of April 28, 2010
This information is current as
Lisbeth A. Guethlein and Peter Parham
Bharati Sanjanwala, Monia Draghi, Paul J. Norman,
HLA-B with KIR3DL1+Bw4
Epitope That Affect Interaction of
Polymorphic Sites Away from the Bw4
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The Journal of Immunology
on April 28, 2010
Polymorphic Sites Away from the Bw4 Epitope That Affect
Interaction of Bw4?HLA-B with KIR3DL11
Bharati Sanjanwala,2Monia Draghi,3Paul J. Norman, Lisbeth A. Guethlein,4
and Peter Parham4
KIR3DL1 is a polymorphic, inhibitory NK cell receptor specific for the Bw4 epitope carried by subsets of HLA-A and HLA-B
allotypes. The Bw4 epitope of HLA-B*5101 and HLA-B*1513 is determined by the NIALR sequence motif at positions 77, 80, 81,
82, and 83 in the ?1helix. Mutation of these positions to the residues present in the alternative and nonfunctional Bw6 motif
showed that the functional activity of the Bw4 epitopes of B*5101 and B*1513 is retained after substitution at positions 77, 80, and
81, but lost after substitution of position 83. Mutation of leucine to arginine at position 82 led to loss of function for B*5101 but
not for B*1513. Further mutagenesis, in which B*1513 residues were replaced by their B*5101 counterparts, showed that poly-
morphisms in all three extracellular domains contribute to this functional difference. Prominent were positions 67 in the ?1
domain, 116 in the ?2domain, and 194 in the ?3domain. Lesser contributions were made by additional positions in the ?2domain.
These positions are not part of the Bw4 epitope and include residues shaping the B and F pockets that determine the sequence and
conformation of the peptides bound by HLA class I molecules. This analysis shows how polymorphism at sites throughout the HLA
class I molecule can influence the interaction of the Bw4 epitope with KIR3DL1. This influence is likely mediated by changes in
the peptides bound, which alter the conformation of the Bw4 epitope. The Journal of Immunology, 2008, 181: 6293–6300.
KIR2DL, with specificity for HLA-C, and the inhibitory KIR3DL,
with specificity for HLA-A and HLA-B (1). KIR3DL1 is a highly
polymorphic inhibitory receptor that recognizes the Bw4 epitope
carried by ?20% of HLA-A allotypes and ?33% of HLA-B al-
lotypes. In most human populations ?50% of the HLA haplotypes
encode an HLA-A and/or HLA-B allotype carrying the Bw4
epitope (2). Consequently, ?75% of people have a cognate ligand
for KIR3DL1. During NK cell development the KIR gene family
is expressed in a variegated manner and, in combination with
CD94:NKGA, an HLA-E receptor, it establishes a repertoire of
cells expressing different inhibitory HLA class I receptors (3).
Cognate interactions between inhibitory MHC class I receptors,
such as KIR3DL1, and their ligands determine the extent to which
mature NK cells respond to the loss of HLA class I expression that
frequently accompanies cellular infection, malignancy, and other
iller cell Ig-like receptors (KIR),5a family of inhibitory
and activating HLA class I receptors, are principally ex-
pressed by NK cells. Prominent KIR are the inhibitory
Bw4, the epitope recognized by KIR3DL1, is determined by five
polymorphic positions in the helical part of the ?1domain (resi-
dues 77, 80, 81, 82, and 83) (4). In HLA-B, Bw4 bears an allotypic
relationship with the Bw6 epitope carried by a majority (?67%) of
HLA-B allotypes. Eight Bw4 variants are defined by polymor-
phism at positions 77, 80, and 81 (5). In contrast, positions 82 and
83 are invariant within the set of Bw4?HLA-A and HLA-B al-
lotypes. Several studies indicate that Bw4 variants having isoleu-
cine or threonine at position 80 are distinguished by NK cells and
exhibit different clinical associations (6–8). Such effects could be
mediated by different KIR3DL1 allotypes or by KIR3DS1, an ac-
tivating receptor that is structurally similar to KIR3DL1 and seg-
regates as an allele of the same locus, KIR3DL1/S1 (2).
from the heterogeneous peptides bound by Bw4?HLA-A and
KIR2DL interacts with residues 7 and 8 of the bound peptide, as well
as with the segment of the ?1helix containing residue 80, for which
and the conservation of key structural features in both the KIR2DL
and KIR3DL1 sequences suggest that KIR3DL1 interacts with
HLA-A and HLA-B in the same way that KIR2DL interact with
HLA-C (10). Supporting this model were observations that peptides
bound to the Bw4?allotype B*2705 do not permit interaction with
KIR3DL1 if they have a charged residue at either position 7 or 8 (11).
That ?25% of B*2705-binding peptides have charged residues at
position 7 or 8 suggests that this is no trivial effect (12, 13). Also,
analysis of the binding of four Bw4?A*2402 tetramers to four
KIR3DL1 allotypes revealed even greater discrimination: only 6 of
the 16 possible interactions occurred (14).
The goal of the study described herein was to determine the
contribution of variable residues in the Bw4 motif to binding of
KIR3DL1. This approach also led us to show that polymorphisms
outside of the Bw4 epitope, and which determine peptide binding,
also alter the interaction of Bw4?HLA-B with KIR3DL1.
Department of Structural Biology, Stanford University, Stanford, CA 94305
Received for publication July 1, 2008. Accepted for publication September 1, 2008.
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.
1This work was supported by National Institutes of Health Grants AI064520 and
2Current address: Department of Pediatrics, Stanford University; Stanford,
3Current address: Department of Pediatrics, Division of Immunology and Transplan-
tation Biology, Stanford University School of Medicine, Stanford, CA 94305.
4Address correspondence and reprint requests to Dr. Peter Parham or Dr. Lisbeth A.
Guethlein, Department of Structural Biology, Stanford University, Fairchild D-157, 299
Campus Drive West, Stanford, CA 94305-5126. E-mail addresses: email@example.com
5Abbreviation used in this paper: KIR, killer cell Ig-like receptors.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
on April 28, 2010
Materials and Methods
PBMC were prepared from buffy coats (Stanford Blood Center) by sepa-
ration on a Ficoll-Hypaque (GE Healthcare) gradient. Samples were ob-
tained with the informed consent of the subjects. All blood collection pro-
tocols were approved by the Stanford University Institutional Review
Board. Genomic DNA was prepared using a QIAamp Blood Kit (Qiagen)
following the manufacturer’s recommendations. KIR3DL1 allele typing
was performed by pyrosequencing (2). NK cell clones were derived from
the PBMCs of a heterozygous donor expressing KIR3DL1*005 and
*01502. PBMCs were stained with PE-Cy5-conjugated anti-CD3, PE-Cy5-
conjugated anti-CD85j mAb specific for LILRB1, FITC-conjugated anti-
CD56, PE-conjugated DX9 specific for KIR3DL1 (BD Biosciences), and
propidium iodide (PI) (Sigma-Aldrich).
Two populations of DX9-reactive NK cells were distinguished by flow
cytometry: the low binding population (DX9low) consists of cells express-
ing 3DL1*005, and the high binding population (DX9high) consists mainly
of NK cells expressing 3DL1*01502, but also the small proportion of cells
expressing both 3DL1*005 and 3DL1*01502. DX9lowNK cells (CD3?
CD85?CD56?KIR3DL1*005?) and DX9highNK cells (CD3?CD85?
CD56?KIR3DL1*01502?) were cloned at one cell per well using a FACStar
flow cytometer (BD Biosciences). Clones were established and main-
tained as described (15, 16) with minor modification. Briefly, NK cell
clones were cultured in IMDM (Invitrogen) containing 200 U/ml re-
combinant IL-2. At the start of culture and weekly thereafter the clones
were cocultured with 1 ? 106/ml irradiated PBMCs from three donors.
All clones had the cell surface phenotype CD3?CD85j?CD56?
Mutagenesis of HLA-B
Initial mutagenesis of B*5101 and B*1513 was performed in a two-step
process as described (17). Briefly, complementary primers containing the
mutation to be introduced were used in separate PCR reactions, with each
paired with a primer specific for the 5? or 3? end of the sequence to be
amplified. The resultant amplicons were gel-purified and used as template
in a second PCR reaction. This reaction used primers specific for the 5? and
3? ends of the sequence. These primers were also used to generate the
full-length wild-type sequence. The final, full-length amplicons were cloned
into the pEF6-V5-His expression vector (Invitrogen). Primer sequences are
available on request to L. Guethlein.
Subsequent mutation of B*1513-L82R was performed using the
QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene) following
the manufacturer’s recommendations. All mutations were confirmed by
complete sequencing of the insert DNA.
Transfection of 221 target cells
Plasmid DNAs containing HLA-B*5101, B*1513, and mutants of these two
alleles in the expression vector pEF6 were transfected into the class I-de-
ficient 221 cell line (18) by electroporation using a Gene Pulser (Bio-Rad
Laboratories) (19). Transfected 221 cells were cultured and maintained
under selection by blasticidine (5 ?g/ml) (Invitrogen). FITC-conjugated
W6/32 mAb (eBioscience), which recognizes all HLA class I isoforms with
similar avidity (20–22), was used to regularly monitor the expression of
HLA-B on transfected 221 cells and to sort for high-expressing cells using
a FACStar instrument. Propidium iodide was used to discriminate dead
cells. Bw4-FITC and Bw6-FITC mAbs (One Lambda) were used to eval-
uate the effect of mutagenesis on the Bw4 and Bw6 serological epitopes.
Assay of the IFN-? response of NK cells to target cells
PBMCs (5 ? 105) were incubated with 221 cells or 221 cells transfected
with HLA class I at a ratio of 1:1 in the presence of 2500 U/ml IL-2 at 37°C
for 14 h. GolgiPlug (BD Biosciences) was added after 1 h of incubation to
inhibit IFN-? secretion. Staining procedures were performed as described
(23). In brief, following incubation with the target cell, PBMCs were
stained with Dead Cell Discrimination reagent (Miltenyi Biotec), PE-Cy5-
conjugated anti-CD3, PE-Cy5-conjugated anti-CD85j, and PE-conjugated
DX9 mAb. The cells were then fixed, permeabilized, and stained with
FITC-conjugated anti-IFN-? mAb (BD Biosciences). Dead cells and cells
expressing CD3 or CD85j were excluded, allowing the DX9?NK cells to
be analyzed for the intracellular accumulation of IFN-?. The frequency of
DX9?NK cells producing IFN-? was determined using FlowJo software.
In Ab-blocking experiments, anti-class I mAb DX17 (BD Biosciences) was
used at a final concentration of 10 ?g/ml. Percentage IFN-? secretion was
calculated using the formula: (frequency IFN-?-secreting DX9?cells in
the presence of 221 transfectant)/(frequency IFN-?-secreting DX9?cells
in the presence of 221) ? 100.
Bw4 motifs. A, B*5101 mutants studied: four point mutants and the B*5101-
Bw6 mutant, in which the Bw4 motif was replaced by the Bw6 motif. B,
Top-down view of the ?1and ?2domains of B*5101 (PDB 1E28). The lo-
cation of the residues defining the Bw4/Bw6 motif are shown by black shad-
ing. C, Binding of the mAb W6/32, which is specific for a common epitope of
HLA class I molecules, to class I-deficient 221 cells transfected with B*5101
by flow cytometry. (Mutant B*5101-A81L was made and transfected, but was
not expressed at the cell surface.) D, Comparison of the binding of anti-Bw4
and anti-Bw6 mAbs to the transfected 221 cells. The binding of Bw4 mAb or
Bw6 mAb was normalized to that of W6/32 mAb binding and then calculated
as relative intensity of B*5101-wild-type (WT) binding for Bw4 and B*5101-
Bw6 binding for Bw6. Since first described (20) and characterized (21, 22),
W6/32 has been extensively used and reproducibly found to be insensitive to
the natural variation in HLA class I, including the Bw4/Bw6 difference. Thus,
the swapping of natural substitutions into the Bw4 motif of B*5101 is highly
unlikely to alter the epitope recognized by W6/32, although that possibility
cannot be ruled out. Experiments were performed at least 10 times with similar
results. Data from a representative experiment are shown.
Transfected 221 cells expressing B*5101 mutants with altered
6294PEPTIDE-BINDING RESIDUES AFFECT KIR-HLA INTERACTION
on April 28, 2010
The cytotoxicity of NK cell clones was measured in a standard 4-h51Cr-
release assay using 221 cells and 221 transfectants expressing HLA class
I as targets (24). In all experiments the E:T ratio was varied from 1:1 to
10:1. In Ab-blocking experiments the anti-KIR3DL1 mAb DX9 was used
at 5 ?g/ml final concentration. Percentage specific lysis was calculated
using the formula: (experimental
lease)/(total51Cr in target cells ? spontaneous51Cr-release) ? 100.
51Cr-release ? spontaneous
Mutation at positions 82 and 83 of HLA-B*5101, but not
mutation at positions 77 and 80, perturb the interaction with
To identify which residues in the Bw4 motif of B*5101 are nec-
essary for binding to KIR3DL1, we made point mutations at po-
sitions 77, 80, 81, 82, and 83 that distinguish Bw4 from Bw6 (Fig.
1A), the alternative motif that is not permissive for KIR3DL1 bind-
ing. A mutant B*5101 carrying the complete Bw6 motif was also
made (Fig. 1B). For each mutant the corresponding residue from
the Bw6 motif was introduced. Mutant cDNAs were transfected
into class I-deficient 721.221 cells and the transfected cells were
assayed for their capacity to bind mAbs specific for Bw4
(FH0007), Bw6 (FH0038), and an epitope shared by all HLA class
I isoforms (W6/32). Mutants substituted at positions 77, 80, 82,
and 83 were expressed similarly to B*5101 (Fig. 1C), but although
the mutant at position 81 was successfully transfected, no HLA
class I protein was detected at the cell surface (data not shown).
Reactivity with the anti-Bw4 mAb was retained in the mutants
substituted at positions 77, 80, or 83, but lost by the position 82
mutant. Complete substitution of the Bw4 motif in B*5101 with
the Bw6 motif gave a strong reaction with anti-Bw6. In contrast,
four of the point mutants failed to react with anti-Bw6 and the one
positive reaction, with the mutant substituted at position 80, gave
a level of anti-Bw6 binding that was ?20% of that seen with the
Bw6 mutant (Fig. 1D).
The capacity of the B*5101 mutants to function as ligands for
four KIR3DL1 allotypes (3DL1*001, 002, 005, and 1502) was
examined (Fig. 2). Populations of NK cells expressing these four
allotypes were obtained from three donors, as shown in Fig. 2A.
Two donors expressing only one 3DL1 allotype permitted analysis
of 3DL1*001- and 3DL1*002-expressing NK cells, and the third
donor expressed 3DL1*005 and 3DL1*1502 on different NK cell
populations that were separable based on the amount of DX9 anti-
KIR3DL1 Abs they bound.
We compared the IFN-? response of 3DL1-expressing NK cells
to class I-deficient 221 cells with their response to 221 cells trans-
fected with natural and mutant HLA class I (23). The specificity of
the reactions was shown by performing the assays in the presence
and absence of a blocking anti-HLA class I mAb (DX17). Similar
results were obtained with the four KIR3DL1 allotypes (Fig. 2B).
The inhibitory capacity of B*5101 was unperturbed by mutation at
1 10 100 1000
1 10 100 1000
1 10 100 1000
Specific Lysis (%)
050 100150 0 50 100 150
0 50100 150
NK cells with intracellular IFN
No mAb DX17
0 50 100150
3DL1*0013DL1*002 3DL1*005 3DL1*01502
* ** * *
cytometric analysis with the anti-KIR3DL1 mAb DX9 of NK cells from donors expressing different KIR3DL1 allotypes. NK cells from the 3DL1*005:
*01502 heterozygous donor form a bimodal distribution in which the cells binding DX9 at low level express 3DL1*005 and the cells binding DX9 at high
level express 3DL1*01502. The latter cells also include the small fraction of cells expressing both 3DL1*01502 and 3DL1*005; to signify this, they are
labeled as 3DL1*01502†cells. B, IFN-? response of NK cells cultured with 221 cells or 221 cells expressing wild-type (WT) or mutant HLA-B*5101.
Cultures were performed in the absence or presence of the anti-HLA class I Ab DX17. Data shown here are the means from seven experiments. Statistically
significant differences between mutant and wild-type are indicated by ???, p ? 0.001, ??, p ? 0.01, and ?, p ? 0.05 as determined by Student’s t test. Error
bars shown are SEM. The Ab-blocking experiment was performed twice with similar results and data from one of those experiments are shown. C, Results
of cytotoxicity assays in which NK cell clones expressing KIR3DL1*005 or KIR3DL1*01502†were incubated with 221 target cells expressing wild-type
or mutant B*5101. The data were obtained at an E:T ratio of 5:1 and are representative of five experiments.
Mutations at positions 82 and 83 in the Bw4 motif of HLA-B*5101 reduce the capacity to engage KIR3DL1 and inhibit NK cells. A, Flow
6295The Journal of Immunology
on April 28, 2010
position 77 and only slightly reduced by mutation at position 80.
KIR3DL1*002 and *1502 allotypes were more affected by muta-
tion at position 80 than the other two allotypes tested. In contrast,
mutants substituted at positions 82 and 83 lost much of the inhib-
itory capacity for all allotypes tested. Only substitution at position
82 affected both the reactivity with KIR3DL1 and the anti-Bw4
mAb. The effects of some combinations of KIR3DL1 allotype and
HLA class I inhibitor were also examined using cytotoxicity as-
says, giving results similar to those observed for the cytokine re-
sponse (Fig. 2C). The relative insensitivity of the inhibition to
mutation at position 80 was unexpected, because dimorphism at
this position determines the specificity of the HLA-C epitopes rec-
ognized by KIR2DL, and Bw4?HLA-B allotypes with isoleucine,
but not threonine, at position 80 correlate with slower progression
to AIDS in KIR3DS1?individuals infected with HIV (7, 8). To
investigate this point further, we made and characterized a B*5101
mutant with alanine at position 80. This mutant also retained the
inhibitory capacity of B*5101 (data not shown).
Mutation at position 83 of HLA-B*1513 perturbs interaction
with KIR3DL1, but not mutation at positions 80 and 81
We chose the B*5101 allotype for analysis because it is common,
widespread, and a strong ligand for KIR3DL1 (25). Previously
same Bw4 motif as B*5101 but differs elsewhere in the molecule
(Fig. 3). In that study of positions 82 and 83, mutation of arginine
83 in B*1513 to glycine gave the same disruptive effect as seen
here for B*5101 (26). In contrast, mutation of leucine 82 to argi-
nine in B*1513 had no effect, whereas in B*5101 that mutation
abrogated interaction with KIR3DL1. Side-by-side comparison in
assays to measure the IFN-? response (Fig. 4A) and cytotoxicity
(Fig. 4B) confirmed this difference, indicating that one or more of
the substitutions that distinguish B*1513 from B*5101, and which
are outside of the Bw4 motif (Fig. 3), influence interaction with
As we had failed to obtain a B*5101 mutant at position 81, this
mutation was made in B*1513. Replacing alanine 81 with leucine
gave a mutant B*1513 that was well expressed at the cell surface
(Fig. 4C). In functional analysis it behaved similarly to the position
80 mutant of B*5101, retaining full reactivity with monoclonal
anti-Bw4 and most reactivity with KIR3DL1 (Fig. 4D). Wild-type
B*1513 and its mutants at positions 81 and 82 mediated greater
inhibition through 3DL1*001 than the other KIR3DL allotypes.
From the combined analysis of B*5101 and B*1513, we find that
arginine 83 is the one residue within the Bw4 motif that is essential
for interaction with KIR3DL1. At position 83 the difference be-
tween the Bw4 and Bw6 motifs is extreme, with the large posi-
tively charged arginine residue in Bw4 being replaced by the
small, neutral glycine in Bw6.
Polymorphisms at sites in the extracellular domains that are
not part of the Bw4 motif influence interaction of Bw4?HLA-B
HLA-B*5101 and B*1513 differ by 13 amino acid substitutions: 2
in the leader peptide, 3 in the ?1domain, 7 in the ?2domain, and
1 in the ?3domain (Fig. 3). To identify which of these substitu-
tions allowed mutation at position 82 in B*1513 (but not B*5101)
to retain KIR binding, we introduced further mutations into B*
1513-L82R by replacing B*1513 residues with the substitutions
present in B*5101.
In a first set of four mutants, the leader peptide and each of the
three extracellular domains of the B*1513-L82R mutant were in-
dividually converted to the B*5101 form. Changing the leader pep-
tide had no affect on the capacity of B*1513 to interact with
KIR3DL1 and inhibit NK cell functions. In contrast, all three mu-
tants having a B*5101 extracellular domain exhibited a reduced
have identical Bw4 motifs but differ at 13
positions throughout the rest of the se-
quence. A, Table showing the positions of
amino acid sequence difference between
the Bw4?B*5101 and B*1513 allotypes.
B, Top-down view of the ?1and ?2do-
mains of B*5101 with bound peptide
(PDB 1E28). The location of the residues
differing between B*5101 and B*1513 are
indicated by black shading. C, Side view
of B*5101 with bound ?2-microglobulin
(?2-m). The location of the residues differ-
ing between B*5101 and B*1513 are indi-
cated by black shading.
HLA-B*5101 and B*1513
6296 PEPTIDE-BINDING RESIDUES AFFECT KIR-HLA INTERACTION
on April 28, 2010
capacity to engage KIR3DL1 and inhibit cytokine secretion by NK
cells (Fig. 5). The greatest perturbation was seen for the ?2domain
conversion, with equivalent and lesser effects for the ?1and ?3
domain conversions. As the ?3domain of B*1513 only differs
from that of B*5101 by substitution of isoleucine for valine at
position 194, these results demonstrate the contribution of isoleu-
cine 194 to the interaction of B*1513-L82R with KIR3DL1.
Residues 67 in the ?1domain, 116 in the ?2domain, and 194
in the ?3domain account for most, but not all, of the capacity
of B*1513-L82R to engage KIR3DL1
A second set of mutants was designed to examine the effects of
lone substitutions and clusters of substitutions in the ?1and ?2
domains (Fig. 3). Distinguishing B*1513 and B*5101 in the ?1
domain is the pair of substitutions at positions 45 and 46 and the
lone substitution at position 67. Mutation at position 67 reduced
the inhibitory function of B*1513-L82R, whereas it was preserved
when positions 45 and 46 were mutated (Fig. 6A). Thus, polymor-
phism at position 67 was responsible for the functional effect con-
tributed by the ?1domain.
Distinguishing B*1513 and B*5101 in the ?2domain is one
cluster of three substitutions at positions 94, 95, and 97, another at
positions 113, 114, and 116, and a lone substitution at position
171. To investigate their contribution to preserving KIR3DL1 in-
teraction with B*1513-L82R, mutants containing these three sets
of differences were constructed. The triple mutant at positions 94,
95, and 97 partially reduced the inhibition, an effect that was sta-
tistically significant for 3DL1*005 but not for 3DLl*01502 (Fig.
6A). Mutation at position 171 also had a minor effect but did not
response of NK cells expressing defined KIR3DL1 allotypes cultured with 221 cells expressing wild-type (WT) or L82R mutants of HLA-B*5101 or
HLA-B*1513. B, Comparison of the capacity of the B*1513-L82R and B*5101-L82R mutants to engage 3DL1*01502†and inhibit the cytolytic response
of NK cells to class I-deficient 221 cells. The experiment was performed three times in duplicate with similar results. Data from a representative experiment
are shown in the figure. C, Comparison of the binding of anti-Bw4 and anti-Bw6 mAbs to wild-type and mutant B*1513. The experiment was performed
eight times with similar results. Data from a representative experiment are shown. D, Capacity of wild-type and mutant B*1513 to bind different KIR3DL1
allotypes and inhibit NK cells. Data shown are the average of three experiments. Statistically significant differences between mutant and wild-type are
indicated by ???, p ? 0.001, ??, p ? 0.01, and ?, p ? 0.05 as determined by paired t test of means. Error bars shown are SEM. Cell surface expression
levels of B*1513 and its mutants were monitored with W6/32, with all transfectants expressing similar levels (data not shown).
Unlike B*5101, substitution of leucine for arginine at position 82 of B*1513 retains interaction with KIR3DL1. A, Comparison of the IFN-?
the differential engagement of KIR3DL1 by B*5101-L82R and B*1513-
L82R. Further mutation of B*1513-L82R generated four mutants in which
the leader peptide, the ?1domain, the ?2domain, or the ?3domain, had the
B*5101 sequence. The capacity of these mutants to engage KIR3DL1*005
and *01502 on NK cells and inhibit the IFN-? response to 221 cells was
measured in the presence and absence of anti-HLA class I Ab DX17. Data
shown are the average of five experiments. Statistically significant differ-
ences between mutant and wild-type are indicated by ???, p ? 0.001,
??, p ? 0.01, ?, and p ? 0.05 as determined by paired t test of means. Error
bars shown are SEM. The Ab-blocking experiment was performed twice
with similar results, and data from one of those experiments are shown.
Polymorphisms in the ?1, ?2, and ?3domains contribute to
6297The Journal of Immunology
on April 28, 2010
reach significance. Because the triple mutant at positions 113, 114,
and 116 failed to be expressed, we mutated these three residues
individually. The mutant at position 114 failed to be expressed,
whereas the mutants at positions 113 and 116 were well expressed,
indicating that failed expression of the triple mutant was due to the
substitution at position 114. Individual mutations at positions 113
and 116 both significantly reduced the inhibition, but the effect was
greater for the 116 mutant. In conclusion, all four mutants with
substitutions in the ?2domain showed diminution of inhibitory
capacity (Fig. 6A). The strongest effect was due to mutation at
residue 116, which reduced interaction with 3DL1 to a level com-
parable to that seen with the position 67 mutant. Smaller effects
were observed for mutation at residues 94, 95, 97, 113, and 171.
The contribution of residue 114 could not be assessed directly
because of the failure of mutants at this position either to be ex-
pressed at the surface of successfully transfected cells or to be
recognized by the W6/32 Ab.
To test the hypothesis that substitution at positions 67 in the ?1
domain, 116 in the ?2domain, and 194 in the ?3domain all con-
tribute to the functional effect, we generated a triple mutant with
all three substitutions and double mutants having the three pair-
wise combinations. Of these four mutants, the triple mutant
showed the weakest interaction with KIR3DL1 (Fig. 6B), demon-
strating that polymorphism at all three positions contributes to the
differential interaction of the B*5101-L82R and B*1513-L82R
mutants with KIR3DL1. Combined mutation at positions 67, 116,
and 194 did not completely abrogate the interaction of B*1513-
L82R with KIR3DL1, showing that one or more polymorphic res-
idues additionally contributed to the capacity of B*1513-L82R to
engage KIR3DL1 and inhibit NK cells. As an alternative approach
to assess the role of position 114, as the point mutant was not
expressed, we made a mutant combining substitution at positions
67, 113, 114, 116, and 194. That this mutant preserved greater
interaction with KIR3DL1 than the double mutant having only the
substitutions at positions 67 and 194 suggests that position 114
does not add to the effects contributed by positions 67, 116, and
194 (Fig. 6B). In summary, our results show that residues 67 in the
?1domain, 116 in the ?2domain, and 194 in the ?3domain make
a major contribution to distinguishing KIR3DL1 recognition of
B*5101-L82R and B*1513. Smaller contributions are due to other
residues in the ?2domain, which include 113, 171, and at least one
residue from 94, 95, and 97.
The Bw4 motif comprises three variable (77, 80, and 81) and two
conserved residues (82 and 83). By swap mutagenesis, in which
Bw4 residues were replaced by their Bw6 counterparts, we find
that none of the variable residues is essential for binding to
KIR3DL1 and inhibiting NK cells. Both conserved residues are
essential for the binding of Bw4?B*5101 to KIR3DL1, but only
residue 83 is essential for the binding of Bw4?B*1513 to
KIR3DL1. In a model of the binding of KIR3DL1 to HLA-B*
5101, arginine 83 is shown to contact the KIR (27). Of note, the
isoleucine at position 80 that has been correlated with progression
of HIV infection (8) is not essential for binding to KIR3DL1.
As B*5101 and B*1513 have identical Bw4 motifs, one or more
of the 13 substitutions that distinguish the two allotypes were af-
fecting the capacity of the mutant at position 82 to bind KIR3DL1.
Further swap mutagenesis showed that substitutions in the leader
peptide (-8 and -11) and at positions 45 and 46 in the ?1domain
are not involved, and by implication neither was residue 114 in the
?2domain. Major contributions are made by positions 67 in the ?1
domain, 116 in the ?2domain, and 194 in the ?3domain, with
smaller contributions from residues 94, 95, 97, 113, and 171.
Throughout this study similar effects and trends were seen with
four KIR3DL1 allotypes: 3DL1*001, *002, *005, and *1502.
Three-dimensional structures for two complexes of antigenic
peptides bound to HLA-B*5101 have been compared with struc-
tures for the serologically related HLA-B*3501 and B*5301 allo-
types (27). B*5101 was seen to bind peptides in a different, non-
standard manner to B*3501 and B*5301, due to an F pocket of
decreased size and altered conformations for both ends of the pep-
tide and its central region. The substitutions responsible for these
differences were at positions 95, 97, 116, and 171 in the ?2domain
(Fig. 7). At these four positions B*1513 has the same residues as
B*1513-L82R is principally due to residues 67, 116, and 194, but has
contributions from other ?2domain residues. Further mutation of B*1513-
L82R was performed to identify individual substitutions and combinations
of substitution within the ?1, ?2, and ?3domains that contribute to the
differential engagement of KIR3DL1 by B*5101-L82R and B*1513-L82R.
Assays were as described in Fig. 5. A, Effects of mutation at single posi-
tions or clusters of positions within the ?1and ?2domains. B, Effect of
combinations of mutations involving positions in different extracellular do-
mains. The results are plotted as the percentage of IFN-? secretion. Data
shown here are averages of three experiments. Statistically significant dif-
ferences between mutant and wild-type are indicated by ???, p ? 0.001,
??, p ? 0.01, and ?, p ? 0.05 as determined by paired t test of means. Error
bars shown are SEM. Cell surface expression level of B*1513 and its
mutants was monitored with W6/32, with all transfectants expressing sim-
ilar levels (data not shown).
Differential KIR3DL1 engagement by B*5101-L82R and
6298 PEPTIDE-BINDING RESIDUES AFFECT KIR-HLA INTERACTION
on April 28, 2010
B*3501 and B*5301, and differs from them at only two other po-
sitions (113 and 152) within the ?2domain. These similarities first
predict that B*1513 binds peptide in a manner more like B*3501
and B*5301 and different from B*5101; second, they point to dif-
ferences in the conformation of bound peptide being responsible
for the differential capacity of the L82R mutants of B*5101 and
B*1513 to engage KIR3DL1. The contribution of residue 67 in the
?1domain, which affects the architecture of the B pocket, also
invokes a peptide-mediated effect. In summary, this study, which
shows how peptide-binding residues of the MHC class I molecule
can affect the interaction of KIR3DL1, complements previous
analyses showing that amino acid sequence differences in the pep-
tides bound by a class I molecule can create complexes that are
permissive or nonpermissive to KIR3DL1 binding (14, 28).
The effect of substitution at position 194 on receptor engage-
ment was unexpected. Residue 194 is located on the membrane-
proximal end of the ?3domain at a considerable distance from the
sites in the ?1and ?2domains that bind peptides and are predicted
to contact KIR3DL1. Residue 194 does contribute to the site on
HLA class I that binds to the LILRB1 NK cell receptor (29), rais-
ing the possibility that LILRB1 contributed to the inhibitory effects
we studied. However, in the design of our experiments we delib-
erately excluded the participation of NK cells expressing LILRB1.
What we cannot exclude is a possible contribution from another
member of the LILR family. Alternatively, the effect of substitu-
tion at position 194 may be to change the conformation of HLA-B
so that it interacts less efficiently with KIR3DL1. Additional ex-
periments will be needed to distinguish these possibilities.
Structural and biochemical studies demonstrate that HLA-B*
5101 is unusual compared with related HLA-B allotypes. Peptide
assembly with B*5101 is slow (30) and the peptides bind in a
nonstandard manner (27) and with low affinity (31, 32). Our results
point to the possibility that the interaction of B*5101 with
KIR3DL1 is also nonstandard, as exemplified by comparison of
B*5101 to B*1513. Again, B*5101 seems less fit, with its inter-
action with KIR3DL1 being highly sensitive to mutation at posi-
tion 82, whereas that of B*1513 is not. This suggests that the
conformation of the Bw4 epitope is significantly perturbed in B*
5101, but not in B*1513, by replacement of leucine 82 with argi-
nine. Such conformational lability in the Bw4 epitope might be a
direct consequence of the low-affinity binding of peptides to B*
5101 and the reduced stabilization they bring to the MHC class I
structure. Pertinent to this point, identical mutation of position 80
prevented expression of B*5101 but not B*1513, and the binding
of different peptides to B*5101 changed the conformation of the
Bw4 epitope as detected by mAbs (33).
pockets that bind peptide anchor resi-
dues are correlated with differential
KIR3DL1 engagement by B*5101-
L82R and B*1513-L82R. Shown are
the nine residues that distinguish B*
1513 and B*5101 and influence the
binding to KIR3DL1. Also shown is
their polymorphism in B*1501, B*
5301, and B*3501. Identities with B*
1513 are shown by a dash. B*5301 and
B*3501 differ only in the Bw4/Bw6
motif. Crystallographic structures have
been determined for all these allotypes,
except for B*1513 (27, 42–44). Al-
though B*5101 has serological similar-
unusual peptide binding site as summa-
rized under “Structural differences be-
tween B*5101 and B*5301/B*3501”.
At key positions that cause these differ-
ences (95, 97, 116, and 171), B*1513 is
identical to B*3501. Thus, the peptide
binding site of B*1513 is likely to be
more like those of B*5301 and B*3501
and distinct from that of B*5101.
Changes in the B and F
6299The Journal of Immunology
on April 28, 2010
A further property that distinguishes B*5101 from other HLA-B
allotypes is its association with Behc ¸et’s disease, a chronic and
systemic inflammatory disease correlated with several genetic and
environmental factors (34). NK cells have been studied in patients
with Behc ¸et’s disease (35–37) and perturbations in KIR3DL1-ex-
pressing NK cells observed (38). Polymorphisms in the genes for
HLA-E and its cognate NK cell receptor CD94:NKG2A were also
correlated with disease (39). Given the complementary roles that
CD94:NKG2A and inhibitory KIR play in the development and
function of human NK cell repertoires (40), it is plausible that KIR
may also contribute to disease susceptibility. Supporting this con-
tention, patients with Behc ¸et’s disease are more likely to have the
compound genotype of Bw4 and KIR3DL1 than do matched con-
trols (41). Thus, the success or failure of the distinctive Bw4
epitope of B*5101 to interact with KIR3DL1 could be a factor that
contributes to the incidence and progression of Behc ¸et’s disease.
The authors have no financial conflicts of interest.
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