HLA-F Is a Predominantly Empty,
Intracellular, TAP-Associated MHC Class Ib
Protein with a Restricted Expression Pattern
This information is current as
of June 18, 2017.
Shane D. Wainwright, P. Andrew Biro and Christopher H.
Holmes
J Immunol 2000; 164:319-328; ;
doi: 10.4049/jimmunol.164.1.319
http://www.jimmunol.org/content/164/1/319
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References
HLA-F Is a Predominantly Empty, Intracellular,
TAP-Associated MHC Class Ib Protein with a Restricted
Expression Pattern1
Shane D. Wainwright,* P. Andrew Biro,† and Christopher H. Holmes2*
T
he HLA-F gene is located at the telomeric end of the
human MHC on chromosome 6 and is highly homologous
with the other HLA class I genes (1, 2). Members of
the MHC class I gene family code for transmembrane glycoprotein
H chains3 that associate with an invariant light chain, b2-microglobulin (b2m). This protein complex contains a characteristic
groove capable of accommodating peptide ligands (3, 4). MHC
class I genes are conventionally divided into two groups: the classical or class Ia genes HLA-A, -B, -C characterized by their
marked polymorphism, and the nonclassical or class Ib genes
HLA-E, -F, -G that exhibit little or no polymorphism (2, 5, 6).
Class Ia molecules are abundantly expressed on the surface of
most somatic cells, and their role in immunity is well established
(reviewed in Refs. 4 and 5). Peptides destined for class Ia are
usually derived from cytosolic proteins by proteasome degradation
and transported into the lumen of the endoplasmic reticulum (ER)
by TAP for loading into empty H chain/b2m dimers. Properly assembled H chain/b2m/peptide trimers are then exported to the cell
surface for inspection by ab CD81 T cells. In this way, class Ia
*Department of Clinical Medicine, Division of Obstetrics and Gynaecology, University of Bristol, St. Michael’s Hospital, Bristol, United Kingdom; and †Department of
Immunology, St. Bartholomew’s and Royal London School of Medicine and Dentistry, Queen Mary Westfield College, London, United Kingdom
Received for publication May 24, 1999. Accepted for publication October 18, 1999.
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.
1
This work was supported by a grant from the Medical Research Council (Grant
G9403577).
2
Address correspondence and reprint requests to Dr. C. H. Holmes, University of
Bristol, Division of Obstetrics and Gynaecology, St. Michael’s Hospital, Southwell
Street, Bristol BS2 8EG, U.K. E-mail address: [email protected]
3
Abbreviations used in this paper: H chain, heavy chain; IEF, isoelectric focusing;
1D-IEF, one-dimensional IEF; B-LCL, EBV-transformed B-lymphoblastoid cell line;
b2m, b2-microglobulin; ER, endoplasmic reticulum; Endo-H, endoglycosidase-H;
MAP, multiantigenic peptide; RAHC, rabbit antiserum against monomorphic determinants on denatured HLA class I heavy chains.
Copyright © 2000 by The American Association of Immunologists
proteins allow cells to be continuously monitored for their repertoire of internal proteins so that virally infected, transformed, or
allografted cells reveal themselves to the immune system and are
eliminated. MHC polymorphism appears to be maintained by overdominant selection to increase both the diversity and number of
peptides presented to T cells, thereby maximizing the immune responsiveness of populations to evolving pathogens.
The relatively nonpolymorphic MHC class Ib gene products are
believed to have more specialized functions in Ag presentation (5,
7). HLA-G is largely confined to placental trophoblast, binds a
similar set of peptides to class Ia proteins, and acquires them in a
TAP-dependent manner (8, 9). This class Ib product may therefore
have a role in Ag presentation at the feto-maternal interface. Also,
by acting as a NK receptor ligand, HLA-G may protect HLA-A,
-B-negative trophoblast cells from the NK-like large granular lymphocytes that populate the maternal decidua during pregnancy (10,
11). By contrast with HLA-G, the HLA-E gene appears to be transcribed in all cells and tissues (12). Studies using transfectants
indicated that HLA-E has a low level of cell surface expression.
This has been suggested to be due to inefficient peptide loading in
the ER and to a low affinity interaction with b2m (13–15). The
peptide-binding groove of HLA-E shares homologies with the murine class Ib protein Qa-1, which has been found to bind class I
leader peptide sequences (16). In common with Qa-1, HLA-E is
now known to bind a restricted set of peptides, including the signal
sequences of some class I molecules, together with peptides derived from some viral proteins (17–19). It has also been demonstrated that class I signal peptides bound to HLA-E confer protection from NK cell-mediated lysis via the CD94/NKG2A receptor
(20, 21). Thus, both HLA-E and HLA-G are involved in controlling NK cell function.
HLA-F is currently the most enigmatic of the human MHC class
Ib genes. DNA cloning and sequencing studies have revealed that
mature HLA-F mRNA lacks exon 7, which normally encodes part
of the class I cytoplasmic domain (22, 23). The m.w. of the predicted HLA-F H chain is consequently some 2 kDa less than the
0022-1767/00/$02.00
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HLA-F is currently the most enigmatic of the human MHC-encoded class Ib genes. We have investigated the expression of HLA-F
using a specific Ab raised against a synthetic peptide corresponding to amino acids 61– 84 in the a1 domain of the predicted HLA-F
protein. HLA-F is expressed as a b2-microglobulin-associated, 42-kDa protein that shows a restricted tissue distribution. To date,
we have detected this product only in peripheral blood B cells, B cell lines, and tissues containing B cells, in particular adult tonsil
and fetal liver, a major site of B cell development. Thermostability assays suggest that HLA-F is expressed as an empty heterodimer devoid of peptide. Consistent with this, studies using endoglycosidase-H and cell surface immunoprecipitations also
indicate that the overwhelming majority of HLA-F contains an immature oligosaccharide component and is expressed inside the
cell. We have found that IFN-g treatment induces expression of HLA-F mRNA and HLA-F protein, but that this does not result
in concomitant cell surface expression. HLA-F associates with at least two components of the conventional class I assembly
pathway, calreticulin and TAP. The unusual characteristics of the predicted peptide-binding groove together with the predominantly intracellular localization raise the possibility that HLA-F may be capable of binding only a restricted set of peptides. The
Journal of Immunology, 2000, 164: 319 –328.
320
HLA-F IS EMPTY, INTRACELLULAR, TAP ASSOCIATED, AND RESTRICTED
G-401 and the human embryonal kidney cell line 293 were obtained from
the European Collection of Animal Cell Cultures (ECACC, Porton Down,
U.K.). All cell lines except BeWo were grown in RPMI 1640 supplemented
with 10% FCS and antibiotics. BeWo was grown in Ham’s F12K supplemented with 10% FCS, antibiotics, and MEM nonessential amino acid
solution.
The following mAb were produced from hybridoma cell lines obtained
from ECACC: W6/32 against monomorphic class I H chains associated
with b2m (30); BB7.7 against a combinatorial determinant of HLA-A, -B,
-C and b2m (31); ME1 against HLA-Bw22, -B7, -B27 (32); MA2.1 against
HLA-A2 and B-17 (33); and BBM1 against b2m (34). The mAb DT9
against HLA-E and HLA-C (27) was produced from the hybridoma cell
line obtained from Dr. Douglas Wilson, Department of Pathology and Microbiology, University of Bristol. Rabbit anticalreticulin was obtained from
Affinity Bioreagents (Golden, CO). The following reagents were generously provided by Dr. Jacques Neefjes, Het Nederlands Kanker Instituut
(Amsterdam, The Netherlands): HC10, a murine mAb against a monomorphic epitope on denatured HLA-B, -C H chains (35); RAHC, a rabbit
antiserum against monomorphic determinants on denatured HLA class I H
chains (36); and rabbit antisera against TAP1 and TAP2 (37).
Tissue and cell preparation
Peptides were synthesized on a multiantigenic peptide core (MAP) using
the facilities of the University of Bristol Centre for Molecular Recognition
(Bristol, U.K.), then adsorbed onto aluminum hydroxide adjuvant. For
polyclonal antisera, rabbits were immunized s.c. at four sites with a total of
1 mg of peptide at two weekly intervals. For mAb production, BALB/c
mice were immunized i.p. on four occasions at two weekly intervals with
100 mg of peptide. The final boost consisted of 100 mg of peptide in PBS
in the absence of adjuvant. Spleen cells were fused with the murine myeloma cell line Sp2/0-Ag14 (a gift from Dr. B. J. Randle, University of
Bristol). Hybridomas secreting Ab against the immunizing peptide were
identified by ELISA. For this, each well of a 96-well microtiter plate was
coated with 1 mg of peptide in 50 mM sodium bicarbonate buffer, pH 9.6.
After a blocking step (10% (w/v) bovine skimmed milk powder, 0.2% (v/v)
Tween-20 in PBS), the wells were incubated with culture supernatant for
1 h at room temperature. After washing in 0.2% (v/v) Tween-20 in PBS
(washing buffer), plates were incubated with peroxidase-conjugated goat
anti-mouse IgG (Bio-Rad, Richmond, CA) and Ab binding was detected
using orthophenylenediamine (Sigma, Poole, U.K.). Positive hybridomas
were subjected to three rounds of cloning by limiting dilution. The isotype
of the selected mAb, designated Fpep1.1, was determined using a commercial kit (Sigma).
Tonsil cells were prepared by previously described methods (38). Briefly,
white lymphoid tissue was removed from freshly excised tonsil, cut into
small pieces, and transferred to HBSS (Sigma), and the cells were released
by shaking. The suspension was passed through a cell dissociation sieve
fitted with a 40-gauge mesh screen (Sigma) and tissue fragments were
pressed through the mesh with a pestle. Remaining tissue fragments were
discarded following gravity sedimentation. Supernatants were pooled and
centrifuged at 400 3 g for 10 min, and the cell pellets were resuspended
at 1.5–2.5 3 107/ml. The suspension was layered onto Histopaque-1077
(Sigma) and centrifuged at 400 3 g for 30 min. Tonsil cells were harvested
from the interface and washed three times in HBSS by centrifugation at
400 3 g.
Purified T and B cell populations were prepared from peripheral blood
by sorting on magnetic microbeads using a commercially available kit,
according to the manufacturer’s instructions (MACS; Miltenyi Biotec, Bergisch Gladback, Germany). Briefly, freshly drawn heparinized blood was
layered onto Histopaque-1077 and centrifuged at 400 3 g. PBMC were
recovered from the interface and washed three times in HBSS by centrifugation at 400 3 g. Selection of B cells was achieved by two cycles of
sorting on beads coated with a mAb to CD19. To isolate T cells, the B
cell-depleted population was subjected to two further cycles of sorting on
beads coated with a mAb to CD3. The mononuclear cell population depleted of B cells and T cells was retained.
Membranes were prepared from cells and tissues using a modification of
a method previously described for EBV-transformed PBL (39). Briefly,
cells were sonicated in 10 mM Tris-HCl, pH 8, containing 20 mM PMSF,
50 mg/ml leupeptin, and 50 mg/ml antipain. The sonicate was subjected to
centrifugation at 100,000 3 g, and the pellet was resuspended in the above
buffer. This method was used to retain ER associating with the nuclear
membrane. Membrane preparations were standardized using a commercial
protein assay kit (BCA; Pierce and Warriner, Chester, U.K.).
Tissues, cell lines, and Ab
Northern analysis of IFN-g-treated cells
All tissues and cells were obtained following informed consent and with
the approval of the Research Ethics Committee of the United Bristol
Healthcare NHS Trust. Blood samples were obtained from healthy adult
volunteers. Tonsils were collected within 1 h of surgical excision. Fetal
liver was obtained following elective termination of pregnancy.
The EBV-transformed B-LCL T244, T245, T248, CD79, CD164,
CD165, CD166, YY, HOM-2, and K205 encompassing a variety of MHC
specificities, and the T cell lines HUT-78 and Jurkat were obtained from
the Department of Immunology, St. Bartholomew’s and the Royal London
School of Medicine and Dentistry, Queen Mary Westfield College (London, U.K.). B-LCL721.221 was a gift from Dr. Nick Holmes, Department
of Pathology, University of Cambridge (Cambridge, U.K.). B-LCL and
fibroblast cell lines derived from the same individuals, designated SF-LCL/
SF-FIB and DW-LCL/DW-FIB, respectively, were obtained from Dr.
Douglas Wilson, Department of Pathology and Microbiology, University
of Bristol. The erythroleukemia cell line K-562 was obtained from Dr.
Frances Spring, Blood Group Reference Laboratories (Bristol, U.K.). The
choriocarcinoma cell line JAR was obtained from Dr. C. F. Graham, Department of Zoology, Oxford University (Oxford, U.K.). The colonic adenocarcinoma cell line HT-29, the human choriocarcinoma cell lines JEG-3
and BeWo, and the T cell line MOLT-4 were obtained from the American
Type Culture Collection (Manassas, VA). The Wilm’s tumor cell line
The cell lines .221, HOM-2, and JEG-3 were cultured in the presence or
absence of 1000 U/ml IFN-g (Serotec, Oxford, U.K.) for 72 h. RNA was
extracted by the RNA-sol B method (Cinna/Biotex, Houston, TX), according to the manufacturer’s instructions, separated on a 1% (w/v) formaldehyde agarose gel at 10 mg/track, transferred to Hybond N1 (Amersham
International, Little Chalfont, U.K.), according to the manufacturer’s nonalkaline protocol, and fixed in 50 mM NaOH. Probes were prepared by
filling in an annealed oligonucleotide primer/template pair. The HLA-F
probe GGGAGTGGACCACAGGGTACGCCAAGGCCAACGCAC and
its primer GTGCGTTGG corresponded to the sequence coding for amino
acids 61–72 of the a1 domain of HLA-F. The universal class I probe
CAGTGTGATCTCCGCAGGGTAGAA was annealed to GTGCTGGGC
CCTGGGCTTCTACCCT and corresponded to the sequence coding for
amino acids 203–215 of the a3 domain of the human class I consensus
sequence. The primers were annealed to 10 ng of their respective template
at a 3:1 molar ratio in 20 ml of 50 mM NaCl, and extended using DNA
polymerase I (Klenow; New England Biolabs, Beverly, MA) in 10 mM
MgCl2, 10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 100 mM dATP, dGTP,
dTTP, and 50 mCi of [a-32P]dCTP (Amersham), for 1 h at room temperature. The reaction was terminated by desalting through a 1 ml Sephadex
G-50 column, and the double-stranded probe was denatured by boiling
before use. Hybridization was conducted overnight at 67°C in 43 SSC, 5%
Materials and Methods
Ab production
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m.w. of class Ia H chains (22). Evidence to date suggests that
expression of HLA-F is limited. HLA-F mRNA has been reported
in B cell lines, PBL, resting T cells, skin, and fetal liver; however,
transcripts have not been detected in T cell lines, adult liver, fibroblasts, myelomonocytic leukemia cells, amnion cells, or placental trophoblast cells (22–25). Studies using transfectants have
suggested that HLA-F is a b2m-associated protein of ;40 – 41 kDa
that could not be detected at the cell surface (22, 23, 26). On the
other hand, it has recently been suggested that a low level of cell
surface class I protein detected in the HLA-A, -B, -C-null mutant
B-LCL721.221 (.221) may be HLA-F (27). Expression of HLA-F
protein in human cells and tissues other than .221 has not been
reported, and its functional role remains unknown.
Studies on the biology of MHC class Ib proteins in human cells
and tissues have been hampered by a lack of suitable reagents.
Recently, McMaster et al. (28, 29) were successful in generating
mAb against synthetic peptides corresponding to the a1 domain of
HLA-G. We have used a similar approach to raise Ab to HLA-F.
In this study, we describe the use of one such reagent to examine
the distribution and cellular localization of HLA-F in some human
cells and tissues. We have also examined the status of HLA-F for
peptide binding in thermostability assays and investigated whether
HLA-F associates with TAP.
The Journal of Immunology
(w/v) dextran sulfate, and 13 Denhardt’s solution. Washes were conducted
under stringent conditions, with a final wash at 67°C in 0.13 SSC. The
filters were exposed to Kodak XAR-5 x-ray film at 270°C using intensifying screens.
Immunoprecipitation
Immunoblotting
Samples were subjected to SDS-PAGE following the method of Laemmli
(40) and transferred to Immobilon-P (Millipore, Bedford, MA) using a
Trans-blot SD semidry electrophoretic transfer cell (Bio-Rad). The membrane was blocked with PBS containing 5% (w/v) bovine skimmed milk
powder and 0.2% (v/v) Tween-20 (blocking buffer), and incubated overnight at 4°C in the primary Ab diluted in blocking buffer. The membrane
was washed extensively in PBS containing 0.2% (v/v) Tween-20 (washing
buffer). Binding was detected by incubating the membranes with peroxidase-conjugated goat anti-mouse IgG (Bio-Rad), peroxidase-conjugated
goat anti-rabbit IgG (Bio-Rad), or biotin-conjugated protein G (Sigma),
followed by StreptABComplex/HRP (Dako, Carpenteria, CA). Binding
was visualized using the ECL Western blotting system (Amersham).
IEF and immunoblotting
Cell lysates were subjected to immunoprecipitation with mAb W6/32. The
immunoprecipitates were digested with sialidase (Oxford GlycoSciences)
following the manufacturer’s method. IEF was then performed using a
Bio-Rad Protean II system run overnight at 800 V, 10 mA, according to the
method of Neefjes et al. (41). Immunoblotting was conducted according to
the method of Kao and Riley (42). Briefly, the gel was washed with agitation four times for 15 min each in 1% (w/v) SDS, 50% (v/v) methanol,
and 5 mM Tris-HCl, pH 8. A final 15-min wash was conducted in electrophoretic transfer buffer before the gel was immunoblotted with mAb
Fpep1.1, as described above. Membranes were stripped of Ab complexes
in 100 mM 2-ME, 2% (w/v) SDS, and 62.5 mM Tris-HCl, pH 6.7, for 30
min at 50°C. The stripped membranes were then incubated successively
with washing buffer and blocking buffer as above, and reprobed with
RAHC. For both Fpep1.1 and RAHC, binding was detected using biotinconjugated protein G, followed by StreptABComplex/HRP, and developed
using the ECL system.
MHC class I stability assay
The assay was conducted according to methods described by Benham et al.
(43). Cells were solubilized, cleared of insoluble material, and precleared,
as described above. Lysates were then divided into two equal aliquots that
were incubated at either 4°C or 37°C for 2 h. The remaining steps were
conducted at 4°C. The lysates were incubated for 1 h with W6/32 and then
with protein G-Sepharose for an additional 1 h. The pellets were washed
extensively with 0.1% (v/v) Triton X-100 in Mg21-free and Ca21-free
PBS, separated by SDS-PAGE, and subjected to immunoblotting, as described above.
Results
Identification of HLA-F protein
A peptide corresponding to amino acids 61– 83 of the a1 domain
in the deduced protein sequence of HLA-G has been successfully
used to raise mAb against HLA-G (28, 29). The peptide sequence
EWTTGYAKANAQTDRVALRNLLR corresponding to amino
acids 61– 83 of the deduced protein sequence of the HLA-F gene
product was used to search the SwissProt protein sequence database held at SEQNET (Daresbury, U.K.). No homologies greater
than five linear amino acids were found with any other human
protein, including class I. Peptides corresponding to amino acids
61– 83 of the predicted sequences of HLA-F, HLA-E, and HLA-G
were synthesized on a MAP core and used to raise rabbit antisera
designated aF, aE, and aG, respectively. In addition, the HLA-F
peptide was used to raise mAb: one IgG2b reagent was selected for
its specific binding to the immunizing peptide by ELISA and designated Fpep1.1. The reactivity of all these Ab on dot blots of the
immunogens is shown in Fig. 1A. Antiserum aF and mAb Fpep1.1
reacted with the HLA-F peptide, but not with HLA-E or HLA-G
peptides. The aE and aG antisera also reacted specifically with
their respective immunogens and are awaiting further characterization. The reactivity of these reagents only with their respective
immunogens also shows that they do not bind to the MAP core.
The mutant cell line .221 does not express HLA-A, -B, -C, or -G
(26, 44), but does express HLA-E (17). It has been suggested that
a b2m-associated HLA-F H chain may be expressed at a very low
level in .221 (26), and therefore, this cell line was used as a potential source of HLA-F protein. Antiserum aF and mAb Fpep1.1
both detected a 42-kDa component, the expected m.w. of HLA-F,
in immunoblots of SDS-PAGE-separated .221 cell membranes
(Fig. 1B, results for Fpep1.1 only are shown). To confirm that the
42-kDa product was indeed a class I protein, Fpep1.1 and aF were
used to immunoblot the b2m-associated class I proteins precipitated from .221 using the monomorphic mAb W6/32 and BB7.7.
Both reagents precipitated a 42-kDa product reactive with aF and
Fpep1.1 (Fig. 1C, left panel, results for Fpep1.1 only are shown).
Thus, Fpep1.1 and aF detect b2m-associated class I proteins in
.221. To examine the class I proteins expressed by .221, the W6/32
precipitates were subjected to immunoblotting with RAHC, a rabbit antiserum recognizing a monomorphic determinant on all denatured class I H chains (36). RAHC identified three components
of ;46, 44, and 42 kDa in .221 (Fig. 1C, right panel): only the
lower, 42-kDa component comigrated with the 42-kDa class I
product detected by Fpep1.1. Thus, .221 appears to express three
b2m-associated class I products, and only one of these is detected
by Fpep1.1.
The W6/32-immunoprecipitated class I proteins identified by
Fpep1.1 and RAHC in .221 were examined further by one-dimensional IEF (1D-IEF). By immunoblotting, Fpep1.1 identified two
acidic bands in .221 (Fig. 2, .221 panel). When this blot was
stripped and reprobed with RAHC, however, three bands were
detected (Fig. 2, .221 panel). In addition to the two acidic components detected by Fpep1.1, RAHC also identified a further, more
alkaline band (Fig. 2). The profile of RAHC-reactive bands observed in Fig. 2 appears to resemble closely the 1D-IEF profile
described for W6/32 immunoprecipitates obtained from metabolically labeled .221 cells in a previous report (17). In this case, the
single alkaline band was identified as HLA-E. On this basis, the
RAHC-positive/Fpep1.1-negative alkaline band in Fig. 2 appears
to represent HLA-E. We confirmed that this was indeed HLA-E by
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Cells were solubilized at 1 3 107 cells/ml in Mg21-free and Ca21-free PBS
containing 1% (v/v) Triton X-100, 20 mM PMSF, 50 mg/ml leupeptin, and
50 mg/ml antipain, and incubated on ice for 30 min. Insoluble material was
removed by centrifugation at 64,000 3 g for 10 min at 4°C. The lysate was
incubated at 4°C under rotation for 30 min with mouse IgG agarose (Sigma), followed by protein G-Sepharose (GammaBind G; Pharmacia, Uppsala, Sweden). The precleared lysate was incubated for 1 h at 4°C with the
relevant Ab. Protein G-Sepharose was added and the incubation continued
for an additional 1 h, after which the pellet was washed five times with
0.1% (v/v) Triton X-100 in Mg21-free and Ca21-free PBS.
Immunoprecipitates to be digested with Endo-H were resuspended in 20
ml of 0.2% (w/v) SDS, 2 mM PMSF, and 100 mM sodium citrate phosphate, pH 5.5, and boiled for 1 min. The tubes were cooled on ice for 5 min
and then digested overnight at 37°C with 8 mU of Endo-H (Oxford GlycoSciences, Abingdon, U.K.). All immunoprecipitates were solubilized by
boiling in Laemmli (40) sample buffer containing 5% (v/v) 2-ME, and
separated by SDS-PAGE.
Total cellular class I expression was compared with class I expression
at the cell surface by immunoprecipitation. Cells were washed three times
in ice-cold HBSS by centrifugation at 400 3 g at 4°C and divided into two
equal aliquots. One aliquot, designated cell surface, was incubated with
W6/32, while the second, designated cell lysate, was incubated in the absence of mAb. Incubations were conducted for 2 h at 4°C under rotation.
Cells were washed extensively in ice-cold HBSS and solubilized for 30
min, as described above. Each aliquot was incubated for 1 h at 4°C under
rotation either in the absence of Ab (intact cells) or in the presence of
W6/32 (cell lysate). Immune complexes were recovered with protein GSepharose, as described above.
321
322
HLA-F IS EMPTY, INTRACELLULAR, TAP ASSOCIATED, AND RESTRICTED
FIGURE 1. Reactivity of Ab raised against the HLA-F peptide. A, Rabbit antisera raised against MAP synthetic peptides corresponding to amino
acids 61– 83 of HLA-E (designated aE), HLA-G (aG), and HLA-F (aF),
together with appropriate preimmune sera (designated pIE, pIG, and pIF,
respectively), were used to probe dot blots (1 mg/spot) of the immunizing
peptides. The blots were also probed with the murine mAb Fpep1.1 raised
against the above HLA-F MAP peptide, and with the negative control mAb
HC10 against HLA-B, -C H chains. Binding was detected using HRPconjugated swine anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG,
as appropriate, and developed using the ECL system. B, Immunoblot of
.221 cell membranes probed with increasing dilutions of mAb Fpep1.1, as
indicated at the top. Proteins were separated at 50 mg/track on a 10%
polyacrylamide gel, and binding was detected using HRP-conjugated goat
anti-mouse IgG, as above. Arrows on the left indicate the positions of
marker proteins at 66, 45, 31, and 21.5 kDa. C, Immunoblot of MHC class
I proteins immunoprecipitated from .221 and probed with Fpep1.1 (left
panel) and RAHC, a rabbit antiserum against human class I H chains (right
panel). Immunoprecipitating mAb are indicated at the top. As a negative
control, immunoprecipitations were also conducted using a mixture of
mAb ME1 and MA2.1 against HLA-A, -B Ag. Precipitated proteins were
separated on an 8% polyacrylamide gel. Binding was detected using HRPconjugated protein G and developed using the ECL system. The position of
the 45-kDa marker protein is indicated by the arrow.
repeating the above experiments using mAb DT9 reported to detect HLA-E in .221 (27): only the upper RAHC-reactive component was precipitated from .221 by DT9 (data not shown). Taken
together, these results show that .221 expresses HLA-F protein in
addition to HLA-E protein, and that HLA-F in common with some
other class I products migrates as a doublet in 1D-IEF.
HLA-F mRNA has previously been detected in B cell lines, but
not in T cell lines (22, 23). The above experiment was repeated
using the B-LCL SF-LCL and the T cell line Jurkat. Fpep1.1 de-
tected the characteristic HLA-F doublet in SF-LCL, but did not
react with the abundant HLA-A, -B, -C proteins precipitated from
these cells by W6/32 (Fig. 2, SF-LCL panel, compare Fpep1.1 and
RAHC tracks, respectively). As expected for a T cell line, Jurkat
expressed considerably less class Ia protein than B-LCL, and
RAHC detected only one major class Ia product in this cell line
(Fig. 2, Jurkat panel, RAHC track). Fpep1.1 showed no reactivity
with Jurkat (Fig. 2, Jurkat panel, Fpep1.1 track). Thus, Fpep1.1
specifically detects HLA-F in the B cell lines .221 and SF-LCL.
Expression of HLA-F in different cell lines and tissues
To date, we have been unable to detect the HLA-F protein efficiently by immunoprecipitation or immunohistochemistry using
the Fpep1.1 or aF Ab, most likely because these reagents can
detect their target protein only under reducing conditions. The expression of HLA-F in different cells and tissues was therefore examined by immunoblotting SDS-PAGE-separated cell membranes
with Fpep1.1. The 42-kDa HLA-F product was detected by
Fpep1.1 in all 12 B-LCL tested (the different B-LCL, which encompass a variety of MHC specificities, are listed in Materials and
Methods). Two of these, SF-LCL and DW-LCL, are shown in Fig.
3A. For these two B-LCL, fibroblast cell lines derived from the
same individuals and designated SF-FIB and DW-FIB, respectively, were also available. These showed no reactivity with
Fpep1.1 (Fig. 3A). In addition, Fpep1.1 did not react with the T cell
lines MOLT-4, Jurkat, and HUT-78, or with the erythroleukemic
cell line K-562 (Fig. 3B). Similarly, the epithelial cell lines HT-29
(colonic adenocarcinoma), 293 (embryonal kidney), G-401
(Wilm’s tumor), and JEG-3 (choriocarcinoma) were unreactive
(Fig. 3B): the choriocarcinoma cell lines BeWo and Jar were also
negative (data not shown). In these experiments, it is possible that
HLA-F may be expressed below the level of detection in non-BLCL. To examine this, class I proteins were purified by immunoprecipitation using W6/32 and the anti-b2m mAb BBM1 and then
subjected to immunoblotting with Fpep1.1. Although the mAb
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FIGURE 2. Analysis by 1D-IEF and immunoblotting of MHC class I
proteins immunoprecipitated from .221, SF-LCL, and Jurkat. MHC class I
proteins were immunoprecipitated using W6/32, subjected to sialidase digestion, separated by 1D-IEF, and immunoblotted with mAb Fpep1.1.
Binding was detected using biotin-conjugated protein G and StreptABComplex/HRP and developed using the ECL system. Membranes were
then stripped of Ab and reprobed with RAHC. Binding was detected as
above. Immunoblotting Ab are indicated at the top, and cell lines are indicated at the bottom. Additional weak bands evident in these blots represent murine IgG, as they are strongly detected using HRP-conjugated goat
anti-mouse IgG in the absence of primary Ab (not shown).
The Journal of Immunology
HC10 readily detected HLA-B, -C H chains in these immunoprecipitates, there was no reactivity with Fpep1.1: results for Jurkat
only are shown in the lowest panel of Fig. 4B.
Freshly isolated PBL were prepared and fractionated into B cell,
T cell, and B/T cell-depleted populations using magnetic beads
coated with mAb to CD19 (B cells) and CD3 (T cells). The 42-kDa
HLA-F product was readily detected by immunoblotting in B cell
membranes (Fig. 4A). However, Fpep1.1 did not react with an
equivalent amount of membrane material prepared either from T
cells or from the B/T cell-depleted populations (Fig. 4A).
We previously detected HLA-F mRNA in human fetal liver tissue (24). To examine the expression of HLA-F protein in this
tissue, Fpep1.1 was used to immunoblot the class I products immunoprecipitated by W6/32 and BBM1 from detergent lysates of
first and second trimester fetal liver. Fpep1.1 detected the 42-kDa
HLA-F product in BBM1 immunoprecipitates from both first and
second trimester fetal liver (Fig. 4B, left panels). However, no
reactivity was observed with the class I products precipitated by
W6/32, even though HC10 readily detected HLA-B, -C proteins in
these precipitates (Fig. 4B, compare W6/32 tracks in left (Fpep1.1)
and right (HC10) panels, respectively). This result was unexpected
because W6/32 immunoprecipitated HLA-F from B-LCL (see Fig.
2). It is possible that HLA-F is expressed with different characteristics in cultured compared with freshly derived cells, or in fetal
compared with adult tissues. To explore this further, we examined
cells prepared from fresh adult tonsil, a rich source of B cells. The
42-kDa HLA-F protein was detected by immunoblotting in both
W6/32 and BBM1 immunoprecipitates made from adult tonsil
cells (Fig. 4B, left panel). These data show that W6/32 can readily
detect its target epitope on HLA-F expressed in B cell lines and
tonsil cells, but that this epitope is not available on HLA-F expressed in fetal liver.
Previous studies have shown that HLA-F mRNA is expressed at
much lower levels than mRNA for HLA-A, -B, -C (23). Our data
also suggest that the HLA-F protein is expressed at much lower
levels than classical class I. This is evident for SF-LCL in Fig. 2,
in which the HLA-F doublet identified by Fpep1.1 in 1D-IEF is
FIGURE 4. Reactivity of Fpep1.1 with freshly isolated cells and tissues.
A, Immunoblot showing the reactivity of Fpep1.1 with an equivalent
amount of membrane material prepared from peripheral blood B cells, T
cells, and mononuclear cells depleted of B and T cells (designated non-B,
-T cells). A total of 50 mg protein/track was separated on an 8% polyacrylamide gel. Binding was detected using HRP-conjugated goat antimouse IgG and developed using the ECL system. Arrow at the left indicates the position of the 45-kDa marker protein. B, Immunoblots showing
the reactivity of Fpep1.1 and HC10 with class I proteins immunoprecipitated from first trimester (1st tri) and second trimester (2nd tri) fetal liver
and from adult tonsil cells by W6/32, BBM1, and a mixture of ME1/
MA2.1. The T cell line Jurkat shown in the bottom panel was used as a
negative control. Precipitates were separated on 8% polyacrylamide gels.
Binding was detected using biotin-conjugated protein G and StreptABComplex/HRP, and developed using the ECL system. Immunoprecipitating
mAb are indicated across the top and immunoblotting mAb at the bottom.
Arrows on the right identify the position of the 45-kDa marker protein.
Note that the m.w. of HLA-F in both fetal liver and tonsil tissue is 42 kDa:
the apparent difference in migration of the HLA-F protein in tonsil and
liver when compared with the 45-kDa marker protein reflects variation
between different gels.
below the level of detection by RAHC. However, RAHC readily
identified the abundant HLA-A, -B, -C in these cells, as well as
identifying HLA-F in .221. Data presented in Fig. 4B also suggest
that the level of HLA-F protein detected by Fpep1.1 in fetal liver
and tonsil is low compared with the expression of HLA, -B, -C
detected by HC10. (In Fig. 4B, the HC10 immunoblot for tonsil
was underexposed to allow visualization of discrete 45-kDa
products.)
Cellular localization of HLA-F
MHC-encoded class I molecules bind peptides and carry them to
the cell surface for interaction with effector T cells. However, previous studies have failed to detect HLA-F at the cell surface of
HLA-F transfectants (22, 23, 26). We investigated the cellular localization of HLA-F in .221, B-LCL, and tonsil cells. In the first
instance, we set out to determine whether HLA-F can be detected
among the class I proteins immunoprecipitated from intact cells.
For this, W6/32 was used to immunoprecipitate class I proteins
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FIGURE 3. Reactivity of mAb Fpep1.1 with different cell lines.
Fpep1.1 was used to probe immunoblots of membranes prepared from the
cell lines indicated at the top. In each case, 50 mg protein/track was separated on an 8% polyacrylamide gel. Binding was detected using HRPconjugated goat anti-mouse IgG and developed using the ECL system. The
position of the 45-kDa marker protein is indicated at the left. A, Immunoblot of cell membranes prepared from the EBV-transformed B cell lines
SF-LCL and DW-LCL, and the fibroblast cell lines SF-FIB and DW-FIB
derived from the two individuals SF and DW, respectively. B, Immunoblots of cell membranes prepared from the T cell lines MOLT-4, Jurkat,
and HUT-78; the erythroleukemic cell line K-562; and the epithelial cell
lines HT-29 (colonic adenocarcinoma), 293 (embryonal kidney), G401
(Wilm’s tumor), and JEG-3 (choriocarcinoma).
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HLA-F IS EMPTY, INTRACELLULAR, TAP ASSOCIATED, AND RESTRICTED
from an equivalent number of intact and detergent-solubilized
cells, and the precipitates immunoblotted using Fpep1.1. Experiments were conducted at 4°C to inhibit intracellular transport. To
control for membrane integrity, parallel precipitations were conducted using an antiserum to the intracellular protein calreticulin
(45). We also compared the expression of HLA-B, -C proteins on
intact cells and detergent extracts of SF-LCL by immunoblotting
the W6/32 precipitates with mAb HC10. HLA-F was detected
among the class I proteins immunoprecipitated by W6/32 from
intact .221, SF-LCL, and tonsil cells (Fig. 5, upper panels). In each
case, however, the amount of HLA-F immunoprecipitated from
intact cells was low when compared with that precipitating from
cell lysates. By contrast with HLA-F, a much higher proportion of
the total HLA-B, -C was precipitated from intact SF-LCL (Fig. 5,
middle panel). In control experiments, calreticulin was detected in
immunoprecipitates from cell lysates, but not from intact cells
(Fig. 5, lower panels). However, because the amount of apparent
cell surface HLA-F was low in these experiments, we could not
exclude the possibility that HLA-F precipitating from intact cells
could actually originate from dead or dying cells.
Maturing class I proteins acquire complex N-linked oligosaccharides only when they pass through the medial Golgi, at which
point they become resistant to digestion with Endo-H. Class I proteins within the ER are therefore immature and Endo-H sensitive.
To further examine the cellular localization of HLA-F, class I proteins were immunoprecipitated from detergent lysates of .221, SFLCL, and tonsil cells, subjected to Endo-H digestion, and immunoblotted with Fpep1.1. Endo-H digestion reduced the m.w. of
HLA-F from ;42 to 40 kDa in equivalent numbers of .221, SFLCL, and tonsil cells: no Endo-H-resistant HLA-F was detected
(Fig. 6A, left panel). The amount of HLA-F precipitated from SFLCL was low when compared with the amounts precipitated from
.221 and tonsil cells (Fig. 6A). To increase the amount of HLA-F
FIGURE 6. Endo-H sensitivity of HLA-F. Class I proteins purified by
W6/32 immunoprecipitation from detergent lysates (panels A and C) or
intact cells (panel B) were incubated in the presence (1) or absence (2) of
Endo-H. The precipitates were separated on 8% polyacrylamide gels before
immunoblotting. Binding was detected using biotin-conjugated protein G
and StreptABComplex/HRP and developed using the ECL system. A, Immunoblot probed with mAb Fpep1.1 showing the Endo-H sensitivity of
HLA-F prepared from lysates of .221, SF-LCL, and tonsil cells at 1 3
107cells/track (left panel). A low level of an apparently Endo-H-resistant
HLA-F protein detected on one occasion only using 1 3 108 SF-LCL cells
is also shown (right panel). B, Immunoblot probed with Fpep1.1 to show
the Endo-H sensitivity of HLA-F proteins immunoprecipitated from intact
.221 and SF-LCL. C, Endo-H sensitivity of class I proteins prepared from
.221 cell lysates and identified by immunoblotting with RAHC. Arrows at
the left in all panels identify the position of the 45-kDa marker protein.
precipitating from SF-LCL, the input cell number was raised 10fold, and the conditions for W6/32 precipitation were adjusted accordingly. Endo-H-resistant HLA-F was not detected in three separate experiments conducted under these conditions, although, in a
further experiment, an apparently Endo-H-resistant HLA-F product was detected at very low level in SF-LCL (Fig. 6A, right
panel).
Our inability to consistently detect Endo-H-resistant HLA-F
prompted us to examine the Endo-H sensitivity of the HLA-F proteins apparently precipitated from intact cells (see Fig. 5A). Class
I proteins precipitated from intact .221 and SF-LCL by W6/32
were subjected to Endo-H digestion and then immunoblotted with
Fpep1.1. In both cell lines, the HLA-F proteins apparently immunoprecipitated from the cell surface were found to be Endo-H sensitive: no Endo-H-resistant HLA-F was detected in immunoprecipitates from either intact .221 or SF-LCL (Fig. 6B). These results
suggest that the HLA-F detected in cell surface immunoprecipitations does not represent mature cell surface HLA-F. When taken
together, therefore, our data show that HLA-F is a predominantly
intracellular protein.
Previous investigators have reported that W6/32 does not bind
to the cell surface in .221 (26, 44). More recently, however, a low
level of W6/32 binding has been reported at the surface of these
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FIGURE 5. Cellular localization of HLA-F. W6/32 immunoprecipitations were conducted on an equivalent number of detergent-solubilized
(designated lysate) and intact (designated surface) .221, SF-LCL, and tonsil
cells. Parallel immunoprecipitations were conducted using an antiserum to
the ER resident protein calreticulin (aCRT). Immunoprecipitates were separated on 8% polyacrylamide gels and subjected to immunoblotting with
Fpep1.1 (upper panels), HC10 (middle panel), or an antiserum to calreticulin (lower panels). Immunoprecipitating Ab are shown at the top and
immunoblotting Ab at the left. Binding was detected using biotin-conjugated protein G and StreptABComplex/HRP and developed using the ECL
system. Arrows at the left indicate the position of the 45-kDa (upper and
middle panels) and 66-kDa (lower panels) marker proteins.
The Journal of Immunology
cells, and this reactivity was suggested to represent a sialated form
of HLA-F (27). In contrast, our data consistently show that HLA-F
expressed in .221 is Endo-H sensitive and intracellular: we have
been unable to detect mature, Endo-H-resistant HLA-F characteristic of a cell surface-expressed product in this cell line. To investigate this further, RAHC was used to detect both HLA-E and
HLA-F in Endo-H-digested class I proteins purified from .221 by
W6/32. Of the three components identified by RAHC, only the
46-kDa product was Endo-H resistant: the 44- and 42-kDa products were both Endo-H sensitive (Fig. 6C). Because the 42-kDa
product is HLA-F, the 46- and 44-kDa products most likely represent HLA-E proteins having both mature and immature oligosaccharides, respectively. These data suggest that the W6/32 reactivity previously reported at the surface of .221 is not HLA-F, but
rather that it represents a mature 46-kDa class I protein, most likely
HLA-E.
Effect of IFN-g on HLA-F expression
We set out to examine whether IFN-g stimulation induces HLA-F
and could lead to cell surface expression of this protein. By Northern analysis, IFN-g was found to increase HLA-F mRNA in both
.221 and the B-LCL HOM-2 (Fig. 7A, left panel). Exposure to
FIGURE 8. Thermostability of HLA-F. Detergent lysates prepared from
.221, SF-LCL, and tonsil cells were incubated at either 4°C or 37°C before
immunoprecipitation with mAb W6/32. Immunoprecipitated class I proteins were separated on an 8% polyacrylamide gel and subjected to immunoblotting with either Fpep1.1 (left panels) or RAHC (right panels).
Binding was detected using biotin-conjugated protein G and StreptABComplex/HRP and developed using the ECL system. Arrows indicate the
position of the 45-kDa marker protein. Note that the SF-LCL and tonsil cell
precipitates immunoblotted with RAHC (right-hand panels) were underexposed to allow visualization of discrete 45-kDa class I proteins. Under
these conditions, HLA-F is below the level of detection for RAHC.
IFN-g also resulted in an increase in HLA-F protein in membranes
prepared from .221 and SF-LCL, as assessed by immunoblotting
using Fpep1.1 (Fig. 7B, left panel). This increase was in line with
the IFN-g-stimulated increase in HLA-B, -C in SF-LCL, but was
modest when compared with the increase in HLA-C observed in
IFN-g-treated JEG-3 choriocarcinoma cells (Fig. 7B, right panels).
We next used Endo-H digestion to determine whether IFN-g-induced HLA-F could reach the cell surface. IFN-g increased the
amount of W6/32-precipitable HLA-F heterodimers in both .221
and SF-LCL (Fig. 7C, compare IFN-g (1)/Endo-H (2) tracks with
IFN-g (2)/Endo-H (2) tracks, respectively). However, Endo-Hresistant HLA-F heterodimers were not detected in IFN-g-treated
cells (Fig. 7C, see IFN-g (1)/Endo-H (1) tracks). Thus, the increase in HLA-F induced by IFN-g does not result in cell surface
expression of the HLA-F protein.
Peptide loading of HLA-F
Under normal conditions, class I proteins can reach the cell surface
only if the peptide-binding groove is occupied. HLA-F may be
predominantly intracellular because it fails to acquire peptide or
because, having acquired peptide, it is unable to exit the ER. Peptide loading of class I molecules results in a change in their thermostability: at 37°C, loaded class I molecules are stable, while
empty class I molecules are unstable (43, 46). To examine the
thermostability of HLA-F, detergent lysates of .221, SF-LCL, and
tonsil cells were incubated at either 4°C or 37°C, precipitated with
W6/32, and immunoblotted with Fpep1.1. In control experiments,
thermostable class I molecules were detected in all these cell populations when precipitates were immunoblotted with RAHC (Fig.
8, right panels). However, the 42-kDa HLA-F protein evident in
.221, SF-LCL, and tonsil cells at 4°C was not detected at 37°C
(Fig. 8, left panels). These results suggest that the peptide-binding
groove of HLA-F is empty in .221, SF-LCL, and tonsil cells.
Interestingly and consistent with our previous Endo-H results
(see Fig. 6), the mature 46-kDa HLA-E product identified by
RAHC in .221 was detected at 37°C, while the immature 44-kDa
HLA-E product was not detected at 37°C (Fig. 8, top right-hand
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FIGURE 7. Effect of IFN-g on HLA-F expression. Cells, as indicated,
were grown for 72 h in the presence (1) or absence (2) of 1000 U/ml of
IFN-g. A, Northern analysis was conducted on RNA prepared from .221,
JEG-3, and HOM-2 cells using an HLA-F-specific oligonucleotide probe
(HLA-F, left panel) and a pan class I oligonucleotide probe (Pan class I,
right panels). B, Immunoblot of membranes prepared from IFN-g-treated
JEG-3, .221, and SF-LCL cells and probed with mAb Fpep1.1 (left panel)
and HC10 (right panels). A total of 50 mg protein/track was separated on
an 8% polyacrylamide gel before immunoblotting. Binding was detected
using HRP-conjugated goat anti-mouse IgG and developed using the ECL
system. C, Endo-H sensitivity of IFN-g-induced HLA-F. Class I proteins
immunoprecipitated from IFN-g-treated and untreated .221 and SF-LCL
cells were incubated in the presence (1) or absence (2) of Endo-H. Immunoprecipitates were separated on an 8% polyacrylamide gel and immunoblotted with Fpep1.1. Binding was detected using biotin-conjugated protein G and StreptABComplex/HRP and developed using the ECL system.
Arrows in B and C indicate the position of the 45-kDa marker protein.
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HLA-F IS EMPTY, INTRACELLULAR, TAP ASSOCIATED, AND RESTRICTED
Fpep1.1 (Fig. 9B). These results suggest that HLA-F can associate
with the multimeric complex involved in peptide loading.
Discussion
panel). This suggests that .221 contains immature and empty
HLA-E together with a mature HLA-E product having an occupied
peptide-binding groove.
Peptide loading generally occurs in a multimeric complex containing class I H chain, b2m, TAP1, TAP2, tapasin, and calreticulin (47). HLA-F may be devoid of peptide because it does not
participate in this complex. To determine whether HLA-F associates with TAP, .221 and SF-LCL were solubilized in digitonin to
maintain class I/TAP interactions, immunoprecipitated with antisera to TAP1 and TAP2, and immunoblotted using Fpep1.1. To
confirm the preservation of TAP associations, we first showed that
anti-TAP Ab were capable of coprecipitating calreticulin from
these lysates (Fig. 9A, top panels). The 42-kDa HLA-F protein
coprecipitated with both TAP1 and TAP2 from .221 (Fig. 9A, middle panel at left). Similar results were obtained in SF-LCL, although the amount of coprecipitating HLA-F was relatively low
(Fig. 9A, middle panel at right). This may be due to competition
for TAP between HLA-F and class Ia molecules, the latter identified by HC10 in these precipitates (Fig. 9A, bottom right panel).
Immunoprecipitations were also conducted on Triton X-100 lysates prepared from .221 and SF-LCL using an antiserum to calreticulin. The 42-kDa HLA-F protein coprecipitated with calreticulin in both cell lines, as assessed by immunoblotting with
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FIGURE 9. Association of HLA-F with TAP and calreticulin. A, Immunoblot of proteins coprecipitated from digitonin lysates of .221 and
SF-LCL cells by rabbit antisera against TAP1 (aTAP1) and TAP2
(aTAP2), and by normal rabbit serum (NRS). Coprecipitated proteins were
immunoblotted with an antiserum to calreticulin (aCRT) and with mAb
Fpep1.1 and HC10. Precipitating and immunoblotting Ab are indicated at
the top and left-hand side, respectively. The positions of the 66-kDa
(aCRT panel) and 45-kDa (Fpep1.1 and HC10 panels) marker proteins,
respectively, are indicated at the right. B, Immunoblot of proteins coprecipitated from Triton X-100 lysates of .221 and SF-LCL by an antiserum
to calreticulin (aCRT) and by normal rabbit serum (NRS). Coprecipitated
proteins were immunoblotted with Fpep1.1. The position of the 45-kDa
marker protein is indicated by the arrow. In both A and B, coprecipitated
proteins were separated on an 8% polyacrylamide gel. Anti-CRT binding
was detected using biotin-conjugated protein G and StreptABComplex/
HRP, and Fpep1.1 and HC10 binding was detected using HRP-conjugated
rabbit anti-mouse IgG. Immunoblots were developed using the ECL system.
We have shown that the HLA-F protein can be detected in B cells,
B cell lines, and B cell-containing tissues, in which it occurs in a
predominantly intracellular, unstable, and immature form. Our
studies have also shown that HLA-F can associate with TAP, but
that the protein does not appear to bind peptide and is expressed in
an empty configuration.
Previous investigators have drawn attention to unusual features
in the predicted peptide-binding groove of HLA-F (22, 23). Despite their variability, human class Ia molecules possess 10 highly
conserved amino acid residues that point into the Ag recognition
site. The class Ib proteins HLA-E and HLA-G retain 8 and 9 of
these residues, respectively. However, only 5 of the 10 residues are
conserved in HLA-F. On this basis, it has been suggested that
HLA-F may have a different biological function from that of other
class I proteins. Some non-MHC-encoded class Ib products have
modified peptide-binding grooves that reflect their specific biological functions. The peptide-binding groove of the IgG transporter
FcRn, for example, is closed, while that of CD1b is specialized to
accommodate nonpeptide ligands (48, 49). However, a recent
structural analysis concluded that the residues lining the putative
binding groove of HLA-F are consistent with peptide binding (6).
Our own preliminary modeling analysis (unpublished observations) also supports this view. We therefore believe that the HLA-F
peptide-binding groove is likely to be a peptide receptor.
MHC-encoded class I proteins are expressed at the cell surface
in a mature, Endo-H-resistant form only after they have acquired
peptide in the ER lumen. In previous studies, HLA-F was not
detected at the cell surface in HLA-F transfectants (22, 23, 26). To
date, however, there has been no information on the cellular localization of HLA-F in normal cells. This requires reagents that
can specifically identify HLA-F among the abundant classical
class I proteins normally expressed in somatic cells. The antiHLA-F reagent Fpep1.1 has allowed us to examine the cellular
localization of HLA-F both in cell lines and in freshly isolated
human cells. Comparison of the class I proteins immunoprecipitated from intact cells and cell lysates indicated that the overwhelming majority of HLA-F is expressed inside .221, SF-LCL,
and tonsil cells. Nevertheless, the detection of limited amounts of
HLA-F among the class I proteins immunoprecipitating from intact cells raised the possibility that some HLA-F may reach the cell
surface. Surprisingly, however, we were consistently unable to detect Endo-H-resistant HLA-F, characteristic of cell surface class I
expression, in whole lysates of .221 and tonsil cells. Similar results
were obtained in SF-LCL, although, on one occasion only, we did
identify at low level an apparently Endo-H-resistant HLA-F product in these cells. The significance of this latter observation is
currently unclear, especially since all the apparent cell surface
HLA-F observed immunoprecipitating from intact cells, including
SF-LCL, was found to be Endo-H sensitive. Moreover, results
from thermostability assays were also entirely consistent with an
intracellular localization, indicating that the peptide-binding
groove of HLA-F is unoccupied in .221, SF-LCL, and tonsil cells.
We cannot rule out the possibility that, in contrast with other class
I molecules, HLA-F may be capable of escaping the ER lumen and
reaching the cell surface in the form of empty immature heterodimers, but this seems inherently unlikely. Also, further work is
required to determine whether HLA-F can, under some circumstances, acquire mature oligosaccharides and reach the cell surface. Nevertheless, when taken together, our data clearly indicate
The Journal of Immunology
calreticulin and TAP. It therefore seems likely that HLA-F participates in the multimeric complex involved in class I peptide loading. This raises the possibility that HLA-F is not being loaded, and
hence released, from TAP. Interestingly, it has recently been found
that some alleles of HLA-C exhibit a stable interaction with TAP
(50). This appears to occur because HLA-C molecules are more
selective than HLA-A, -B in the range of peptides they bind. Because of this restricted peptide binding, most HLA-C molecules
are retained in the ER and not transported to the cell surface. It has
been reported that the amounts of intracellular HLA-C are similar
to those of HLA-A, -B (50). In contrast, previous studies have
shown that the level of HLA-F mRNA is low (23), and our own
work now shows that HLA-F protein expression is also low. A
combination of low level expression and restricted peptide binding
could therefore account for the predominantly intracellular, empty
HLA-F observed in the present study.
HLA-F shows a restricted tissue distribution. To date we have
detected HLA-F protein only in B cells, B cell lines, and tissues
containing B cells, in particular adult tonsil and fetal liver, a major
site of B cell development. Our results are broadly in line with two
previous studies in which the distribution of HLA-F mRNA was
examined using RNase protection assays (22, 23). In each case,
HLA-F transcripts were detected in B cell lines, but not T cell
lines. These studies also raised the possibility that HLA-F mRNA
was expressed in peripheral blood T cells. In contrast, we did not
detect HLA-F protein in T cells. In one study, the HLA-F mRNA
detected in PBMC was found to be reduced following PHA activation, and this was attributed either to a drop in the proportion of
B cells or to the expression of HLA-F mRNA by resting, but not
activated, T cells (23). In another report, HLA-F mRNA was detected in T cells enriched from PBMC by nylon wool fractionation
(22). It is possible that T cells may express untranslated HLA-F
mRNA or, alternatively, the previous detection of HLA-F transcripts in T cell preparations may reflect low level B cell contamination. Taken together, however, our results show that the HLA-F
protein has a restricted tissue distribution that, by analogy with
some other class Ib proteins, may indicate a specialized function
for this molecule.
Our studies have revealed an apparent difference in the characteristics of HLA-F expression between fetal and adult life. We
found that W6/32 immunoprecipitated HLA-F from B cell lines
and tonsil cells, but not from fetal liver. By contrast, W6/32 readily
immunoprecipitated other MHC class I proteins from this tissue,
and mAb to b2m also precipitated HLA-F from fetal liver. This
suggests that the W6/32-defined epitope on HLA-F is unavailable
in fetal liver. Our own previous work together with that of others
has shown that W6/32 epitopes are not available in class I/calreticulin complexes (45, 47). One possibility therefore is that, by
contrast with HLA-F in adult cells, HLA-F in fetal liver is associated with calreticulin, or with an as yet unidentified protein that
masks the W6/32 epitope. Alternatively, a conformational change
in the HLA-F proteins expressed in fetal liver may abrogate W6/32
binding. It remains to be determined whether these differences are
reflected in the function of HLA-F during fetal and adult life.
The possibility cannot be excluded that HLA-F has an intracellular function. For example, it might be speculated that, like HLADM, which has a role in peptide loading of MHC class II, HLA-F
could be involved in peptide loading of other HLA molecules in
the TAP complex of B cells. It is also possible that, instead of
entering the class I secretory pathway, HLA-F may carry ligands
to another cellular compartment, although our inability to detect
loaded HLA-F makes this unlikely. Our data show that HLA-F
associates with TAP, and it is therefore possible that this class I
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that HLA-F is an empty and intracellular class I protein in normal
cells.
Our results on the cellular localization of HLA-F in .221 are in
contrast with a recent report by Braud et al. (27), who suggested
that HLA-F is expressed at the surface of these cells. Unlike previous investigators (18, 26), these authors reported a low level of
W6/32 reactivity at the surface of .221 by flow cytometry. This
was proposed to be cell surface HLA-F because it was stated that
a sialated, mature HLA-F protein was detectable in these cells
using pulse-chase and IEF analysis, although the criteria used to
identify HLA-F in these studies were not given. By contrast, the
present study used an HLA-F-specific reagent to demonstrate that
.221 expresses only immature and empty HLA-F, characteristic of
an intracellular class I protein. Our study further reveals that sialidase-treated (immature) HLA-F migrates as a doublet in IEF gels.
It is possible that in pulse-chase experiments analyzed by IEF, in
which sialidase is not used, these products may appear to represent
immature and sialated forms of HLA-F.
Although these data show that HLA-F is not expressed at the
cell surface in .221, our studies reveal that this cell line does express low levels of an Endo-H-resistant, thermostable 46-kDa class
I protein. This protein shows no reactivity with the mAb Fpep1.1,
and therefore does not represent HLA-F. Because the 46-kDa
product has the properties of a mature cell surface class I protein,
it may be responsible for the low level of W6/32 reactivity observed by Braud et al. on the cell surface of .221 (27). Moreover,
the low level expression of this product could explain why other
groups have reported that .221 is W6/32 negative by flow cytometry (18, 26). On the other hand, we have not used flow cytometry
and cannot rule out the possibility that this apparently loaded class
I product fails to reach the cell surface in .221.
We have not identified the 46-kDa class I protein unequivocally,
although, because .221 expresses only HLA-E and HLA-F, it
seems reasonable to propose that it represents cell surface HLA-E.
This observation is surprising given that two recent studies have
reported that HLA-E does not reach the cell surface in .221 (18,
27). In both studies, different mAb detecting HLA-E failed to bind
to .221 in flow cytometry. In addition, in one report, no Endo-Hresistant class I proteins were detected in pulse-chase analysis of
W6/32 precipitates from these cells (18). In the second report,
sialated and hence mature HLA-E was not detected in .221 by
pulse-chase and IEF analysis (27). The reasons for the discrepancy
between our results and those of others are not clear. However, the
46-kDa product may be at the limit of detection in flow cytometry
not only for W6/32, but also for anti-HLA-E reagents, especially
DT9, which was raised against cotton top tamarin MHC class I
molecules (27) and, in our hands, cross-reacts only weakly with
HLA-E. The 46-kDa protein may also fall below the level of detection in pulse-chase analysis: our approach accesses the entire
population of W6/32-precipitable class I molecules, while pulsechase experiments identify only the proportion of class I proteins
synthesized during isotopic labeling.
Our detection of an apparently mature HLA-E protein in .221 is
intriguing given that HLA-E is incapable of binding peptides derived from the signal sequences of either HLA-E itself or of
HLA-F (17). On this basis, HLA-E would not be expected to reach
the cell surface in .221. However, the detection of apparently
loaded and mature HLA-E in .221 suggests that this molecule can
bind peptides other than those derived from MHC class I signal
sequences, as already suggested by others (19).
HLA-F may be empty because it is unable to interact with or
acquire peptides from TAP. Although we have not yet conducted
pulse-chase experiments to determine the steps involved in its assembly, our studies nevertheless show that HLA-F associates with
327
328
HLA-F IS EMPTY, INTRACELLULAR, TAP ASSOCIATED, AND RESTRICTED
protein is awaiting appropriate ligands to allow cell surface expression in B cells. Such putative ligands may not normally be
present in B cells, but may become available following infection of
B cells, or during normal B cell differentiation or activation.
Acknowledgments
We thank Karen Simpson, Jim Houlihan, Poppy Fotiadou, Nick Holmes,
and Kuo-Jang Kao for helpful discussions, and the staff of St. Michael’s
Hospital (Bristol, U.K.) for their help and cooperation in providing material
for this study.
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