ligands.
Significant differences between mouse and rat NK allorecognition
systems can be seen from a careful evaluation of available data
from each species. To date, all known stimulatory MHC targets of
rat alloreactive NK cells are encoded within the RT1-C/E/M class
Ib region (5, 10, 19), while alloinhibitory rat receptors appear to
recognize RT1-A class Ia ligands (27, 28, 31). In the mouse, both
inhibitory and activating Ly-49 receptors recognize class Ia molecules. In addition, the most widely studied activating Ly-49 receptor, Ly-49D, recognizes MHC-encoded xenogeneic ligands
(36, 37). Unlike the broadly alloreactive rat Ly-49s3 receptor,
mouse Ly-49D has a narrow allogeneic specificity for the classical
mouse Dd allele of mouse MHC-I (38), whereas the Ly-49P, Ly49R, and Ly-49W receptors all recognize Dd and Dk (13, 14).
Thus, in mice, individual activating Ly-49 receptors display narrow specificities against MHC class Ia ligands, as well as against
MHC-related ligands encoded by other mammalian species,
whereas Ly-49s3 is broadly reactive with ligands from several
MHC haplotypes. Another notable difference between mouse and
rat is that whereas mouse-activating Ly-49 receptors generally are
specific for allo-MHC-I molecules, the Ly-49s3 receptor apparently also reacts with a self-MHC ligand in the PVG strain from
which it was isolated. This suggests that Ly-49s3⫹ PVG NK cells
may be potentially autoreactive. This is in line with previous functional data showing the expression of an activating class Ib NK
allodeterminant (ALC-2) in a syngeneic PVG strain setting, in
which self-tolerance was ensured by a dominant inhibitory influence from the PVG class Ia region (RT1-Ac) (28) No xenogeneic
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a reduced Ly-49s3 surface expression level maps close to the RT1C/E/M region in intra-MHC recombinant strains between the a-a-av1
and u-u-u haplotypes, although we could not formally exclude class II
(RT1-B/D)-specific effects due to the lack of informative PVG recombinants. As shown in Fig. 7B, NK cells from PVG.R8 (Aa-B/Du-Cu or
a-u-u) expressed 2- to 3-fold more Ly-49s3 than did NK cells from
PVG.R23 (u-a-av1) rats. Since these data were generated in NK gene
complex identical congenic strains differing only in the MHC, the data
suggest that activating as well as inhibitory rat Ly-49s may calibrate
expression levels in the presence of their corresponding MHC ligands.
Ly-49s3⫹ NK cells from ligand-bearing rats exhibited not only
reduced cell surface receptor levels, their relative numbers were
also significantly diminished in the same set of MHC congenic
PVG strains. Thus, ligand-negative PVG.1U rats had a significantly higher fraction (38%) of Ly-49s3⫹ splenic NK cells than
did the ligand-positive strains, PVG (29%), PVG.1AV1 (28%),
PVG.1LV1 (32%), and PVG.1N (23%) (Table II). Notably, relative numbers of Ly-49s3⫹ NK cells were lowest in the PVG.1N
strain, which again correlates with the presence of an apparent
prominent Ly-49s3 ligands in the n haplotype.
The Journal of Immunology
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33. Günther, E., and L. Walter. 2001. The major histocompatibility complex of the rat
(Rattus norvegicus). Immunogenetics 53:520.
34. Wang, C.-R., A. Livingstone, G. W. Butcher, E. Hermel, J. C. Howard, and
K. F. Lindahl. 1991. Antigen presentation by neoclassical MHC class I geneproducts in murine rodents. In NATO ASI Series, Molecular Evolution of the Major
Histocompatibility Complex, Vol. H 59. J. Klein and D. Klein, eds. SpringerVerlag, Berlin, pp. 441– 462.
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ligands for rat Ly-49 receptors have yet been identified, but a limited
study of different class I-negative target lines and lymphoblasts failed
to reveal an influence of mouse H2 on rat NK lysis (39).
The physiologic functions of activating mouse and rat Ly-49
receptors are difficult to discern. Typically, an individual mouse
expresses only a limited range of activating Ly-49 receptors
(BALB/c expresses only inhibitory functional Ly-49 proteins) with
limited specificities for MHC-I. An individual NK cell expressing
an activating Ly-49 will often also express an inhibitory Ly-49
against the same MHC-I ligand. For example, the mouse class I
molecule H2-Dd is recognized by the stimulatory Ly-49D receptor
as well as by the inhibitory Ly-49A and Ly-49G2 receptors, and
nearly all Ly-49D⫹ NK cells coexpress either Ly-49A or -G2 in
selected strains of mice (17). Since inhibitory signals are thought
to predominate over stimulatory signals, the MHC-dependent activation of natural killing of H2-Dd⫹ targets by, for instance, Ly49D, might be easily abrogated by inhibitory signals generated
through Ly-49A. In rats, the classical MHC class Ia ligands for
inhibitory Ly-49 receptors are distinct from the nonclassical MHC
class Ib ligands recognized by activating Ly-49 receptors. Classical and nonclassical MHC-I ligands differ in their tissue-specific
expression, and may be differentially regulated in response to cellular stress or inflammation. Inflammation or stress-induced upregulation of nonclassical class Ib ligands on rat hemopoietic or
inflammatory cells might lead to enhanced NK activation, overriding tonic inhibitory effects mediated through inhibitory Ly-49
receptors. In these ways, activating rat Ly-49 receptors may play a
role in controlling ongoing cellular immune responses during inflammatory states.
It is also possible that the physiologic significance of activating
Ly-49 receptors lies in their recognition of foreign cells or of cells
expressing virally encoded xenogeneic ligands. Xenorecognition
by Ly-49s3 has not yet been studied, but the activating mouse
Ly-49D and Ly-49H receptors each appear to recognize xenogeneic ligands. Mouse Ly-49D binds to mouse H2-Dd, but it also
displays xenogeneic cross-reactivity against an MHC-encoded
molecule in RT1l and RT1lv1 haplotype rats, as well against a target
structure on Chinese hamster cells (36 –38). Mouse Ly-49H does
not react with common mouse MHC haplotypes (40), but appears
to be involved in the strain-specific resistance to murine CMV (41,
42), presumably through the recognition of a novel virus-encoded
MHC-like homologue (m157) on infected cells (43). Xenorecognition of foreign MHC may simply be a fortuitous coincidence, but
certainly the recognition of CMV-infected cells by Ly-49H serves
an evolutionary advantage in mice. It is intriguing to speculate that
many activating Ly-49 receptors might recognize foreign or pathogenic proteins, and, as such, activating Ly-49 receptors may play
important physiologic roles in innate immune surveillance against
foreign cells or against selected infectious agents. The true physiologic functions of polymorphic activating Ly-49 receptors have
yet to be fully defined, however, and are the subject of ongoing
investigation.
29
30
Ly-49s3, PROMISCUOUS ACTIVATING MHC RECEPTOR ON RAT NK CELLS
35. Leong, L. Y. W., A.-F. Le Rolle, E. V. Deverson, S. J. Powis, A. P. Larkins,
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G. W. Butcher. 1999. RT1-U: identification of a novel, active, class Ib alloantigen
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36. Nakamura, M. C., C. Naper, E. C. Niemi, S. C. Spusta, B. Rolstad,
G. W. Butcher, W. E. Seaman, and J. C. Ryan. 1999. Natural killing of xenogeneic cells mediated by the mouse Ly-49D receptor. J. Immunol. 163:4694.
37. Idris, A. H., H. R. C. Smith, L. H. Mason, J. R. Ortaldo, A. A. Scalzo, and
W. M. Yokoyama. 1999. The natural killer gene complex genetic locus Chok
encodes Ly-49D, a target recognition receptor that activates natural killing. Proc.
Natl. Acad. Sci. USA 96:6330.
38. Nakamura, M. C., P. A. Linnemeyer, E. C. Niemi, L. H. Mason, J. R. Ortaldo,
J. C. Ryan, and W. E. Seaman. 1999. Mouse Ly-49D recognizes H-2Dd and
activates natural killer cell cytotoxicity. J. Exp. Med. 189:493.
39. Torgersen, K. M., M. Salcedo, J. T. Vaage, C. Naper, B. Rolstad, H.-G. Ljunggren, and P. Höglund. 1997. Major histocompatibility complex class I-independent killing of xenogeneic targets by rat allospecific natural killer cells. Transplantation 63:119.
40. Ryan, J. C., C. Naper, S. Hayashi, and M. R. Daws. 2001. Physiologic functions
of activating natural killer (NK) complex-encoded receptors on NK cells. Immunol. Rev. 181:126.
41. Brown, M. G., A. O. Dokun, J. W. Heusel, H. R. C. Smith, D. L. Beckman,
E. A. Blattenberger, C. E. Dubbelde, L. R. Stone, A. A. Scalzo, and
W. M. Yokoyama. 2001. Vital involvement of a natural killer cell activation
receptor in resistance to viral infection. Science 292:934.
42. Daniels, K. A., G. Devora, W. C. Lai, C. L. O’Donnell, M. Bennett, and
R. M. Welsh. 2001. Murine cytomegalovirus is regulated by a discrete subset of
natural killer cells reactive with monoclonal antibody to Ly49H. J. Exp. Med.
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43. Arase, H., E. S. Mocarski, A. E. Campbell, A. B. Hill, and L. L. Lanier. Direct
recognition of cytomegalovirus by activating and inhibitory NK cell receptors.
Science In press.
44. Naper, C., S. Hayashi, E. Joly, G. W. Butcher, B. Rolstad, J. T. Vaage, and J. C.
Ryan. Ly49i2 is an inhibitory rat natural killer cell receptor for a major histocompatibility complex class Ia molecule (RT1-A1c). Eur. J. Immunol. In press.
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