The Novel Inhibitory NKR-P1C Receptor and
Ly49s3 Identify Two Complementary,
Functionally Distinct NK Cell Subsets in Rats
This information is current as
of June 14, 2017.
Lise Kveberg, Camilla J. Bäck, Ke-Zheng Dai, Marit
Inngjerdingen, Bent Rolstad, James C. Ryan, John T. Vaage
and Christian Naper
J Immunol 2006; 176:4133-4140; ;
doi: 10.4049/jimmunol.176.7.4133
http://www.jimmunol.org/content/176/7/4133
Subscription
Permissions
Email Alerts
This article cites 48 articles, 18 of which you can access for free at:
http://www.jimmunol.org/content/176/7/4133.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2006 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
References
The Journal of Immunology
The Novel Inhibitory NKR-P1C Receptor and Ly49s3 Identify
Two Complementary, Functionally Distinct NK Cell Subsets
in Rats1
Lise Kveberg,* Camilla J. Bäck,* Ke-Zheng Dai,† Marit Inngjerdingen,* Bent Rolstad,*
James C. Ryan,‡ John T. Vaage,2† and Christian Naper†
A
superfamily of killer cell lectin-like receptors (KLRs)3
was originally discovered in NK cells, but certain family
members are also variably expressed by other cell types
(1–3). In rats, the KLRs are encoded by the NK gene complex
(NKC) located on chromosome 4 (4), and syntenic chromosomal
regions have been identified in many other species. Several KLR
gene families are clustered together in specific subregions of the
NKC, and there is considerable species-to-species variation in the
number, expression patterns, and putative functions of the genes
encoded by these different KLR families. Some families, like Ly49
(Klra), are polygenic in rodents (4, 5) while there is only a single
family member in humans (6). The Ly49 genes are located in the
distal (telomeric) part of the rat NKC (7). They encode both activating and inhibitory Ly49 molecules which are expressed in
subsets of rat NK cells. Although inhibitory Ly49 receptors
react with both classical (RT1-A) and nonclassical (RT1-CE/
N/M) MHC class I molecules, their activating counterparts
mainly recognize nonclassical MHC (8 –11). Furthermore, the
activating Ly49 receptors can be instrumental in triggering rat
NK alloreactivity (10, 11).
*Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo,
Oslo, Norway; †Institute of Immunology, Rikshospitalet University Hospital, Oslo,
Norway; and ‡Veterans Affairs Medical Center, Northern California Institute for Research and Education and University of California, San Francisco, CA 94121
Received for publication August 3, 2005. Accepted for publication January 19, 2006.
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
This work was supported by the Norwegian Cancer Society and the Research Council of Norway and Mjåland’s Fund for Cancer Research. K.-Z.D. is a Fellow of the
Norwegian Cancer Society.
2
Address correspondence and reprint requests to Dr. John T. Vaage, Institute of
Immunology, Rikshospitalet University Hospital, NO-0027 Oslo, Norway. E-mail
address: [email protected]
3
Abbreviations used in this paper: KLR, killer cell lectin-like receptor; Clr, C-type
lectin-related; NKC, NK gene complex; FCM, flow cytometry; PVDF, polyvinylidene
difluoride; LN, lymph node; KIR, killer cell Ig-like receptor.
Copyright © 2006 by The American Association of Immunologists, Inc.
The distantly related KLRH1 molecule, which is encoded by a
gene just centromeric of the Ly49 region, shows slightly greater
amino acid homology with Ly49 than with other KLR family
members (12). It is possible that KLRH1 also functions as a receptor for MHC-encoded molecules on the basis of its expression
pattern in MHC congenic strains. A putative ortholog of KLRH1
exists in the mouse but nothing is known about its cellular expression or function (12). The Cd69, Clec, Klre, Cd94, Nkg2, and Klri
gene families are all located centromeric of Klrh1, i.e., in the central region of the rat NKC (13), while the Nkr-p1 and C-type lectinrelated (Clr)/Ocil (14, 15) genes map to its most centromeric region. This organization of the rat NKC is similar to that in the
mouse (1).
As with many of the other KLR families, the NKR-P1 (KLRB)
family consists of both activating and inhibitory members. The
prototypical NKR-P1A molecule has been used as a marker for rat
NK cells and is an activating receptor (16 –18). The NKR-P1B
receptor (4), in contrast, has inhibitory structural features but it has
not been possible to prove NKR-P1B-specific inhibitory functions
on primary cells because available anti-NKR-P1 mAb fail to distinguish NKR-P1A from NKR-P1B (our unpublished data and Ref.
19). The anti-NKR-P1A/B mAb 3.2.3 (16) reacts with NK cells
from a broad range of rat strains, but not from the AUG strain
where there is only a very faint staining (our unpublished data).
This suggests some allelic variation of the NKR-P1 molecules.
Despite their putative roles in the recognition of certain tumor
cells (20), the natural ligands of NKR-P1 molecules have long
remained elusive. Recently, it was shown, however, that members
of the Clr family function as specific ligands for mouse NKR-P1
molecules. The Clr genes are interspersed between the Nkr-p1
genes. Thus, specific receptor-ligand pairs are therefore not inherited separately, but rather en bloc (21, 22). The recent findings of
Clr ligands have created a renewed interest in the NKR-P1 family,
because these ligands and their receptors represent a form of selfnonself discrimination that is independent of MHC class I, a principle that can now be extended to other NK cell receptors (23). In
0022-1767/06/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
The proximal region of the NK gene complex encodes the NKR-P1 family of killer cell lectin-like receptors which in mice bind
members of the genetically linked C-type lectin-related family, while the distal region encodes Ly49 receptors for polymorphic
MHC class I molecules. Although certain members of the NKR-P1 family are expressed by all NK cells, we have identified a novel
inhibitory rat NKR-P1 molecule termed NKR-P1C that is selectively expressed by a Ly49-negative NK subset with unique
functional characteristics. NKR-P1Cⴙ NK cells efficiently lyse certain tumor target cells, secrete cytokines upon stimulation, and
functionally recognize a nonpolymorphic ligand on Con A-activated lymphoblasts. However, they specifically fail to kill MHCmismatched lymphoblast target cells. The NKR-P1Cⴙ NK cell subset also appears earlier during development and shows a tissue
distribution distinct from its complementary Ly49s3ⴙ subset, which expresses a wide range of Ly49 receptors. These data suggest
the existence of two major, functionally distinct populations of rat NK cells possessing very different killer cell lectin-like receptor
repertoires. The Journal of Immunology, 2006, 176: 4133– 4140.
4134
A NOVEL NKR-P1C⫹, Ly49-NEGATIVE NK CELL SUBSET IN RATS
the present paper, we have cloned and characterized a novel inhibitory NKR-P1 member in the rat, which we have termed NKRP1C. It has an unusual cellular distribution, in that it is mainly
expressed by a Ly49-negative NK cell subset with unique functional characteristics.
Materials and Methods
Rats
Rats were housed in compliance with institutional guidelines and sacrificed
at 8 –12 wk of age. The MHC congenic and intra-MHC recombinant rat
strains PVG.1N (RT1n or n; i.e., RT1-An-B/Dn-CE/N/Mn (class Ia-class IIclass Ib), abbreviated n-n-n), PVG.1L (l-l-l), PVG.1LV1 (l-l-lv1), PVG.7B
(c-c-c), and PVG.R23 (u-a-av1) were bred in Oslo, Norway. The PVG.7B
strain possesses a CD45 allotype (RT7.2) but is otherwise used interchangeably with the standard PVG strain (RT7.1). PVG.1U (u-u-u), BN
(n-n-n), AO (u-u-u), and DA (a-a-av1) rats were from Harlan U.K., and
Buffalo (b-b-b) and WAG (u-u-u) rats from Harlan Netherlands.
Flow cytometry (FCM)
Generation of the anti-NKR-P1C hybridoma STOK27 and
biochemical characterization
One BN rat was immunized twice s.c. with IL-2-activated NK cells (106
and 5 ⫻ 106) from PVG.1N (RT1n) rats with an interval of ⬃2 mo, and
boosted i.v. with 10 ⫻ 106 cells 3 days before fusion. Mononuclear splenocytes were fused with NS0 cells using Polyethylene Glycol 4000 and cultured in hypoxanthine/aminopterin/thymidine selection medium. Supernatants were screened for staining of subsets of IL-2-activated NK cells
by FCM.
Immunoprecipitation, deglycosylation, and Western blotting
NK cells were surface biotinylated at 2.5 ⫻ 107 cells/ml in PBS (pH 8), with
0.5 mg/ml EZ-Link Sulfo-NHS-LC-Biotin (Pierce) for 30 min at room temperature and lysed in 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, protease
inhibitors, and 1% Triton X-100. Lysates were precleared and immunoprecipitated with STOK27 precoupled Sepharose 4B beads (Amersham Biosciences) at 2 h at 4°C. For deglycosylation, the immunoprecipitates were
solubilized in 40 mM sodium phosphate (pH 6), with 1% Triton X-100 and
0.1% SDS, digested overnight at 37°C with 1 U of PNGase F/N-Glycosidase
F (complemented with 20 mM EDTA, 1% 2-ME), 1 mU O-glycosidase, 50
mU Clostridium perfringens neuraminidase, or a combination of Oglycosidase and neuraminidase (all enzymes were from Roche Diagnostics).
The samples were resolved by 10 or 12.5% Criterion Tris-HCl gels (Bio-Rad)
and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore). After blocking with 5% dry milk in TBS with 0.05%
Tween 20, the PVDF membranes were incubated with a streptavidin-HRP
conjugate before detection by chemiluminescence (Supersignal West Pico
Chemiluminescent Substrate; Pierce).
Lysates from 20 ⫻ 106 RNK-16.NKR-P1C transfectants, stimulated for
5 min at 37°C with pervanadate or left unstimulated, were precleared before incubation for 2 h at 4°C with 2 ␮g of mAb STOK27 or STOK2
(isotype-matched control). Immunoprecipitations were performed with
GammaBind G-Sepharose Beads (Amersham Biosciences) for 1 h at 4°C.
After washes (0.1% Triton X-100), the immunoprecipitates were resolved
by Criterion Tris-HCl gels as above. After transfer to PVDF membranes
and blocking, membranes were incubated with a polyclonal rabbit antiSH-PTP1 (C-19; Santa Cruz Biotechnology) for 2 h at room temperature,
washed, and incubated with peroxidase-conjugated goat anti-rabbit IgG
and developed with chemiluminescence.
Cells, cytotoxicity assay, and cytokine measurement
The YAC-1, 293T, and P388.D1 cell lines were grown in RPMI 1640, 10%
FCS, and antibiotics, and the RNK-16 line in complete RPMI (cRPMI;
RPMI 1640, 10% FCS, 5 ⫻ 10⫺5 M 2-ME, L-glutamine, and antibiotics).
The generation of Con A lymphoblasts and 4-h 51Cr release assay was
Expression cloning and transfection
cDNAs encoding NKR-P1C were isolated by eukaryotic expression cloning as previously described using mAb STOK27 and a cDNA library from
KLRH1⫹ PVG NK cells (12). After four rounds of immunoenrichment, the
final sublibrary induced the transient expression of a molecule that stained
with mAb STOK27 on ⬃20% of the 293T cells. Four positive clones of
384 clones analyzed were sequenced on both strands.
RNK-16 cells (3 ⫻ 106) were transfected with a full-length NKR-P1C
cDNA subcloned into the EMCV.SR␣ expression vector, as described (29).
Electroporation with 20 ␮g of ScaI-linearized construct was performed at
120 mV, 960 ␮F, and with capacitance extender (Bio-Rad Electroporator).
The cells were grown for 24 h in cRPMI, plated out in 96-well plates at 10
000 cells/well in cRPMI with 1 mg/ml active G418. Cells appeared in 10%
of the wells and were tested for STOK27 staining by FCM after 2–3 wk.
RT-PCR
NKR-P1C⫹ and Ly49s3⫹ single-positive NK cells from the spleen were
obtained by FACS sorting (FACSDiva; BD Biosciences), total RNA was
isolated with TriReagent (Sigma-Aldrich), and cDNA was generated with
Moloney murine leukemia virus reverse transcriptase (Promega). PCRs
were typically performed on a GeneAmp PCR thermocycler (Applied Biosystems) using hot start for 3 min at 96°C. Pyrococcus woesi polymerase
was added at 80°C before running for 35 cycles at 95°C 20 s, 54°C 30 s,
72°C 30 s. The following upper and lower primers, respectively, were used:
Ly49si3, 5⬘-AGGCAATGATCTTCTGCT-3⬘ and 5⬘-TCAATCAGG
GAATTTCTCA-3⬘; Ly49s2, 5⬘-GTGTTTCTTTCGGCTAGTAT-3⬘ and
5⬘-AGGCAAGTTTAGATGGGTT-3⬘; Ly49s7/s1, 5⬘-TCGTAAATTCCT
CACCACAT-3⬘ and 5⬘-CCAGTAATTGTCTGGAATAAG-3⬘; Ly49i8,
5⬘-AGACAAGAGAAACATGAACAT-3⬘ and 5⬘-GAAGTTGGGACT
TAAGGAAG-3⬘; Cd94, 5⬘-TCTCATGGCAGTTTCTAGGA-3⬘ and 5⬘CCTGGACCTTTCTTGTTCATA-3⬘; Nkg2a, 5⬘- GTAATAGAGCAG
GAAATCGTT-3⬘ and 5⬘- TGGTAGGTTCTCGTCATTGA-3⬘; Nkg2d, 5⬘TCCTCCAGAGATGAGCAAAT-3⬘ and 5⬘-TTCTGTGGAGGG
CGCAGTA-3⬘; Cd45, 5⬘-CGGGGTTGTTCTGTGCTCTGTTC-3⬘ and 5⬘CTTTGCTGTCTTCCTGGGCTTTGT-3⬘. PCR products were resolved by
agarose gel electrophoresis (1% Tris-borate-EDTA).
Results
Generation of the monoclonal alloantibody STOK27 reacting
with a major subset of rat NK cells
In an attempt to identify novel alloreceptors on rat NK cells, we
immunized low NK-alloresponder BN strain rats with alloreactive
and MHC-matched PVG.1N NK cells. The supernatant of one hybridoma, STOK27, labeled a subpopulation of NKR-P1A/B⫹ NK
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
A total of 2.5–10 ⫻ 105 IL-2-activated NK cells or mononuclear cells
(depleted for Ig⫹ cells) from the indicated organs were labeled with different combinations of the following conjugated mAbs: FITC-conjugated
3.2.3 (anti-NKR-P1A/B; Ref. 16), DAR13 (anti-Ly49s3; Ref. 10), STOK2
(anti-Ly49i2; Ref. 24), or STOK27 (anti-NKR-P1C); PE-conjugated G4.18
(anti-CD3; Ref. 25), or 10/78 (anti-NKR-P1A/B; both from BD Pharmingen); and biotinylated STOK27, STOK9 (anti-KLRH1; Ref. 12), DAR13,
or Fly5 (anti-Ly49i5/s5; Ref. 11) followed by RPE-Cy5-conjugated
streptavidin.
performed as described (26). Four micrograms per well of mAb STOK27
or the isotype control mAbs TIM2 (anti-mouse TIM2; a gift from Dr. M.
Daws, Oslo, Norway) or STOK2 was added to plated effector cells 20 –30
min before the addition of target cells. Spontaneous release was usually
below 10% of the total cpm in the cells. Results are presented as mean
values from triplicates for each E:T cell ratio, error bars representing one
SD. A two-sided unpaired t test was used for statistical comparisons of
triplicate values for each E:T ratio (by the built-in analysis package in the
GraphPad Prism software).
IL-2-activated NK cells were generated by positive selection of NKRP1A/B-expressing splenocytes, as described (27). Purified NKR-P1C⫹ and
Ly49s3⫹ NK cells were obtained either by positive or negative selection.
For positive selection, splenocytes were stained with biotinylated mAb
STOK27 or DAR13, washed, incubated with anti-biotin microbeads, separated by a SuperMACS magnetic cell separator (Miltenyi Biotec), and
cultured for 1–2 wk with cRPMI with IL-2. For negative selection, IL-2activated NK cells were generated by adherence to plastic (28). At the day
of the experiment, NKR-P1C⫹ cells were depleted of Ly49s3-expressing
cells using DAR13-precoated rat anti-mouse IgG1 Dynabeads (Dynal).
Conversely, the Ly49s3⫹ cells were depleted of NKR-P1C-expressing
cells using STOK27-precoated sheep-anti rat Dynabeads. Purity was generally ⬎80%.
Concentrations of cytokines were measured by Luminex using the BioPlex cytokine assay (Bio-Rad). NKR-P1C⫹ and Ly49s3⫹ NK cells were
obtained by negative selection, as described above, to avoid mAb-mediated
stimulatory effects before the assay. They were cultured at 1–2 ⫻ 106
cells/well at 37°C for 20 h in 24-well plates before the collection of supernatants. The wells were either left uncoated or were precoated with
purified mAb 3.2.3 alone or in combination with a secondary rabbit
anti-mouse IgG.
The Journal of Immunology
cells by FCM that differed from that seen when staining with available mAbs against the Ly49s3, Ly49i2, and KLRH1 receptors
(Fig. 1A and our unpublished data). These data suggested that the
STOK27⫹ population constituted a novel subset of NK cells in
PVG strain rats. Similar results were obtained in two other high
NK alloresponder strains, AO and WAG. Cells from BN strain rats
as well as from two other low NK-alloresponder strains, DA and
LEW, failed to react with mAb STOK27 (our unpublished data).
A minor population (⬃5%) of freshly isolated mononuclear
splenocytes from PVG.7B rats stained with the mAb STOK27 by
single-color FCM (Fig. 1B). Lymph node (LN) T and B cells and
4135
Characterization of the STOK27-Ag as a novel inhibitory NKRP1 receptor (NKR-P1C)
FIGURE 1. The monoclonal alloantibody STOK27 reacts with a homodimeric membrane glycoprotein expressed by a novel subset of rat NK
cells. mAb STOK27 stains a large subset of IL-2-activated NKR-P1A/B⫹
NK cells (A) as well as minor fractions of mononuclear cells from the
spleen and cervical LNs (B) from PVG.7B strain rats by single-color FCM.
Three-color FCM analysis of mononuclear splenocytes (C) show that a
majority of the NK cells (CD3⫺NKR-P1A/B⫹) and a minor population of
the NK-T cells (CD3⫹NKR-P1A/B⫹) are STOK27⫹, while all the “conventional” T cells (CD3⫹NKR-P1A/B⫺) are negative. D, Immunoprecipitated STOK27-Ag from surface-biotinylated IL-2-activated NK cells migrates as a homodimeric glycoprotein, with single bands at ⬃90 and 45
kDa under nonreducing (Non-Red.) and reducing (Red.) conditions. E, The
STOK27-Ag contains N-linked carbohydrates as revealed by PNGase F
treatment. Incubation in digestion buffer alone (Ctr.) or digestion with Oglycosidase (O-Glycos.), neuraminidase (Neur.), or a combination of the
two (O ⫹ Neur.) was without effect.
Initial biochemical characterization suggested that the STOK27-Ag
exists as a homodimeric membrane glycoprotein in IL-2-activated NK
cells. Protein bands migrated at ⬃45 and 90 kDa under reducing and
nonreducing conditions, respectively, after immunoprecipitation with
mAb STOK27 (Fig. 1D). The 45-kDa band was reduced to ⬃28 kDa
by PNGase F treatment (Fig. 1E), suggesting the existence of abundant N-linked carbohydrates. Treatment with neuraminidase or Oglycosidase was without any apparent effect.
Four cDNAs encoding the STOK27-Ag were isolated by expression cloning in eukaryotic 293T cells using a cDNA library
from PVG NK cells (12). The four cDNAs were identical in the
open reading frame (669 nt) and encoded a novel NKR-P1 protein
of 223 aa that we have termed NKR-P1C (Fig. 2A). At the amino
acid level, this receptor is ⬃70 and 85% identical with the respective NKR-P1A and NKR-P1B receptors. In the lectin-like domain,
NKR-P1C and NKR-P1A or NKR-P1B are 82 and 78% identical,
respectively. Identity is much lower for NKR-P1D being only 48%
(for the lectin-like domain 45%). As shown in Fig. 2B, transient
transfection of 293T cells with an Nkr-p1c construct induced surface expression of the STOK27-Ag, whereas the Nkr-p1a control
construct did not (the latter construct induced NKR-P1A expression in the same cells as judged by staining with mAb 3.2.3; data
not shown). It should be noted that the Nkr-p1c designation has
been used once previously, for an unpublished partial Nkr-p1 clone
from the F344 strain (30). The corresponding gene is present in the
BN strain genome and is most likely a pseudogene, and we have
therefore adopted the Nkr-p1c name for the present sequence
instead. A phylogenetic tree depicts how the full-length NKRP1C protein relates to selected members of other rat KLR families (Fig. 2C).
The cytoplasmic domain of NKR-P1C contains an ITIM, VVYADL (motif-specific amino acids underlined), suggesting inhibitory function (Fig. 2A). It also lacks the positively charged amino
acid in the transmembrane region required for the binding to activating signaling adapters (31, 32). Inhibitory signaling was confirmed by functional studies of the rat NK line, RNK-16, stably
transfected with an Nkr-p1c construct (RNK-16.NKR-P1C transfectants; Fig. 3A). Four individual RNK-16.NKR-P1C clones were
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
thymocytes, in contrast, were mainly STOK27-negative (Fig. 1B
and our unpublished data). Three-color FCM analysis of the
splenocytes revealed that ⬎60% of the NKR-P1A/B⫹CD3⫺ NK
cells were STOK27⫹. There were also a few heterogeneously
stained STOK27⫹ cells among the NKR-P1A/B⫹CD3⫹ T cells
(hereafter referred to as NK-T cells), whereas the “conventional”
NKR-P1A/B⫺CD3⫹ T cells were negative (Fig. 1C).
As can be deduced from Fig. 1, A and C, the proportion of
STOK27⫹ NK cells was consistently higher among freshly isolated splenic NK cells than among IL-2-activated NK cells. Therefore, we considered whether expression of the STOK27-Ag could
be labile, being switched off (or on) during culture. Purified
STOK27⫹ NK cells, however, maintained their positive phenotype
(⬎80%) during the standard culture period of 2 wk. Furthermore,
STOK27⫹ cells failed to reappear within 1 wk after cellular depletion (our unpublished data). Therefore, we concluded that the
STOK27-Ag most likely is a stable marker whose surface expression is fixed during culture. This further implied that the relative
reduction of STOK27⫹ cells among the IL-2-activated NK cells
was likely caused by a preferential expansion of STOK27⫺
(Ly49s3⫹) NK cells.
4136
A NOVEL NKR-P1C⫹, Ly49-NEGATIVE NK CELL SUBSET IN RATS
RNK-16 transfectants, but not in untreated or control transfectants
(Fig. 3C).
The NKR-P1C⫹ NK cell subset is predominantly Ly49 negative
and is complementary to the Ly49s3⫹ NK subset
FIGURE 2. The STOK27-Ag is a novel NKR-P1 molecule, termed
NKR-P1C. A, Deduced amino acid sequence of NKR-P1C. The three
known rat NKR-P1 molecules (NKR-P1A, -B, and -D) are shown as a
comparison. A putative ITIM (VVYADL; motif-specific amino acids underlined) is marked with a dotted line and is present in NKR-P1C and
NKR-P1B. The predicted transmembrane region is underlined; note the
lack of a basic amino acid (Arginine/R), marked with an asterisk (ⴱ), in the
transmembrane region of the two ITIM-containing NKR-P1 molecules. B,
mAb STOK27 stains 293T cells transiently transfected with an NKR-P1C
(clone S27.2.8) expression construct, but not with NKR-P1A as a control.
C, Phylogenetic tree based on analysis of selected rat KLR molecules
(whole protein). It is generated by the neighbor joining method with exclusion of positions with gap and correction for multiple substitutions (essentially the same tree was generated without this exclusion and correction). Bootstrap values are based on 1000 replications and those above 50%
are shown above their corresponding branches. Proteins with the following
accession/version numbers are included in the analysis: NKR-P1A
(AAA41710.1), NKR-P1B (AAB01986.1), NKR-P1D (CAA66111.1),
Ocil (AAK50880.1), Ly49si3 (AAV68593.1), Ly49s2 (AAV74512.1),
Ly49i2 (AAM56042.1), Ly49i8 (AAV74509.1), NKG2A (AAC40050.1),
NKG2C (AAC40049.1), CD94 (AAC10220.1), KLRH1 (AAM22620.1),
KLRE1 (AAO49445.1), NKG2D (AAC40092.1), MAFA (CAA56208.1),
KLRI1 (AAQ20111.1), and KLRI2 (AAR00557.1). Accession number of
the novel Nkr-p1c cDNA sequence is DQ157010 (protein identification
ABA40404). It is identical to the open reading frame of an uncharacterized
Nkr-p1c gene from the BS strain (AF525533).
obtained which killed the FcR⫹ P388.D1 mouse macrophage line
(our unpublished data). Addition of purified mAb STOK27 to the
killing assay resulted in a variable reduction of cytotoxicity, i.e.,
redirected inhibition, as shown in Fig. 3B. Accordingly, immunoprecipitation experiments showed an association of NKR-P1C
with the SHP-1 phosphatase (⬃72 kDa) in pervanadate-treated
We have recently shown that some inhibitory and activating Ly49
receptors are coexpressed in certain subsets of rat NK cells (11). A
striking finding in the present study is that NKR-P1C was expressed mainly by the Ly49-negative fraction of NK cells. Although 64% of splenic NK cells were STOK27⫹, only 13% of the
Ly49s3⫹ cells coexpressed NKR-P1C (Fig. 4A, upper left plot).
The NKR-P1C⫹/Ly49s3⫹ double-positive fraction represented
only 4.5% of the NK cells which is much less than would be
expected from random expression of these two receptors (0.34 ⫻
0.64 ⫻ 100 ⫽ 22%). Similar results were obtained for the Ly49i2
and Ly49i5/s5 receptors, as well as for KLRH1 (Fig. 4A). The
KLRH1 receptor, encoded by a gene located just proximal (centromeric) to the Ly49 region, is slightly more related to the Ly49
receptors than to other KLRs, as visualized by the phylogenetic
tree in Fig. 2C.
Because we only have mAbs to a group of related Ly49 receptors encoded from the same block of Ly49 genes (block 2, 3⬘UTR
type 4) (7), we extended the analysis to more distant Ly49 members by RT-PCR. In these studies, we analyzed Ly49s3⫹ and
NKR-P1C⫹ single-positive splenocytes obtained by FACS sorting,
which together account for ⬃90% of the splenic NK cells (Fig. 4A,
upper left plot). mRNA levels of the Ly49si3 (block 1), Ly49s2
(block 2, 3⬘UTR type 2), and Ly49s1/s7 (block 3) genes were
much higher, by a factor of 100 or more, in the Ly49s3⫹ than the
NKR-P1C⫹ single-positive NK subset (Fig. 4B). The same was
also the case for Ly49i8 gene, which is located in the distal (telomeric) end of the Ly49 region. Ly49i8 is phylogenetically the most
distant Ly49 member, and the only one with a putative homolog in
the mouse genome (Ly49B) (7, 33). The expression of Cd94,
Nkg2a, and Nkg2d was comparable in the two NK subsets (Nkg2a
message was somewhat increased in the NKR-P1C⫹ subset, Fig.
4B). These studies show that the NKR-P1C⫹ NK subset expresses
little Ly49 (and KLRH1) receptors, suggesting the presence of a
common regulatory mechanism for gene expression within the
Ly49 region of the rat NKC. Furthermore, most Ly49-expressing
NK cells apparently coexpress the Ly49s3 receptor which identifies a complementary subset of rat NK cells.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
FIGURE 3. NKR-P1C is an inhibitory receptor associating with the tyrosine phosphatase SHP-1. A, mAb STOK27 stains brightly RNK-16 cells
stably transfected with NKR-P1C (RNK-16.NKR-P1C cells) as detected by
single-color FCM. B, Lysis of FcR⫹ P388 target cells by the NKR-P1CRNK-16 transfectants is reduced by the addition of mAb STOK27 (redirected inhibition), as compared with the isotype control (Ctr.) mAb
STOK2. C, NKR-P1C associates with the 72-kDa tyrosine phosphatase
SHP-1 in pervanadate-treated (Stim.) RNK-16.NKR-P1C transfectants following immunoprecipitation with mAb STOK27, and not with the control
(Ctr.) mAb STOK2. The last lane shows detection of SHP-1 in whole cell
lysate (Wcl).
The Journal of Immunology
NKR-P1C⫹ NK cells secrete cytokines and are cytolytic, but fail
to kill allogeneic Con A lymphoblasts
IL-2-activated NKR-P1C⫹ and Ly49s3⫹ NK cells from PVG.7B
(RT1 haplotype Ac-B/Dc-Cc or c-c-c) rats, obtained by negative
selection were tested for their ability to lyse MHC-allogeneic Con
A lymphoblasts. In accordance with previous findings (10),
Ly49s3⫹ cells effectively lysed the allogeneic PVG.1N (n-n-n),
PVG.R23 (u-a-av1), and DA (a-a-av1) targets, while sparing syngeneic PVG.7B control cells. By contrast, neither allogeneic nor
syngeneic lymphoblasts were killed by NKR-P1C⫹ NK cells (Fig.
5A). Their inability to lyse the allotargets was not caused by a
general nonresponsiveness as they lysed YAC-1 tumor target cells
equally well as the Ly49s3⫹ NK cells (Fig. 5B). Also, they showed
a brisk secretory response of the TNF-␣, GM-CSF, and IFN-␥
cytokines upon receptor stimulation with mAb 3.2.3 (anti-NKRP1A/B; Fig. 5C). Therefore, it is likely that the general lack of
activating Ly49 receptors (see above) explains why they do not
have alloreactive specificities.
FIGURE 5. The NKR-P1C⫹ NK cells are not alloreactive, but kill
YAC-1 tumor cells and secrete cytokines upon stimulation. A and B, Cytolytic activity of the complementary NKR-P1C⫹ (f) and Ly49s3⫹ (E)
PVG.7B NK populations against MHC-mismatched (PVG.R23, PVG.1N,
and DA) Con A lymphoblasts, syngeneic (PVG.7B) control (Ctr.) lymphoblasts, and YAC-1 tumor targets. The NKR-P1C⫹ subset fails to lyse the
allotargets but effectively lyses YAC-1 tumor cells. Results presented are
representative of two to six experiments. The cytolytic activity of NKRP1C⫹ vs Ly49s3⫹ NK cells is statistically different (p ⬍ 0.01) against
PVG.R23, PVG.1N, and DA targets, but not against PVG.7B or YAC-1. C,
Cytokine production by NKR-P1C⫹ and Ly49s3⫹ NK cells. Cells were
stimulated with mAb 3.2.3, either alone (mAb 3.2.3) or cross-linked with
a secondary anti-mouse Ab (3.2.3 ⫹ anti-mIg), or they were kept in medium as a control (Ctr.). Concentration (picograms per milliliter) of the
indicated cytokines was determined in the supernatant, which was harvested after 20 h culture and analyzed by a multiplex bead assay (Luminex). Values above scale are indicated with an asterisk (ⴱ).
NKR-P1C⫹ NK cells bind a conserved ligand on Con A
lymphoblasts
In mice, the Clr ligands for mouse NKR-P1 receptors are present
on normal hemopoietic cells (21, 22). Therefore, we tested normal
lymphoblast targets for a functional inhibitory ligand for NKRP1C. The NKR-P1C⫹ NK cells did not kill syngeneic (PVG.7B) or
MHC-allogeneic (PVG.1U, PVG.1L, PVG.R23, PVG.1N, and
DA) lymphoblasts. Cytotoxicity against all the targets was induced
by addition of mAb STOK27 irrespective of the target MHC haplotype (Fig. 6A and our unpublished data). By contrast, mAb
STOK27 had no effect on the cytolytic activity of Ly49s3⫹ NK
cells (Fig. 6B). These results suggested that NKR-P1C binds a
monomorphic ligand, possibly a Clr ligand, on the Con A blasts.
NKR-P1C⫹ NK cells appear early during ontogeny and show a
distinct in vivo tissue distribution as compared with Ly49expressing NK cells
Previous studies in mice have shown that NK cells acquire Ly49
receptors relatively late in ontogeny (34). This also appears to be
the case in rats. As visualized in Fig. 7A, the percentage of
Ly49s3⫹ cells was low (6.3%) among freshly isolated splenic NK
cells from 3-day-old rats. It increased to 28% at 4 wk of age,
similar to the levels seen in young adult (8-wk-old) rats (Fig. 7B).
By contrast, the proportion of NKR-P1C⫹ NK cells was normal,
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
FIGURE 4. The NKR-P1C⫹ NK subset expresses little Ly49 receptors
and is complementary to the Ly49s3⫹ NK subset. A, NK cells (NKR-P1A/
Bbright cells) from the spleen of PVG.7B rats were analyzed by three-color
FCM for coexpression of NKR-P1C, Ly49 molecules, and KLRH1. B,
NKR-P1C⫹ and Ly49s3⫹ single-positive splenic NK cells were analyzed
for relative expression levels of the indicated mRNAs by semiquantitative
RT-PCR. The two complementary subpopulations account for almost 90%
of the NK cells and were obtained by FACS-sorting using the gates indicated in the upper left FACS plot in A. Ten-fold dilutions (⫺1, ⫺2, ⫺3,
and ⫺4) of cDNA were analyzed, with CD45 used as a cDNA loading
control.
4137
4138
A NOVEL NKR-P1C⫹, Ly49-NEGATIVE NK CELL SUBSET IN RATS
FIGURE 6. NKR-P1C⫹ NK cells bind a monomorphic ligand on Con A lymphoblasts. A, Blocking of the NKR-P1C receptor with mAb STOK27 (4
␮g/well) induces killing of both syngeneic (PVG.7B) and MHC-allogeneic (PVG.1U, PVG.1L, and PVG.R23) Con A lymphoblasts by NKR-P1C⫹ NK
cells from PVG.7B rats, as compared with an isotype control (Ctr.) mAb. B, Addition of mAb STOK27 has no effect on the cytolytic activity of Ly49s3⫹
NK cells against either MHC-matched (PVG.7B) or MHC-mismatched (PVG.R23) blast targets. Results presented are representative of two to six
experiments. Addition of mAb STOK27 statistically alters (p ⬍ 0.01) cytolytic activity of NKR-P1C⫹ NK cells against all targets, but not of the Ly49s3⫹
NK cells (no significant difference).
FIGURE 7. NKR-P1C⫹ NK cells
appear earlier during ontogeny and
have a different tissue distribution compared with Ly49s3⫹ NK cells. A, NK
cells (NKR-P1A/Bbright cells) from the
spleen of PVG.7B rats at the indicated
ages were analyzed by three-color FCM
for expression of NKR-P1C (C⫹) and
Ly49s3 (s3⫹), results are summarized
in B. The rats were used at 1, 3, and 7
days (d1, d3, d7) and 4 and 8 wk (4 and
8 wk.) after birth. C, Expression of
NKR-P1C and Ly49s3 by NK cells
(NKR-P1A/Bbright cells) from the
spleen, bone marrow (BM), cervical
and mesenteric lymph nodes (LN) of
2-mo-old PVG.7B rats. Mean fluorescence intensities (x- and y-mean) of the
NKR-P1C and Ly49s3 single-positive
cells are shown. In A and C are shown
plots/values representative of two and five
experiments, respectively. In B are presented median values from two to four
experiments.
NKR-P1C, in contrast, was higher in mesenteric LNs than in the
other three tissues (y-mean intensity 126 vs 55–71; Fig. 7C). Together, with the results presented above, these data suggest that the
two complementary NK cell subsets may perform different functions in vivo.
Discussion
We have identified an inhibitory NKR-P1 molecule (NKR-P1C)
with a new monoclonal alloantibody generated in BN rats. This
strain was chosen for immunization because it has a limited NK
allorecognition repertoire compared with PVG strain rats. Thus,
while BN strain NK cells only kill allogeneic lymphoblasts expressing the u-u-u rat MHC haplotype (35), PVG.1N NK cells lyse
targets from haplotypes u-u-u, l-l-lv1, l-l-l, and a-a-av1 (our unpublished data). This difference could not be attributed to an MHC
effect during NK maturation as the two strains are MHC identical.
Rather, we suspected that it was due to the lack of certain activating alloreceptors, reminiscent of the functionally described Nka
alloresponder gene(s) in the Ly49 region of PVG but not DA rats
(4). Although the immunization protocol was aimed at identifying
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
i.e., with more than two-thirds being positive in neonatal rats, and
remained at comparable levels until they reached the adult stage at
8 wk of age. At all ages, there were few Ly49s3⫹/NKR-P1C⫹
double-positive cells (Fig. 7, A and B). These data suggest that
maturation of the NKR-P1C⫹ NK subset precedes that of the Ly49
subset(s) during ontogeny.
In addition, in vivo distribution of the two complementary NK
cell subsets differed markedly. In BM, the proportion of Ly49s3⫹
NK cells was consistently increased compared with the spleen (41
vs 30%) and that of the NKR-P1C⫹ NK cells was comparably
reduced (53 vs 65%). The opposite pattern was observed in mesenteric LNs where there was a striking reduction of the Ly49s3⫹
NK subset (14 vs 30%) as well as a marked increase of the NKRP1C⫹ NK subset vs the spleen (85 vs 65%). In cervical LNs, the
composition was approximately the same as that seen in the spleen
(Fig. 7C). The distribution of the Ly49i2 and Ly49i5/s5 NK subsets followed that of the Ly49s3⫹ cells (our unpublished data).
There were also associated differences in receptor expression
levels at the different locations. Ly49s3⫹ cells from the spleen and
BM expressed higher levels of Ly49s3 than from mesenteric and
cervical LNs (x-mean intensity ⬃680 vs 360). Expression level of
The Journal of Immunology
methylation, histone modification, or changes in higher-order
chromatin structure (45).
The NKR-P1C-encoding gene is lacking in the genome from the
BN rat strain, explaining why we were able to generate a mAb to
NKR-P1C in BN rats. The BN strain harbors the three previously
published Nkr-p1 genes encoding the NKR-P1A (activating; Refs.
17 and 18), NKR-P1B (inhibitory; Refs. 4 and 19), and NKR-P1D
(presumably activating; Ref. 46) molecules, as well as a novel
fourth Nkr-p1 gene with inhibitory structural features (our unpublished data). Hence, there are at least five different Nkr-p1 genes in
rats, but little is known about their cellular distribution and function. As shown in the present study, NKR-P1C appears to be restricted to NK cells, and mainly to a Ly49-negative fraction with
unique functions. The two available anti-NKR-P1 mAbs (3.2.3 and
10/78) do not distinguish between the NKR-P1A and NKR-P1B
molecules. They are generally used as pan-NK cell reagents, but
they also stain a significant proportion of CD8⫹ T cells. Generation of locus-specific reagents will aid the characterization of the
rat NKR-P1 family.
The identity of the NKR-P1 ligands has long remained elusive.
Candidates have been both histocompatibility Ags (20) and simple
saccharide moieties, but the natural ligands for NKR-P1 receptors
were recently shown to be the Clr molecules, which are encoded
within the NKR-P1 genomic region. Although the ligand for NKRP1C has not yet been identified, the functional data suggest it will
be one of the 5–10 uncharacterized Clr molecules present in the
BN strain genome. Blocking of NKR-P1C with mAb STOK27
induced modest lysis of allogeneic and syngeneic lymphoblasts
irrespective of their MHC haplotype, pointing to the recognition of
a monomorphic target cell ligand. This is compatible with NKRP1C functioning as a “missing self” receptor for a non-MHC ligand, likely a conserved Clr molecule. Assuming that this is the
case, its negative regulation of NK cytotoxicity must operate independently of any MHC-dependent inhibitory receptors present
on the same cells. Comparable levels of Cd94 and Nkg2a message
were observed in the NKR-P1C⫹ and Ly49s3⫹ NK subsets, suggesting that many of the NKR-P1C⫹ cells coexpress a conserved
inhibitory CD94/NKG2A receptor which is likely to bind the rat
ortholog of mouse Qa1 (i.e., RT-BM1). NKR-P1C may be important for the maintenance of self-tolerance in the NKR-P1C⫹ subset, and the ontogenetic data suggest that it may be important already at an early stage of development.
It is likely that the observed functional differences between the
NKR-P1C⫹ and Ly49s3⫹ NK cells are a reflection of their functions in vivo. This is compatible with their distinct tissue distribution. The marked predominance of the NKR-P1C⫹ subset in the
mesenteric LNs is striking, but it can only be speculated as to
whether this is due to preferential recruitment of these cells, exclusion of the Ly49s3⫹ cells, or enhanced in situ development. The
involvement of various chemokines and their receptors in the trafficking of these NK subsets remains to be determined. It is also
tempting to draw a comparison with subsets in other species, notably the CD56bright and CD56dim cells in humans (47). The
CD56bright cells lack KIR expression, and are preferentially located in the LNs, where they apparently can develop from precursors (48). Future studies will have to be performed to further clarify the functional and phenotypic differences between the two
unique subsets in rats.
Acknowledgments
We thank Bente Kahrs Omdal for excellent technical assistance,
Dennis Buurman (Bio-Rad) for help with multiplex cytokine measurement,
and Dr. Michael Daws (Oslo, Norway) for valuable discussions and critical
comments of the manuscript.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
these activating alloreceptors, we instead obtained the anti-NKRP1C mAb STOK27, reactive against a unique subset of nonalloreactive, Ly49-negative NK cells.
The NKR-P1C⫹ and Ly49s3⫹ NK cell subsets are largely complementary, i.e., with rare cells (⬍5%) being NKR-P1C/Ly49s3
double positive. Together, the two subsets account for ⬎90% of
the NK cell pool in PVG strain rats. They also express very different KLR repertoires, which may explain their observed functional differences, such as the inability of the NKR-P1C⫹ subset to
kill allogeneic cells. It is unlikely that the NKR-P1C receptor specifically inhibits alloactivation of this subset, because blockade
with mAb STOK27 equally affects killing of syngeneic and allogeneic targets, and fails to restore allokilling to levels seen for
Ly49s3⫹ NK cells. Rather, the defect in killing of allogeneic targets by the NKR-P1C⫹ subset is likely related to the generally
limited expression of Ly49 receptors in this subset.
We have recently estimated the number of functional, nonallelic
Ly49 genes in the rat to be ⬃25 (7). In the present study, we tested
for 12 of them by FCM or RT-PCR, and they were all preferentially expressed in the Ly49s3⫹ NK subset. In aggregate, our available mAbs react with all block 2 Ly49 molecules (7), i.e., Ly49i2/
i3/i4/i5 and Ly49s3/s4/s5, except for Ly49s2, which was assayed
by RT-PCR together with selected genes from block 1 (Ly49si3),
block 3 (Ly49s1/s7), and with Ly49i8 (orthologous to mouse
Ly49b). It can be concluded from these studies that NKR-P1C⫹
NK cells express little Ly49 receptors and that Ly49s3 is a good
overall marker for Ly49-expressing NK cells, at least in high NK
alloresponder rat strains such as PVG.
The Ly49 receptors in rodents and the killer cell Ig-like receptors (KIRs) in humans are functional analogs and bind polymorphic MHC class I epitopes. Similar to the marked haplotypic variation of the KIRs, the Ly49 repertoire shows marked differences
both in gene and allele content between inbred strains of rats and
mice (7, 36, 37). Such polymorphisms are a prerequisite for NK
alloreactivity. Previous studies have shown a marked enrichment
of NK alloreactivity in selected Ly49 NK subsets both in rats (10,
24) and mice (38, 39). Similarly, NK alloreactivity in humans is
associated with the individually specific repertoire of KIR receptors (40). The precise contribution of inhibitory and activating
Ly49 and KIR receptors to NK-mediated alloreactivity remains to
be determined. Studies in rats have pointed toward a central role
for the activating receptors (10, 11). Therefore, it is possible that
transplant patients lacking activating KIRs have a reduced risk of
rejection of NK-incompatible (KIR/HLA) stem cell grafts in the
clinical setting, especially under nonmyeloablative regimens. This
is relevant as the activating Ly49 and KIR receptors are often
inactivated by mutation in different individuals of the species (7,
36), possibly because their potential involvement in autoimmunity
has led to negative selection during evolution (41).
In the mouse, Ly49 receptors are expressed by a stochastic
mechanism (34, 42). Recently, it has been shown that they have an
upstream bidirectional promoter “switch” that is active during
early NK development and determines expression of individual
Ly49 receptors in a stochastic fashion (43, 44). Other mechanisms
must be sought, however, to explain why the NKR-P1C⫹ NK cells
show such limited expression of Ly49 (and KLRH1) receptors
spanning ⬃1.8 Mb of the rat genome, i.e., from the telomeric
(Ly49i8) to the centromeric end (KLRH1) of the Ly49 genomic
region. This suggests the presence of some dominant regulatory
mechanism affecting gene expression of this region of the rat
NKC. This could be a suppressor molecule binding to a common
regulatory element or some sort of epigenetic process inhibiting
access of transcription factors to the regulatory regions, e.g., DNA
4139
4140
A NOVEL NKR-P1C⫹, Ly49-NEGATIVE NK CELL SUBSET IN RATS
Disclosures
The authors have no financial conflict of interest.
References
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
1. Yokoyama, W. M., and B. F. M. Plougastel. 2003. Immune functions encoded by
the natural killer gene complex. Nat. Rev. Immunol. 3: 304 –316.
2. Lanier, L. L. 2005. NK cell recognition. Annu. Rev. Immunol. 23: 225–274.
3. Raulet, D. H., R. E. Vance, and C. W. McMahon. 2001. Regulation of the natural
killer cell receptor repertoire. Annu. Rev. Immunol. 19: 291–330.
4. Dissen, E., J. C. Ryan, W. E. Seaman, and S. Fossum. 1996. An autosomal
dominant locus, Nka, mapping to the Ly-49 region of a rat natural killer (NK)
gene complex, controls NK cell lysis of allogeneic lymphocytes. J. Exp. Med.
183: 2197–2207.
5. Brown, M. G., A. A. Scalzo, K. Matsumoto, and W. M. Yokoyama. 1997. The
natural killer gene complex: a genetic basis for understanding natural killer cell
function and innate immunity. Immunol. Rev. 155: 53– 65.
6. Westgaard, I. H., S. F. Berg, S. Ørstavik, S. Fossum, and E. Dissen. 1998. Identification of a human member of the Ly-49 multigene family. Eur. J. Immunol.
28: 1839 –1846.
7. Nylenna, Ø., C. Naper, J. T. Vaage, P. Y. Woon, D. Gauguier, E. Dissen,
J. C. Ryan, and S. Fossum. 2005. The genes and gene organization of the Ly49
region of the rat natural killer cell gene complex. Eur. J. Immunol. 35: 261–272.
8. Naper, C., J. C. Ryan, R. Kirsch, G. W. Butcher, B. Rolstad, and J. T. Vaage.
1999. Genes in two major histocompatibility complex class I regions control
selection, phenotype, and function of a rat Ly-49 natural killer cell subset. Eur.
J. Immunol. 29: 2046 –2053.
9. Naper, C., S. Hayashi, E. Joly, G. W. Butcher, B. Rolstad, J. T. Vaage, and
J. C. Ryan. 2002. Ly49i2 is an inhibitory rat natural killer cell receptor for an
MHC class Ia molecule (RT1–A1c). Eur. J. Immunol. 32: 2031–2036.
10. Naper, C., S. Hayashi, L. Kveberg, E. C. Niemi, L. L. Lanier, J. T. Vaage, and
J. C. Ryan. 2002. Ly-49s3 is a promiscuous activating rat NK cell receptor for
nonclassical MHC class I-encoded target ligands. J. Immunol. 169: 22–30.
11. Naper, C., K.-Z. Dai, L. Kveberg, B. Rolstad, E. C. Niemi, J. T. Vaage, and
J. C. Ryan. 2005. Two structurally related rat Ly49 receptors with opposing
functions (Ly49 stimulatory receptor 5 and Ly49 inhibitory receptor 5) recognize
nonclassical MHC class Ib-encoded target ligands. J. Immunol. 174: 2702–2711.
12. Naper, C., S. Hayashi, G. Løvik, L. Kveberg, E. C. Niemi, B. Rolstad, E. Dissen,
J. C. Ryan, and J. T. Vaage. 2002. Characterization of a novel killer cell lectinlike receptor (KLRH1) expressed by alloreactive rat NK cells. J. Immunol. 168:
5147–5154.
13. Saether, P. C., I. H. Westgaard, L. M. Flornes, S. E. Hoelsbrekken, J. C. Ryan,
S. Fossum, and E. Dissen. 2005. Molecular cloning of KLRI1 and KLRI2, a novel
pair of lectin-like natural killer-cell receptors with opposing signalling motifs.
Immunogenetics 56: 833– 839.
14. Zhou, H., V. Kartsogiannis, Y. S. Hu, J. Elliott, J. M. W. Quinn, W. J. McKinstry,
M. T. Gillespie, and K. W. Ng. 2001. A novel osteoblast-derived C-type lectin
that inhibits osteoclast formation. J. Biol. Chem. 276: 14916 –14923.
15. Plougastel, B., C. Dubbelde, and W. M. Yokoyama. 2001. Cloning of Clr, a new
family of lectin-like genes localized between mouse Nkrp1a and Cd69. Immunogenetics 53: 209 –214.
16. Chambers, W. H., N. L. Vujanovic, A. B. DeLeo, M. W. Olszowy,
R. B. Herberman, and J. C. Hiserodt. 1989. Monoclonal antibody to a triggering
structure expressed on rat natural killer cells and adherent lymphokine-activated
killer cells. J. Exp. Med. 169: 1373–1389.
17. Giorda, R., W. A. Rudert, C. Vavassori, W. H. Chambers, J. C. Hiserodt, and
M. Trucco. 1990. NKR-P1, a signal transduction molecule on natural killer cells.
Science 249: 1298 –1300.
18. Ryan, J. C., E. C. Niemi, R. D. Goldfien, J. C. Hiserodt, and W. E. Seaman. 1991.
NKR-P1, an activating molecule on rat natural killer cells, stimulates phosphoinositide turnover and a rise in intracellular calcium. J. Immunol. 147:
3244 –3250.
19. Li, J., B. A. Rabinovich, R. Hurren, J. Shannon, and R. G. Miller. 2003. Expression cloning and function of the rat NK activating and inhibitory receptors NKRP1A and -P1B. Int. Immunol. 15: 411– 416.
20. Ryan, J. C., E. C. Niemi, M. C. Nakamura, and W. E. Seaman. 1995. NKR-P1A
is a target-specific receptor that activates natural killer cell cytotoxicity. J. Exp.
Med. 181: 1911–1915.
21. Iizuka, K., O. V. Naidenko, B. F. M. Plougastel, D. H. Fremont, and
W. M. Yokoyama. 2003. Genetically linked C-type lectin-related ligands for the
NKRP1 family of natural killer cell receptors. Nat. Immunol. 4: 801– 807.
22. Carlyle, J. R., A. M. Jamieson, S. Gasser, C. S. Clingan, H. Arase, and
D. H. Raulet. 2004. Missing self-recognition of Ocil/Clr-b by inhibitory NKR-P1
natural killer cell receptors. Proc. Natl. Acad. Sci. USA 101: 3527–3532.
23. Kumar, V., and M. E. McNerney. 2005. A new self: MHC-class-I-independent
natural-killer-cell self-tolerance. Nat. Rev. Immunol. 5: 363–374.
24. Naper, C., J. C. Ryan, M. C. Nakamura, D. Lambracht, B. Rolstad, and
J. T. Vaage. 1998. Identification of an inhibitory MHC receptor on alloreactive rat
natural killer cells. J. Immunol. 160: 219 –224.
25. Nicolls, M. R., G. G. Aversa, N. W. Pearce, A. Spinelli, M. F. Berger,
K. E. Gurley, and B. M. Hall. 1993. Induction of long-term specific tolerance to
allografts in rats by therapy with an anti-CD3-like monoclonal antibody. Transplantation 55: 459 – 468.
26. Vaage, J. T., C. Naper, G. Løvik, D. Lambracht, A. Rehm, H. J. Hedrich,
K. Wonigeit, and B. Rolstad. 1994. Control of rat natural killer cell-mediated
allorecognition by a major histocompatibility complex region encoding nonclassical class I antigens. J. Exp. Med. 180: 641– 651.
27. Naper, C., J. T. Vaage, D. Lambracht, G. Løvik, G. W. Butcher, K. Wonigeit, and
B. Rolstad. 1995. Alloreactive natural killer cells in the rat: complex genetics of
major histocompatibility complex control. Eur. J. Immunol. 25: 1249 –1256.
28. Vujanovic, N. L., R. B. Herberman, A. A. Maghazachi, and J. C. Hiserodt. 1988.
Lymphokine-activated killer cells in rats. III. A simple method for the purification
of large granular lymphocytes and their rapid expansion and conversion into
lymphokine-activated killer cells. J. Exp. Med. 167: 15–29.
29. Ryan, J. C., E. C. Niemi, and M. C. Nakamura. 2000. Functional analysis of
natural killer cell receptors in the RNK-16 rat leukemic cell line. Methods Mol.
Biol. 121: 283–295.
30. Ryan, J. C., and W. E. Seaman. 1997. Divergent functions of lectin-like receptors
on NK cells. Immunol. Rev. 155: 79 – 89.
31. 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 –137.
32. Lanier, L. L. 2001. On guard—activating NK cell receptors. Nat. Immunol. 2:
23–27.
33. Hao, L., and M. Nei. 2004. Genomic organization and evolutionary analysis of
Ly49 genes encoding the rodent natural killer cell receptors: rapid evolution by
repeated gene duplication. Immunogenetics 56: 343–354.
34. Kubota, A., S. Kubota, S. Lohwasser, D. L. Mager, and F. Takei. 1999. Diversity
of NK cell receptor repertoire in adult and neonatal mice. J. Immunol. 163:
212–216.
35. Løvik, G., J. T. Vaage, C. Naper, H. B. Benestad, and B. Rolstad. 1995. Recruitment of alloreactive natural killer cells to the rat peritoneum by a transfected cell
line secreting rat recombinant interleukin-2. J. Immunol. Methods 179: 59 – 69.
36. Anderson, S. K., K. Dewar, M.-L. Goulet, G. Leveque, and A. P. Makrigiannis.
2005. Complete elucidation of a minimal class I MHC natural killer cell receptor
haplotype. Genes Immun. 6: 481– 492.
37. Makrigiannis, A. P., D. Patel, M.-L. Goulet, K. Dewar, and S. K. Anderson. 2005.
Direct sequence comparison of two divergent class I MHC natural killer cell
receptor haplotypes. Genes Immun. 6: 71– 83.
38. Lindberg, J., A. Martı́n-Fontecha, and P. Höglund. 1999. Natural killing of MHC
class I⫺ lymphoblasts by NK cells from long-term bone marrow culture requires
effector cell expression of Ly49 receptors. Int. Immunol. 11: 1239 –1246.
39. Olsson, M. Y., K. Kärre, and C. L. Sentman. 1995. Altered phenotype and function of natural killer cells expressing the major histocompatibility complex receptor Ly-49 in mice transgenic for its ligand. Proc. Natl. Acad. Sci. USA 92:
1649 –1653.
40. Valiante, N. M., M. Uhrberg, H. G. Shilling, K. Lienert-Weidenbach,
K. L. Arnett, A. D’Andrea, J. H. Phillips, L. L. Lanier, and P. Parham. 1997.
Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors. Immunity 7: 739 –751.
41. Parham, P. 2005. MHC class I molecules and KIRs in human history, health and
survival. Nat. Rev. Immunol. 5: 201–214.
42. Held, W., and D. H. Raulet. 1997. Expression of the Ly49A gene in murine
natural killer cell clones is predominantly but not exclusively mono-allelic. Eur.
J. Immunol. 27: 2876 –2884.
43. Saleh, A., A. P. Makrigiannis, D. L. Hodge, and S. K. Anderson. 2002. Identification of a novel Ly49 promoter that is active in bone marrow and fetal thymus.
J. Immunol. 168: 5163–5169.
44. Saleh, A., G. E. Davies, V. Pascal, P. W. Wright, D. L. Hodge, E. H. Cho,
S. J. Lockett, M. Abshari, and S. K. Anderson. 2004. Identification of probabilistic transcriptional switches in the Ly49 gene cluster: a eukaryotic mechanism
for selective gene activation. Immunity 21: 55– 66.
45. Wilson, C. B., K. W. Makar, M. Shnyreva, and D. R. Fitzpatrick. 2005. DNA
methylation and the expanding epigenetics of T cell lineage commitment. Semin.
Immunol. 17: 105–119.
46. Appasamy, P. M., T. W. Kenniston, C. S. Brissette-Storkus, and
W. H. Chambers. 1996. NKR-P1dim/TCR␣␤⫹ T cells and natural killer cells
share expression of NKR-P1A and NKR-P1D. Nat. Immun. 15: 259 –268.
47. Cooper, M. A., T. A. Fehniger, and M. A. Caligiuri. 2001. The biology of human
natural killer-cell subsets. Trends Immunol. 22: 633– 640.
48. Freud, A. G., B. Becknell, S. Roychowdhury, H. C. Mao, A. K. Ferketich,
G. J. Nuovo, T. L. Hughes, T. B. Marburger, J. Sung, R. A. Baiocchi, et al. 2005.
A human CD34⫹ subset resides in lymph nodes and differentiates into CD56bright
natural killer cells. Immunity 22: 295–304.