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Service de Recherches en Hémato-Immunologie, Commissariat à l’Energie Atomique-Direction des Sciences du Vivant-Department de Recherche Médicale, Institut Universitaire d’Hématologie, Hôpital Saint Louis, and
Fondation Jean Dausset, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1, 2,
and 3), a transmembrane region, and a cytoplasmic tail
(4). Three other spliced transcripts encode membrane-bound
proteins that lack 2 (HLA-G2), 3 (HLA-G4), or both the 2 and
3 domains (HLA-G3). The full-length HLA-G1 isoform, which has a
classical HLA class I structure (5), has been widely
investigated over the past few years. First identified on extravillous
cytotrophoblasts (6, 7), HLA-G is also expressed in HLA
class I-positive tissues such as oocytes (8), embryos
(9), amnion (10), thymic epithelial cells
(11), cytokine-activated monocytes (12, 13),
and some tumors (14). HLA-G1 has been identified as a key
molecule in maternal-fetal tolerance (15), since it
inhibits the lytic activity of both decidual and peripheral blood NK
cells (16, 17, 18, 19, 20) as well as Ag-specific CTL
(21) and allogeneic proliferative responses
(22). Additional evidence that HLA-G can be expressed in
heart after transplantation suggests that it may participate in graft
tolerance (23). In contrast to well-characterized HLA-G1, studies of both the expression and function of HLA-G2, -G3, and -G4 isoforms have been hampered by a lack of suitable reagent. Accordingly, we separately transfected the cDNA of HLA-G1, -G2, -G3, and -G4 into the HLA-A-, -B-, -C-, and -E-positive M8 cell line, which we have previously described as lacking HLA-G transcription (24). Stable transfectants were then obtained for each HLA-G isoform. The original approach of this work was to investigate the biochemical and functional features of HLA-G2, -G3, and -G4 isoforms 1) without interfering with the proper folding of these truncated isoforms (i.e., untagged HLA-G isoforms), and 2) in which HLA-G is coexpressed with the other HLA class I molecules. Using flow cytometric analysis, cell surface protein biotinylation, endoglycosidase-H (Endo-H)4 treatments, and pulse-chase experiments, we show here that HLA-G2, -G3, and -G4 isoforms can reach the cell surface with an immature glycosylation pattern after a 2-h chase period. Moreover, cytotoxicity experiments indicate that the transfection of each HLA-G isoform in M8 cells leads to inhibition of polyclonal NK cells and NK clone lytic activity. Furthermore, evidence is provided that each HLA-G isoform also impairs the Ag-specific CTL response. Taken together, these results strongly suggest that, like the full-length HLA-G1, the HLA-G2, -G3, and -G4 truncated isoforms are expressed at the cell surface and exhibit an important role in modulating immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M8 is an HLA-A-, -B-, -C-, and -E-positive (HLA-A1, -A2, -B12, and -B40/male), but HLA-G-negative, melanoma cell line (24). HLA-G cDNA was obtained by RT-PCR from the HLA-G-positive JEG-3 choriocarcinoma cell line cloned into the pcDNA3–1/hygromycin expression vector (Invitrogen, San Diego, CA), and transfected by electroporation into M8 cells, as previously described (25). Stable transfectant cells were named M8-HLA-G1, M8-HLA-G2, M8-HLA-G3, M8-HLA-G4, and M8-HLA-G5, according to the HLA-G isoform cDNA transfected (25). M8 cells transfected with the pcDNA3–1 vector alone were used as a negative control cell line (M8-pcDNA). All transfectants were controlled by RT-PCR and Southern blot analysis using specific primers and probes (25). The NK-like YT2C2-PR subclone and the M58–66 influenza virus matrix peptide-specific CTL line (HC12) were obtained as previously described (21, 26). Cells were maintained in RPMI 1640 medium supplemented with 10% inactivated FCS, 2 mM L-glutamine, 1 µg/ml gentamicin, and fungizone. HLA-G transfectants were selected in medium containing hygromycin (Sigma, St. Louis, MO).
Monoclonal Abs and flow cytometric analysis
The following mAbs were used: 4H84, IgG1 anti-denatured
HLA-G heavy chain (provided by S. Fisher and M. McMaster); HC10, IgG2a
anti-HLA-B and -C (provided by H. Ploegh); 87G, IgG2a
anti-HLA-G1 and -G5 (provided by D. Geraghty); Z199, IgG2b
anti-NKG2A (Immunotech, Marseilles, France); PAG 1, a rabbit
polyclonal Ab that we raised against peptide 61–83 of the HLA-G 1
domain that recognizes denatured HLA-G -chain of all HLA-G
isoforms (25, 27); and B-5-1-2 anti--tubulin
(Sigma-Aldrich reagent). Flow cytometry assays were conducted
as previously described (26). In addition, we performed
propidium iodide staining of dying or dead cells, which
allowed us to gate only nonpermeabilized cells.
Western blot analysis
Aliquots of total proteins from M8 transfectants (lysates) or precipitated proteins from M8 transfectants were separated in 12% SDS-PAGE. The gels were blotted onto nitrocellulose membranes (Hybond; Amersham, Buckinghamshire, U.K.), and the membranes were blocked by incubation with PBS containing 0.2% Tween 20 and 5% nonfat dry milk. The membranes were then probed with the corresponding Ab overnight at 4°C and washed in PBS containing 0.2% Tween 20. In biotinylation experiments, membranes were first incubated with the 4H84 mAb together with the anti-tubulin, and a second revelation was conducted by incubating membranes with the HC10 mAb. Then, the membranes were subsequently incubated for 30 min at room temperature with peroxidase-conjugated sheep anti-mouse IgG Ab (Sigma), washed thoroughly, stained with enhanced chemiluminescence reagent (Amersham), and exposed to x-ray film.
Cell surface protein biotinylation
Cell surface proteins of viable M8 transfected cells were biotinylated as monolayers in tissue culture flasks (75 cm2). Cells were first washed twice with PBS and treated with sulfo-succinimidyl-6-(biotinamido)hexanoate-biotin (Pierce, Rockford, IL; 200 µg/ml of PBS) for 4 min at room temperature. The cells were washed twice with PBS, treated with 50 mM glycine for 5 min, and again washed twice with PBS. Dead or dying cells (nonadherent cells) were thus removed by washing the cells. Cells were then collected in tubes, recovered with PBS, and washed five times at 4°C. Biotinylation experiments in which intracellular proteins were voluntarily biotinylated, cells were collected in tubes and treated with 1 mg/ml of biotin for 2 h at room temperature. Cells were then centrifuged, and the supernatant was removed before cell lysis.
Immunoprecipitation of cell surface protein
Cells were lysed in 1 ml of lysis buffer containing 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS; Sigma). Insoluble material was removed by centrifugation at 14,000 rpm for 30 min at 4°C. Biotinylated proteins were then precipitated with 100 µl of 50% streptavidin-agarose slurry (Bio-Rad, Richmond, CA) for 1 h at 4°C. After extensive washing of streptavidin-agarose beads in 0.05% CHAPS, 0.1% SDS, 300 mM NaCl, and 10 mM Tris-HCl, pH 7.5, they were resuspended in 40 µl of sample buffer and boiled for 5 min.
Enzymatic treatments
Total proteins or cell surface precipitated proteins from M8 transfectants were incubated for 16 h at 37°C in 80 µl of 0.2% SDS, 1% 2-ME, 100 mM sodium citrate, and 0.2 M PMSF with or without 8 mU of peptide-N-glycosidase (PNGase; Roche, Indianapolis, IN) or Endo-H (Roche). Subsequently, the samples were examined by Western blot analysis.
Pulse-chase experiments
M8 cells were grown in RPMI 1640 medium supplemented with 10% FCS (Sigma). Radiolabeling was accomplished by incubating cells that had reached 90% confluence. Cells (10 x 107) were first starved for 30 min at 37°C in RPMI 1640-methionine/cysteine (Life Technologies, Rockville, MD) supplemented with dialyzed 5% FCS, 300 mg/ml L-glutamine, 50 mg/ml L-leucine, 200 mg/ml L-arginine, 200 mg/ml glucose, and 35 mg/ml inositol. Cells were then labeled for 20 min at 37°C with [35S]ProMix (250 µCi/ml; Amersham, Arlington Heights, IL). Labeled cells were extensively washed in RPMI 1640 medium containing 10% FCS and an excess of cold methionine/cysteine and incubated at 37°C in 5% CO2 for 0, 2, or 4 h in fresh cell culture medium. At these different time points, cells were biotinylated, and cell surface proteins were precipitated with streptavidin-agarose beads as described above. At this time, lysates were centrifuged, and both beads and supernatant were collected separately and treated in parallel as described in the following steps 1 and 2, respectively. 1) After washing, precipitated molecules were eluted from the beads in 50 µl of 2% SDS in lysis buffer at 95°C for 5 min and overnight at 37°C. The proteins were diluted in 1 ml of lysis buffer and reprecipitated with the anti-HLA-G, PAG 1 rabbit polyclonal Ab for 1 h at 4°C. Surface HLA-G proteins were precipitated with protein A-Sepharose beads and analyzed by SDS-PAGE. 2) The supernatant was reprecipitated with rabbit polyclonal anti-HLA-G, and then unbiotinylated HLA-G proteins were analyzed by SDS-PAGE.
Cytotoxicity assays
The cytolytic activity of PBMC from healthy adult volunteer donors (men and women, aged 30–60 years) obtained by Ficoll/Histopaque density gradient, of YT2C2-PR, or of the HC12 CTL line used as effectors was assessed in 4-h 51Cr release assays in which effector cells were mixed with 5 x 103 51Cr-labeled targets (100 µCi of 51Cr sodium chromate; 1 Ci = 37 GBq; Amersham) at various E:T cell ratios in U-bottom microtiter plates, as previously described (26). For cytotoxicity assays in which the HC12 CTL line was used as the effector, targets were pulsed with M58–66 peptide for 1.5 h. After 4 h at 37°C in a humidified 5% CO2 incubator, 50 µl of the supernatant was collected for scintillation counting (Wallac 1450 Microbeta; Pharmacia, Piscataway, NJ). The percentage of specific lysis was calculated as follows: % specific lysis = [(cpm experimental well - cpm spontaneous release)/(cpm maximum release - cpm spontaneous release)] x 100. Spontaneous release was determined by incubation of labeled target cells with medium. Maximum release was determined by solubilizing target cells in 0.1 M HCl. In all experiments spontaneous release was <10% of maximum release.
Ab blocking assays
In experiments in which anti-NKG2A mAb was used to block the HLA-E inhibitory receptor CD94/NKG2A, effector cells were firstly preincubated for 15 min at room temperature in culture medium containing 10% human AB serum and then preincubated for 15 min at room temperature with Z199 (mouse anti-human NKG2A; Immunotech, Westbrook, ME) before the NK cell cytotoxicity assay. The mAb was present in the culture medium during the entire assay period. In experiments in which mAbs were used in an attempt to block HLA-G-NK interaction, target cells were incubated with the optimal concentration of either the 4H84 (1/100) or the 87G mAb (2 µg/ml), then washed and incubated with an F(ab')2 goat anti-mouse IgG Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) to prevent Ab-dependent cell cytotoxicity by interaction of NK cells with the first mAb used. mAb toxicities were checked in each assay and were always <3%.
Statistical analysis
The statistical significance (p < 0.05) of cytotoxic assays was analyzed using Student’s t test to compare the percentage of specific lysis of M8-HLA-G transfectants to that of the M8-pcDNA control cell line. Assays were conducted in triplicate for each experiment, and the SD of the mean triplicate value was <5%. Note that all HLA-G-positive M8 transfectants inhibit effector lysis, whatever the E:T cell ratio.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether the HLA-G2, -G3, and -G4 transcripts are
translated into proteins, we transfected the corresponding cDNA into
the M8 HLA class I-positive melanoma cell line. Western blot analysis
of all HLA-G transfectants was then conducted using the 4H84 mAb, which
specifically detects denatured HLA-G via the 1 domain epitope common
to all HLA-G isoforms (27). In addition to the expected
translation of the HLA-G1 transcript into a 39- to 40-kDa protein, we
found that the HLA-G2, -G3, and -G4 transcripts were, respectively,
translated into 30-/31-, 22-, and 29-/30-kDa proteins, respectively
(Fig. 1
A). However, these
molecular masses were higher than predicted, indicating that HLA-G
isoforms may have been glycosylated. After PNGase F treatment of cell
lysates obtained from M8 transfectants, the 4H84 mAb revealed bands
that matched the predicted molecular masses (36, 27, 17, and 25 kDa,
respectively, for HLA-G1, -G2, -G3, and -G4; Fig. 1B). These
results demonstrate that, like HLA-G1, the HLA-G2, -G3, and -G4
isoforms are indeed translated as glycoproteins. Additional smaller
bands corresponding to glycosylated proteins were observed for HLA-G1
(i.e., band at 30 kDa) and HLA-G2 (i.e., band at 17 kDa) in the lysate
of the corresponding M8 transfectant (Fig. 1A). Furthermore,
in other Western blot experiments, additional smaller bands were also
revealed for HLA-G1 (i.e., 26 kDa) and HLA-G2 (i.e., band at 28 kDa) as
well as for HLA-G3 (i.e., 17 kDa) and HLA-G4 (i.e., 26 kDa; data not
shown). Whether these smaller migrating bands are due to in vivo
protein degradation remains to be determined.
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Although the HLA-G2, -G3, and -G4 proteins were observed in the
corresponding M8 transfectant, they were undetectable on the cell
surface by flow cytometry with the currently available specific
anti-HLA-G1 87G (5) and 01G (25) mAbs
that otherwise positively stain HLA-G1 at the cell surface of M8-HLA-G1
(data not shown). Failure to detect these smaller isoforms could have
been due to their inability to form the conformational epitope
recognized by these mAbs. We then asked whether the anti-HLA-G
heavy chain, 4H84 mAb, could detect the HLA-G isoforms at the cell
surface. To exclude intracellular HLA-G staining by 4H84 mAb, we gated
on nonpermeabilized cells, as revealed by negative propidium iodide
staining. As shown in Fig. 2, M8-HLA-G1
cells are positively stained by both 87G and 4H84 mAbs. All M8-HLA-G1
cells are stained by 87G, but not by 4H84, which may be due to the
distinct affinity and/or specificity between these mAbs. Interestingly,
M8-G2, -G3, and -G4 are positively stained by the 4H84 mAb, showing
that all HLA-G isoforms are expressed on the cell surface of the
corresponding M8 transfectant.
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shows, using 4H84 mAb, immunoblotting of HLA-G into the lysates
(lanes 1) or after precipitation of biotinylated
proteins (lanes 3). As expected, while no HLA-G
protein was detected on the M8-pcDNA control cell line (Fig. 3A, lane 3), a band corresponding to surface
HLA-G1 was revealed on M8-HLA-G1 transfected cells (Fig. 3E,
lane 3). Under similar experimental conditions, bands
corresponding to HLA-G2, -G3, and -G4 isoforms were also revealed on
the corresponding transfectant (Fig. 3, B–D, lanes
3), showing that these isoforms reach the cell surface.
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F,
lane 3), although a high level of HLA-G5 protein synthesis
was observed in the lysate of the M8-HLA-G5 transfectant (Fig. 3F, lane 1); and 2) highly expressed
intracellular protein (i.e., tubulin), which was indeed detected
in all transfectants lysates (Fig. 3, lanes 1), but was not
precipitated by streptavidin after biotinylation (Fig. 3, lanes
3). Furthermore, we have compared the abilities of HLA-G2, -G3,
and -G4 and tubulin to be biotinylated. For this purpose we have
conducted similar experiments in which intracellular proteins from
M8-HLA-G2, -G3, and -G4 cells were voluntarily biotinylated and
precipitated by streptavidin-agarose beads. These precipitated proteins
were compared with lysates from unbiotinylated cells after Western blot
analysis using both anti-tubulin and anti-HLA-G (i.e., 4H84
mAb) Abs. As shown in Fig. 4, both
tubulin and HLA-G2, -G3, or -G4 are equally biotinylated, while in the
lysate of the corresponding M8 transfectant, the tubulin is either
equally revealed compared with HLA-G2 and -G3 or expressed less
compared with HLA-G4. Altogether, these results highlight that tubulin
exhibits a similar capacity for biotinylation as HLA-G2, -G3, and -G4,
leading to the conclusion that in our experiments, we may underestimate
the amounts of HLA-G2, -G3, and -G4 surface protein. Furthermore,
experiments performed on unbiotinylated M8 transfectants also confirmed
that streptavidin-agarose beads did not precipitate any nonspecific
proteins (Fig. 3, A–F, lanes 5). Taken together
these results show that under our conditions biotinylated proteins
correspond to membrane-bound proteins, leading to the conclusion that
HLA-G2, -G3, and -G4 isoforms can reach the cell surface as
membrane-anchored glycoproteins.
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Having demonstrated that HLA-G2, -G3, and -G4 are expressed at the
cell surface, we then asked whether they are expressed as mature
glycoproteins. The acquisition of complex N-linked sugar
during maturation of HLA class I glycoproteins occurs when the proteins
pass through the medial Golgi, at which point they become resistant to
digestion with Endo-H. Thus, the presence of Endo-H resistance
indicates that protein maturation has proceeded past the endoplasmic
reticulum (ER). To further examine whether the N-linked
carbohydrate side chains of the membrane-bound HLA-G2, -G3, and -G4
glycoproteins are modified, both lysates and surface proteins (i.e.,
biotinylated proteins) were subjected to Endo-H digestion, followed by
SDS-PAGE analysis. By using the anti-HLA-G 4H84 mAb, intracellular
HLA-G1 molecules contain oligosaccharides both sensitive (immature HLA)
and resistant (mature HLA) to Endo-H (Fig. 3E, lane
2), while biotinylated HLA-G1 molecules are totally Endo-H
resistant (Fig. 3E, lane 4). In contrast, HLA-G2,
-G3, and -G4 isoforms, whether they are expressed (i.e., biotinylated)
or not at the cell surface remain completely sensitive to Endo-H (Fig. 3, B—D, lanes 4 and 2, respectively).
As a control, the same membrane was rehybridized with the
anti-HLA-B and -C HC10 mAb, showing that, as expected, the HLA-B
and -C biotinylated molecules are totally resistant to Endo-H treatment
(Fig. 3, A–D and F, lanes
4). This attests that newly synthesized proteins are
undetectable in our biotinylation experiments. Taken together, these
results indicate that in contrast to classical HLA class I molecules,
and to the full-length HLA-G1, the glycosylation pattern of the
truncated HLA-G2, -G3, and -G4 isoforms is altered and is consistent
with their cell surface expression as immature glycoproteins.
HLA-G2, -G3, and -G4 isoforms are transported to the cell surface after a 2-h chase period
To investigate the time during which HLA-G isoforms reach the cell
surface, cell surface protein biotinylation experiments of
[35S]methionine/cysteine pulse-labeled
M8-HLA-G2, -G3, and -G4 cells were conducted at various chase times.
Surface biotinylated molecules were then precipitated with
streptavidin-agarose beads, and HLA-G isoforms were reprecipitated
using a rabbit Ab, namely PAG 1, which is directed against the HLA-G
161–83 epitope, and analyzed by SDS-PAGE
(Fig. 5, biotinylated). Concomitantly,
after streptavidin-agarose precipitation, the unbiotinylated proteins
were collected from the remaining supernatant, and HLA-G isoforms were
reprecipitated using PAG 1 and analyzed by SDS-PAGE (Fig. 5, unbiotinylated). Results show that cell surface labeled HLA-G isoforms
are detected at low levels after the pulse (Fig. 5, B–D,
0-h chase time, biotinylated), reach a maximum 2 h after the pulse
(Fig. 5, B–D, 2-h chase time, biotinylated) and decreased
4 h after the pulse (Fig. 5, B–D, 4-h chase time,
biotinylated). In contrast, the amounts of unbiotinylated labeled HLA-G
proteins were similar at all time point (Fig. 5, B–D,
unbiotinylated). No band was detected in the M8-pcDNA control cell line
(Fig. 5A). Thus, we conclude that HLA-G2, -G3, and -G4
isoforms reach the cell surface within a 2-h chase period.
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Previous data have shown that the full-length HLA-G1 isoform impairs the lytic activities of both NK and CTL cytotoxic effectors (21, 28). To determine whether the truncated HLA-G isoforms, like HLA-G1, were functional molecules with respect to CTL and NK effectors, we conducted chromium release assays using M8 transfected cells as targets.
To test whether HLA-G2, -G3, and -G4 isoforms impaired T cell cytolytic
function, we used an in vitro model of the T cell anti-virus
response. This model consists of CD8+ CTL cells
that are specific for the influenza virus matrix epitope, M58–66,
presented by HLA-A2 (21). These CTL effectors were induced
in vitro from the PBMC of a healthy donor by incubation for 1 wk with
the HLA-A2.1-restricted M58–66 peptide, giving rise to the HC12 cell
line. Carrying out CTL cytotoxicity assays using HLA-A2-positive M8
transfectants sensitized with M58–66 as the target cells, we found
that lysis of M8 HLA-G1, -G2, -G3, and -G4 targets by peptide-specific
CTL was significantly reduced compared with lysis of M8-pcDNA cells
(Fig. 6). Since all M8 transfectants
exhibited similar levels of HLA-A2 cell surface expression (data not
shown), we conclude that the CTL inhibitory effect was not due to
decreased peptide presentation, but to HLA-G surface expression on the
target cells. Considered together, these results suggest that all HLA-G
isoforms have the capacity to inhibit CTL lysis, thus counteracting the
positive CTL lytic signal resulting from TCR recognition of the HLA
class I/peptide complex.
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To determine whether the truncated HLA-G isoforms were functional
molecules with respect to NK cells, cytotoxicity assays were conducted
using freshly isolated polyclonal NK cells obtained from PBMC as the
effectors against M8 transfectants, which were used as targets.
Twelve experiments were conducted, each recorded with polyclonal NK
cells from a different donor. We observed that lysis of M8 HLA-G1, -G2,
-G3, and -G4 targets by fresh NK cells obtained from all 12 donors
tested was significantly reduced compared with lysis of the M8-pcDNA
control cell line (Fig. 7A).
Taken together, these results show that all HLA-G isoforms are able to
inhibit the lytic activity of ex vivo-isolated polyclonal NK cells.
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The NK lysis inhibition of HLA-G2, -G3, and -G4 isoforms does not occur through an HLA-E/CD94NKG2A pathway
To question the implication of HLA-E in the HLA-G isoform-mediated
NK lysis inhibition, we conducted cytotoxicity experiments using as
target cells the M8 transfectants facing polyclonal NK cells treated
with an mAb specific for NKG2A. Fig. 7B shows that NK lysis
of M8-pcDNA is enhanced by blocking NKG2A on polyclonal NK cells,
showing that cell surface HLA-E molecules interact with CD94/NKG2A and
act as NK inhibitory molecules. In contrast, HLA-G isoform-mediated NK
lysis inhibition is not enhanced by blocking the CD94/NKG2A receptor.
These results show that the HLA-G isoform-mediated NK lysis inhibition
does not occur through HLA-E/CD94/NKG2A interactions.
To confirm that the HLA-G isoforms can inhibit NK cytolysis through an
HLA-E-independent pathway, we tested the ability of the CD94-negative
YT2C2-C2 NK clone to be inhibited by each HLA-G isoform expressed on M8
cells. We have previously reported that the lytic activity of the
YT2C2-PR NK clone was inhibited by HLA-G1 through an unknown
killer-Ig-like receptor (KIR) (26), but not
inhibited by the nonclassical HLA-E and by the classical HLA-A, -B, and
-C class I molecules (31). This was prompted by our
observation that neither CD94/NKG2A, nor p140, p70, p58, and Ig-like
transcript 2 (ILT2) or leukocyte Ig-like receptor-1 (LIR-1) receptors
are detected on this clone (31). As shown in Fig. 7C, while the M8-pcDNA control cell line was efficiently
lysed by YT2C2-PR, lysis of M8-HLA-G2, -G3, and -G4 transfectants was
significantly reduced. Thus, like HLA-G1, the other membrane-bound
isoforms also have an inhibitory effect on this CD94/NKG2A-negative NK
subclone. This result confirms that such HLA-G-mediated inhibition
occurs through a CD94/NKG2A-independent pathway.
Interestingly, we recently found that among the HLA class I inhibitory
receptors characterized to date, the only receptor that could be
detected at the cell surface of YT2C2-PR is an HLA-G1-specific receptor
belonging to the KIR103 gene family (data not shown), namely KIR2DL4
(18, 32). Unfortunately, the KIR2DL4 antiserum is not
functional in cytotoxicity experiments, which leaves us unable to
directly implicate this receptor as the inhibitory receptor involved in
HLA-G-mediated inhibition. However, since KIR2DL4 is the only
inhibitory receptor expressed on this NK cell, we suggest that the
HLA-G inhibitory effect is most likely manifested through interaction
between KIR2DL4 and the HLA-G isoforms, probably via their common
extracellular 1 domain.
The NK lysis of M8-HLA-G2, -G3, and -G4 cells is not restored by anti-HLA-G mAbs
Since the 87G mAb reacts specifically with HLA-G1 and the 4H84 mAb
reacts with all the HLA-G protein isoforms, we next asked whether these
Abs could restore NK lysis of M8-HLA-G1, -G2, -G3, and -G4 target
cells. For this purpose, Ab blocking assays were conducted using as
effectors, polyclonal NK cells in the presence of target cells
preincubated, or not, with 4H84 or 87G mAb (Fig. 8). The results show that, as described
above, M8-HLA-G1, -G2, -G3, and -G4 are protected from lysis of
polyclonal NK cells in the absence of Ab (Fig. 8A;
p < 0.02). By using mAb-treated targets it was found
that while the 87G mAb restores NK lysis of the M8-HLA-G1 cells
(p > 0.4), it does not restore lysis of
M8-HLA-G2, -G3, and -G4 targets (Fig. 8C; p
< 0.02). This result is in good agreement with the specificity of 87G,
which reacts only with the full-length HLA-G1 isoform and consequently
has no effect on the function of HLA-G2, -G3, and -G4. In contrast,
although the 4H84 mAb reacts with HLA-G1, -G2, -G3, and -G4, it does
not restore lysis of all M8-HLA-G transfectants (Fig. 8B).
Similar results were obtained using the YT2C2-PR subclone as effector
(data not shown). Taken together, these data show that HLA-G2, -G3, and
-G4 isoforms are able to inhibit NK lytic activity, although neither
87G nor 4H84 mAb are able to restore such inhibition.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As a first question, we asked whether these truncated HLA-G2, -G3, and -G4 isoforms, the sequences of which retain exons encoding a transmembrane region and a cytoplasmic tail, could be expressed at the cell surface as proteins formed by one or two extracellular domains. By carrying out cell surface flow cytometry, biotinylation, and pulse-chase experiments, our results provide evidence that HLA-G2, -G3, and -G4 isoforms are able to reach the cell surface within 2 h after synthesis, as Endo-H-sensitive glycoproteins. In contrast, HLA-G1 and classical HLA class I molecules are well defined as Endo-H-resistant, cell surface glycoproteins. Interestingly, although this feature of proteins containing immature oligosaccharides that can reach the cell surface is unusual, it has been reported in the literature for other proteins, such as the HLA class I-like molecule, CD1d (35). An escape from ER retention has also been described for the mouse CMV glycoprotein, gp34 which associates with folded class I MHC molecules and bears two oligosaccharides that are Endo-H sensitive at the cell surface (36). Similarly, the cell surface expression of HLA-G isoforms may be due to association of the truncated HLA-G isoforms with a chaperoning protein, such as classical HLA class I molecules, allowing their escape from ER retention. In this regard, mouse nonclassical Qa-1 molecules form heterodimers with mouse classical H-2Ld molecules (37).
Previous reports in discrepancy with our results show that only HLA-G1, but not the other alternative forms of HLA-G, is expressed at the cell surface of transfected cells (33, 34). In these studies the authors used HLA-G cDNA linked to a tag molecule that can be detected with a specific Ab to track HLA-G isoform expression. Several explanations may account for such an inability to detect cell surface expression of HLA-G2, -G3, and -G4 proteins. 1) Since it is well established that the addition of a tag molecule to proteins can affect protein structure, we may expect that the tagged HLA-G2, -G3, and -G4 isoforms exhibit a misfolded protein structure. In this case, energy-dependent cytoplasmic proteases will eliminate the misfolded proteins, thus compromising their forward transport to the cell surface. The fact that tagged HLA-G protein may behave differently from its untagged HLA-G counterpart is supported by the absence of HLA-G5 protein in the lysate of tagged HLA-G5 transfectants (33), while it is well described that untagged HLA-G5 protein is easily detectable in transfected cell lysates (5), as also reported in our study. 2) The putative association between the short HLA-G isoforms and a chaperoning protein required for their cell surface expression may be prevented by the conformation adopted by tagged HLA-G isoforms. 3) The cell lines used for HLA-G transfection (i.e., HLA class I-negative JAR, J26, and C1R) may not express the chaperoning molecule to which HLA-G2, -G3, and -G4 would be associated. Accordingly, the cell surface expression of the short HLA-G isoforms will depend on the cell type in which they are expressed.
In view of the presence of HLA-G isoforms at the cell surface, our further efforts were concentrated on the functional role of these truncated HLA-G2, -G3, and -G4 isoforms. Our results show that all HLA-G isoforms inhibit both acquired (Ag-specific CTL) and innate (NK cells) immune effector cells. Indeed, we found that HLA-G1, -G2, -G3, and -G4 isoforms impair the cytolytic activity of Ag-specific CTL. This may provide new insight into the ability of a nonclassical HLA ligand to inhibit virus-specific CTL activation through inhibitory receptors normally found on NK cells, resulting in a less efficient immune control of viral replication.
Using freshly isolated polyclonal NK cells, we have also demonstrated
that transfection of the cDNA corresponding to HLA-G2, -G3, and –G4
isoforms significantly inhibits the NK cytolysis of M8 cells. These
results show that expression of even one HLA-G isoform on HLA class
I-positive targets constitutes an additional protective mechanism
against NK cytolysis. To control the implication of the other HLA class
I molecules present on the M8 transfectants in the inhibitions
observed, we checked by flow cytometry that all the M8 transfectants
exhibited a similar level of HLA-A, -B, -C, and -E expression, as
attested by their similar staining using anti-HLA-A, -B, -C, and -E
mAbs (data not shown). Furthermore, previous reports show that
HLA-G1-mediated NK inhibition can be indirectly due to HLA-E cell
surface up-regulation and its interaction with the CD94/NKG2A
inhibitory receptor (29, 30). However, we show here that
the HLA-G isoform-mediated NK lysis inhibition does not occur through
HLA-E, since 1) M8-HLA-G2, -G3, and -G4 are still protected from
polyclonal NK lysis despite the blockage of HLA-E/CD94NKG2A
interactions; and 2) the lytic activity of theYT2C2-PR, which does not
express CD94/NKG2A, is inhibited by each HLA-G isoform. Our recent
evidence that the HLA-G1-specific inhibitory receptor, namely KIR2DL4,
is expressed on YT2C2-PR strongly suggests that HLA-G isoform-mediated
inhibition occurs through KIR2DL4 and more probably through the 1
domain common to all HLA-G isoforms. This conjecture is supported by
the ability of the HLA-G3 isoform, which consists only of the 1
domain, to inhibit the KIR2DL4-positive YT2C2-PR NK subclone cytolysis.
It should be noted that in the classical HLA class I 1 domain,
residues 77–80 have been described as an important KIR recognition
site (38). In this region, Met76 and
Glu79 are unique to HLA-G (in all alleles
described to date) (39) and might be involved in KIR2DL4
recognition. However, since the KIR2DL4 antiserum is not functional in
cytotoxicity experiments, we cannot rule out that other inhibitory
receptors may be implicated in this HLA-G-mediated inhibition. Besides
KIR2DL4, another described HLA-G inhibitory receptor present on
peripheral blood NK cells is ILT2 (or LIR-1) (16, 19, 20).
However, it is unlikely that HLA-G3 and -G4 isoforms inhibit NK
cytolysis through interaction with this KIR, since a recent report
showed that ILT2 (LIR-1) interacts with the 3 domain of the HLA
class I ligand (40), which is not found in HLA-G3 and -G4
isoforms. Moreover, we cannot exclude that HLA-G isoforms mediate NK
and CTL cytolysis inhibition through another indirect pathway distinct
from HLA-E. Indeed, in an attempt to provide direct evidence that the
inhibition of NK lysis was due to the presence of HLA-G2, -G3, and -G4
molecules on the cell surface of M8 cells, we conducted cytotoxicity
experiments using as effectors polyclonal NK cells or the YT2C2-PR
subclone against the M8-HLA-G1, -G2, -G3, and -G4 targets that had been
previously treated, or not, with 87G (which reacts specifically with
the HLA-G1 isoform) or 4H84 mAb (which reacts with all HLA-G isoforms).
Unfortunately, while the 87G mAb restores NK lysis of the M8-HLA-G1
cells, neither 87G nor 4H84 mAbs restore lysis of M8-HLA-G2, -G3, and
-G4 targets. Thus, although the 4H84 mAb recognizes HLA-G1, -G2, -G3,
and -G4, it does not reverse their NK inhibitory function.
Interestingly, a distinct behavior between 87G and 4H84 is also
observed in flow cytometry experiments, in which all M8-HLA-G1 cells
are stained by 87G mAb, but not by 4H84 mAb. Taking these findings
together, one could postulate that the distinct ability of 87G and 4H84
mAbs to react with M8-HLA-G1 and to restore M8-HLA-G1 lysis may be
explained by 1) mAb affinity: the 4H84 mAb presents a lower affinity
than the 87G mAb to detect HLA-G1 cell surface molecules; and/or 2) mAb
specificity: the epitope recognized by the 4H84 mAb (i.e., the sequence
61–83 of the HLA-G 1 domain) on HLA-G1 is not involved in
HLA-G1-mediated NK lysis inhibition. Furthermore, while 87G recognizes
the 
2-microglobulin
(2m)-associated form, 4H84 may not. Thus, 87G
and 4H84 mAbs may not recognize the same form of cell surface HLA-G1
molecules (i.e., native vs denatured form), leading to the conclusion
that conformational forms recognized by 87G are functional, while
denatured forms recognized by 4H84 are not. As for HLA-G1, similar
conclusions can be extrapolated for HLA-G2, -G3, and -G4 concerning the
4H84 mAb.
In HLA-A- and -B-negative trophoblast tissue, HLA-G is the predominant
HLA class I molecule expressed (7). The placental
expression of HLA-G2, -G3, and -G4 (41) would be
particularly important in situations in which HLA-G1 expression is
altered, such as mutation of the HLA-G gene or TAP deficiencies.
Indeed, the cell surface expression of HLA-G1, as a trimolecular
-chain/2m/peptide complex, is TAP and
2m dependent (5, 42). In this
regard, in homozygous TAP-negative fetuses (43) HLA-G1
placental expression should be impaired by a lack of peptide supply in
the ER of fetal tissue. Therefore, HLA-G2, -G3, and -G4 isoforms, whose
expression at the cell surface is probably independent of peptide
loading, may substitute for the loss of HLA-G1 surface expression and
thus contribute to the survival of these fetuses. Consistently,
intracellular transport of HLA-G1 was previously found to be impaired
upon ICP47 transfection, which blocks peptide transport from the
cytosol to the endoplasmic reticulum via TAP (44).
Instead, a small 18-kDa molecule, which may correspond to the HLA-G3
isoform, is efficiently expressed, is transported to the cell surface,
and is capable of protecting the HLA-G transfectant from NK lysis.
Furthermore, the survival of fetuses homozygous for the deleted
HLA-G*0105N null allele, which is predicted to prevent HLA-G1
expression (45), may also be due to the expression of
truncated HLA-G forms assuming the protective HLA-G function.
Consistently, it has been suggested that in homozygous HLA-G*0105N
individuals, HLA-G2 may be expressed at both mRNA and protein levels
(45, 46). Moreover, by performing blocking experiments
using anti-HLA class I Abs, previous studies have concluded that
HLA class I molecules present on trophoblastic tissue do not protect it
from decidua NK lysis (47, 48). However, the fact
that trophoblastic cells are still protected from NK lysis in the
presence of anti-HLA class I Abs did not take into account the
possible cell surface expression of the truncated HLA-G isoforms, which
can still assume protection of trophoblast cells.
Besides its selective physiological placental expression, HLA-G has also been found in some HLA class I-positive tumors, such as melanoma (14, 24), colon (49), and lung carcinoma (50). Such expression in tumors would favor malignant progression by allowing HLA-G-positive tumors to escape from both NK and CTL immunosurveillance. As mentioned above in pregnancy disorders in which the smaller HLA-G isoforms can substitute for the loss of HLA-G1 expression, such substitution may also be extrapolated to tumor situations. Indeed, when HLA-A, -B, -C, and -E are down-regulated in tumor cells as the result of a transport defect (51), HLA-G1 expression should also be impaired. In such deficient tumors, which become good targets for NK lysis, expression of the other membrane-bound HLA-G isoforms might still occur, thus facilitating the immune escape of HLA-G-positive tumors from NK immunosurveillance.
Taken together, our results strongly support the idea that expression
of at least one HLA-G isoform on the cell surface may favor immune
tolerance by inhibiting both NK and CTL effectors during pregnancy,
tumor progression, or transplantation. This is particularly interesting
for HLA-G3, which contains the expected functional epitope in its only
1 monomorphic domain.
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Acknowledgments |
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Footnotes |
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2 B.R and N.R.F. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Edgardo D. Carosella, Service de Recherches en Hémato-Immunologie, Commissariat à l’Energie Atomique-Direction des Sciences du Vivant-Department de Recherche Médicale, Institut Universitaire d’Hématologie, Hôpital Saint Louis, 1 avenue C. Vellefaux, 75010 Paris, France.
4 Abbreviations used in this paper: Endo-H, endoglycosidase H; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate; PNGase-F, peptide-N-glycosidase F; ER, endoplasmic reticulum; ILT, Ig-like transcript; LIR, leukocyte Ig-like receptor; 2m, 2-microglobulin; KIR, killer-Ig-related receptor.
Received for publication October 20, 2000. Accepted for publication February 13, 2001.
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E. C. Ibrahim, N. Guerra, M.-J. T. Lacombe, E. Angevin, S. Chouaib, E. D. Carosella, A. Caignard, and P. Paul Tumor-specific Up-Regulation of the Nonclassical Class I HLA-G Antigen Expression in Renal Carcinoma Cancer Res., September 1, 2001; 61(18): 6838 - 6845. [Abstract] [Full Text] [PDF]
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N. Lila, N. Rouas-Freiss, J. Dausset, A. Carpentier, and E. D. Carosella Soluble HLA-G protein secreted by allo-specific CD4+ T cells suppresses the allo-proliferative response: A CD4+ T cell regulatory mechanism PNAS, October 9, 2001; 98(21): 12150 - 12155. [Abstract] [Full Text] [PDF]
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 N. Lila, C. Amrein, R. Guillemain, P. Chevalier, C. Latremouille, J.-N. Fabiani, J. Dausset, E. D. Carosella, and A. Carpentier Human Leukocyte Antigen-G Expression After Heart Transplantation Is Associated With a Reduced Incidence of Rejection Circulation, April 23, 2002; 105(16): 1949 - 1954. [Abstract] [Full Text] [PDF]
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