HLA-F Complex without Peptide Binds to
MHC Class I Protein in the Open Conformer
Form
This information is current as
of June 16, 2017.
Jodie P. Goodridge, Aura Burian, Ni Lee and Daniel E.
Geraghty
J Immunol 2010; 184:6199-6208; Prepublished online 5 May
2010;
doi: 10.4049/jimmunol.1000078
http://www.jimmunol.org/content/184/11/6199
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References
The Journal of Immunology
HLA-F Complex without Peptide Binds to MHC Class I
Protein in the Open Conformer Form
Jodie P. Goodridge, Aura Burian, Ni Lee, and Daniel E. Geraghty
I
n the human system, MHC class I (MHC-I) proteins comprise
a group of molecules that perform a variety of functions,
extending from the classical Ag-presenting molecules HLA-A,
-B, and -C to distantly related proteins including MHC-I–related
chain A that resides in the MHC and interacts with the activating
immune receptor NKG2D (1–4). Classical MHC-I molecules are
expressed as trimeric complexes that consist of H chain (HC), b2microglobulin (b2m) and peptide, usually 8–10 aas in length, embedded in the polymorphic binding groove of the HC. Bound peptides are derived from intracellular proteins that have been cleaved
by the proteosome and other cytoplasmic peptidases and transported
into the endoplasmic reticulum (ER), usually by the transporters
associated with Ag processing (TAPs). In the ER, peptides are
loaded onto MHC-I via an assembly complex, and the trimeric
complex is subsequently expressed on the cell surface (5, 6).
Whereas a large majority of studies have focused on MHC-I
complexes as the structural component, MHC-I molecules are also
expressed on proliferating lymphoid cells as a stable pool of MHC-I
HC (7). These HCs are expressed devoid of peptide and/or b2m and
appear to be derived from fully mature MHC-I molecules rather than
arising from protein misfolding (8, 9). They were originally detected
using mAbs that do not react with MHC-I complex, but do react with
denatured MHC-I. The reactivities of most of these Abs have been
mapped to epitopes located between residues 55 and 87 in the a1
Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA
98109
Received for publication January 11, 2010. Accepted for publication March 29, 2010.
This work was supported by National Institutes of Health Grant HD045813 (to D.E.G.).
Address correspondence and reprint requests to Dr. Daniel E. Geraghty, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, D4-100, Seattle, WA
98109-1024. E-mail address: [email protected]
Abbreviations used in this paper: b2m, b2-microglobulin; B-LCL, B lymphoblastoid
cell line; ER, endoplasmic reticulum; HC, H chain; IP, immunoprecipitation; MHC-I,
MHC class I; NEM, N-ethyl maleimide; SEC, size-exclusion chromatography; SPR,
surface plasmon resonance; TAP, transporter associated with Ag processing.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000078
domain, covering sequences that directly interact with peptide in
the complex structure and thus not available as Ab epitopes when
peptide is bound. Whereas functional studies of MHC-I molecules
have understandably focused on MHC-I complex structures for
several decades, recent work has begun to examine potential alternative functions for MHC-I HC. These so-called open conformers
have been implicated in a number of interactions with other receptors both in trans and in cis, including the formation of homodimers, on the cell surface (10).
Also resident in the MHC, and more recently diverged from the
classical MHC-I than is MHC-I–related chain A, are the nonclassical MHC-I molecules HLA-E, HLA-F, and HLA-G. HLA-G
has been studied extensively after expression was identified exclusively in the placental trophoblast in alternative protein forms,
each form derived from alternative splicing of the primary mRNA
(11–13). A function for HLA-G as an inhibitor of NK activity
through interaction with ILT2 or ILT4 NK receptors has been
suggested (14, 15). HLA-E function has been elucidated through
its interaction with CD94/NKG2 receptors (16). The interaction
with such CD94/NKG2 heterodimers can augment, inhibit, or
have no effect on NK cell-mediated cytotoxicity and cytokine
production (17). Surface expression of HLA-E requires nonamer
peptides—including those derivative from the signal sequences of
other HLA class I molecules including HLA-A, -B, -C, and -G,
but not HLA-F (18)—and also forms stable complexes with limited numbers of structurally unrelated peptides (19). Similar to
MHC-I, both HLA-G and HLA-E are expressed in complex forms
with bound peptide, but are also expressed as HC under certain
circumstances. Indeed, HLA-G free HC is expressed on the surface of trophoblasts and may modulate the efficiency of the
CD85J/LIR-1 and HLA-G interaction (20). An Ab specific for
HLA-E free HC has been described, and expression has been
observed on activated lymphocytes and B lymphoblastoid cell lines
(B-LCLs) although no function has yet been suggested (18, 21).
The third nonclassical molecule HLA-F has been less studied,
and neither its native structure nor function is known. Evidence of
a physical association of HLA-F and TAP was reported (22), but
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
HLA-F has low levels of polymorphism in humans and is highly conserved among primates, suggesting a conserved function in the
immune response. In this study, we probed the structure of HLA-F on the surface of B lymphoblastoid cell lines and activated
lymphocytes by direct measurement of peptide binding to native HLA-F. Our findings suggested that HLA-F is expressed independently of bound peptide, at least in regard to peptide complexity profiles similar to those of either HLA-E or classical MHC class I
(MHC-I). As a further probe of native HLA-F structure, we used a number of complementary approaches to explore the interactions
of HLA-F with other molecules, at the cell surface, intracellularly, and in direct physical biochemical measurements. This analysis
demonstrated that HLA-F surface expression was coincident with MHC-I H chain (HC) expression and was downregulated upon
perturbation of MHC-I HC structure. It was further possible to directly demonstrate that MHC-I would interact with HLA-F only
when in the form of an open conformer free of peptide and not as a trimeric complex. This interaction was directly observed by
coimmunoprecipitation and by surface plasmon resonance and indirectly on the surface of cells through coincident tetramer and
MHC-I HC colocalization. These data suggest that HLA-F is expressed independently of peptide and that a physical interaction
specific to MHC-I HC plays a role in the function of MHC-I HC expression in activated lymphocytes. The Journal of Immunology,
2010, 184: 6199–6208.
6200
Materials and Methods
Cells and cell culture
Cell lines NKL, OSP2, Daudi, MT2, Jurkat, Hut78, THP-1, Supt1, H9,
Molt3, and U937 were all obtained from the American Type Culture
Collection (Manassas, VA) and cultured according to the product information sheet provided. B-LCLs (AMAI, BM9, BM15, Boleth, BSM, JY,
Steinlin) were previously collected and studied by the International Histocompatibility Workshops and Conference and obtained directly from the
International Histocompatibility Working Group in Seattle (29). B-LCL
721.221 was obtained from the American Type Culture Collection and
maintained in RPMI 1640 medium supplemented with 10% v/v FCS, 2
mM L-glutamine, and 1 mM sodium pyruvate. Other B-LCLs were grown
in a culture medium of RPMI 1640 supplemented with 10% FBS, 100U/ml
penicillin, and 100U/mL streptomycin.
Abs
MHC-I mAbs used in this study included: W6/32, specific for pan HLA
class I MHC complex; HCA2, specific for a subset of allelic HLA class I
HCs (30); LA45, specific for a different subset of HLA class I HCs (31);
3D12, specific for HLA-E complex; 7G3, specific for HLA-E HC without
peptide (18); 3D11 and 4A11, HLA-F specific mAbs previously described
(23); 4B4, specific for intracellular HLA-F as described in this study; and
16G1, reactive with soluble HLA-G COOH terminus (32) and used as
a control Ab for IgG1 isotype mAbs.
Acid treatment of cells was performed by resuspension of ∼2.0 3 106
cells in 1 ml RPMI 1640 plus 10% FBS (pH 2.2) for 90 s, followed by the
addition of 13 ml RPMI 1640 plus 10% FBS (pH 7.4). The cells were spun
down, washed two times, and resuspended in cold FACS buffer (PBS plus
2% BSA, 0.1% NaN3) for staining. CD3 stimulation was performed using
solid phase bound anti-CD3 mAb UCHT1 (Biolegend, San Diego, CA);
1.0 3 105 cells were incubated for 20 h in separate wells of a 96-flat well
plate coated with anti-CD3 or isotype control Ab. After 20 h, the cells were
washed twice in cold FACS buffer and stained with the appropriate reagents. N-ethyl maleimide (NEM) treatment was performed by culture of
∼1.0 3 105 cells in culture medium plus 100 mM NEM for 20 min (35),
with or without the presence of mAb HCA2 or isotype control. The cells
were spun down, washed an additional two times, and resuspended in
FACS buffer for staining.
Measurement of peptides bound to HLA-F
HLA-F was isolated from the detergent lysate of PLH cells as described
previously (18), using three different HLA-F specific immunoaffinity
columns constructed with each of 3D11, 4A11, and 4B4, an HLA-E specific 3D12 column, and a W6/32 column. A total of three separate isolations were performed using different affinity column combinations
connected in series: 1) 4A11, 4B4, and 3D12; 2) 3D12, 3D11, and 4B4;
and 3) 4A11, 4B4, 3D12, and W632. MHC complexes were eluted with 2N
acetic acid, and peptides were recovered in 2.5 N acetic acid by ultrafiltration using a Microcon filter with a 10,000 M.W. cutoff (Millipore,
Billerica, MA) and concentrated by vacuum centrifugation. Eluted peptides were subjected to tandem mass spectrometry (MS/MS) performed by
the Fred Hutchinson Cancer Research Center Proteomics Resource.
Mass spectrometry
The liquid chromatography/mass spectrometry setup consisted of a trap
column (100 mm 3 2 cm) made from an IntegraFrit (New Objective, Woburn, MA) packed with Magic C18AQ resin (5 mm, 200Å particles; Michrom
Bioresources, Auburn, CA), followed by an analytical column (75 mm 3 25
cm) made from a PicoFrit (New Objective) packed with Magic C18AQ resin
(5-mm, 100Å resin; Michrom Bioresources). The columns were connected
in-line to an Eksigent 1D+ nano-HPLC (Eksigent Technologies, Dublin, CA)
in a vented column configuration to allow fast sample loading (36). The
HPLC column setup was coupled to an LTQ-Orbitrap (Thermo Fisher Scientific, Waltham, MA) hybrid mass spectrometer using the nano-electrospray source. The MHC class peptide samples were analyzed by a liquid
chromatography-quadrupole mass spectrometer using a 60-min gradient
from 2 to 35% acetonitrile with 0.1% formic acid (against water with 0.1%
formic acid) at a flow rate of 300 nl/min. A spray voltage of 2.25 kV was
applied to the nanospray tip. The mass spectrometer experiment consisted of
a full MS scan in the Orbitrap (AGC target value 1e6, resolution 60K, one
microscan, and injection time 500 ms) followed by up to 5 MS/MS spectra
acquisitions in the linear ion trap. The five most intense ions from the Fouriertransform full MS scan were selected for fragmentation in the linear ion trap
by collision-induced dissociation with a normalized collision energy of 35%
(isolation width of 2 m/z, target value of 1e4, and injection time of 100 ms).
Selected ions were dynamically excluded for 15 s with a list size of 100,
a repeat count of 1, and a repeat duration of 15 s. The dynamic exclusion mass
width around each precursor ion was 20.55 m/z to +1.55 m/z. Charge-state
screening was used, allowing any ions with an identified charge state (+1 or
higher) to be selected for MS/MS.
Immunofluorescence staining and FACS analysis
Database identification
Cell surface expression of proteins was measured by indirect immunofluorescence staining as previously described (18). Cells were preincubated with
saturating concentration of mAb followed by washing and labeling with FITCconjugated goat F(ab9)2 anti-mouse Ig (BioSource, Camarillo, CA). Samples
were analyzed on a FACScan cytometer (Becton Dickinson, Mountain View,
CA) and analysis was performed using FlowJo (Tree Star, Ashland, OR). For
tetramer staining, 1 mg refolded H2-Db (control) or HLA-F monomer was
incubated with 1.5 mg R-PE conjugated streptavidin (SAPE; Invitrogen,
Carlsbad, CA) per reaction at room temperature for 30 min. H2-Db and HLA-F
tetramers were synthesized in vitro using previously described protocols and
constructs (23, 33, 34). Recombinant MHC-I HC and beta2-microblobulin were
expressed in Escherichia coli and purified from the inclusion body. The peptide/
MHC complex was refolded by dilution of the proteins, and subsequently purified by gel-filtration. The complex was biotinylated using BirA enzyme
(Affinity, Denver, CO) and the biotinylated complex was repurified by gel filtration. Tetramer was formed by mixing biotinylated peptide/MHC complex
with PE or Alexa Fluor 647-conjugated Streptavidin (Invitrogen) at a 4:1 ratio.
The liquid chromatography-quadrupole mass spectrometer data files were
converted to the mzXML format and subsequently searched using the
database search program X!Tandem (www.thegpm.org) (37) contained in
the CPAS analysis system (38). The default scoring algorithm in X!Tandem was replaced with the k-score scoring algorithm contained in CPAS
(39). The data were searched against the IPI human database (v. 3.59;
European Bioinformatics Institute, Hinxton, England, U.K.), in which
three trypsin (porcine and bovine) sequences have been added (80,131 total
sequences). The database search was performed with no enzyme specificity
and a maximum peptide length of 50 aas. The database search results were
analyzed using PeptideProphet (Institute for Systems Biology, Seattle,
WA) (40) and also manually filtered for likely MHC class peptides using
the following criteria: mass accuracy ,10 ppm; peptide length #13 aas;
X!Tandem expectation value ,2. The purpose of the filtering criteria was
to identify candidate peptide MS/MS scans that were then manually inspected to determine whether the MS/MS fragment ions matched the
identified peptide sequence.
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surface expression was not reduced in TAP-negative mutant lines
(23). Unlike classical MHC-I, the HLA-F cytoplasmic tail can be
required for export from the endoplasmic reticulum, implicating
a function for HLA-F independent of peptide-loading in the ER
(24). Our work using new mAbs reactive with HLA-F showed
that, although HLA-F was not surface expressed on most cell lines
that contained intracellular protein, HLA-F was expressed on the
surface of B and some monocyte cell lines and in vivo on extravillous trophoblasts that had invaded the maternal decidua (12,
23). Further examination of PBLs demonstrated intracellular
HLA-F protein expression in all resting lymphocyte subsets, including B, T, NK, and monocyte cells, and HLA-F surface expression was upregulated upon activation for all cell types (25).
HLA-F has low levels of polymorphism in humans and is highly
conserved among primates (26, 27); combined with the structural
similarities of HLA-F and other HLA class I, this suggests
a conserved function in the immune response possibly along
broadly similar lines to those of HLA-E or -G. In this study, we
explored the structure of HLA-F on the surface of B-LCLs and
activated lymphocytes by testing for similarities in peptide binding between native HLA-F and classical class I and its closer
cousins HLA-E and HLA-G, which bind relatively restricted sets
of peptides (18, 28). As a further probe of native HLA-F structure,
we used a number of complementary approaches to explore the
interactions of HLA-F with other molecules, at the cell surface,
intracellularly, and in direct physical biochemical interactions.
HLA-F BINDS MHC CLASS I H CHAIN
The Journal of Immunology
Immunoprecipitation and Western blotting
Immunoprecipitation was performed as described (18, 23) with modifications. Briefly, 1 3 109 PLH cells were lysed in 1% NP-40, 140 mM
NaCl, 200 mM PMSF, 10 mg/ml pepstatin, 14 mg/ml aprotinin in 10 mM
Tris pH 7.8, precleaned by control Ab, equally divided into three parts.
Each part was subjected to a sequential immunoprecipitation by HLA-F
specific Ab coupled sepharose 4B in three distinct sequences: 1) 4B4,
3D11 then 4A11; 2) 4A11, 3D11 then 4B4; 3) 3D11, 4A11 then 4B4.
After extensive washing with lysis buffer, Ag was eluted by 0.05 M diethylamine pH 11.2, neutralized by 1M Tris pH 6.8, separated on a 10%
Tris-Glycine gel (Novex, San Diego, CA), and electroblotted as described
(18). HLA protein was detected by mAb followed by HRP-labeled goat
anti-mouse Igs (BioSource) at 1:5000 dilution and finally with an ECL
system (Amersham Biosciences, Arlington Heights, IL). Densitometry
was performed by scanning the radiograph film with a Sharp JX-320
scanner (Sharp Electronics, Mahwah, NJ) and quantified with Image
Quant 5.0 software (University of Virginia ITC–Academic Computing
Health Sciences).
Expression and purification of recombinant HLA proteins
Biosensor analysis of the interactions between HLA-F and HCs
SPR measurements were conducted in HBS-EP buffer using a Biacore 3000
system (Biacore AB). For SPR binding analysis of HLA-F/b2m complex,
10 mg/ml HLA-F/b2m in 10 mM sodium acetate (pH 4.5) and 10 mg/ml
HLA-E/b2m (without peptide) in 10 mM sodium acetate (pH 5.0) were
covalently immobilized on a CM5 research-grade sensor chip (Biacore
AB) by standard amine coupling chemistry. The reference flow cell was
left blank. Immobilization of 1700 response units for F/b2m and 2200
response units for E/b2m (without peptide) resulted in optimal responses
for subsequent analyses. Injections of 85 ml containing various concentrations of HC and control solutions were performed at 20 ml/min, followed by a dissociation phase in buffer. Raw sensorgrams were corrected
by subtracting the reference flow cell response.
Results
Peptide elution suggests that native HLA-F is expressed as an
empty MHC-I
Previous molecular modeling of HLA-F suggested a structure similar
to other MHC-I complexes with possibly a partially open-ended
peptide binding groove (46). However, an examination of the sequence of HLA-F suggested fundamental structural differences from
other MHC-I, because 5 of 10 residues conserved in both human and
mouse MHC-I and pointing into the Ag-recognition site were substantially altered in HLA-F (47). In addition, the altered cytoplasmic
domain of HLA-F can direct surface expression through pathways
independent of loading with peptides in the ER (24). In these respects,
HLA-F is unique among MHC-I HCs, indicating that HLA-F might
also be unique in regard to peptide binding characteristics.
To examine HLA-F for peptide binding, we used B-LCLs because they express HLA-F on the cell surface (23). LCL PLH was
chosen because we had measured surface levels of HLA-F that
were highest among several LCLs examined. These cells also
express HLA-E and HLA-A, B, and C class I abundantly, providing convenient and important controls for the experimental
variables. HLA-E and MHC-I also provided examples of two
distinct ranges of peptide binding of MHC-I, where HLA-E had
been shown to bind small numbers of highly specific peptides that
are derived from the available HLA class I expressed by the LCL
being examined (18). MHC-I as assessed by pan–MHC-I Ab W6/
32 will contain hundreds to thousands of distinct peptides with
peptide motifs predicted from those HLA class I expressed in the
LCL (48).
In the first experiment, lysate from 2 3 1010 cells was passed
through columns constructed with Abs 4A11 (anti–HLA-F) and
4B4 (anti–HLA-F intracellular form) arranged in series. After acid
treatment and elution, samples were analyzed by mass spectrometry to characterize eluted peptides. To confirm these results, we
repeated the experiments two additional times using different sequential Ab columns and with the addition of the anti–HLA-F reagent 3D11 (anti–HLA-F all forms), which was not tested in the
first experiment. Positive controls were provided by the addition of
W6/32 at the end of the third series, and 3D12 (anti–HLA-E) in the
second and third series. Sequential column arrangements and the
number of specific peptides identified in each experiment are reported in Table I.
After filtering nonspecific results from complex mixtures of
peptides, 789 putative peptides eluted from the W6/32 column were
Table I.
Peptide elution summary
Number of Peptidesa Identified
(mAb)
Serial MHC-I Isolation
4A11 → 4B4
3D12 → 3D11 → 4B4
4A11 → 4B4 → 3D12 → W632
Peptide intersection
4A11 4B4 3D11 3D12 W6/32
6
ND
24
0
13
3
53
0
ND
17
ND
ND
ND
17
37
8
ND
ND
789
ND
a
Selection criteria were dMass ,0.001; scan no. #12,000; peptides with
length # 13.
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
Construction of plasmid, protein expression, and refolding was described
previously for both HLA-E and HLA-F (21, 23). The DNA sequences used
for the glycine-serine linker and a BirA substrate peptide have been described (41). b2m in pHN1+ was provided by D.C. Wiley (Harvard University, Cambridge, MA) and expressed in E. coli strain XA90.
Both HC and L chain b2m inclusion bodies were isolated from cell
pellets, washed repeatedly in detergent, and solubilized in 8 M urea, 25
mM MES (pH 6.0), 10 mM EDTA, and 0.1 mM DTT (solubilization
buffer) as described (42). Refolding was accomplished using a variation of
the method by O’Callaghan et al. (43), by dilution of 12 mg b2m (in 2 ml
solubilization buffer) into 500 ml of 400 mM L-arginine, 100 mM Tris (pH
8.0), 2 mM EDTA, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, and 0.2 mM PMSF (refolding buffer). After 1 h at 4˚C, 18.5 mg HC
(in 30 ml solubilization buffer) and 17 mg peptide (in DMSO at 1.7 mg/ml)
were added (or no peptide in the cases of HLA-E open conformer refoldings). The initial molar ratio of HC:b2m:peptide was 1:2:30. The refolding mixture was pulsed three times with additional HC at 12 h
intervals. After 48 h, the refolding mixture was concentrated initially on
a stir cell (Amicon, Beverly, MA) and subsequently in a 10-kDa cutoff
Centriprep ultrafiltration unit (Amicon). Refolded HLA-E or HLA-F was
separated from aggregates and buffer exchanged into 50 mM pipes (pH
7.0), 150 mM NaCl, 1mM EDTA, and 0.02% NaN3 on a Superdex 200
prep-grade size-exclusion chromatography column (Amersham Pharmacia,
Piscataway, NJ). Properly refolded complexes were purified by size-exclusion chromatography (SEC) to yield protein .95% pure (21).
HLA-E complex was produced as above with two different peptides:
VMAPRTLVL from HLA-A2 and VMAPRTLFL from HLA-G. HLA-A2
and HLA-A3 HCs were refolded with b2m in the presence of CMV pp65
(495–503) (NLVPMVATV) peptide and HIV Nef (73–82) (QVPLRPMTYK)
peptide, respectively (42). Refolding HC with L chain in the absence of
peptide failed to produce a well-defined peak, as assessed by FPLC for all
MHC-I attempted with the exceptions of HLA-E and HLA-F. Instead, for
HLA-A2 and HLA-A3 the generation of putative empty or peptide-free
complexes was performed using minor modifications of the method using
conditional ligands in MHC-I complexes (44). The optimal conditional
ligands used for refolding A2 and A3 HCs with b2m were KILGFVFJV
and RIYRJGATR, respectively, where J indicates the modified amino acid
that is light sensitive and labile (45). Putative empty complexes were
generated after a 5-min UV exposure and purified on an analytical Superdex 200 SEC column (Amersham Biosciences) in HBS-EP buffer (10
mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.005% v/v P-20
surfactant [Biacore AB, Uppsala, Sweden]). The proteins were injected on
the surface plasmon resonance (SPR) machine immediately after UV exposure and purification.
All the other proteins used for SPR binding studies were repurified by
SEC in HBS-EP buffer within 24 h of use. All purified proteins showed the
appropriate disulfide bonding state by comparative reducing and nonreducing SDS-PAGE and were monomeric by analytical SEC. Protein
concentrations were determined by a bicinchoninic acid protein assay
(Pierce, Rockford, IL).
6201
6202
HLA-F BINDS MHC CLASS I H CHAIN
MHC-I HC modulates HLA-F surface expression
Because it was possible to refold HLA-F with b2m we initially
attempted to identify a receptor for HLA-F by analyzing differential patterns of HLA-F tetramer binding to a diverse panel of
cell lines, followed by attempts to generate Abs that would replicate these patterns and block tetramer binding. Despite repeated
attempts, we were unable to obtain any Abs producing the expected binding patterns. At the same time, we were investigating
blocking of tetramer using existing Abs, and we observed a potential interaction between MHC-I HC Abs and HLA-F. Reactivities of two of these Abs, HCA2 and LA45, have been well
characterized with HCA2 reacting preferentially with a subset of
HLA-A locus HCs (30), where the LA45 epitope includes approximately half the alleles of both HLA-A and -B loci HCs (31).
Our initial experiments indicated that HCA2 or LA45 Ab binding
interfered with HLA-F Ab binding, because prior addition of either Ab resulted in reduced binding of the anti–HLA-F reagents
Table II. Peptides eluted from HLA-E and pan MHC-I
Ab
Peptides
3D12
VMAPRTLLL (HLA-A*0201)
VMAPRTLIL (HLA-B*3501)
ILGPPPPSFa
ILQKNVPILa
VMSKLASFLa
RFPPTPPLFb
YVAVGMILb
VMSVGFLLb
265 with motif x(MVL)xxxxx(YK) (HLA-A*0201)
98 with motif xDxxxxxxL (HLA-C*0602)
W6/32
a
Confirmed binding by elution.
b
Confirmed binding by refolding.
(Fig. 2A). However, neither HC Ab blocked the levels of F tetramer binding, and none of the anti–HLA-F reagents interfered
with HCA2 or LA45 binding (Fig. 2A). The effect of HC Abs was
specific, because LCLs that expressed different combinations of
HCA2 and LA45 epitopes affected HLA-F detection on these cells
in accord with the presence or absence of those epitopes (Fig. 2B).
The observation that 3D11 did not block HCA2 binding and that
HCA2 did not interfere with F-tetramer binding suggested the
possibility that HCA2 and LA45 were instead downmodulating
HLA-F or changing surface levels through other means, such as
HLA-F proteolytic release.
To test for proteolytic release of HLA-F, we examined supernatants for HLA-F protein after treatment with HCA2 incubation.
No HLA-F protein was detected in supernatants of cells treated
with HCA2, as assessed with an HLA-F–specific ELISA, suggesting the alternative possibility that HLA-F is internalized in
response to HC Ab. Accordingly, we repeated these experiments
in the presence of 100 mM NEM, which inhibits transcytosis and
endocytosis and prevents the uptake of surface proteins (50).
Whereas HCA2 substantially downmodulated HLA-F expression
on B-LCL AMAI, the same experimental conditions resulted in no
change of HLA-F levels when NEM was present at appropriate
concentrations (Fig. 2C). These results further suggested that
HLA-F is downmodulated upon the addition of HCA2 rather than
HCA2 blocking 3D11 binding.
Increased HCA2 binding on cells correlates with increased
HLA-F tetramer binding
Mild acid treatment of resting cells is known to destabilize class I
MHC complexes on the cell surface, causing peptide and b2m to
dissociate generating MHC-I HC (51). We thus reasoned that if
HLA-F is interacting with MHC-I HC, specific binding of the F
tetramer should increase with increased levels of MHC-I HC. We
first examined cells that are negative for HLA-F expression and for
MHC-I HC, including the T cell lymphoma cell lines Jurkat, Molt-3,
HUT-78 and H9, and monomyelocytic cell line U937, as well as
class I bare, b2m deficient Burkitt’s lymphoma cell line Daudi. All
these cells were analyzed for HCA2 binding and HLA-F tetramer
binding before and after mild acid treatment (Fig. 3A, 3B). On untreated cells, no HLA-F tetramer binding was observed, whereas
after mild acid treatment all cell lines except class I bare cell line
Daudi became both HCA2 reactive and bound HLA-F tetramer in
a manner closely associated with the intensity of HCA2 staining
(Fig. 3B). The control HLA-Ap0201 tetramer did not bind to either
untreated or acid treated cells (not shown).
A second measure of coincidence between HCA2 expression and
HLA-F tetramer binding was taken by activation of cell line OSP2,
which can be activated upon treatment with anti-CD3 Ab. Consistent with the results obtained after acid treatment, upregulation
of HCA2 was observed and was found coincident with increased F
tetramer binding (Fig. 3C). Essentially similar results were obtained with a T cell clone after activation and expansion, and
activated T cell clone 1C7-7 stained with both HCA2 and HLA-F
tetramer showed coincident expression of the two signals, as did
H9 cells after acid treatment (Fig. 3D). These latter experiments
showed that although HCA2 is not always coincident with HLA-F
Abs or with F tetramer, the latter are always found overlapping
with HC.
Coimmunoprecipitation of HLA-F with MHC-I HC including
HLA-E
If MHC-I HC does bind directly to HLA-F it might be possible to
isolate both proteins as part of a complex, depending on the strength
of the interactions. To test this possibility, we performed sequential
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identified (Table I). Given the HLA type of PLH as Ap0301,
Bp4701, Cwp0602, these peptides were analyzed using the HLA
Peptide Binding Predictions Web site (www-bimas.cit.nih.gov/
molbio/hla_bind/), which provides access to software that ranks
potential 8-mer, 9-mer, or 10-mer peptides based on a predicted
half-time of dissociation to HLA class I molecules (49). Of the
789 putative peptides eluted from the W6/32 column, 265 had the
motif for HLA-Ap0301 and 98 showed the HLA-Cwp0602 motif.
Several of these peptides were confirmed by MS/MS analysis. The
motif for Bp4701 was not available for comparison. In two experiments, we were able to unambiguously identify the expected
peptides bound to HLA-E, including nonamers derived from HLAAp0301 and Bp4701 both expressed on the target LCL (Table II). In
addition, six peptides not previously reported as binding to HLA-E,
some of which have motifs substantially distinct from the class I
derived nonamers, were identified independently from both separate
anti–HLA-E columns. Six of these peptides were confirmed as
binding to HLA-E through in vitro refolding and three of these
through subsequent peptide elution analysis (data not shown).
Using the same filtering criteria, from 3 to 53 peptides were
identified from the repeat runs of the total of six anti-F columns, on
the order of the numbers identified from the HLA-E columns.
However, no peptides with a molar abundance relative to HC within
20-fold of those obtained for HLA-E were found from any of the
anti–HLA-F columns (Fig. 1). In addition, none of the peptides
identified in anti–HLA-F Ab columns were identical between
different runs, neither between Abs in the same serial isolation nor
between the same Ab from different isolations (Table I). The results for HLA-E and pan MHC indicate a sound technical approach, supporting these negative results from the anti–HLA-F
columns analyzed within the same experiments.
The Journal of Immunology
6203
immunoprecipitations (IPs) of HLA-F from LCL PLH with three
anti-F reagents (Fig. 4). Earlier experiments had not detected any
other MHC-I protein coprecipitating with HLA-F, but were limited
in their experimentation with different detergents for lysis and
had not included all available Abs that were reactive with HLA-F
(23). One Ab that we isolated, 4B4, does not react with surface
HLA-F on any cell lines tested, but does IP HLA-F protein. In addition, Abs 3D11 and 4A11 also have distinct reactivities at specific
times after activation of lymphocytes (25), indicating that each of
the three anti–HLA-F Abs react with distinct forms of HLA-F.
These considerations motivated experimentation with a variety of
detergents and further testing of available anti-F reagents. In an
experiment designed under conditions similar to the peptide isolations described above, we performed sequential IPs with Abs 4B4,
3D11, and 4A11 in each of the three possible sequences followed by
Western blot analysis with anti-HC Abs. Representative results of
these experiments are presented in Fig. 4. Three features of these
results are noteworthy. First, within each of the HLA-F Ab IP series,
it was possible to coprecipitate HCA2-reactive proteins with Ab
4B4 regardless of the order in IP sequence. This IP product included
HLA-F (Fig. 4, left) and three bands reactive with HCA2. Control
experiments showed that HLA-F is not reactive with HCA2 under
the conditions used for Western blot analysis (data not shown). Of
the three HCA2 reactive bands observed, the lower HCA2 reactive
band corresponds in size to soluble HLA class I and can be produced
in cells as a result of proteolytic cleavage as described (52). Second,
blotting of an identical IP with anti–HLA-E–HC Ab 7G3 showed
that the middle, fainter band identified by HCA2 was in fact HLA-E
(Fig. 4, right). A third feature is the higher m.w. band that is reactive
with 3D11, found only in the 3D11 IP, and is present despite running
samples under reducing conditions.
Direct measurement of HLA-F and MHC-I HC interactions
As an additional and more direct method for measuring HLA-F and
MHC-I HC interactions, we used label-free SPR-based technology
for studying these biomolecular interactions in real time. Our
hypothesis to this point was that HLA-F physically interacts with
MHC-I HC but not with the complex form. This was based on the
coincidence of HLA-F and MHC-I HC in cell binding assays and
modulation of HLA-F surface levels as described above. However,
a direct measurement required producing the HC open conformer
presumably without peptide in a homogeneous and reproducible
manner. Because it was not possible to refold sufficient quantities
of HLA-A2 or -A3 without peptide, we took advantage of the
method of conditional MHC ligands to produce the appropriate
empty class I molecules (44). This method was developed to allow
for refolding of the stable complex, which can then be treated with
UV to produce empty or at least peptide-receptive MHC molecules that can then be charged with a peptide of choice (45). After
refolding HLA-A2 and -A3 with peptides that can form corresponding complexes but that can be cleaved upon ultraviolet irradiation, we were able to produce homogeneous preparations of
putative empty MHC molecules after purification by SEC. It is
possible that a small fragment remains, depending on its affinity,
after UV treatment. However, considerable data on MHC-I peptide binding indicates that no peptides of 5 aas have been demonstrated to stabilize classical MHC-I, supporting the likelihood
that our UV-treated complexes are indeed empty. But even if
complexes do have some portion of peptide bound, when contrasted with peptide-bound complexes where there is little or no
binding, the affinity for HLA-F is clearly and markedly changed
(Fig. 5). Furthermore, these results are consistent with the HLA-E
binding in which we have direct evidence of empty HLA-E.
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FIGURE 1. Peptide elution from MHC-I. MS elution times are charted against relative abundance for each of three MHC-I Abs W6/32 (pan class I), 4B4
(anti–HLA-F), and 3D12 (anti–HLA-E) as indicated. The relative intensity of the highest peak within each profile is indicated to the right of each
chromatogram. The positions and sequences of peptides that had confirmed sequences via MS/MS are indicated within each profile. None of the putative
peptides in the 4B4 profile were found in more than one run, and it was not possible to confirm any via MS/MS.
6204
HLA-F BINDS MHC CLASS I H CHAIN
To examine direct binding, we tested a surface coupled with
HLA-F refolded with b2m. Because we were interested in distinguishing between MHC-I in complex with peptide versus empty
MHC-I binding, we passed HLA-A2 refolded with conditional
ligand before and after UV treatment over the HLA-F/b2m surface. HLA-A2 UV-treated protein showed binding to the HLA-F/
b2m surface and increasing amounts (2.5 and 7.5 mM) of the
HLA-A2 UV-treated protein resulted in proportional increases in
SPR signals (Fig. 5A, left). Furthermore, no binding was observed
when HLA-A2 was used before the UV treatment or when the
HLA-A2 was refolded with a conventional ligand. To demonstrate
the specificity of the binding to HLA-F/b2m, a second surface
coupled with HLA-EG/b2m was used, and 2.5 mM HLA-A2 was
injected after UV treatment and over both surfaces, resulting in
significantly stronger binding to the HLA-F/b2m surface (Fig. 5A,
right). The HLA-EG/b2m surface signal accounts for nonspecific
binding of HLA-A2 to the surface. Essentially similar results were
obtained with HLA-A3, in which the signal from the UV-treated
complex was measured with increasing protein amounts, and no
background binding to HLA-F was observed without UV treatment or when conventional peptide was used for refolding. Again,
the binding was specific for HLA-F as evidenced by background
levels of HLA-A3 binding to the empty HLA-E coated surface
(Fig. 5B).
Motivated by the identification of HLA-E in the coimmunoprecipitations described above, we extended these results to include
a comparative binding of HLA-E refolded with and without
peptide. Our prior work had shown that it was possible to refold
sufficient quantities of HLA-E without peptide, obviating the need
for a similar conditional ligand strategy (21). Refolded forms of
both allelic variants were available for analysis. When either allele
was refolded with conventional nonamer peptides derived from
either HLA-A2 or HLA-G signal sequences (18), binding was
detected at background levels on either HLA-F or control HLA-E
surfaces, whereas strong binding was evident when empty versions of either HLA-E allele were exposed to the HLA-F surface
(Fig. 5C, 5D). The binding patterns of the HLA-EG and ER alleles
were essentially similar at the level of analysis performed. Although binding of HLA-ER appears quantitatively stronger in the
results shown, variation in protein preparation and purification
prevent a quantitative comparison between experiments.
Discussion
Our findings combined with previous data demonstrate that HLA-F
is expressed independent of bound peptide, at least in regard to
peptide complexity profiles similar to those of either HLA-E or
classical MHC-I. In addition, we were able to demonstrate that not
only was HLA-F surface expression coincident with MHC-I HC
expression, but that HLA-F surface expression was downregulated
upon perturbation of the MHC-I HC structure. It was further
possible to directly demonstrate that MHC-I would only interact
with HLA-F in the form of an open conformer, probably free of
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FIGURE 2. HLA-F surface expression is downregulated in response to MHC-I HC mAb. A, B-LCL SAVC cells with the indicated mAb or tetramer (solid
lines) and following addition of MHC-I HC mAbs HCA2, LA45, or anti–HLA-F 3D11 indicated within brackets (dotted lines). The reagent used for
staining in each FACS profile is indicated immediately above the bracketed mAb with control mAb (gray). B, Differential downregulation of HLA-F in
response to mAb HCA2 and LA45 in B-LCLs with and without reactive MHC-I. Cell line names and reactivities with the respective mAb are indicated
directly beneath each set of bars. Anti-F mAb 3D11 reactivity is plotted after the addition of HCA2, LA45, and W6/32 with bars according to the key on the
right. C, Relative staining of anti-F mAb 3D11 on B-LCL AMAI cells without HCA2 (solid lines) and following HCA2 addition (dotted lines) with control
mAb (gray). Cells were untreated (left) and treated with 100 mM NEM (right) as indicated.
The Journal of Immunology
6205
peptide and not as peptide bound complex. This interaction was
directly observed through coimmunoprecipitation and SPR and
indirectly on the surface of cells through coincident tetramer and
MHC-I HC colocalization. These data suggest that HLA-F is
expressed independently of peptide and that a physical interaction
specific to MHC-I HC plays a role in the functional consequence of
MHC-I HC expression in activated lymphocytes.
Although our results suggest that HLA-F is expressed as HC
without peptide, IPs using our panel of HLA-F specific Abs
coprecipitate b2m in subequimolar amounts (23, 25). Based on
these data and the differential coimmunoprecipitation experiments
reported in this study (Fig. 4), it appears likely that more than one
complex form of HLA-F is expressed, with a portion complexed
with b2m, and some or all complexed with MHC-I HC. In total
there is evidence of at least three different forms of HLA-F based
on differential staining of surface HLA-F using Abs 4A11 and
3D11 over the course of lymphocyte activation and the unique
4B4 binding pattern. In light of this, it is also possible that HLA-F
FIGURE 4. MHC-I HC, including HLA-E HC, coprecipitate with HLA-F. Cells were lysed directly in NP40 buffer containing freshly prepared 50 mM
iodoacetamide. Three sets of sequential immunoprecipitations of total cell lysate from LCL PLH were performed and separated on a reducing 10% T-G gel.
Each set of sequential IPs were performed, starting and finishing with different mAbs of the group of three as indicated above each lane. The first serial set
included in the leftmost three lanes of each gel started with IP using 4B4 and loaded in lane 1, after which the remaining material was subjected to IP with
3D11 and run in lane 2, and again the remaining lysate subjected to IP with 4A11 and run in lane 3. Two additional serial IPs were performed and run in
lanes 4–6 and 7–9 using mAbs as indicated above each lane and grouped together with bars immediately below each mAb designation. After fractionation,
each gel was blotted and analyzed via Western analysis as described in Materials and Methods using the mAb for detection indicated immediately beneath
each image. All three gels shown were identically constructed using the order of sequential IPs as indicated above each lane. The positions of m.w. markers
are indicated (left and right).
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FIGURE 3. Acid-induced free class I HC
expression facilitates HLA-F tetramer binding.
HLA-F negative and free Class I HC negative
T cell lines Jurkat, Molt-3, HUT78, H9 and
monocytic cell line U937, and Class I bare cell
line Daudi were acid treated and subsequently
stained for free Class I HC (HCA2) and for
HLA-F tetramer binding. A, HCA2 reactivity
(solid line) does not increase upon acid treatment (dashed line) for class I bare cell line
Daudi, whereas T cell line H9 becomes both
HCA2 positive (left panel, dashed line) and
HLA-F tetramer reactive (right panel, dashed
line) after acid treatment. B, Comparison of
HCA2 reactivity with HLA-F tetramer staining
in the six cell lines upon acid treatment reveals
a strong association between free class I HC
expression and HLA-F tetramer binding.
Shaded diamonds designate cells before acid
treatment and open diamonds after acid treatment. C, Cell line OSP2 examined before
(solid line) and after (dotted line) CD3 + CD28
mAb treatment and examined for MHC-I HC
expression and F tetramer binding as indicated.
D, Immunofluorescent staining (original magnification 3100) of CTL clone 1C7-7, which
expresses levels of HLA-F and HCA2 reactive
MHC-I HC, and H9 cells after acid treatment
(from A) stained with HLA-F tetramer and
HCA2 individually as indicated with overlapping profiles (right).
6206
can bind larger peptides, beyond the limits of our analysis of 13
mers, or it can be stabilized by interactions with proteins other
than MHC-I or other biomolecules such as phospholipids similar
to CD1d (53). Interestingly, the HLA-F protein is entirely dependent on its cytoplasmic tail for export from the ER, and the
HLA-F cytoplasmic sequence contains motifs that are overlapping
with those of CD1d (24, 54).
It is possible to refold HLA-F with b2m without peptide which
forms a stable structure that can be used in tetramer binding (55)
and Fig. 3). Open conformer MHC-I are relatively unstable in
comparison but are upregulated by cold treatment, whereas HLA-F
is not, suggesting that there is likely a threshold of stability that
MHC-I HC must reach to traffic to and remain on the cell surface.
The possibility that HLA-F is required for the formation of the open
conformer is raised considering their coincident surface expression
and physical interaction. Because HLA-F is otherwise expressed
intracellularly in resting lymphocytes, it is also possible that the
formation of MHC-I HC upon activation induces complex formation between HLA-F and MHC-I HC, which then signals transport
to the surface.
When free HC Abs bind to MHC-I HC on the cell surface, the
conformation of the HC may be changed to more closely resemble
MHC complex with peptide. Whether HLA-F recycles with MHC-I
HC, as suggested by internalization upon addition of HC Abs,
would clearly add an important dimension to studies of the trafficking of MHC-I HC. As an interesting supplement, HLA-F does have
a tyrosine-based internalization motif (TSQA) similar to that proposed to regulate MHC-I endocytosis and intracellular trafficking,
whereas HLA-C and HLA-G do not (56). This might be relevant to
HLA-C and HLA-G cycling in trophoblast cells in light of the coincident expression of these molecules in conjunction with HLA-F
in the placental environment (12). It is not known whether HLA-C is
expressed as a free HC in trophoblasts, but evidently HLA-G is (15,
20) in tropohoblasts that have invaded the maternal decidua, HLA-F
and HLA-G are coexpressed on the surface. It would be interesting
to explore the idea that the lack of the internalization motif on HLAC and HLA-G was directly related to their expression in the placental environment.
Our data do not address whether HLA-F interacts with all MHC-I
HC, because we were able to analyze only those MHC-I for which
a conditional ligand was available (HLA-A2 and -A3) or that we
were able to refold without peptide (HLA-E). Considering the
extensive polymorphism of MHC-I, it is possible that HLA-F
interacts with only a subset of MHC-I alleles or, more generally
stated, that the affinity between HLA-F and different MHC-I alleles
will differ substantially. Our analysis of the three MHC-I HC was
not performed under conditions that allowed for precise quantitative measurement; however, differences could be detected between HLA-A2 or HLA-A3 and HLA-E. We do not have detail on
the interaction points between HLA-F and MHC-I HC, but a logical
presumption from this work would be that they are restricted to
regions exposed upon removal of peptide.
The reactivities of MHC-I HC-specific mAbs have been mapped to
epitopes formed by residues within positions 57–84 in the a1 domain
that are specific to the open conformer (10). These residues are in
contact with peptide or otherwise hidden in the peptide-bound molecule, but become exposed in the MHC-I HC conformer. HCA2 has
been mapped to residues 77–84, probably ruling this segment out
because HLA-F and HCA2 do not compete for binding to MHC-I, but
a similar comparative analysis with other MHC-I HC specific Abs
could be useful in mapping the HLA-F contact residues. Further insight might come from comparative sequence analysis with HLA-F
homologs expressed in other primates. For example, HLA-F homologs are found in macaques with as few as 10 aa differences in the a1
to a3 domains (26).
These findings leave open the possibility that HLA-F binds to
a specific receptor or that HLA-F and MHC-I HC interactions can
occur in trans between cells. Addressing the former possibility,
whereas our immunoprecipitations with 3D11 and 4A11 did not
coprecipitate equimolar amounts of MHC-I as did 4B4, it is unlikely that this evidence argues against an interaction of HLA-F and
MHC-I HC on the surface. Subequimolar amounts were detected
with both Abs, and it is not clear that this was due to experimental
conditions (e.g., the detergent used) and possibly compounded by
Ab specificities. In fact, there is a strong correlation with binding of
HLA-F tetramer and the presence of surface MHC-I HC (Fig. 3). In
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FIGURE 5. MHC-I HC, but not H complex, binds directly and specifically to HLA-F. Surface plasmon resonance binding sensorgrams were
analyzed using surfaces coated with refolded Fb2m on the left or with two
channels simultaneously, containing Fb2m and EGb2m separately, shown
in the rightmost panels. A, HLA-A2 refolded with conditional ligand as
described in Materials and Methods was injected over the Fb2m surface
after UV exposure at 2.5 mM (red line) and 7.5 mM (purple line), and
HLA-A2 refolded with conditional ligand with no UV at 5.0 mM (green
line) and refolded with CMV pp65 (495–503) peptide at 4mM (orange
line), all as indicated. HLA-A2 refolded with conditional ligand and
treated with UV was passed over the separate and marked Fb2m (red line)
and EGb2m (blue line) surfaces simultaneously in the panel immediately to
the right. B, HLA-A3 refolded with conditional ligand was injected after
UV exposure at 6 mM (red line) and 9.5 mM (purple line) and HLA-A3
refolded with conditional ligand with no UV at 14 mM (green line) and
refolded with HIV Nef (73–82) peptide at 10 mM (orange line), all as
indicated. HLA-A3 refolded with conditional ligand and treated with UV
was passed over the Fb2m (red line) and EGb2m (blue line) surfaces simultaneously as indicated. C, HLA-EG + b2m refolded without peptide at
4mM (purple line) and refolded with nonamer from HLA-A2 (orange line)
and HLA-G (green line) were injected over the Fb2m surface. HLA-EG +
b2m refolded without peptide at 4 mM was recorded over the Fb2m (red
line) and EGb2m (blue line) surfaces simultaneously as indicated. D,
Identical to C, using refolded HLA–ER HC.
HLA-F BINDS MHC CLASS I H CHAIN
The Journal of Immunology
Acknowledgments
We thank Ofer Majdic for providing mAb LA45, Huib Ovaa and Ruiwert
Hoppes for conditional ligands, Jianhong Cao for tetramers, Wei-Dang Li
for help producing mAb 4B4, Julio Vasquez for microscopy, Thomas Spies
for HCA2, and Roland Strong for help with SPR.
Disclosures
The authors have no financial conflicts of interest.
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HLA-F BINDS MHC CLASS I H CHAIN