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IMMUNOBIOLOGY
KLRL1, a novel killer cell lectinlike receptor, inhibits natural killer cell
cytotoxicity
Yanmei Han, Minghui Zhang, Nan Li, Taoyong Chen, Yi Zhang, Tao Wan, and Xuetao Cao
Natural killer (NK) cell inhibitory receptors play important roles in the regulation
of target susceptibility to natural killing.
Here, we report the molecular cloning and
functional characterization of a novel NK
cell receptor, KLRL1, from human and
mouse dendritic cells. KLRL1 is a type II
transmembrane protein with an immunoreceptor tyrosine-based inhibitory motif
and a C-type lectinlike domain. The KLRL1
gene is located in the central region of the
NK gene complex in both humans and
mice, on human chromosome 12p13 and
mouse chromosome 6F3, adjacent to the
other KLR genes. KLRL1 is preferentially
expressed in lymphoid tissues and immune
cells, including NK cells, T cells, dendritic
cells, and monocytes or macrophages.
Western blot and fluorescence confocal microscopy analyses indicated that KLRL1 is
a membrane-associated glycoprotein, which
forms a heterodimer with an as yet unidentified partner. Human and mouse KLRL1 are
both predicted to contain putative immunoreceptor tyrosine-based inhibitory motifs
(ITIMs), and immunoprecipitation experi-
ments demonstrated that KLRL1 associates
with the tyrosine phosphatases SHP-1 (SH2domain-containing protein tyrosine phosphatase 1) and SHP-2. Consistent with its
potential inhibitory function, pretreatment
of target cells with human KLRL1-Fc fusion
protein enhances NK-mediated cytotoxicity.
Taken together, our results demonstrate that
KLRL1 belongs to the KLR family and is a
novel inhibitory NK cell receptor. (Blood.
2004;104:2858-2866)
© 2004 by The American Society of Hematology
Introduction
Natural killer (NK) cells are crucial for innate host defense against
certain tumor cells and pathogens and, in particular, against viral
infections.1 The susceptibility of tumor targets to natural killing is
inversely related to target-cell expression of major histocompatibility complex (MHC) class I molecules, which formed the basis for
the “missing-self ” hypothesis.2 Missing-self is now explained by
the expression of NK cell inhibitory receptors specific for MHC
class I molecules.3,4 In humans, there are 3 distinct families of
inhibitory receptors for HLA class I molecules: (1) killer cell
Ig-like receptors (KIRs), which are type I transmembrane molecules belonging to the immunoglobulin (Ig) superfamily5; (2)
immunoglobulin-like transcripts (ILTs), which are expressed mainly
on B, T, and myeloid cells, although some members are also
expressed on NK cells6; (3) killer cell lectinlike receptors (KLRs),
which are type II transmembrane glycoproteins encoded by the NK
gene complex (NKC).7-9 Inhibitory receptors mediate their effects
through the immunoreceptor tyrosine-based inhibitory motif(s)
(ITIM) present in their cytoplasmic domain,3 which become(s)
tyrosine phosphorylated by a src-family tyrosine kinase on ligand
binding. The src-family tyrosine kinases include SH2-domain–
containing protein tyrosine phosphatase 1 (SHP-1), SHP-2, and
SH2-domain–containing inositol polyphosphate 5⬘ phosphatase
(SHIP1). SHP-1, in particular, has been demonstrated to associate
with phosphorylated ITIMs and to mediate inhibition of NK cell
cytotoxicity.
Several different NKC-encoded KLR families have thus far
been identified; members are generally activating, inhibitory, or
costimulatory receptors. With the exception of NKG2 (natural
killer group 2), most KLRs are orphan receptors, whose physiologic ligands or functions remain undefined or have not been
directly determined. The human NKG2A-CD94 (KLRC1) and
NKG2C-CD94 (KLRC2) heterodimers recognize the nonclassic
MHC class I molecule, HLA-E (Qa-1 in mice), which primarily
displays peptides derived from the signal peptides of classic MHC
class I molecules.10 The interactions of NKG2-CD94 heterodimers
with HLA-E or Qa-1 molecules allow NK cells to indirectly
monitor the expression of classic MHC class I molecules. Human
and mouse KLRs orthologs have a broad expression pattern, which
includes both NK and T-cell subsets.11 NKG2A-CD94 receptor
expression is up-regulated by antiviral CD8⫹ T cells during acute
polyoma infection; this is responsible for down-regulating their
antigen-specific cytotoxicity during both viral clearance and virusinduced oncogenesis.12 CD94/NKG2 expression is also observed
on antigen (Ag)–specific CD8⫹ T cells following infection with
influenza virus and Listeria monocytogenes, but in these infections
binding of the CD94/NKG2A receptor by its ligand (Qa-1b) does
not significantly inhibit CD8⫹ T cells. CD94/NKG2A-mediated
inhibition of T cells may thus be limited to particular circumstances
or may involve synergy with other receptors that are similarly
up-regulated.13,14 NKG2D (KLRK1) molecules are also expressed
by T cells, mediating costimulatory functions dependent on the
availability of the adaptor protein DAP10,15 and emerging findings
also indicate that primed T cells might express other NKC
molecules, including the Ly49 family and KLRG1.16,17 However,
From the Institute of Immunology, Second Military Medical University,
Shanghai, China.
Reprints: Xuetao Cao, Institute of Immunology, Second Military Medical
University, 800 Xiangyin Rd, Shanghai 200433, China; e-mail:
[email protected].
Submitted March 8, 2004; accepted June 29, 2004. Prepublished online as
Blood First Edition Paper, July 6, 2004; DOI 10.1182/blood-2004-03-0878.
Supported by grants from the National Natural Science Foundation of China
(30121002), the National Key Basic Research Program of China
(2001CB510002), and the National High Biotechnology Development Program
of China (2002BA711A01).
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The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2004 by The American Society of Hematology
BLOOD, 1 NOVEMBER 2004 䡠 VOLUME 104, NUMBER 9
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BLOOD, 1 NOVEMBER 2004 䡠 VOLUME 104, NUMBER 9
the role of these molecules in modulating the function of antigenspecific T cells requires further evaluation.
In this study, we report the molecular cloning, tissue and cell
distribution, chromosome arrangement, and functional analysis of a
novel NK cell receptor, KLRL1, derived from human or mouse
dendritic cells. The predicted protein is a type II transmembrane
protein that contains an ITIM in the cytoplasmic tail and belongs to
the KLR family. We demonstrate that KLRL1 associates with the
tyrosine phosphatases SHP-1 and SHP-2 and inhibits NK cell
cytotoxicity.
Materials and methods
Cell culture
Unless stated otherwise, cell lines were obtained from the American Type
Culture Collection (Manassas, VA) and maintained in RPMI 1640 medium
(Invitrogen, Carlsbad, CA) supplemented with 2 mM glutamine, penicillin
(100 U/mL), streptomycin (100 ␮g/mL), and 10% (vol/vol) heatinactivated fetal calf serum (FCS; Hyclone, Logan, UT) in a 37°C 5% CO2
atmosphere. The NK-92 cell line, a kind gift of Prof Zhigang Tian (School
of Life Sciences, University of Science and Technology of China, Hefei, PR
China), was cultured in Minimum Essential Medium Alpha Medium
(Invitrogen) containing 12.5% horse serum, 12.5% heat-inactivated FCS,
0.1 mM 2-ME (2-mercaptoethanol), 0.2 mM inositol, 0.02 mM folic acid,
and 100 IU/mL human interleukin 2 (IL-2) (Sigma, St Louis, MO). Human
polyclonal NK cells were isolated from peripheral mononuclear cells of
healthy donors by using an NK cell isolation kit (Miltenyi Biotec, Auburn,
CA). Human NK cells were cultured in Iscove modified Dulbecco medium
(IMDM; Hyclone) supplemented with 500 IU/mL IL-2, 10% heatinactivated FCS, and 2 mM glutamine. Mouse NK cells, CD4⫹ T cells, and
CD8⫹ T cells were isolated from mouse splenocytes by positive selection
with CD45R (B220), CD49b (DX5), CD4, and CD8a⫹ MicroBeads
(Miltenyi Biotec).
Cloning of human and mouse KLRL1 full-length cDNA
The main expression sequence tagged (EST) of human KLRL1 (hKLRL1)
was directly isolated from a human dendritic cell (DC) cDNA library by
large-scale random sequencing as described previously.18,19 Full-length
cDNA was cloned from human DCs by using the polymerase chain reaction
(PCR) primers 5⬘-ACGAATTCATGTCTGAAGAAGTTACTTA-3⬘ (sense)
and 5⬘-TCAAGCTTGCCTCC CTAAAATATGTAG-3⬘ (antisense) and Advantage polymerase (Clontech, Palo Alto, CA). The PCR product was
cloned into the vector pcDNA3.1/Myc-His (⫺) B (Invitrogen) and sequenced. The full-length sequence is available in GenBank under the
accession no. AF247788. The murine homolog of hKLRL1 cDNA,
obtained by reverse transcriptase (RT)–PCR from mouse DC using the
primers 5⬘-ACGAATTCAATGTCTGAAGAAATTGTT-3⬘ (sense) and 5⬘TCAAGCTTCTG TATCCTCTGGGAGGC-3⬘ (antisense) was designated
as murine KLRL1 (mKLRL1). The full-length sequence of mKLRL1 is
available in GenBank under the accession no. NM_177686.
Cellular and tissue distribution of human and mouse KLRL1
Total cellular RNA was isolated using Trizol reagent (Invitrogen), and
first-strand cDNA was prepared by using the Superscript II system with an
Oligo(dT)15 primer (Invitrogen). cDNA synthesis was checked by PCR,
with human or mouse ␤-actin primers as a positive control. Human adult
multiple tissue cDNA (MTC) panels were purchased from Clontech.
RT-PCR was performed with primers specific for hKLRL1 and mKLRL1 as
described earlier. The reaction was subjected to denaturation (94°C for 30
seconds), annealing (55°C for 30 seconds), and extension (72°C for 30
seconds) for 30 cycles, and PCR products were confirmed by DNA
sequencing.
KLRL1 INHIBITS NATURAL KILLER CELL CYTOTOXICITY
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Eukaryotic expression vector construction and
cell transfection
To express Flag-tagged hKLRL1 and mKLRL1 proteins (hKLRL1-Flag
and mKLRL1-Flag) in eukaryotic cells, coding regions of human and
mouse KLRL1 were cloned in frame with the Flag tag in the expression
vector pcDNA3.1/mic-His (⫺) B (Invitrogen) to generate phKLRL1/Flag
and pmKLRL1/Flag vectors. The coding regions of both molecules were
also cloned in frame with green fluorescent protein (GFP) coding sequence
in pcDNA3.1/mic-His (⫺) B to generate GFP-fused hKLRL1 and mKLRL1
expression vectors, phKLRL1-GFP, and pmKLRL1-GFP. 293T and L929
cells were transfected with human and mouse KLRL1 vectors, respectively,
using PoLyFect transfection reagent (Qiagen, Valencia, CA) in accordance
with the manufacturer’s instructions. Forty-eight hours after transfection, cells were harvested for Western blot and fluorescence confocal
microscopy analysis.
Generation of anti-hKLRL1 mAb
Monoclonal antibodies (mAbs) against hKLRL1 were produced by immunizing BALB/c mice (BK Experimental Animal Co, Shanghai, China) with
NIH/3T3 cells transfected with phKLRL1/Flag vector. Spleen cells were
fused with murine SP2/0 myeloma cells by using polyethylene glycol-1000
and cultured in 96-well plates by using standard procedures. Hybridoma
supernatants were screened for their reactivity against hKLRL1-Flag fusion
protein by enzyme-linked immunosorbent assay (ELISA). Selected hybridomas were cloned by limiting dilution, and mAbs were produced in ascites
fluids and purified. Flow cytometric analysis showed that the obtained
anti-hKLRL1 mAbs recognized NIH/3T3 cells transfected with the fulllength cDNA encoding hKLRL1 but not mock-transfected cells (data not
shown). Data presented in the present study were obtained with the
anti-hKLRL1 mAb HK13 of isotype IgG1, ␬.
Western blot and immunoprecipitation analysis
Harvested cells were lysed in cell lysis buffer (Cell Signaling, Beverly, MA)
containing proteinase inhibitors (Sigma). Cell lysates were fractionated by
12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes.
Membranes were probed with primary antibodies anti–SHP-1, anti–SHP-2,
anti-SHIP (Santa Cruz Biotechnology, Santa Cruz, CA), or anti-Flag
(Sigma), then incubated with appropriate horseradish peroxidase (HRP)–
coupled secondary antibodies (New England Biolabs, Mississauga, ON,
Canada), and relevant protein bands were visualized by using LumiGLo
reagent (Cell Signaling). For sodium pervanadate stimulation, 100 ⫻ 106
cells in 0.5 mL phosphate-buffered saline (PBS) were preincubated at 37°C
for 5 minutes, then 5 ␮L of a 100⫻ sodium pervanadate solution was added
to 0.03% H2O2, 100 ␮M Na3VO4 (Sigma) and incubated for 5 minutes at
37°C. The stimulation was stopped by adding ice-cold 2 ⫻ 1% digitonin
lysis buffer (25 mM Tris (tris(hydroxymethyl)aminomethane)–HCl, 150
mM NaCl, pH 7.5, 1% digitonin, 1 mM NaF, 1 mM PMSF (phenylmethlsulfonyl fluoride), 1 mM Na3VO4, 10 ␮g/mL leupeptin, and 10 ␮g/mL
aprotinin), and the cells were lysed for 30 minutes at 4°C, then centrifuged
15 minutes at 16 000g in a microcentrifuge. For immunoprecipitation, the
supernatant was collected and precleared by incubating for 1 hour with 20
␮L protein A beads (Santa Cruz), then centrifuged for 2 minutes at 2300g.
The supernatant was collected and incubated for 1 hour at 4°C with
anti-hKLRL1 mAb HK13. Protein A beads (30 ␮L) or anti-Flag M2agarose beads (Sigma) were added, and precipitation was performed for 8
hours at 4°C. The beads were washed 3 times with 0.5% digitonin lysis
buffer and centrifuged for 3 minutes at 2300g, resuspended in SDS sample
buffer, and boiled for 2 minutes. Samples were run on SDS-PAGE,
transferred to PVDF membranes, and analyzed as described above.
Fluorescence confocal microscopy analysis
293T cells and L929 cells growing on glass coverslips placed in 6-well
plates were transiently transfected with KLRL1/GFP expression vectors.
Forty-eight hours after transfection, cells were observed by fluorescence
confocal microscopy (LSM 510 confocal microscope; Carl Zeiss, Atlanta,
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2860
HAN et al
GA). All cell images were obtained using a 40 ⫻ 1.2 water CApochromat
objective on the confocal microscopy with laser scanning microscope
(LSM) 510 software (version 3.2). Images were analyzed using Adobe
Photoshop 7.0.
N-glycoside F digestion
293T cells (10 ⫻ 106) transiently transfected with phKLRL1/Flag expression vector were lysed in 10 mM sodium phosphate buffer, pH6.5,
containing 0.1% SDS and 50 mM ␤-mercaptoethanol. To denature the
proteins, the cell lysates were heated for 5 minutes at 95°C, and then
Nonidet P-40 (final concentration, 1%) and protease inhibitor mixture
(Sigma) were added. Aliquots of these preparations were treated with
N-glycosidase F (5 mU/mL; Calbiochem, Darmstadt, Germany) for 8 hours
at 37°C. Reactions were stopped by the addition of SDS-PAGE loading
buffer, and samples were subjected to Western blot analysis as described in
“Western blot and immunoprecipitation analysis.”
Expression and purification of soluble hKLRL1-Fc
fusion protein
Coding regions of human interleukin-2 signal peptide, the extracellular
domain (residues 133-265) of hKLRL1 and human IgG4 CH2 and CH3
fragments were cloned in frame into pcDNA3.1/mic-His (⫺) B for
expression of secreted human extracellular KLRL1-Fc fusion protein
(hexKLRL1-Fc). COS-7 cells were transfected by using the diethylaminoethanol (DEAE)–dextran method with minor modifications. After overnight recovery in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FCS, cells were cultured in DMEM plus 1% FCS for 6
days.20 The supernatant was harvested, and secreted hexKLRL1-Fc protein
was purified by using Affi-Gel protein A–agarose columns (Bio-Rad,
Hercules, CA).
Flow cytometry
For single-color analysis, 50 ␮L cells (10 ⫻ 106 cells/mL) were incubated
with 5 ␮L mAb HK13 or human extracellular KLRL1-Fc fusion protein
(hexKLRL1-Fc) for 30 minutes on ice. After 3 washes, labeled cells were
incubated with 5 ␮L fluorescein isothiocyanate (FITC)–conjugated sheep
antimouse Ig or goat antihuman IgG (Sigma). Stained cells were analyzed
by fluorescence activated cell sorting (FACS; FACSCalibur; Becton
Dickinson, Mountain View, CA).
BLOOD, 1 NOVEMBER 2004 䡠 VOLUME 104, NUMBER 9
cDNA was obtained by PCR from human DCs. The 1566-bp cDNA
contained a single open reading frame (ORF) of 798 bp with 3
in-frame stop codons upstream of the initial codon and a putative
polyadenylation signal located 15 bp upstream of the poly (A)
stretch. The 3⬘-untranslated region also contained a number of
potential rapid degradation signals, including 2 repeats of the
consensus sequence ATTTA.22 The ORF encoded a 265–amino
acid protein with a theoretical molecular mass of 30.8 kDa and an
isoelectric point of 8.77. The presence of 6 putative N-glycosylation sites within the stalk and C-type lectin domains indicated that
the protein might be a glycoprotein. No signal sequence was
detected, but a putative transmembrane domain of 23 residues
extending from residue 44 to residue 66 was identified (Figure 1A).
The N-terminal was oriented on the cytoplasmic side of the
membrane, indicating that the full-length sequence encoded a type
II transmembrane protein. Sequence comparison revealed high
homology with members of the KLR subfamily of receptors. The
overall protein sequence showed 34% identity and 51% similarity
with hCLEC1 (human C-type lectinlike receptor 1), 30% identity
and 47% similarity with hCLEC2, 29% identity and 43% similarity
with hLOX-1 (endothelial receptor for oxidized low-density lipoprotein), and 24% identity and 43% similarity with hCD94 (Figure
1B). Sequence comparisons showed that it belongs to the KLRs but
constitutes the first member of a separate, novel family (family L),
and it was, thus, designated human Killer cell C-type Lectinlike
Receptor L 1 (hKLRL1; Figure 1C). The full-length sequence is
available in GenBank under the accession no. AF247788.
Blast searches of a mouse EST database (GenBank dbEST)
using the predicted polypeptide sequence of hKLRL1 lead to the
cloning of a mouse homolog of hKLRL1, designated mKLRL1
(accession no. NM_177686). The 2181-bp full-length cDNA,
obtained from mouse DCs, encoded a 267-residue type II transmembrane protein with a typical C-type lectin domain, with features
similar to those of hKLRL1, including 4 potential N-glycosylation
sites. Overall, human and mouse KLRL1 shared 50% identity and
65% similarity (Figure 1A). A rat ortholog of KLRL1 (rKLRL1;
accession no. XM_232420), identified from rat EST databases,
shared 70% similarity with hKLRL1 and 86% with mKLRL1.
Cytolytic assay
Cytolytic activity of NK cells was measured with the CytoTox 96
Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI),21 based on
detection of lactate dehydrogenase (LDH) activity released from damaged
cells. Released LDH in culture supernatant is measured with a 30-minute
coupled enzymatic assay, which results in the conversion of a tetrazolium
salt into a red formazan product, which can be equated to percentage lysis.
Exponentially growing A549, HeLa, MCF-7, and K562 cells were harvested as target cells and preincubated with hKLRL1-Fc fusion protein or
control IgG 20 ␮g/mL for 8 hours. Target cells (2 ⫻ 104 cells/mL) in a
volume of 50 ␮L were then placed in wells of a 96-well round-bottom plate,
then 50 ␮L effector cells (either NK-92 cells or IL-2–stimulated human NK
cells), at various concentrations, were added to each assay well. Plates were
centrifuged for 5 minutes at 250g and incubated in a 37°C 5% CO2
atmosphere for 4 hours, and supernatants were harvested and tested
according to manufacturer’s instructions. The results are presented as
median values from triplicate assays for each effector-target cell ratio.
Results
Identification and sequence analysis of human and
mouse KLRL1
An EST for a potential novel C-type lectin protein was originally
identified by large-scale random sequencing,18,19 and full-length
KLRL1 possesses structural features characteristic of the
KLR family
The predicted hKLRL1 protein consisted of 4 structural regions:
cytoplasmic (residues 1-43), transmembrane (44-66), stalk (67132), and a C-type lectinlike domain (CTLD; 133-265), a key
feature of the KLR family. Many other structural features of KLR
subfamily receptors were present in hKLRL1. All KLR subfamily
receptors contain CTLD motifs and, thus, structurally belong to the
C-type lectin family. Thirteen invariant residues (including 6 Cys,
which play a crucial role in forming disulfide bridge frameworks)
are relatively conserved among CTLD sequences of different
C-type lectins.23 A putative CTLD motif (133-249), in the Cterminal region of hKLRL1, contains 11 of the 13 invariant
residues, including all the 6 Cys residues (Figure 1B). CTLD
homology was compared with those of other KLRs. The CTLD of
hKLRL1 was most similar to those of human CD94 (29%
similarity and 42% identity) and human LOX-1 (26% similarity
and 31% identity) (Figure 1 B). In common with other KLRs, the
CTLDs of human and mouse KLRL1 lack the motifs required for
Ca2⫹ binding and showed little sequence similarity to the ligandbinding loops of classic C-type lectins. Of note, a putative
immunoreceptor tyrosine-based inhibitory motif (ITIM)24-26 was
identified close to the N-terminus of the cytoplasmic domain of
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BLOOD, 1 NOVEMBER 2004 䡠 VOLUME 104, NUMBER 9
KLRL1 INHIBITS NATURAL KILLER CELL CYTOTOXICITY
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Figure 1. Multiple alignment of KLRL1 with closely related KLR family receptors. Alignment was performed with the GCG package and minimally adjusted manually.
Identical residues are boxed in black, and similar residues are in gray. (A) Alignment of human and mouse KLRL1. Approximate domain boundaries are indicated for the
cytoplasmic, transmembrane, stalk, and C-type lectinlike domains (CTLDs). Putative N-glycosylation sites are shown by boxes. (B) Multiple alignment of CTLDs of KLRL1 and
closely related KLR family members. Asterisks indicate positions of the conserved invariant residues characteristic of CTLDs. (C) Dendrogram displaying total amino acid
identity among some KLR family members. Human, mouse, and rat KLRL1 constitute a separate branch of the KLRs, most closely related to CLEC2 and CLEC1. The GenBank
accession numbers of the analyzed sequences are AF247788 (hKLRL1), NM_177686(mKLRL1), XM_232420 (rKLRL1), NM_016509 (hCLEC2), AF201457 (mCLEC2),
AF200949 (hCLEC1), AF023840 (hNKG2A), AJ001684 (hNKG2C), AY100458 (mKLRE1), AF486186 (rKLRE1), U30610 (hCD94), AF030311 (mCD94), NM_005810
(hKLRG1), NM_016970 (mKLRG1), NM016523 (hKLRF1), NM_002258 (hKLRB1), AF133299 (hLLT1), and AF416564 (rKLRH1).
both hKLRL1 and mKLRL1 (VTYADL and IVYANL, respectively, conserved amino acids underlined; Figure 1 A). The
structural features characteristic of KLRs present in KLRL1
suggested that KLRL1 might function as a killer cell receptor, with
its actions mediated by interactions with other signaling molecules.
KLRL1 gene is located in the NK gene complex
Chromosome mapping analysis of KLRL1 showed conserved gene
localization among human, mouse, and rat KLRL1. The hKLRL1
gene is located on chromosome 12p13.31, along with several other
KLR family receptors (Figure 2A), including KLRG1,27 KLRB1
(NKRP1A), LLT1, DCAL1, CD69,28 KLRF1,29 C-type lectin
superfamily member 2 (CLECSF2), CLEC1, CLEC2,30
CLECSF12,1 LOX-1,17 KLRD1 (CD94),31 KLRK1 (NKG2D),
KLRC4 (NKG2F), KLRC3 (NKG2E), KLRC2 (NKG2C), KLRC1
(NKG2A), and KLRA1 (Ly49). The short arm of chromosome 12
is now recognized as a region that is particularly enriched for genes
encoding C-type lectinlike receptors important for NK cell functions, and this region has been designated NK gene complex. These
receptors are all involved in activating, inhibitory, or costimulatory
signaling functions in immune cells. Similar conservation of
KLRL1 gene location was observed for mouse and rat: mKLRL1
and rKLRL1 genes were located within the NK gene complex on
mouse and rat chromosomes 6F3 (Figure 2B) and 4q42 (data not
shown), respectively, accompanied by Klrg1,32 C lectin-related
protein A (ClrA),33 CD69, Clec2, Clecsf12, LOX-1, KLRE1,34 D1,
C3, C2, C1, and ly49.35 The location of the KLRL1 gene in the KLR
family gene cluster indicated that there might have been multiple
gene duplications within the KLR family during its evolution from
a common ancestral gene.
KLRL1 is preferentially expressed in lymphoid tissues and
immune cells
In human multiple tissue cDNA panels, hKLRL1 mRNA was
highly expressed in lymphoid tissues, such as spleen and
peripheral blood leukocytes, and present at lower levels in
thymus, placenta, pancreas, and small intestine (Figure 3A).
Expression was not detected in heart, brain, lung, liver, skeletal
muscle, kidney, prostate, testis, ovary, or colon. A similar
expression pattern was observed for murine KLRL1, with
mKLRL1 mRNA preferentially expressed in peripheral blood
leukocytes; less frequent in thymus, spleen, heart, brain, and
lung; and undetectable in other tissues (Figure 3B). As KLRL1
was expressed preferentially in lymphoid tissues, we further
examined its cellular distribution by RT-PCR analysis of hematopoietic cell lines and freshly isolated cells. As shown in
Figure 3C-D, both human and mouse KLRL1 expression is
restricted to immune cells. hKLRL1 mRNA could be detected in
myelomonocytic cells, including THP-1 (monocytes) and U937
(monocytic leukemia) cells, peripheral blood mononuclear cell
(PBMC)–derived DCs, NK cells, and CD8⫹ T cells but not in B
cells (Raji, Ramos, and Daudi cells), CD4⫹ T cells, or solid
tumor cell lines (A431, A549 and HeLa cells). RT-PCR analyses
of murine cells revealed that mKLRL1 was highly expressed in
mouse bone marrow–derived DCs, NK cells, CD4⫹ T cells,
CD8⫹ T cells, and macrophages (J774, RAW264.7). Unexpectedly, mKLRL1 was also expressed in B16 melanoma cells.
Taken together, the findings suggested that KLRL1 was preferentially expressed in lymphoid tissues and immune cells and
that in comparison to hKLRL1, mKLRL1 had a more comprehensive tissue and cellular distribution, which may be related to
the respective functions of these 2 proteins.
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HAN et al
BLOOD, 1 NOVEMBER 2004 䡠 VOLUME 104, NUMBER 9
Figure 2. Chromosomal location of the KLRL1 gene.
Physical map of the region constituting the KLR family
gene cluster within (A) the human NK gene complex on
chromosome 12p13 and (B) the mouse NK gene complex on chromosome 6F3. The organization of the KLR
gene and an expanded view of KLR gene structure are
also shown. Arrows indicate direction of transcription.
Human KLRL1 is a membrane-associated glycoprotein
To study KLRL1 expression in mammalian cells, phKLRL1/Flag
expression vector was transfected into 293T cells, and the expression of Flag-tagged human KLRL1 protein was examined by
Western blot analysis with anti-Flag antibody. An approximately
75-kDa protein was detected in both nonreducing and reducing
conditions (Figure 4A), and no specific band was observed in mock
vector transfected cells. The apparent molecular mass was considerably larger than that predicted from the deduced amino acid
Figure 3. Tissue and cellular expression pattern of KLRL1. RT-PCR was
performed with human and mouse KLRL1-specific primers on the following tissue
and cells: (A) human adult multiple tissue cDNA (MTC) panels, (B) adult mouse
normal tissues, (C) human hematopoietic cells and cell lines and solid tumor cell
lines, and (D) mouse hematopoietic cells and cell lines and solid tumor cell lines. All
the samples were similarly positive for ␤-actin.
sequence (30.8 kDa), suggesting that hKLRL1-Flag protein was
very likely to be modified after its translation. Glycosylation was
the most likely posttranslational modification, as hKLRL1 contained 6 putative N-glycosylation sites within the stalk and C-type
lectin domains. To confirm the glycosylation of hKLRL1, we
treated the cell lysate of phKLRL1/Flag-transfected 293T cells
with peptide N-glycosidase F. As shown in Figure 4A, the apparent
molecular mass of the N-glycosidase F–treated hKLRL1-Flag
protein was reduced to about 31 kDa, consistent with the calculated
molecular mass of hKLRL1, indicating that mature hKLRL1
protein is highly glycosylated.
Because human KLRL1 was predicted to be a type II transmembrane protein, we examined the cellular localization of GFP-fused
KLRL1 to determine whether it localized to the cell surface.
phKLRL1/GFP and pmKLRL1/GFP expression vectors were transiently transfected into 293T and L929 cells, respectively. Fortyeight hours after transfection, cells were subjected to fluorescence
confocal microscopy analysis. As shown in Figure 4B, specific
signals of both GFP-fused hKLRL1 and mKLRL1 were restricted
to the cell membrane, whereas the control GFP signal was diffused
throughout the cytoplasm. This result confirmed the structural
prediction that KLRL1 was a cell membrane-associated protein.
KLRL1 associates with protein tyrosine phosphatases SHP-1
and SHP-2
As a rule, the inhibitory NK receptors transmit their signals by
way of protein-tyrosine phosphatases, in particular SHP-1, which
dock onto phosphorylated ITIMs following receptor engagement.
To examine the ability of the ITIM-containing hKLRL1 protein to
associate with the protein-tyrosine phosphatases SHP-1, SHP-2,
and SHIP, important inhibitory regulators of immunoreceptor
signal transduction, NK92 and U937 cells were pretreated with
pervanadate to prevent the tyrosine dephosphorylation of cellular
proteins, and KLRL1 was immunoprecipitated by using HK13
mAb and protein A beads. Western blotting showed that SHP-1 and
SHP-2 coprecipitated with hKLRL1. Supporting this finding,
probing of digitonin lysates of pervanadate-stimulated NK92 and
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BLOOD, 1 NOVEMBER 2004 䡠 VOLUME 104, NUMBER 9
KLRL1 INHIBITS NATURAL KILLER CELL CYTOTOXICITY
2863
stimulated polyclonal NK cells toward the target cells. In contrast,
treatment with hexKLRL1-Fc did not influence lysis of K562 cells.
To test whether targets cells expressed hKLRL1 ligands, A549,
HeLa, MCF-7, and K562 cells were stained with hexKLRL1-Fc
and FITC-conjugated goat antihuman IgG. As shown in Figure 6C,
68.99%, 69.36%, and 34.22% of A549, HeLa, and MCF-7 cells,
respectively, stained positive with hexKLRL1-Fc, whereas K562
cells remained unstained. These results implied that hexKLRL1-Fc
neutralized the potential ligands of hKLRL1 expressed on the
surface of target cells, thus preventing engagement by hKLRL1
receptors present on NK cells and releasing effector cells from the
inhibitory effect of hKLRL signaling, leading to enhanced target
cell lysis. hKLRL1 thus appeared to be a novel functionally
inhibitory NK cell receptor that reduces NK cell activity by ways of
interaction with cell-surface ligands of target cells.
Discussion
The functions of NK cells are modulated by the balance between a
number of activating and inhibitory receptors. KIRs are mostly
Figure 4. KLRL1 is expressed as a membrane-associated glycoprotein. (A)
hKLRL1 transiently expressed in 293T cells is an N-linked glycoprotein. Lysates of
293T cells transiently transfected with phKLRL1/Flag or mock vector were analyzed
by Western blot under nonreducing (Non-R, in the absence of 2-mercaptoethanol),
reducing (Red, in the presence of 2-mercaptoethanol), or N-glycosidase F digestion
(N-Gly) conditions. (B) Both human and mouse KLRL1 is expressed as a transmembrane protein. 293T cells and L929 cells were transiently transfected with phKLRL1GFP (i), pmKLRL1-GFP expression vectors (iii), or GFP-alone control vectors (ii,iv),
respectively, and fluorescence confocal microscopy analysis was performed 48
hours after transfection. Original magnification, ⫻900.
U937 cells with anti-hKLRL1 mAb HK13 revealed prominent
bands of approximately 110 kDa (Figure 5A-B). For murine
KLRL1, L929 cells transiently transfected with pmKLRL1/Flag
vector were treated with pervanadate, and Flag-tagged mKLRL1
was precipitated with anti-Flag M2-agarose beads. As shown in
Figure 5C, SHP-1 and SHP-2 were coprecipitated in the anti-Flag
precipitate of cells transfected with pmKLRL1/Flag but not in
precipitates derived from control vector-transfected cells or parental cells. The interaction was tyrosine phosphorylation dependent
because the 2 proteins could not be coprecipitated from cells not
pretreated with pervanadate. SHIP recruitment was not detected in
NK-92, U937, or pmKLRL1/Flag-transfected L929 cells (data not
shown). These results indicated that on phosphorylation of tyrosine, presumably that located within the ITIM, KLRL1 recruited
both protein-tyrosine phosphatases SHP-1 and SHP-2, suggesting
likely involvement in negative regulation of signaling.
Human KLRL1 inhibits NK cell cytotoxicity against target cells
To investigate whether hKLRL1 functions as an inhibitory receptor
of NK cells, chimeric proteins with the extracellular domain of
KLRL1 fused with IgG4 CH2 and CH3 fragments (hexKLRL1-Fc)
were expressed in COS-7 cells, purified, and used to examine the
role of hKLRL1 in NK cell cytotoxicity. The expression of a
37-kDa hexKLRL1-Fc protein was confirmed by Western Blot
analysis (Figure 6A). As shown in Figure 6B, pretreatment of target
cells (A549, HeLa, and MCF-7 cells) with hexKLRL1-Fc fusion
protein enhanced the cytotoxicity of NK-92 cells and IL-2–
Figure 5. KLRL1/partner heterodimer associates with SHP-1 and SHP-2. (A)
NK-92 and U937 cells were pretreated with pervanadate (⫹) or not (⫺), then digitonin
lysates of the cells were incubated with HK13 and protein A beads, and precipitates
were subjected to Western blot analysis with anti–SHP-1 and anti–SHP-2. (B)
hKLRL1 forms a functional heterodimer with a putative partner molecule. Pervanadate stimulated (5 minutes at 37°C) (⫹) and unstimulated (⫺) NK-92 and U937 cells
were lysed in 1% digitonin buffer and tested by Western blotting, using anti-hKLRL1
mAb HK13. (C) L929 cells transiently transfected with mouse KLRL1/Flag expression
vector were pretreated with pervanadate (⫹) or untreated (⫺), then digitonin lysates
of the cells were incubated with anti-Flag M2-agarose beads, and precipitates were
subjected to Western blot analysis with anti–HP-1, anti–SHP-2, and anti–Flag
antibodies. Crude lysates (right hand panels) served as a control. The data shown are
representative of 3 independent experiments.
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2864
HAN et al
Figure 6. hKLRL1 inhibits NK cell cytotoxicity. (A) The purified hexKLRL1-Fc
protein is expressed as an approximately 37-kDa fusion protein. The fusion protein
was harvested from the supernatants of COS-7 cells transiently transfected with the
phexKLRL1-Fc expression vector and purified by using Affi-Gel protein A–agarose
columns. Protein expression in supernatants of untransfected COS-7 cells and
phexKLRL1-Fc–transfected COS-7 cells was examined by Western Blot analysis by
using anti-Fc antibody. (B) Pretreatment of target cells with hexKLRL1-Fc fusion
protein enhances NK cell cytotoxicity, except in the case of K562 cells. Target cells
(A549, HeLa, MCF-7, and K562 cells) were preincubated for 8 hours with hexKLRL1-Fc
fusion protein (hexKL-Fc) or control IgG (20 ␮g/mL). Cell-mediated cytolysis was
assayed by using IL-2–stimulated human polyclonal NK cells and NK-92 cells as
effector cells. Target cells untreated with mAb were included as an additional control.
All data represent median values of triplicate samples and are representative of 3
independent experiments. (C) Potential hKLRL1 ligands were present on the surface
of A549, HeLa, and MCF-7 target cells but not on K562 cells. Flow cytometric analysis
of A549, HeLa, MCF-7, and K562 cells stained with hexKLRL1-Fc fusion protein.
inhibitory receptors that play a critical role in recognizing selfclass–I MHC molecules and, thus, protect healthy host cells from
NK-targeted lysis. They have the following interesting features:
highly divergent structures, varied immune functions, and concentration in one genetic location. Of the 14 groups of CTLDcontaining proteins, KLRs belong to group V, which possess the
BLOOD, 1 NOVEMBER 2004 䡠 VOLUME 104, NUMBER 9
“nonclassic” C-type lectin domain, but not “classic” C-type lectin
domain. Classic C-type lectins bear carbohydrate-recognition domains (CRDs) that bind to glycan ligands in a calcium-dependent
manner, whereas nonclassic C-type lectins share structural homology with their classic counterparts but have evolved to bind
nonsugar ligands. Several KLR family members have been identified thus far, designated KLRA through KLRK. In this study, we
have described a novel NK cell inhibitory receptor belonging to the
KLR family, KLRL1, which is preferentially expressed in lymphoid tissues and immune cells, including NK cells, T cells,
dendritic cells, and monocytes or macrophages. The hKLRL1 gene
is located within the NK gene complex on chromosome 12p13, just
between hKLRF1 and hCLEC2. mKLRL1 and rKLRL1 share a
similar chromosomal arrangement. In humans, NK gene complex
members at this locus include CD69,28 CLEC1, CLEC2,30 DCAL1
(dendritic cell–associated lectin-1),36 KLRG1,27 KLRF1,29 CD94,
and the NKG2 family; in rodents, the NKC contains Clr,33 Klrg1,32
Klre1,34 Cd69, Cd94, and Ly49 family members.35 The KLRL1
protein is a CTLD-containing type II transmembrane glycoprotein;
it is most closely related to CLEC1 and CD94 and shares modest
similarity with all known members of the KLR family. The
conservation in sequence, structure, and chromosome arrangement
is reminiscent of the leukocyte receptor complex on chromosome
19q13.3-13.4, which includes the KIRs expressed on NK cells and
subsets of T cells, the gp49 family of receptors expressed on mast
cells and natural killer cells, and sialic acid–binding Ig-like lectins
(Siglec-3, -5, -6, -7, -8, -9, and -10) expressed on myeloid cells and
DCs.19 This suggests structural and functional coevolution of these
receptors on NK, myeloid, and dendritic cells.
The stalk section of the hKLRL1 molecule contains 2 conserved
cysteines that have been shown to form disulfide-linked dimers.37,38
We would, therefore, expect that native hKLRL1 also formed
homodimers or heterodimers in cells, as is the case with the other
KLRs. However, species of approximately 75 kDa in hKLRL1/Flagtransfected cell lysates, although much larger than the molecular
size predicted from the amino acid sequence, was observed under
both nonreducing and reducing conditions. Given that N-Glycoside
F treatment reduces the apparent molecular mass to about 31 kDa,
consistent with the calculated molecular mass of hKLRL1, high
levels of glycosylation, rather than dimerization, appear to be
responsible for the high molecular weight of the mature hKLRL1
protein. However, closer examination by Western blotting with the
anti-hKLRL1 mAb HK13 revealed weak approximately 110-kDa
hKLRL1 bands in NK-92 and U937 cell lysates, in addition to the
approximate 75-kDa bands. Interestingly, the approximate 110kDa bands became significantly stronger in digitonin lysates of
pervanadate-treated NK-92 and U937 cells. Together, the presented
findings indicate that hKLRL1 may form a functional heterodimer
with an as yet unidentified partner that 293T cells lack.
hKLRL1 contains a putative ITIM (VTYADL) in its cytoplasmic domain, which predicts inhibitory receptor function. Immunoprecipitation analysis demonstrated that hKLRL1 was physically
associated with SHP-1 and SHP-2 in lysates of pervanadate-treated
NK-92 and U937 cells. These results suggest an inhibitory role for
hKLRL1 in NK and myelomonocytic cells. Notably, during
revision of the present manuscript, Marshall et al39 reported a novel
inhibitory C-type lectinlike receptor, designated MICL and identical to KLRL1, which negatively regulates granulocyte and monocyte function. Among the KLRs, NKG2A contains 2 functional
ITIMs that recruit both SHP-1 and SHP-2, but not SHIP, by way of
their SH2 domains40; NKG2F (KLRC4) also has both a cytoplasmic ITIM-like sequence and a charged transmembrane amino acid
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BLOOD, 1 NOVEMBER 2004 䡠 VOLUME 104, NUMBER 9
residue, but its exact function is not known. We prepared an
hKLRL1-Fc fusion protein and used it to investigate the role of
hKLRL1 in NK cell cytotoxicity. We demonstrated that blocking of
potential hKLRL1 ligands on target cells enhanced NK cell
cytotoxicity, further supporting the notion that hKLRL1 functions
as an inhibitory receptor of NK cells.
Flow cytometry with hKLRL1-Fc fusion protein showed that
the putative ligand(s) of hKLRL1 might be present on the
membranes of A549, HeLa, and MCF-7 target cells but not on
K562 cells. Although KLRL1 belongs to the C-type lectin superfamily, it does not contain the classic CRD, so ligands are unlikely to be
carbohydrates. The amino acid sequence present in the CRDs of
animal lectins provides information about saccharide-binding specificity. For example, the sequence EPN is found in CRDs known to
bind mannose or glucose derivatives and is present in 2 domains of
the macrophage-mannose receptor (MMR).41 The EPS sequence
present in the CRD-2 of the MMR is believed to contribute only
weakly to binding of polyvalent ligands.42 In contrast, the sequence
QPD is characteristic of CRDs that bind galactose and N-acetylgalactosamine and is present in the hepatic antiasialoglycoprotein
receptor 1 (ASGPR-1) and -2.41 However, these sequences are
lacking from the amino acid sequences of both hKLRL1 and
mKLRL1. Further studies are required to confirm whether the
nonclassic MHC class I molecules are the natural ligands of
KLRL1, because most KLRs are receptors for nonclassic MHC
class I molecules.10,32,43
The discovery of new NK cell receptors will lead to a better
understanding of how NK cells interact with and kill target cells
KLRL1 INHIBITS NATURAL KILLER CELL CYTOTOXICITY
2865
through their complex set of activating and inhibitory receptors that
recognize corresponding ligands on tumor cells; this may, in turn,
reveal new approaches to cancer immunotherapy. Although both
NK cells and CD8⫹ cytotoxic T cell (CTLs) have the ability to kill
susceptible tumor cells, it seems that NK cells are responsible for
controlling a low tumor burden at an initial stage until the adaptive
arm of the immune system plays an important role in mediating
antitumor responses. Therefore, blockade of the inhibitory receptors expressed on a principal subset of NK cells can be a powerful
means to eradicate tumors when the tumor burden is minimal, such
as occurs following cytoreductive therapy. It has been shown that
blockade of inhibitory receptors can effectively augment the
antitumor activity of both allogeneic and syngeneic NK cells,
which may be an improved strategy for NK cell–based
immunotherapy.44-46
In conclusion, we have identified a novel C-type lectin-like
molecule, KLRL1, which has preferential hematopoietic expression and acts as a NK cell inhibitory receptor. Future studies are
required to elucidate the physiologic functions of this receptor and
to discover its putative heterodimeric partner molecule and naturally recognized ligands.
Acknowledgments
We thank Dr J. Rayner for critically reading this manuscript. We
also thank Dr X. Zhou, Mrs Y. Li, Miss Y. Zheng, Miss X. Zuo,
Miss W. Ni, and Mrs M. Jin for their expert technical assistance.
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2004 104: 2858-2866
doi:10.1182/blood-2004-03-0878 originally published online
July 6, 2004
KLRL1, a novel killer cell lectinlike receptor, inhibits natural killer cell
cytotoxicity
Yanmei Han, Minghui Zhang, Nan Li, Taoyong Chen, Yi Zhang, Tao Wan and Xuetao Cao
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