HLA Class I Allelic Sequence and
Conformation Regulate Leukocyte Ig-Like
Receptor Binding
This information is current as
of June 14, 2017.
Des C. Jones, Vasilis Kosmoliaptsis, Richard Apps, Nicolas
Lapaque, Isobel Smith, Azumi Kono, Chiwen Chang, Louise
H. Boyle, Craig J. Taylor, John Trowsdale and Rachel L.
Allen
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2011; 186:2990-2997; Prepublished online 26
January 2011;
doi: 10.4049/jimmunol.1003078
http://www.jimmunol.org/content/186/5/2990
The Journal of Immunology
HLA Class I Allelic Sequence and Conformation Regulate
Leukocyte Ig-Like Receptor Binding
Des C. Jones,* Vasilis Kosmoliaptsis,†,‡ Richard Apps,* Nicolas Lapaque,x
Isobel Smith,* Azumi Kono,{ Chiwen Chang,* Louise H. Boyle,* Craig J. Taylor,†
John Trowsdale,*,1 and Rachel L. Allen‖,1
H
uman leukocyte Ag class I proteins direct the functions
of both adaptive and innate immunity through their recognition by the TCR and leukocyte receptor complexencoded receptors, which include members of the killer Ig-like
receptor (KIR) and leukocyte Ig-like receptor (LILR) families.
LILRs expressed on professional APCs can influence adaptive
immune responses (1) by modulating cytokine release and costimulatory receptor expression (2–6)
LILRs are termed activating (LILRA) or inhibitory (LILRB) on
the basis of their cytoplasmic domains. Inhibitory LILRs possess
a long cytoplasmic tail containing ITIMs (7–9), whereas activating LILRs possess a short cytoplasmic tail and associate with the
adaptor molecule FcεRg (10, 11). The putative soluble protein
LILRA3 has no known signaling ability (12, 13). The best characterized members of the LILR family are the inhibitory receptors
*Immunology Division, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom; †Tissue Typing Laboratories, Addenbrookes Hospital, Cambridge, CB2 0QQ United Kingdom; ‡Department of Surgery, University of
Cambridge, Addenbrooke’s Hospital, Cambridge, CB2 0QQ United Kingdom; xInstitut National de la Recherche Agronomique, Unité Mixte de Recherche, 1319
Micalis, Domaine de Vilvert, F-78352 Jouy-en-Josas, France; {Department of Basic
Medical Science and Molecular Medicine, Tokai University School of Medicine,
Isehara, Kanagawa 259-1143, Japan; and ‖Centre for Infection, St. George’s Hospital,
University of London, London SW17 0RE, United Kingdom
1
J.T. and R.LA. are cosenior authors.
Received for publication September 17, 2010. Accepted for publication December
23, 2010.
This work was supported by Arthritis Research UK Project Grant 17951, the Wellcome Trust, the Medical Research Council, and the National Institute for Health
Research Cambridge Biomedical Research Centre.
Address correspondence and reprint requests to Dr. Des C. Jones, Immunology Division, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: FHC, free H chain; KIR, killer Ig-like receptor;
LD, linkage disequilibrium; LILR, leukocyte Ig-like receptor; b2m, b2-microglobulin; MFI, mean fluorescence intensity; SAB, single Ag bead.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003078
LILRB1 (ILT2/LIR1/CD85j) and LILRB2 (ILT4/LIR2/CD85d),
which recognize a wide range of classical and nonclassical HLA
class I proteins (8, 9, 14–22). The activating receptor LILRA1 has
been shown to bind HLA-B27, the product of which is associated
with diseases such as ankylosing spondylitis (23).
Classical HLA class I proteins are highly polymorphic (24).
KIRs, expressed on NK cells and some T cell subsets, recognize
subsets of HLA class I alleles. Their binding may be influenced
also by peptide bound to class I (25–30). KIR variation, in conjunction with that in HLA class I, is associated with diseases,
including viral infections (31, 32), autoimmunity (33, 34), and
complications of pregnancy (35). The broad specificity of LILRB1
and LILRB2 results from their interaction with the conserved b2microglobulin (b2m) subunit (LILRB1) and/or the a3 domain of
HLA class I (20, 36). Despite this, a study of four different HLA
class I alleles indicated a range of affinities for LILRB1 and
LILRB2 (21). Such differences in the affinity and avidity of ligand
binding can influence signaling through LILR and the activation
of APCs bearing them (5). Consequently, variations in affinity for
individual HLA class I alleles might be detected by genetic association of LILR with disease (37).
Although HLA class I molecules usually require association with
b2m and antigenic peptide before they can progress to the cell
surface (38), they can subsequently dissociate to generate open
confomers that lack peptide and/or b2m (39, 40). Unfolded HLA
class I molecules are a feature of activated lymphocytes (41–47)
and their recognition and functions are of growing interest (45,
48). The inhibitory receptor LILRB2 can bind to b2m-free forms
of both the HLA-B27 allele as well as the nonclassical HLA-G
molecule (20, 23). The activating receptor LILRA1 has also been
shown to engage b2m-free forms of HLA-B27 (23).
We sought to perform what we believe to be the first comprehensive study of LILR binding to the products of different HLA
class I alleles using a panel of .90 single Ag beads (SABs) (49).
We also determined the binding of LILR to “unfolded” or b2mfree forms of HLA class I.
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Leukocyte Ig-like receptors (LILRs) are a family of innate immune receptors predominantly expressed by myeloid cells that can
alter the Ag presentation properties of macrophages and dendritic cells. Several LILRs bind HLA class I. Altered LILR recognition
due to HLA allelic variation could be a contributing factor in disease. We comprehensively assessed LILR binding to >90 HLA class
I alleles. The inhibitory receptors LILRB1 and LILRB2 varied in their level of binding to different HLA alleles, correlating in
some cases with specific amino acid motifs. LILRB2 displayed the weakest binding to HLA-B*2705, an allele genetically associated
with several autoimmune conditions and delayed progression of HIV infection. We also assessed the effect of HLA class I
conformation on LILR binding. LILRB1 exclusively bound folded b2-microglobulin–associated class I, whereas LILRB2 bound
both folded and free H chain forms. In contrast, the activating receptor LILRA1 and the soluble LILRA3 protein displayed
a preference for binding to HLA-C free H chain. To our knowledge, this is the first study to identify the ligand of LILRA3. These
findings support the hypothesis that LILR-mediated detection of unfolded versus folded MHC modulates immune responses
during infection or inflammation. The Journal of Immunology, 2011, 186: 2990–2997.
The Journal of Immunology
2991
Materials and Methods
Ab blocking assay
Cloning of LILR sequences and production of LILR-Fc DNA
constructs
The HLA class I Abs W6/32 and HC10 were assessed for their ability to
block LILRB1, -B2, and -A1 binding to b2-associated and FHC forms of
C*0602 expressed on stably transfected 721.221 cells. Concentrated supernatants containing 0.5 mM LILR-Fc fusion protein were incubated for
1 h at 4˚C with PE-labeled F(ab9)2 goat anti-human Fc Ab (109-116-170;
Jackson ImmunoResearch Laboratories) at a concentration of 5 mg/ml.
Meanwhile, 1 3 105 cells were incubated for 30 min at 4˚C with either
1 mg W6/32 (Sigma-Aldrich), 10 ml HC10 supernatant, 1 mg IgG2a isotype Ab (Sigma-Aldrich), or W6/32 and HC10 in combination and brought
to a volume of 25 ml with PBS/2% BSA. Twenty-five microliters of the
LILR-Fc/secondary Ab solution was then added to the cell/Ab mixture and
incubated for a further 1 h at 4˚C. Cells were washed twice with PBS/2%
BSA followed by a final wash in PBS. The level of LILR-Fc binding was
measured using a FACScan flow cytometer (BD Biosciences). The same
procedure was performed on cells previously treated with pH 3.5 citrate
buffer (0.131 M sodium citrate, 0.066M sodium phosphate, and 2% BSA)
for 30 s at 4˚C followed by two washes with PBS/2% BSA to increase the
level of HLA class I FHC.
Statistical analysis
The relationship between LILR-Fc binding and level of b2m-associated
HLA class I (as determined by W6/32) was assessed by non-linear regression (GraphPad Prism 5) generating a curve of best fit and R2 values.
Differences in LILR-Fc binding between groups of HLA class I alleles
were statistically assessed using a two-tailed Mann–Whitney U test (Graph
pad 5). LILR MFI values were normalized against level of HLA class I by
division using W6/32 (for b2m-associated HLA) or HC10 (for FHC) MFIs
prior to statistical analysis.
Transfections and production of LILR-Fc fusion proteins
Prior to transfection, HEK293T cells were maintained in RPMI 1640
supplemented with 10% FBS and penicillin-streptomycin (50 U/ml) and
incubated at 37˚C with 5% atmospheric CO2. HEK293T cells were
transfected with Signal plgplus vector containing LILR-Fc coding sequence or an empty Signal plgplus control vector using jetPEI transfection
reagent (Polyplus Transfection), following the manufacturer’s recommended protocol. Culture media were replaced with fresh Pro293a serumfree medium (Lonza) 24 h posttransfection. Supernatants were harvested
5 d posttransfection and concentrated using an Amicon Ultra 15 centrifugal filter column (Millipore) with a nominal molecular mass limit of 30
kDa. The retentate was then washed twice with 10 ml PBS while on the
filter column.
The concentration of Fc protein was assessed by ELISA.
MHC SAB binding assay
Fc-fusion proteins at a concentration of 1 mM were screened against
LABScreen HLA class I SAB (LS1A04 lot no. 005; One Lambda, Canoga
Park, CA) and HLA class II-coated beads (LSM12 lot no. 008; One
Lambda), according to the manufacturer’s standard protocol. The HLA
class I SAB panel contained HLA-A (n = 31), -B (n = 50), and -C (n = 16)
alleles, the protein sequences of which are provided in the Supplemental
Materials section (Supplemental Fig. 12). Binding was assessed on
a Luminex LABScan 100 (One Lambda), and the median fluorescence
intensity (MFI) value was obtained. For each bead, the MFI values of each
LILR-Fc were normalized for background binding by subtracting the MFI
values of the Fc-negative control. Results for B*3701 and B*4701 were
omitted due to their high background binding of LILRB3-Fc and the Fcnegative control.
To assess the level of b2m-associated HLA class I on each bead population, the HLA class I-specific mouse IgG mAb W6/32 was used and Ab
binding was detected using PE-conjugated rabbit F(ab9)2 anti-mouse IgG
(both from AbD Serotec, Oxford, U.K.). The level of b2m was measured
using the mouse mAb BBM.1 (Santa Cruz Biotechnology). The presence
of HLA class I free H chain (FHC) was assessed using the mouse mAbs
HC10 (51), HCA2 (52) (provided by Prof. Hidde Ploegh, Cambridge,
MA), and L31 (53, 54) (provided by Dr. Patrizio Giacomini, Rome, Italy).
These FHC-specific Abs vary in their recognition of allelic subsets; alleles
that carry Thr or Asn at residue 80 and Arg82 of the mature protein are
recognized by HCA2 (55). L31 recognizes alleles with Phe67 or Tyr67
(prominently HLA-C alleles) (54), whereas HC10 binds strongly to alleles
carrying Arg62 (mainly HLA-B and -C alleles) (55).
To assess binding of LILR-Fc molecules to HLA class I FHC, b2m was
removed from SABs by incubating the beads in pH 3.0 citrate buffer
(0.131 M sodium citrate, 0.066 M sodium phosphate, and 2% BSA) for 2
min, then washing twice in PBS supplemented with 1% BSA (56).
Results
Differential binding of LILRA1, LILRA3, LILRB1, and LILRB2
to HLA class I locus and allele products
On the basis of sequence similarities with LILRB1, the activating
receptor LILRA1 and the soluble receptor LILRA3 are predicted
to engage with HLA-class I (36). LILRA1 has been shown to bind
HLA B27 (23), but as yet no other alleles of HLA-class I have
been investigated and no binding studies have been reported for
LILRA3. Using Fc fusion proteins, we assessed the binding of
LILRA1, LILRA3, LILRB1, LILRB2, and LILRB3 to beads
coated with allomorphs of either HLA class I or HLA class II.
LILRA1, LILRA3, LILRB1, and LILRB2 bound HLA class I but
not HLA class II. No binding of LILRB3 to either HLA class I or
HLA class II was detected (data not shown). The inability of
LILRB3 to bind HLA class I is in accordance with previous
studies and predictions (22, 23, 36).
SABs were used next to assess the influence of isotypic and allotypic variation of HLA class I on LILR binding. The level of HLA
class I on each SAB was determined using the mAbs BBM.1 (antib2m) and W6/32 (a pan-HLA class I Ab). Their binding profiles
correlated strongly (Supplemental Fig. 1). The binding of LILRA1,
LILRA3, LILRB1, and LILRB2 to the product of each HLA class I
allele was compared with that of W6/32 (Supplemental Fig. 2). Of
all the receptors tested, LILRB1 binding correlated best with level
of HLA class I, as indicated by W6/32 reactivity (Fig. 1). However,
LILRB1 binding to HLA-A alleles displayed considerable variability (Fig. 1). HLA-A alleles with Ala193 and Val194 (numbers
correspond to the mature protein) were significantly associated with
a lower level of LILRB1 binding (Fig. 1, Supplemental Fig. 6B).
These two variable positions correspond to an established binding
site of LILRB1 (36). Two further variations, serine at position 207
and glutamine at 253, are in almost complete linkage disequilibrium
(LD) with Ala193 and Val194 and were also strongly associated with
weaker binding (Fig. 1, Supplemental Fig. 6B). Alleles that carried
a serine at position 246 also displayed lower binding, although this
was less significant (Fig. 1, Supplemental Fig. 6B) and is most
likely due to linkage with the Ala193 and Val194 polymorphisms.
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Full-length LILRB1, -B2, and -A1 were amplified from macrophage cDNA
using the primers listed in Supplemental Table I. RNA extraction and
subsequent cDNA synthesis were performed as previously described (50).
All PCRs were performed using Phusion polymerase (Finnzymes) with the
following cycling parameters: 2 min at 98˚C followed by 11 cycles of 98˚C
for 10 s, 68˚C for 30 s, and 72˚C for 60 s, followed by 21 cycles of 98˚C
for 10 s, 65˚C for 30 s, and 72˚C for 60 s, followed by 10 cycles of 98˚C
for 10 s, 60˚C for 30 s, and 72˚C for 60 s. PCRs were carried out on either
MJ Research (Reno, NV) Dyad DNA engines or MJ research PTC-200
thermal cyclers. PCR products were HindIII and XbaI (New England
Biolabs) digested and then ligated into the p3xFLAG-CMV-9 vector
(Sigma-Aldrich) using a Rapid DNA ligation kit (Roche). The full protein
coding sequence of LILRA3 was amplified from dendritic cells using
primers NK1076 and NK645 (Supplemental Table I) and cloned into
TOPO vector (Invitrogen) following the manufacturer’s recommended protocol.
The extracellular coding region of each LILR was subsequently amplified from the cloned sequences by PCR using the appropriate primer pairs
listed in Supplemental Table I. The extracellular sequence of LILRB3 was
amplified from a clone supplied by Professor Marco Colonna. PCR products
were ligated into the Signal plgplus vector containing the human IgG1-Fc
domain (R&D Systems) following a HindIII and XbaI restriction digest.
Sequences were verified by cycle sequencing using BigDye Terminator
version 3.1 methodology (Applied Biosystems) and an Applied Biosystems 3730xl DNA analyzer.
2992
LILR RECOGNITION OF HLA CLASS I
were unable to identify any HLA-A or -B residue positions that
correlated with the overall binding pattern of LILRB2. However,
the presence of a cysteine at residue 1 (Cys1) and/or an aspartic
acid at position 9 (Asp9) of HLA-C correlated with significantly
stronger LILRB2 binding (Supplemental Fig. 7). Residue 1 is
located on an exposed surface distal from the b2m and LILR
binding sites, whereas position 9 is located on the b-pleated sheet
of the a1 domain. This residue influences peptide loading within
the groove. Variation at positions 1 and 9 of class I did not influence LILRB1 binding (Supplemental Fig. 7).
LILRA1-Fc and LILRA3-Fc binding profiles did not correlate
with levels of HLA class I on SABs as determined by W6/32
reactivity. Both of these LILRs displayed an overall preference
for HLA-C (Fig. 3A, 3B, Supplemental Fig. 6A), binding strongest
to alleles that carry Asp9 (Supplemental Fig. 7B). Direct comparison of the HLA class I binding profiles for LILRA1 and
LILRA3 revealed a strong correlation (Fig. 3C), suggesting that
these two receptors share highly similar HLA recognition sites.
LILRA1, LILRA3, and LILRB2 bind HLA class I FHC
The presence of Val194, in the absence of the other polymorphisms,
as found in several HLA-B alleles (such as B*35, B*51, and B*58)
and most HLA-C alleles, did not influence LILRB1 recognition.
None of the HLA-A polymorphisms correlating with altered
LILRB1 binding appeared to influence LILRB2 recognition (Supplemental Fig. 6C). No other HLA variation correlated with altered
LILRB1 binding.
In contrast to LILRB1, there was a greater degree of variability
in LILRB2 binding to SABs. This protein displayed strongest binding to HLA-A and weakest binding to a subset of HLA-B alleles,
including B*2705 and B5701 (Fig. 2, Supplemental Fig. 6A). We
FIGURE 2. Binding of LILRB2 to HLA class I SABs. The level of
LILRB2 binding was compared with that of W6/32. The binding results
from all the HLA alleles are displayed together and separated by loci.
Two members of the LILR family, LILRA1 and LILRB2, have
previously been shown to bind b2m-free forms of the HLA-B27
allele and the nonclassical HLA-G allele (20, 23, 57). It is possible
that FHCs of other HLA class I alleles that are present on
activated lymphocytes (41–47) may also act as LILR ligands.
Levels of unfolded HLA-class I on SABs were determined using
the Abs HCA2, HC10, and L31, all of which bind unfolded H
chains of allelic subsets of HLA-class I, as described in Materials
and Methods (Supplemental Fig. 3). Binding of LILRA1-Fc,
LILRA3-Fc and LILRB2-Fc positively correlated with HC10 Ab
staining, unlike LILRB1-Fc (Fig. 4A). To further assess LILR
binding to FHCs of HLA class I, b2m was liberated from HLA
class I trimeric complexes using acid treatment. Removal of b2m
was confirmed by the almost complete loss of W6/32 binding
(Supplemental Fig. 4). The presence of the remaining HLA class I
FHC was confirmed by Ab staining (Supplemental Fig. 4). Of the
FIGURE 3. Binding of LILRA1 and -A3 to HLA class I SABs. Binding
of LILRA1 and LILRA3 was compared with that of W6/32 (A, B). Both
LILRA1 and -A3 displayed a preference for HLA-C (red dots). The
binding patterns of these two LILR correlated strongly (C, left) in comparison with LILRB2 which correlated poorly with LILRA3 binding (C,
right).
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FIGURE 1. Binding of LILRB1 to HLA class I SABs. The level of
LILRB1 binding correlates with the level of b2m-associated HLA class I,
as assessed using W6/32. Binding to products of a range of alleles from
individual HLA loci are as indicated. Binding curves were produced by
nonlinear regression. LILRB1 binding to HLA-A correlated with variation
at residues 193, 194, 207, 246, and 253 within the a3 region.
The Journal of Immunology
2993
receptors tested, LILRA1, LILRA3, and LILRB2 all bound FHCs,
whereas LILRB1 binding was abrogated by the loss of b2m (Supplemental Fig. 5). Interestingly, LILRA1 and -A3 displayed increased binding to SABs following the removal of b2m, whereas
LILRB2 binding was slightly reduced (Fig. 4B).
Blocking with mAbs W6/32 and HC10 confirms differential
LILR recognition of alternative forms of HLA class I
The Abs W6/32 and HC10 were used to block the interaction of
LILR-Fc proteins to a 721.221-HLA-C*0602 transfectant, both
before and after the partial removal of b2m (using a mild acid
treatment) to assess further the influence of HLA class I conformation on LILR binding. W6/32 dramatically reduced the binding
of LILRB1 and LILRB2-Fc to untreated cells (Fig. 5A, 5C), in
accordance with previous reports (12, 58), whereas HC10 had
a weak effect on LILRB2 and no effect on LILRB1-Fc binding.
FIGURE 5. Differential blocking of LILR binding
by W6/32 and HC10 mAbs. W6/32, HC10, and an
IgG2a isotype control were assessed for their ability
to block LILR binding to 721.221 HLA-C*0602
transfectants both before and after mild acid treatment. W6/32 and HC10 were used both singly and in
combination. Representative results are shown in A.
The effect of acid treatment on the binding of W6/32
(red), HC10 (blue), and isotype mAb (black) are
shown in B. Combined results of the blocking experiment are displayed in C. LILRA3 binding was
not determined as it gave a high level of nonspecific
binding to cells.
LILRA1-Fc (and the negative control LILRB3-Fc) failed to bind
cells prior to acid treatment.
The level of LILRB1-Fc binding diminished following acid
treatment (reflecting the reduction in the level of b2m-associated
HLA class I; Fig. 5B) and was completely abrogated by W6/32,
whereas HC10 had little influence (Fig. 5A). In contrast, W6/32
had only a moderate effect on LILRB2-Fc binding to acid-treated
cells, whereas HC10 displayed a greater level of blocking (Fig.
5A, 5C). The blocking of LILRB2 binding by either W6/32 or
HC10 is consistent with binding of this receptor to both b2massociated and FHC forms of HLA class I. The increased level
of HC10-mediated blocking, following acid treatment, suggested
that FHC was the dominant LILRB2 ligand on these cells.
LILRA1 binding, which was predominantly to acid-treated cells,
was greatly reduced by HC10 but was unaffected by W6/32 (Fig.
5A, 5C), suggesting that this receptor interacts with FHC.
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FIGURE 4. Differential binding of LILR to folded or unfolded HLA class I molecules. A, Binding of LILRB1, -B2, -A1, and -A3 was compared with the
level of FHC on untreated SABs as assessed using the mAb HC10. B, Using SABs, LILR binding of b2m-associated (folded) HLA class I was compared
with that of FHC denatured by acid treatment.
2994
The ability of HC10 to block LILRB2 and -A1 binding to FHC
suggests that some LILR contact sites are centered on the peptidebinding groove, as the HC10 binding site is located on the edge of
the empty peptide-binding groove within the a1 helix.
HLA class I variation influences LILR binding of FHC
Variation at residue 9 also appeared to influence the binding of
LILRA1, -A3, and -B2 to HLA-B and HLA-C FHC. All three
LILRs displayed strongest binding to HLA-B and -C alleles carrying Asp9 (namely HLA-B*0801, HLA-C*0602, C*0702, and
C*1802) (Fig. 7B, Supplemental Fig. 8). However, the influence of
HLA-C Asp9 was not dependent on the removal of b2m by acid
treatment (Supplemental Fig. 7B).
The presence of a free cysteine at position 1 of HLA-C was also
significantly associated with higher binding of LILRA1, -A3, and
-B2 (Fig. 7C), as it was for LILRB2 binding prior to acid treatment (Supplemental Fig. 7A). Free cysteines enable homodimer
formation for HLA-G and HLA-B27 (23, 57) and the free cysteine
at position 1 of some HLA-C alleles may be acting in a similar
manner. Dimerization of HLA-C could be important in in vivo
interactions by encouraging the clustering of HLA-C and, in doing
so, maximize LILR binding, thereby increasing the avidity of the
interaction with the dimeric LILR-Fc molecules.
Interestingly, most alleles carrying Cys1 are in strong LD with
a polymorphism known as 235C, which is associated with
delayed onset to AIDS (59). Conversely, most alleles carrying
Gly1 display strong LD with the variant associated with rapid
onset (termed 235T). Consequently, LILRA1 bound significantly
higher to the FHC products of 235C-linked alleles (p = 0.038,
Mann–Whitney U test, GraphPad Prism 5) (Supplemental Fig.
11). LILRB2 and LILRA3 also displayed preference for 235Clinked alleles (p = 0.053) (Supplemental Fig. 11).
None of the polymorphic sites that altered LILR binding of FHC
occurred within the recognition sites of the mAbs used for the
purpose of normalization, namely HC10, L31, and HCA2, which
all bind within the a1 helix. Consequently, it is unlikely that these
findings are due to altered mAb binding.
Discussion
FIGURE 6. The binding of LILRB2, -A1, and -A3 to acid treated HLA
class I SABs. A, The level of LILR binding was compared with the overall
level of acid-treated FHC on each bead as assessed by HC10. LILRB2
binding correlated with levels of FHC, whereas LILRA1 and -A3 displayed a preference for HLA-C. Only alleles that bound well to HC10 in
addition to either L31 or HCA2 were analyzed. B, The binding patterns of
LILRA1 and -A3 correlated strongly with each other but not with LILRB2.
All SABs were used in this analysis.
We assessed the binding of four LILR proteins to a panel of
classical HLA class I allelic products. LILR binding was clearly
influenced by HLA allelic variation: LILRB1 exhibited a lower
affinity for a subset of HLA-A alleles, LILRB2 displayed a lower
affinity for several HLA-B alleles, including HLA*B2705, whereas
LILRA1 and LILRA3 generally displayed a greater preference for
HLA-C alleles, particularly following the removal of b2m by acid
treatment. Additionally, HLA allelic variation located within the
peptide-loading groove influenced LILR binding of FHC, altering
LILRB2 binding to HLA-A alleles, perhaps accounting for the
high binding of LILRB2, -A1, and -A3 to HLA-B*0801 FHC.
Unusual recognition of individual HLA class I alleles by LILR
may alter the overall balance between activating and inhibitory
signals within immune cells and subsequently contribute to the
development of disease. LILRB2 displayed considerably weaker
binding to HLA-B*2705 on untreated SABs. B*2705 is genetically associated with several autoimmune conditions, particularly
ankylosing spondylitis (60). Thus, it is possible that the comparatively weak interaction of HLA B*2705 with the inhibitory receptor LILRB2 may influence immune activity during disease. For
example, as LILRs have been shown to regulate cytokine secretion, reduced LILRB2-mediated inhibition of APCs could enhance
the production of inflammatory cytokines such as TNF-a that
strongly contribute to the immunopathology of ankylosing spondylitis. Conversely, an increased tendency for activation could be
of benefit in controlling infection: HLA-B27 is strongly associated
with delayed onset of AIDS in HIV patients, as is HLA-B*5701
(61), another allele that displayed weak binding to LILRB2. These
weaker LILRB2–ligand interactions may promote more effective
immune responses against HIV-infected cells. This is in contrast to
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Using acid-treated SABs, LILRB2-Fc binding correlated well with
overall level of FHC (as assessed with HC10 binding) (Fig. 6A),
with a preference for the FHC of HLA-A compared with that of
HLA-B and -C (Supplemental Fig. 8). Significantly, LILRA1 and
-A3 displayed marked preferential binding to HLA-C FHC (Fig.
6A, Supplemental Fig. 8). These locus-specific preferences were
consistent with those found prior to the acid treatment of SABs
(Supplemental Fig. 6A). The binding patterns of LILRA1 and
LILRA3 to FHC of all HLA class I alleles tested were highly
comparable (Fig. 6B), providing further evidence that these
receptors share highly similar binding sites.
The level of LILRB2 binding to HLA-A correlated with variation
at positions 9, 144, and 145 of the mature protein, as normalized to
HCA2 (Fig. 7A, Supplemental Fig. 9). This HLA-A variation did
not influence LILRB2 binding to SABs prior to acid treatment
(Supplemental Fig. 10). Residue 9 is located on the floor of the
peptide-binding groove, whereas 144 and 145 are located on the
a2 helix. The influence of these polymorphisms on LILR binding
is further evidence of LILR contact sites outside of the characterized b2m and a3 binding regions.
LILR RECOGNITION OF HLA CLASS I
The Journal of Immunology
2995
HLA-B*3503, an allele associated with rapid onset of AIDS that
has previously been found to bind LILRB2 more strongly than the
neutral HLA-B*3501 allele. The presence of HLA-B*3503 results
in greater inhibition and reduced dendritic cell responsiveness
(37). Further work will explore the link between the strength of
LILRB2 interaction with HLA-B27/57 and APC responses.
Variations in LILRB2 binding to HLA-B did not correlate
significantly with HLA polymorphism, which is in contrast to
LILRB1 binding patterns of HLA-A. It is possible that this variation resulted from other factors, such as the nature of the peptides
presented by HLA class I and the levels of their stability as FHCs.
The influence of bound peptide on LILRB2 binding has been
demonstrated previously (5), which raises the possibility that
changes in the MHC-bound peptide repertoire in certain disease
states such as infection, malignancy, and cellular stress could lead
to altered LILR binding and consequent effects on immune function.
LILRA1 and -A3 displayed preference for FHC forms of specific
alleles, most of which belonged to the HLA-C locus (particularly
C*0602 and C*0702). Interestingly, most of those alleles are in
strong LD with a polymorphism termed 235C, located 35 kb
upstream of the HLA-C locus (59). This polymorphism is associated with increased expression of HLA-C and is genetically
linked with delayed onset to AIDS (59). Alleles in LD with the
polymorphic form associated with rapid onset (termed 235T)
overall displayed weaker binding (Supplemental Fig. 11). However, this correlation was not complete, as LILRA1 and LILRA3
bound relatively strongly to HLA-C*0702, an allele associated
with rapid onset. The observed differences in the level of binding
and the resulting modulation of LILRA1 signaling may contribute
to the relative protective or detrimental effects conveyed by some
of these alleles in HIV infection. The potential functional impact
of high-affinity 235C alleles would be further enhanced by their
increased expression. HLA-B*0801 FHC also bound LILRA1 and
-A3 strongly. HLA-B*0801 occurs on the ancestral MHC 8.1
haplotype that is linked to a number of autoimmune conditions
and appears to be associated with an exaggerated Th2-type cytokine response (62, 63).
LILRA1 and LILRA3 displayed highly comparable binding
patterns to HLA class I beads both before and after the removal of
b2m, suggesting that they share similar binding sites. LILRA3 is
predicted to encode a soluble protein (64, 65) and may therefore
compete with LILRA1 to provide a negative regulatory effect, as
previously shown for a soluble form of LILRB1 (50). LILRA3 is
not present on all leukocyte receptor complex haplotypes (64).
LILRA3 deficiency has been associated with the development of
Sjögren’s syndrome and multiple sclerosis (66–68).
Our results are consistent with differential LILR recognition of
alternative forms of HLA class I: LILRB1 binding is solely restricted to b2m-associated HLA class I, LILRB2 is able to bind
both b2m-associated and FHC forms, whereas LILRA1 only
recognizes FHC HLA class I. The LILRB1 and -B2 profiles are in
accordance with their crystal structures, which revealed a b2m
requirement for LILRB1, but not LILRB2, binding (20, 36, 69).
The ability of LILRA1 and LILRB2 to bind FHCs of HLA*B2705
has been reported previously (57, 70). LILRB2 also bound FHC
forms of HLA-G and HLA-Cw4 (20). In this study, we show that
recognition of HLA class I FHC is a feature of LILRA1, LILRA3,
and LILRB2.
The influence of polymorphism located within the peptide
binding groove on the binding of LILRB2, -A1, and -A3 to FHC
would be consistent with LILR binding to this domain. This is
supported by the ability of peptide to alter LILRB2 recognition of
HLA-B27 (5) and HC10 to abrogate LILR binding to FHC (the
HC10 binding site is located within the empty peptide-binding
groove). Consequently, the preferential binding displayed by
LILRA1 and -A3 to FHC may be due to the lack of peptide within
the groove, rather than an absence of b2m. Crystal structure
analysis has solely focused on the interaction between the first and
second Ig domains of LILR and the a3 and b2m regions of the
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
FIGURE 7. HLA class I polymorphism influences LILR binding to FHC on acid-treated SABs. A, Variation at residues 9, 144, and 145 within the
peptide-loading groove of HLA-A FHC influenced binding of LILRB2. LILRB2 binding was normalized to HCA2. Significance was calculated using
a two-tailed Mann–Whitney U test (*p , 0.05, **p , 0.01, ***p , 0.001; ns = p . 0.05; GraphPad Prism 5). B, Variation at residue 9 of HLA-C FHC
influenced LILRB2, -A1, and -A3. LILR binding was normalized to HC10. Similar results were obtained when normalizing against L31 binding (data not
shown). C, Presence of a free cysteine at residue 1 of HLA-C FHC correlated with increased binding of LILRB2, -A1, and -A3. LILR binding was
normalized to HC10. Similar results were obtained when normalizing against L31 (data not shown).
2996
Disclosures
The authors have no financial conflicts of interest.
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