Immunogenetics (2014) 66:467–477
DOI 10.1007/s00251-014-0777-2
ORIGINAL PAPER
The activating Ly49W and inhibitory Ly49G NK cell receptors
display similar affinities for identical MHC class I ligands
Brian J. Ma & Carla M. Craveiro Salvado & Kevin P. Kane
Received: 26 February 2014 / Accepted: 23 April 2014 / Published online: 7 May 2014
# Springer-Verlag Berlin Heidelberg 2014
Abstract The Ly49 receptor family plays an important role in
the regulation of murine natural killer (NK) cell effector
function. They recognize cell surface-expressed class I MHC
(MHC-I) and are functionally equivalent to the killer Igrelated receptors (KIRs) in human NK cells. Ly49s exist in
activating and inhibitory forms with highly homologous extracellular domains, displaying greater variability in the stalk
regions. Inhibitory Ly49s can recognize self-MHC-I and
therefore mediate tolerance to self. The role of activating
Ly49 receptors is less clear. Some activating Ly49 receptors
have been shown to recognize MHC-I molecules. The binding
affinity of activating Ly49 receptors with MHC-I is currently
unknown, and we sought to examine the affinities of two
highly related receptors, an activating and an inhibitory
Ly49 receptor, for their shared MHC-I ligands. The
ectodomain of inhibitory Ly49G of the BALB/c mouse strain
is highly similar to the Ly49W activating receptor in the
nonobese diabetic (NOD) mouse. Recombinant soluble
Ly49G and W were expressed, refolded, and analyzed for
binding affinity with MHC-I by surface plasmon resonance.
We found that Ly49G and Ly49W bound with similar affinity
to the same MHC-I molecules. These results are a first determination of an activating Ly49 receptor affinity for MHC-I
and show that, unlike prior results obtained with activating
and inhibitory KIR receptors, functional homologues to Ly49
receptors, activating and inhibitory Ly49, can recognize
common MHC-I ligands, with similar affinities.
B. J. Ma : C. M. Craveiro Salvado : K. P. Kane
Department of Medical Microbiology and Immunology, University
of Alberta, Edmonton, Alberta T6G 2E1, Canada
K. P. Kane (*)
Department of Medical Microbiology and Immunology, University
of Alberta, 6-020 Katz Group Centre, Edmonton, Alberta T6G 2E1,
Canada
e-mail: [email protected]
Keywords NK cell . Activating Ly49 receptor . Inhibitory
Ly49 receptor . Binding affinity . SPR
Abbreviations
CTLD
FACS
FITC
IgG
IPTG
ITAM
ITIM
KIR
MCMV
MHC
MHC-I
NK
PE
SPR
β2m
C-type lectin-like domain
Fluorescence-activated cell sorting
Fluorescein isothiocyanate
Immunoglobulin G
Isopropyl-1-thio-β-D-galactopyranoside
Immunomodulatory tyrosine-based activating
motif
Immunomodulatory tyrosine-based inhibitory
motif
Killer cell immunoglobulin-like receptor
Murine cytomegalovirus
Major histocompatibility complex
MHC class I
Natural killer
Phycoerythrin
Surface plasmon resonance
β2-Microglobulin
Introduction
Natural killer (NK) cells function as the first line of defense
against tumors and viruses by secreting cytokines or mediating direct cytolysis in response to the altered cells. They
express a large repertoire of activating and inhibiting receptors, for which the balance of integrated signals determines
whether an individual NK cell responds (Lanier 2005). The
activity of NK cells is generally explained by the “missingself” mechanism, where NK cell interaction with a normal cell
will lead to inhibition, unless there is loss of self class I major
histocompatibility complex (MHC-I) molecule expression on
468
the target cell (Ljunggren and Kärre 1990). In the mouse,
inhibitory receptors responsible for such activity include
members of the C-type lectin-like Ly49 receptor family
(Carlyle et al. 2008). Human NK cells do not employ Ly49
receptors, but instead use killer immunoglobulin-related receptors (KIRs) which are structurally unrelated, but functionally equivalent (Campbell and Hasegawa 2013). Ly49s are
encoded by multiple gene loci, and the number of genes varies
among different inbred mouse strains (Anderson et al. 2005).
The Ly49 and MHC-I genes are located on different chromosomes and therefore can segregate independently (Trowsdale
2001). In addition, expression of different Ly49s on any given
NK cell is determined stochastically by probabilistic promoter
switches (Held and Kunz 1998; Makrigiannis et al. 2004;
Saleh et al. 2004).
Inhibitory Ly49s contain an immunoreceptor tyrosinebased inhibitory motif (ITIM) in their cytoplasmic tails that
upon receptor engagement with classical MHC-I on the surface of target cells leads to phosphorylation of the ITIM,
recruitment of SHP-1 phosphatase, and transmission of an
inhibitory signal (Nakamura et al. 1997). Activating Ly49
receptors lack ITIMs in their intracellular domains and instead
have a positively charged transmembrane residue that allows
association with the immunoreceptor tyrosine-based activating motif (ITAM)-containing adapter protein, DAP12 (Smith
et al. 1998; Tomasello et al. 1998). Upon activating Ly49
engagement with a ligand, the DAP12 ITAMs become phosphorylated, leading to recruitment of Syk kinase which propagates an activating signaling cascade (McVicar et al. 1998).
Some activating Ly49s have been shown to contribute to
resistance against murine cytomegalovirus (MCMV) infection. Ly49H, for example, recognizes the MCMV-encoded
decoy m157, while Ly49P can detect MCMV-infected cells
that co-express host H-2Dk, and m04 encoded by MCMV
(Cheng et al. 2008; Kielczewska et al. 2009). In addition,
some activating Ly49s, including Ly49W, have been shown
to directly recognize MHC-I ligands in the absence of virus
infection (Nakamura et al. 1999; Silver et al. 2000, 2001).
Activating Ly49s have ectodomains highly similar to inhibitory Ly49s with the same or similar MHC-I specificity
(Silver et al. 2000, 2001, 2002). The ectodomain, whether of
an activating or inhibitory Ly49, is comprised of a stalk region
linked to a C-terminal C-type lectin-like domain (CTLD).
Disulfide bonds between stalk regions stabilize either activating or inhibitory homodimers. The lectin-like domains are
responsible for binding and interacting with MHC-I. The
ectodomain sequence similarities shared by activating and
inhibitory Ly49s are mostly due to the CTLD region, with a
higher degree of genetic variability in the stalk region. The
contribution made by the stalk to ligand recognition specificity is unknown. A study of MHC-I ligand interaction by Ly49
chimeric receptors, where CTLD and stalk regions are exchanged, supports the possibility of a contribution by the stalk
Immunogenetics (2014) 66:467–477
region, in addition to the CTLD, in determining MHC-I
recognition (Brennan et al. 1996). The similarities observed
in the CTLDs of inhibitory and activating Ly49s may be
explained by a mutation within a primordial inhibitory receptor. A mutation and functional switch of a rodent inhibitory
signaling domain may have resulted in a single activating
signaling domain, the progenitor to mouse activating Ly49
signaling domains (Abi-Rached and Parham 2005).
Furthermore, this genetic occurrence is also observed for rat
activating Ly49 receptors and KIRs (Abi-Rached and Parham
2005).
Given the genetic and evolutionary relationship between
inhibitory and activating Ly49s, and despite the potential for
recognizing the same MHC-I ligands, the inhibitory signal
typically dominates (Moretta et al. 1995; Mandelboim et al.
1996). In agreement, previous results showed that inhibitory
KIR2DL3 binds with higher affinity than activating KIR2DS2
to their shared HLA-Cw7 ligand (Vales-Gomez et al. 1998).
Similarly, the human inhibitory CD94/NKG2A receptor was
shown to have a higher affinity for HLA-E than the activating
CD94/NKG2C (Vales-Gomez et al. 1999; Kaiser et al. 2005).
Thus, in instances where these receptors are expressed simultaneously on the same NK cell, the inhibitory receptor
will bind to the MHC target with higher affinity and
may therefore outcompete the activating receptor for
ligand. This paradigm has been challenged, however, as the
activating CD94/NKG2E heterodimer binds with similar affinity as the inhibitory CD94/NKG2A to a shared ligand,
HLA-E (Kaiser et al. 2005). We therefore wanted to examine
affinity relationships between activating and inhibitory Ly49
receptors.
To date, no study has been performed to compare the
affinities of activating and inhibitory Ly49 receptors for
shared MHC-I ligands. In the present study, we compare the
binding affinity of the activating Ly49W mouse receptor of
the nonobese diabetic (NOD) strain (Ly49WNOD) with its
most highly related inhibitory counterpart, Ly49G mouse
receptor of the BALB/c strain (Ly49GBALB/c). Our findings
demonstrate that inhibitory Ly49G and activating Ly49W
interact with similar affinities for their ligands, which is in
contrast to what has been shown for the KIRs.
Materials and methods
Protein expression The complementary DNA (cDNA)
corresponding to the ectodomains of Ly49G2 BALB/C
(residues 67–267) and Ly49W1NOD (residues 67–268) were
each amplified by PCR with a 5′ BamHI site and a 3′ HindIII
site and inserted into pAN-6 (Avidity, LLC) to introduce an Nterminal biotin holoenzyme synthetase (BirA) recognition
sequence (Schatz 1993; Beckett et al. 1999). These constructs
were subcloned into the pET21a vector and transformed into
Immunogenetics (2014) 66:467–477
the BL21(DE3) strain of Escherichia coli (Stratagene). After
bacterial growth in the presence of biotin and induction with
0.5 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG),
protein-containing inclusion bodies were purified by sonication,
centrifuged, and dissolved with 6 M guanidine hydrochloride.
The cDNA encoding the ectodomains of H-2Dd (residues
26–301), H-2Dk (residues 26–299), and H-2Kb (residues
23–295) and mouse β2-microglobulin (A allele) were
subcloned into the pET21a expression vector. The pET21a
constructs were each transformed into BL21 cells, and the
proteins were each expressed as inclusion bodies after
induction with IPTG. Purified inclusion bodies were
dissolved in 8 M urea.
Antibodies The anti-Ly49G/W antibody (rat IgG2a) was produced from the 4D11 hybridoma cell line (Silver et al. 2001).
FITC-coupled mouse antirat IgG were purchased from
Jackson ImmunoResearch Laboratories.
Refold screen Optimal conditions for protein refolding from
inclusion bodies were determined using the FoldIt Screen
(Hampton Research). In brief, inclusion bodies were diluted
into 16 different refolding buffers in 1 ml total. After 48 h, the
refolding buffer was removed and replaced with 100 mM
NaCl and 100 mM Tris, pH 8.0, by ultrafiltration. Proteins
that remained in solution were captured onto avidin-coated
5-μm latex beads, incubated with 4D11 (anti-Ly49G/W), and
analyzed on a Becton Dickinson FACSCanto flow cytometer
with FACSDiva software. Analyses were performed using
FCS Express (De Novo Software).
Protein refolding and purification The ectodomain of Ly49G
and Ly49W proteins were refolded as previously described
(Natarajan et al. 1999) but with different optimal refolding
buffers. Both receptors were refolded in 55 mM Tris, pH 8.2;
10.56 mM NaCl; 0.44 mM KCl; 2.2 mM MgCl2; 2.2 mM
CaCl2; 550 mM L-Arg, 0.055 % PEG 3350; 5 mM reduced
glutathione; and 0.5 mM oxidized glutathione. The Ly49s
were initially purified by size exclusion chromatography on
a HiLoad 26/60 Superdex 75 column (GE Healthcare), on
an ÄKTAexplorer 100 (GE Healthcare), followed by
immunoabsorbent chromatography on a 4D11-coupled
POROS 20 100×2.1-mm column (Applied Biosystems)
on a BioCAD 700E Workstation (Applied Biosystems).
Soluble MHC-I was refolded by rapid dilution of mixed
chain solubilized inclusion bodies into L-Arg buffer containing peptides as previously described (Garboczi et al. 1992).
The peptides used in MHC-I refolding were RGPGRAFVTI
(HIV gp120 P18-I-10) for H-2D d (Shirai et al. 1992),
RRLGRTLLL (polyoma virus Middle T Protein MT389-397)
for H-2Dk (Lukacher and Wilson 1998), and SIINFEKL
(Ovalbumin OVA257-264) for H-2Kb (Carbone and Bevan
1989) that were commercially synthesized (GenScript Corp.).
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The trimeric MHC-I complex consisting of heavy chain, β2microglobulin, and peptide were isolated by size exclusion
chromatography using a Superdex 75 column (GE Healthcare).
Construction of Ly49 fluorescent multimers The refolded soluble Ly49 receptors were multimerized by mixing 7.56 μg of
monomers in five additions with 2.44 μg of Extravidin-PE
conjugate (Sigma) in 5-min intervals. Fluorescent Ly49
multimers (1 μg) were subsequently used to stain EL4 or
EL4 transfected with H-2Dd or H-2Dk (2.5×105 cells).
EL4 cell transfection The cDNA encoding H-2Dk or H-2Dd
was subcloned into the pEF6 vector (Invitrogen) and subsequently stably expressed in EL4 cells, a mouse T cell lymphoma cell line, via electroporation and blasticidin selection.
Transfected cells were enriched using sheep antimouse IgG
Dynabeads (Invitrogen) and 34-5-8S (monoclonal antimouse
H-2Dd) (Abcam) or 15-5-5 (monoclonal antimouse H-2Dk)
(eBioscience).
Surface plasmon resonance analysis Surface plasmon resonance (SPR) measurements of Ly49 binding with MHC-I
molecules were obtained with a Biacore 3000 (GE
Healthcare). Dimers of Ly49G or W were immobilized on a
CM-5 sensor chip (GE Healthcare) using amide coupling with
target resonance units (RU) of 2,000. All SPR experiments
were conducted at 25 °C in HBS-EP buffer (GE Healthcare)
using a constant flow rate of 30 μl min−1. A concentration
series (0.5, 1, 2, 4, 8, 16, 32 μM) of H-2Dd, H-2Dk, and H-2Kb
was injected over the surface using a randomized order.
Background binding was accounted for in the analysis using
the double referencing method (Myszka 1999). The binding
data were analyzed using BIAevaluation 4.1 (GE Healthcare)
software and the 1:1 Langmuir binding model. Equilibrium
KD values were determined assuming a 1:1 binding interaction
(A+B=AB). The RU measurements at equilibrium (Req) were
plotted against the concentration, and the KD values were
calculated using nonlinear least-squares fit of the Langmuir
binding equation: AB=B×ABmax /(KD +B).
Results
Activating Ly49W and inhibitory Ly49G have highly similar
extracellular domains The existence of receptors having
highly homologous extracellular domains, but opposite effector functions, is a common occurrence in NK cell receptor
biology. Both human KIR and functionally equivalent murine
Ly49 receptor families include highly similar inhibitory and
activating receptors. In addition, some activating and inhibitory receptors have been demonstrated to recognize the same
MHC-I ligands; for example, inhibitory Ly49G and activating
470
Ly49W recognize H-2Dd and H-2Dk (Silver et al. 2001,
2002). A comparison between activating and inhibitory
KIRs showed that inhibitory KIR2DL3 binds with much
higher affinity to HLA-Cw7 than activating KIR2DS2
(Vales-Gomez et al. 1998). Presumably, this higher affinity
of inhibitory binding to the same MHC-I would ensure that
the inhibitory response would dominate. No equivalent study
has been performed with Ly49 receptors.
We sought to compare the binding kinetics and equilibrium
KD of an activating Ly49 receptor with an inhibitory Ly49,
where both recognize the same MHC-I ligand. Previously, we
cloned and sequenced a number of activating and inhibitory
Ly49 receptors from NK cells of the NOD mouse strain
(Silver et al. 2000, 2001), including Ly49W. The activating
Ly49WNOD receptor shares a high extracellular domain similarity with Ly49G and the highest similarity with Ly49G from
the BALB/c mouse strain (Fig. 1). Ly49W NOD and
Ly49GBALB/c have 98 % sequence identity in the CTLD and
are predicted to share all contact residues, should they bind
MHC ligands similarly to Ly49A, based on the Ly49A-H-2Dd
co-crystal (Fig. 1). Therefore, Ly49WNOD and Ly49GBALB/c
are activating and inhibitory receptors that recognize the same
MHC-I ligands with extracellular domains that are highly
similar in the CTLD, differing mostly in the stalk region, yet
the two receptors transmit opposite signals. In addition to their
sequence similarity (Fig. 1), a previous study involving chimeric Ly49GBALB/c, a molecule constructed to have an activator
transmembrane and intracellular domain, but the Ly49GBALB/c
ectodomain, resulted in similar activity towards H-2Dd and
H-2Dk expressing targets as with Ly49WNOD (Silver et al.
2001, 2002). We therefore hypothesized that activating and
inhibitory Ly49s might bind to MHC-I with similar affinity.
Production of soluble Ly49 and MHC-I proteins Preparation
of soluble Ly49s for kinetic analysis was approached by
Fig. 1 Sequence comparison by amino acid alignment of the inhibitory
receptor Ly49GBALB/c with the activating Ly49WNOD receptor. Dots represent sequence identity. Structural features are demarcated by horizontal
arrows and include the cytoplasmic, transmembrane (TM), stalk, and Ctype lectin domain (CTLD). Residues predicted to interact with MHC-I
based on the Ly49A-H-2Dd co-crystal structure are indicated (diamonds)
Immunogenetics (2014) 66:467–477
expressing the entire ectodomains of Ly49GBALB/c and
Ly49WNOD (hereafter referred to as Ly49G and Ly49W, respectively) which included their respective stalks and CTLDs,
with an N-terminal BirA recognition sequence, in inclusion
bodies of E. coli. We then screened a panel of 16 different
conditions for their ability to refold Ly49G and Ly49W, identifying conditions that yielded the highest amount of refolded
protein. With the refolding conditions established, Ly49G and
W were refolded from solubilized inclusion bodies on a larger
scale and then purified by size exclusion chromatography
(Fig. 2a). Both Ly49G and W purified primarily as dimeric
species with masses of approximately 53 kDa, as expected
(Fig. 2a). Dimers were pooled independently and then further
purified by immunoadsorbent chromatography with the 4D11
antibody. The immunoisolated Ly49s were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) to demonstrate purity and show that, after reduction,
the dimers are converted to monomer species, as would be
predicted (Fig. 2c). Three different MHC-I proteins—H-2Dd,
H-2Dk, and H-2Kb—were expressed in E. coli and refolded
with β2m and HIV gp120 P18-I-10, MT389-397, or OVA 257264 (SIINFEKL), respectively, using established methods
(Garboczi et al. 1992; Natarajan et al. 1999). The refolded
MHC-I complexes were isolated by size exclusion chromatography (Fig. 2b) and assessed by SDS-PAGE, which showed the
soluble form of the MHC-I heavy chain and β2m light chain
migrating as expected (Fig. 2d).
Soluble Ly49G and W bind MHC-I ligands on live cells We
wanted to test the functionality of our purified Ly49 proteins
by examining their ability to bind to MHC-I expressed by
cells. A direct assessment of soluble activating Ly49 binding
to MHC-I on cells in comparison to a highly homologous
inhibiting Ly49 has yet to be reported. We used extravidin-Rphycoerythrin to construct fluorescent multimeric staining reagents with the purified Ly49 proteins to test their ability to
interact with MHC-I. Using the same amounts (as determined
by BCA assay), fluorescent multimeric staining reagents were
made with dimers of Ly49G and Ly49W. The multimers were
used to stain EL4 cells stably transfected with either H-2Dk or
H-2Dd (Fig. 3a). Both of the Ly49 multimers did not stain the
untransfected EL4 (H-2b) cells (Fig. 3b). This was expected,
since neither of these Ly49 receptors are known to bind H-2Kb
or H-2Db (Hanke et al. 1999; Silver et al. 2001). Ly49G dimers
stained H-2Dd transfected cells and, to a lesser extent, also
stained H-2Dk. Ly49W dimers stained both H-2Dd and H-2Dk
transfected cells quite effectively. These results are the first
direct biochemical evidence for a substantial interaction of an
activating Ly49 with MHC-I ligands.
Ly49W binds with equal or higher affinity to MHC-I compared
to Ly49G The ability to produce refolded soluble Ly49 molecules allowed us to quantitatively measure the interaction of
Immunogenetics (2014) 66:467–477
471
Fig. 2 Purification of Ly49
proteins and MHC-I proteins for
SPR analysis. a and b
Chromatographs from separation
of refolded Ly49 (a) and MHC-I
(b) proteins by size exclusion
chromatography. The highlighted
fractions contained the refolded
protein of interest and were pooled
and concentrated. Ly49G and W
refolded predominantly as dimers
(according to molecular weight). c
and d SDS-PAGE analysis of
purified Ly49 and MHC-I
proteins. c One microgram of
Ly49 proteins was run on a 10 %
acrylamide reducing gel. d One
microgram of MHC-I proteins
was run on 15 % acrylamide gel.
HC heavy chain, β2m β2microglobulin
an activating Ly49 with MHC-I in comparison with an inhibitory Ly49, by surface plasmon resonance (SPR). Based on the
cell staining results, it appears that the dimers of both Ly49G
and W are effective candidates for SPR analysis. The quantitative study of the interaction of Ly49 with MHC-I by SPR
was performed by immobilizing Ly49 proteins on CM5 chip
through amide coupling. A concentration series (0.5–32 μM)
of soluble MHC-I was flowed over the immobilized Ly49
receptors (Fig. 4). Ly49G and W receptors showed binding
to H-2Dd and H-2Dk, producing measurable sensorgrams
(Fig. 4a). In contrast, there was little or no binding of H-2Kb
to Ly49G or W, as expected based on previous studies (Kane
1994; Silver et al. 2000, 2001, 2002) and cell staining results
(Fig. 3). Ly49G and W each bound to both H-2Dd and Dk
(Fig. 4a). The sensorgrams show that both Ly49G and W
interact with very fast kinetics. The KD of H-2Dk interaction
with Ly49G and W was 22.9 and 13.6 μM, respectively
(Fig. 4b). Comparatively, both Ly49G and W demonstrated
about 2-fold weaker binding to H-2Dd with KD of 46.1 and
48.3 μM, respectively. This pattern of lower-affinity binding
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Immunogenetics (2014) 66:467–477
Fig. 3 Binding to H-2Dd and H-2Dk expressed on EL4 transfectants by
multimeric Ly49G and Ly49W. a Expression of MHC-I on EL4
transfectants. EL4 cells stably transfected with H-2Dd (EL4.Dd) were
stained with 34-5-8. EL4 cells stably transfected with H-2Dk (EL4.Dk)
were stained with 15-5-5. EL4 cells were stained with 34-5-8 or 15-5-5 as
a negative control. Isotype controls are shown in grey histograms. b
Fluorescent multimeric staining reagents were created by capturing equal
amounts of the biotinylated Ly49 molecules onto Extravidin-PE conjugates. Untransfected EL4 and EL4 stably transfected with H-2Dd (EL4.Dd)
or H-2D k (EL4.Dk ) were stained with Ly49G or Ly49W (black
histograms). Cells were also stained with Extravidin-PE as a negative
control (grey histograms). The MFI (mean fluorescence intensity) is
shown in the top right-hand corner of each histogram. These results are
representative of three independent experiments
for H-2Dd compared to H-2Dk by Ly49W is consistent with
what we have previously observed in functional studies
(Silver et al. 2001). In contrast to previous studies involving
activating and inhibitory KIRs (Vales-Gomez et al. 1998),
Ly49W bound with about 1.5-fold higher affinity to H-2Dk
as compared to Ly49G. Furthermore, both Ly49G and W
bound with equal affinity to H-2Dd. This demonstrates that
an activating Ly49 receptor can indeed bind MHC-I with
equal or greater affinity than a related inhibitory receptor for
the same ligand.
human KIR family, as well as the CD94/NKG2 receptors,
both inhibitory and activating versions of the receptors with
highly homologous extracellular domains exist. In addition,
individual NK cells in both humans and mice have the potential to express multiple KIR or Ly49 receptors (Valiante et al.
1997; Kubota et al. 1999). Expression of both KIR and Ly49
receptors on NK cells is, for the most part, randomly determined during NK cell maturation (Wagtmann et al. 1995;
Valiante et al. 1997; Held and Kunz 1998). Thus, it is possible
that activating and inhibitory receptors to the same ligand can
be expressed on the surface of a single cell. Based on previous
work with KIR and CD94/NKG2 activating and inhibitory
receptors, a paradigm has been proposed that inhibiting receptors always bind with higher affinity than their activating
counterparts (Vales-Gomez et al. 1998, 1999). This presumably ensures that an inhibitory response would dominate in the
event that both the activating and inhibitory versions of the
receptors for the same ligand are present on the same NK cell.
Inhibitory signaling dominance has been challenged with the
Discussion
Regulation of NK cell function is controlled by a balance of
signals through engagement of inhibitory and activating receptors with their cognate ligands. In the mouse Ly49 family
and the structurally divergent, but functionally equivalent,
Immunogenetics (2014) 66:467–477
473
Fig. 4 Inhibitory Ly49G and activating Ly49W have similar affinities for
the same MHC-I ligands. a H-2Dd, Dk, and Kb were injected at seven
different concentrations ranging from 0.5 to 32 μM in 2-fold increments
over CM5 biosensor chips coupled with either inhibitory Ly49G or
activating Ly49W. SPR was measured with each interaction, and the
sensorgrams are shown. Injections were all conducted in HBS-EP buffer
at 25 °C and 30 μl/min with about 2,000 RU of immobilized ligand. b
Equilibrium binding curves of Ly49 binding to MHC-I. The binding
responses of H-2Dd and Dk with Ly49G and W at equilibrium (Req) were
plotted against concentration, and steady-state dissociation constants (KD)
were determined by curve fitting and noted at the top left corner of each
plot. These results are representative of three independent experiments
demonstration that inhibitory CD94/NKG2A and activating
CD94/NKG2E bind HLA-E with similar affinities (Kaiser
et al. 2005) (Table 1).
In this report, we quantified the affinities of a pair of highly
homologous activating/inhibitory Ly49 receptors, Ly49WNOD
and Ly49GBALB/c, respectively. Although the activating/
inhibitory pair of Ly49 receptors share highly homologous
CTLDs, since there is variability in the stalk region, and a
previous report suggested a role for the stalk region in ligand
recognition (Brennan et al. 1996), we wanted to test how
their affinities would compare for their mutual MHC-I
ligands. Successful refolding of the soluble Ly49
Table 1 Comparison of various
receptor:ligand binding affinities
KD (μM)
Receptor:ligand
Inhibitory (I)/activating
(A) receptor
KD (μM)
Reference
Ly49GBALB/c:H-2Dd
Ly49GBALB/c:H-2Dk
Ly49WNOD:H-2Dk
Ly49WNOD:H-2Dd
Ly49A:H2Dd
I
I
A
A
I
46.1
22.9
13.6
48.3
1.8–4.4
Ly49C:H2Kb
Ly49C:H2Dd
KIR2DL1:HLA-Cw4
KIR2DL2:HLA-C*03:04
KIR2DL2:HLA-C*03:04
KIR3DS1:HLA-B2705
Ly49I129/J:m157
Ly49H:m157
CD94/NKG2-A:HLA-E
I
I
I
I
I
A
I
A
I
80–102
136
7.2
0.015–0.036
0.03–5.6
6.95
0.166
0.936
0.53–56.6
CD94/NKG2-C:HLA-E
A
3.8–120.0
CD94/NKG2-E:HLA-E
TCR:pMHC
A
–
0.72–22.9
0.1–300
This study
This study
This study
This study
Wang et al. (2002),
Deng et al. (2008)
Dam et al. (2003, 2008)
Deng et al. (2008)
Stewart et al. (2005)
Frazier et al. (2013)
Frazier et al. (2013)
Li et al. (2010)
Adams et al. (2007)
Adams et al. (2007)
Vales-Gomez et al. (1999),
Kaiser et al. (2005)
Vales-Gomez et al. (1999),
Kaiser et al. (2005)
Kaiser et al. (2005)
Bridgeman et al. (2012)
474
molecules from inclusion bodies allowed for the isolation
of Ly49W and Ly49G on conformation-dependent antiLy49 immunoabsorbant columns. Employing SPR, we
measured the affinity of interaction of Ly49W from the
NOD mouse and Ly49G from the BALB/c strain with the
MHC-I ligands H-2Dk and H-2Dd. This is the first report
to compare the affinity of activating and inhibitory Ly49s
for the same MHC-I ligands. In contrast to observations
made with KIR, both inhibitory Ly49G and activating
Ly49W bound H-2D d with similar affinities, while
Ly49W bound H-2Dk with a somewhat higher affinity.
This demonstrates that activating Ly49 receptors can have
similar or higher affinity for MHC-I ligands than their
inhibitory counterparts. Therefore, similar to what was
reported for CD94/NKG2 receptors (Kaiser et al. 2005)
(Table 1), some Ly49 inhibitory receptors with weaker
affinities than some activating Ly49 may not be ensured
of dominance. There are other factors that need to be
considered to dissect the individual contributions of receptors to effector function. Due to antibody crossreactivity, it is currently difficult to access the relative
surface expression levels of activating and inhibitory
Ly49 receptors on NK cells. Furthermore, although
Ly49W and Ly49G are found on different inbred strains,
they, or related receptors, may co-express in wild or
outbred mice. However, the activating receptors Ly49D
and Ly49H, for which specific antibodies are available,
appear to be expressed at the cell surface of NK cells at
similar levels to inhibitory Ly49 receptors (Smith et al.
2000). Another possible factor in determining effector
function outcome is signaling strength. It remains to be
determined what an equal engagement by an activating
and inhibitor receptor will have on outcome. It has been
shown that an activating KIR receptor can overcome the
inhibitory effect of CD94/NKG2A and that the kinetics of
activation and inhibition may follow a biphasic pattern
(Warren et al. 2001; Foley et al. 2008).
The KD values ranging from 13 to 48 μM reported in this
study are within the range of what has been reported for
inhibitory Ly49A and C receptor interactions with MHC-I
interactions (Table 1). In comparison to other MHC-binding
NK receptors with known affinities, these values are also
comparable (Table 1). As examples, human inhibitory KIRs
have been reported to bind MHC-I in the range of 6.9–117 μM
(Valés-Gómez et al. 1998; Maenaka et al. 1999) while human
activating and inhibitory CD94/NKG2 receptors bind HLA-E
in the range of 0.7–120 μM (Vales-Gomez et al. 1999; Kaiser
et al. 2005) (Table 1). The affinities of Ly49G and W for
MHC-I ligands are also within the range for the T cell receptor
(TCR) binding to MHC-I-peptide antigen complexes
(Table 1) but not as avid as that by Ly49H or Ly49I for the
MCMV-encoded MHC-I surrogate, m157 (Table 1). As observed with the KIR and CD94/NKG2 receptors, the Ly49G/
Immunogenetics (2014) 66:467–477
W-MHC-I interactions exhibited very fast kinetics with association and dissociation rates that were beyond our ability to
measure. A previous study compared the affinity of interaction
of Ly49A and Ly49C with H-2Dd and found that Ly49A binds
with approximately 30-fold greater affinity (Deng et al. 2008)
(Table 1). Combining these results with our study, the rank
order of affinity of several Ly49 molecules for H-2Dd can be
listed as follows: Ly49A>Ly49W, Ly49G>Ly49C.
Ly49G and W share 98 % sequence identity in their CTLD
which may explain our findings of their similar affinities for
H-2Dd. Ly49G and W may bind MHC-I ligands solely via the
CTLD, similarly to the related Ly49A, according to cocrystals of Ly49A bound with H-2Dd (Tormo et al. 1999).
The extracellular stalk regions of Ly49G and W are highly
variable, but whether these differences affect their relative
ability to bind H-2Dd remains to be determined. Our findings
here show that Ly49W binds H-2Dk with higher affinity than
H-2Dd by SPR. It should be noted that the binding of
multimers of Ly49G and, to a lesser extent, Ly49W to
H-2D d was more effective than to H-2D k on EL4
transfectants. This differs from the SPR results. These differences may be due to possible influences of MHC-I-bound
peptides, as some other Ly49 receptors are known to be
peptide selective in their interaction with MHC-I (Franksson
et al. 1999; Ma and Kane 2011). This possibility is currently
unknown for Ly49G and W. In SPR experiments, a single
peptide species occupied the peptide groove of each MHC-I
tested, whereas on the cell surface, a diverse array of MHC-I
peptides are bound to MHC-I. It is conceivable that the
affinities of Ly49G and W for H-2Dd and H-2Dk may differ
depending on the particular peptide bound to these MHC-I
molecules; alternatively, there may be posttranslational modification of the MHC-I such as differential glycosylation, in
different cell types, that may influence interaction with Ly49
receptors.
We examined Ly49 exon sequences, as defined by Kubo
et al. (1993; Makrigiannis and Anderson 2001), and this
suggests that Ly49W may be a chimeric gene. Ly49L and M
are Ly49W-related genes encoding activating Ly49 receptors,
which may have arisen by tandem gene duplication (Belanger
et al. 2008). Intact Ly49L and/or M, or gene fragments thereof,
can be found in multiple inbred mouse strains (Makrigiannis
et al. 2000; Silver et al. 2001; Belanger et al. 2008). The NOD
mouse has intact Ly49M and Ly49W coding sequences
resulting in viable transcripts (Silver et al. 2001; Belanger
et al. 2008). Interestingly, through the first five exons, the last
of which is the first of three exons that encode the CTLD,
Ly49W of NOD is identical in nucleotide sequence to Ly49L of
the CBA/J strain (Makrigiannis et al. 2000). However, exons 6
and 7 of Ly49W differ substantially from Ly49L of any mouse
strain, including CBA/J, and thus, by overall sequence comparison, Ly49W does not appear to presently be an allele of
Ly49L. In comparing exon 6 and 7 sequences, it would seem
Immunogenetics (2014) 66:467–477
that these exons of Ly49W were contributed by an Ly49G
gene at some time, possibly by recombination or gene conversion with Ly49L. The Ly49G-related exons 6 and 7 of
Ly49W may contribute to its ability to bind MHC-I ligands
comparably to inhibitory Ly49, as we determined here by
SPR, particularly binding of H-2Dk. Like Ly49GBALB/c,
Ly49W has the amino acid sequence DCGK in the predicted
L6 loop encoded in exon 7, which we have shown promotes
recognition of H-2Dk (Lavender et al. 2004). By contrast, Ly49L of CBA/J and BALB/c strains has NcDK
which may not be as supportive of H-2Dk recognition
(Lavender et al. 2004).
An analogous chimeric gene to Ly49W may be Ly49O that
encodes an inhibitory Ly49 in the 129/J and C57L/J strains
(Makrigiannis et al. 1999, 2002; Mehta et al. 2001). It may
have arisen due to a recombination between Ly49A and
Ly49D, which resulted in a gene that encodes the cytoplasmic,
transmembrane, and stalk regions of the inhibitory Ly49A
receptor connected to the CTLD and ligand recognition region
of the activating Ly49D receptor (Mehta et al. 2001).
Consistent with this possibility, although containing Ly49O,
the 129/J strain lacks Ly49A and Ly49D, which both may have
contributed gene sequences resulting in Ly49O (Mehta et al.
2001). Due to the genetic plasticity of Ly49 genes, including
gene duplication and intergene recombination resulting in
chimeric receptor genes, caution must be exercised in concluding the presence and ligand specificity of specific Ly49
receptors, based solely on antibody binding or incomplete
Ly49 sequence data.
Understanding the affinity of activating Ly49 receptors for MHC-I may be important in the context of
autoimmunity. With the publication of the NOD Ly49
gene cluster sequence, it was noted that NOD mice have
the largest number of activating Ly49 genes among the
four mouse strains that have been sequenced in the Ly49
gene region (Belanger et al. 2008). The susceptibility of
NOD mice to diabetes has been mapped to the NK gene
complex, and this expanded number of activating Ly49
genes could potentially contribute to the susceptibility of
autoimmune disease in NOD mice (Carnaud et al. 2001;
Belanger et al. 2008). Although we report the equilibrium constant of one NOD activating Ly49 receptor in this
report, Ly49W is not known to recognize the H-2Db or
H-2Kd expressed by NOD. It remains to be determined
whether NOD activating Ly49 contributes to the induction of diabetes, even though NK cells can be detected in
the NOD pancreas, prior to T cell infiltration and disease
onset (Brauner et al. 2010).
Acknowledgments This work was supported by an operating grant
from the Canadian Institutes for Health Research (to K.P.K.). B.J.M.
was supported by the Canadian Institutes for Health Research and Alberta
Heritage Foundation for Medical Research studentships.
475
Conflict of interest The authors declare that they have no conflicts of
interest.
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The manuscript does not contain clinical studies or patient data nor were
animals used in this study.