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* Department of Neurology, Medical School, and
Institute of Brain Research, University of Tübingen, Tübingen, Germany; and
Institute of Anthropology and Human Genetics, Ludwig Maximilians University, Munich, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and in 10 of 12
cell lines in the presence of IFN-. HLA-G protein was detected in
four of 12 cell lines in the absence of IFN- and in eight of 12 cell
lines in the presence of IFN-. Immunohistochemical analysis of human
brain tumors revealed expression of HLA-G in four of five tissue
samples. Functional studies on the role of HLA-G in glioma cells were
conducted with alloreactive PBMCs, NK cells, and T cell
subpopulations. Expression of membrane-bound HLA-G1 and soluble
HLA-G5 inhibited alloreactive and Ag-specific immune responses. Gene
transfer of HLA-G1 or HLA-G5 into HLA-G-negative glioma cells (U87MG)
rendered cells highly resistant to direct alloreactive lysis, inhibited
the alloproliferative response, and prevented efficient priming of
cytotoxic T cells. The inhibitory effects of HLA-G were directed
against CD8 and CD4 T cells, but appeared to be NK cell independent.
Interestingly, few HLA-G-positive cells within a population of
HLA-G-negative tumor cells exerted significant immune inhibitory
effects. We conclude that the aberrant expression of HLA-G may
contribute to immune escape in human
glioblastoma. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and the expression of
CD95 ligand (CD95L)4
(2), both acting on
lymphoid cells (reviewed in Ref. 3). MHC class Ia expression is frequently altered in a variety of malignancies. These alterations may have a decisive influence on the immune response and on the metastatic capacity of the tumors (4). HLA-G is a nonclassical MHC class I molecule (class Ib) structurally related to classical MHC class Ia (HLA-A, -B, -C) that—in contrast to class Ia molecules—exhibits a limited tissue distribution. First detected on extravillous cytotrophoblast cells (5), it has now been recognized that HLA-G is also expressed by thymic epithelial cells (6), cytokine-activated monocytes (7), inflamed muscle and myoblasts (8), and some tumors (e.g., Ref. 9).
In contrast to HLA class Ia molecules, HLA-G is characterized by a limited polymorphism and the transcription of alternatively spliced mRNAs that encode at least seven different isoforms, including membrane-bound HLA-G1, -G2, -G3, and -G4 and soluble HLA-G5 (formerly HLA-sG1), -G6 (formerly HLA-sG2), and -G7 proteins (reviewed in Ref. 10). HLA-G binds CD8 and a restricted repertoire of peptides and might therefore act as a peptide-presenting molecule (11, 12, 13, 14). However, HLA-G has so far chiefly been regarded as a mediator of immune tolerance because it protects target cells from NK cytolysis through direct or indirect interaction with several inhibitory receptors expressed on NK cells (15, 16, 17). Furthermore, HLA-G can also modulate Ag-specific lysis through interaction with surface molecules expressed on cytotoxic CD8 T cells (18, 19). Therefore, expression of HLA-G has been proposed to allow immune escape in tumors.
In this work, we report the expression and functional activity of HLA-G in glioblastoma cells. Studying the negative immune regulatory effects of HLA-G on different effector lymphocyte populations in models of primary and secondary immune responses, we propose that HLA-G-dependent suppression of T cell responses represents a novel immune escape pathway of human glioblastoma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following Abs were used: 87G (IgG2b, anti-HLA-G; kindly
provided by D. Geraghty, Fred Hutchinson Cancer Research Center,
Seattle, WA), 4H84 (mIgG1, anti-HLA-G; kindly provided by M.
McMaster, University of California, San Francisco, CA); MEM-G/9 (mIgG1,
anti-HLA-G; Exbio, Prague, Czech Republic); BFL1.1.2 (mIgG2b,
anti-HLA-G; Immunotech, Marseille, France); W6/32 (mIgG2a,
anti-HLA-A, -B, -C, -G, -E and
2-microglobulin (2m);
Biozol, Eching, Germany); L243 (mIgG2a, anti-HLA-DR), BB7.2
(mIgG2b, anti-HLA-A2; American Type Culture Collection, Manassas,
VA); RPA-T4 (mIgG1
, FITC- and PE-labeled anti-CD4); HIT8A
(mIgG1, PE-labeled anti-CD8), Annexin V-FITC (BD PharMingen,
Heidelberg, Germany); B9.11 (mIgG1, FITC-labeled anti-CD8), N901
(NKH-1; mIgG1, PE-labeled anti-CD56) (Immunotech); goat
anti-mouse F(ab')2 (Dianova, Hamburg,
Germany); and peroxidase-labeled rabbit anti-human
2m (DAKO, Hamburg, Germany). The Abs were
titrated for flow cytometry and used at concentrations indicated
(usually 20 µg/ml) in the functional assays. Soluble CD95L was
obtained from CD95L-transfected murine N2A neuroblastoma cells
(20). Propidium iodide (PI) was purchased from
Sigma-Aldrich (St. Louis, MO).
Cell culture
The human malignant glioma cell lines LN-18, U138MG, U87MG, LN-428, D247MG, T98G, LN-319, LN-229, A172, U251MG, U373MG, and LN-308 (kindly provided by Dr. N. de Tribolet, Neurosurgical Service, Centre Hospitalier Universitaire, Vaudois, Lausanne, Switzerland) and SV40 fetal human astrocytes non-neoplastic astrocytes (kindly provided by A. Muruganandam, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada) were cultured in 75-cm3 Falcon plastic flasks (BD Biosciences, Heidelberg, Germany) using DMEM supplemented with 1% glutamine (Life Technologies, Paisley, U.K.), 10% FCS (Biochrom, Berlin, Germany), and penicillin (100 IU/ml)/streptomycin (100 µg/ml) (Life Technologies). The JEG-3 human choriocarcinoma cell line and the K-562 human erythroleukemia cell line (American Type Culture Collection) were maintained in RPMI 1640 supplemented with 1 mM sodium pyruvate, penicillin (100 IU/ml)/streptomycin (100 µg/ml), and 10% FCS. Cells were routinely tested for contamination with mycoplasma. Where indicated, the cells were irradiated using a Gammacell 1000 Elite (Nordion, Ottawa, Ontario, Canada).
Immunohistochemistry
Tumor biopsy specimens were surgically removed from four patients with glioblastoma (grade IV, World Health Organization; male, age range 32–64 years old, mean 46 years old) and one patient with anaplastic oligo-astrocytoma (grade III, World Health Organization; female, 42 years old). Frozen sections 10 µm thick were cut, fixed in acetone, and immunostained with anti-HLA-G Ab (87G, BFL1.1.2). The reaction product was visualized with the streptavidin-biotin method (reagents from DAKO) using diaminobenzidine (Serva, Heidelberg, Germany) as an electron donor. As control tissue, placenta was used.
HLA-G transfectants
HLA-G transfectants were obtained as described elsewhere (18). In brief, HLA-G1 (encoding the full-length HLA-G transcript) and HLA-G5 (coding for the secreted HLA-G H chain) plasmids were generated by cloning HLA-G1 and HLA-G5 cDNAs into a GFP vector (pIRES2-pEGFP; Clontech Laboratories, Palo Alto, CA). GFP vector transfectants (pIRES2-pEGFP) were used as controls. Transfection was done using Effectene transfection reagent (Qiagen, Hilden, Germany). Cells were selected in medium containing 0.5 mg/ml neomycin (G418; Calbiochem, Bad Soden, Germany). The following transfectants were used in the study: K-562-HLA-G1, U87MG-pIRES2-EGFP (referred to as U87MG-pEGFP), U87 MG-HLA-G1, and U87MG-HLA-G5.
PBMCs and purified lymphocyte populations
PBMCs were isolated from the peripheral blood of normal healthy volunteers by density gradient centrifugation using Biocoll Separating Solution (Biochrom). Cells were analyzed for their HLA-A2 isotype by flow cytometry. CD4 and CD8 T cells were purified by positive selection with CD4 or CD8 Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). NK cells were isolated by an NK cell-negative isolation kit (Dynal Biotech, Hamburg, Germany). The purity of the isolated subpopulations used in the experiments was >95% as determined by flow cytometry.
Flow cytometry
Adherent cell lines were collected after treatment with cell dissociation buffer (Life Technologies) at 37°C for 5 min. JEG-3, K-562-HLA-G1, or K-562 cells were used as positive and negative control cell lines for HLA-G or MHC expression when indicated. Cells were washed with PBS containing 0.1% BSA and 0.1% sodium azide and blocked with human Igs (Alphaglobin; Grifols, Langen, Germany) for 10 min at 4°C. After one washing step, the unlabeled first Ab was added at the final concentration. Isotype control mAbs were used at the same concentration as the primary Ab. Incubation was done on ice for 30 min, followed by two washes. Goat anti-mouse IgG (F(ab')2-PE (5 µg/ml; Sigma-Aldrich) or IgG (F(ab')2-di-chlorotriazinyl-fluoresceine (10 µg/ml; Dianova) were used as secondary Abs. After washing, PI was added to the cells at a final concentration of 0.02 µM. PI-positive (nonviable) cells were excluded from analysis. Specific fluorescence indexes (SFIs) were calculated by dividing mean fluorescence obtained with specific Ab by mean fluorescence obtained with isotype control Ab. Analyses of PBMCs and lymphocyte subsets were performed similarly. Cells were blocked with human Igs and stained either with directly labeled Abs or with the respective combinations of an unlabeled first Ab and a fluorescent secondary Ab. Flow cytometry was performed using a FACSCalibur (BD Biosciences, Heidelberg, Germany).
Detection of apoptosis by Annexin V binding
PBMCs or purified lymphocyte subsets undergoing apoptotic cell death were analyzed by staining with FITC-labeled Annexin V (BD PharMingen). After various incubation times with the glioma cells, lymphocytes were collected, washed with PBS, and resuspended in a buffer containing 10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2. Then Annexin V-FITC and PI were added. After incubation for 30 min, the cells were analyzed by flow cytometry. When analysis of lymphocyte subsets was desired, PBMCs were first incubated with respective PE-labeled cell surface Abs (CD4-PE and CD8-PE from BD PharMingen; CD56-PE from Immunotech) before the Annexin V staining was performed.
Primary alloreactive proliferation
To suppress the proliferation of stimulator cells, glioma cells were incubated in serum-free medium for 24 h, irradiated at 50–200 Gy, and maintained in serum-free medium for another 24 h. Cells were detached using cell dissociation buffer (Life Technologies), counted, and seeded in RPMI 1640 medium. HLA-A2-mismatched responder PBMCs (105) were coincubated with 104 irradiated glioma cells in a total volume of 100 µl. The assay was performed in 96-well plates in triplicate. PBMCs without glioma cells, glioma cells without PBMCs, and PHA-stimulated PBMCs (10 µg/ml) were used as controls. After 4 days, the cells were pulsed for 24 h with 0.5 µCi of [methyl-3H]thymidine (Amersham Pharmacia Biotech, Freiburg, Germany). Cells were harvested with a cell harvester (Inotech, Dottikon, Switzerland), and the incorporated radioactivity was bound to a glass fiber filtermat (Wallac, Turku, Finland). The filtermat was wetted with Ultima Gold Scintillation Cocktail (Packard, Dreieich, Germany), and radioactivity was determined in a 1450 Microbeta Plus Liquid Scintillation Counter (Wallac).
Secondary alloreactive lysis (51Cr release)
To test for lytic activity of primed cytotoxic T cells after coculture with HLA-A2-mismatched glioma cells (U87MG-pEGFP, U87MG-HLA-G1, U87MG-HLA-G5) for 5 days, a 51Cr release assay was performed. Growth-suppressed glioma cells were seeded into 25-cm2 flasks to a density of 106 cells. HLA-A2-mismatched PBMCs (1.5 x 107) were added in 5 ml of RPMI 1640 containing 10% FCS. Cells were cocultured for 5 days. Glioma cells (106) were labeled by addition of 100 µCi 51Cr (catalog no. NEZ147; NEN, Boston, MA) in 1 ml, and 104 labeled glioma cells were seeded as target cells in a U-shaped 96-well plate. Primed alloreactive cytotoxic lymphocytes obtained after 5 days of coculturing were removed and added to the labeled target cells at different E:T ratios (ranging from 10 to 80) in a volume of 100 µl. After coincubation for 4 h, 50 µl of the supernatant was transferred to a Luma-Plate 96 (Packard), dried overnight, and measured. To correct for spontaneous release of 51Cr, a control of labeled target cells in medium only was included (0% specific lysis). The maximum 51Cr release possible was determined by addition of Nonidet P-40 (100% lysis). Lysis was calculated as follows: [cpm (effector cells) - cpm (spontaneous)]/[cpm (Nonidet P-40) - cpm (spontaneous)] x 100%.
Primary alloreactive lysis (51Cr release)
Primary alloreactive lytic damage of glioma target cells by HLA-A2-mismatched lymphocytes was measured in a 51Cr release assay. Target glioma cells were radioactively labeled and seeded into 96-well U-bottom wells as described above. For blocking experiments, glioma cells or HLA-G transfectants were preincubated with specific Abs or the corresponding isotype control Abs. After incubation for 1 h at 37°C, cross-linking by a secondary Ab (goat anti-mouse F(ab')2, 10 µg/ml; Dianova) was performed to immobilize the Ab on the glioma cells. Effector cells (PBMCs, CD4, CD8, NK cells) were added at E:T ratios ranging from 1 to 100 in a total volume of 100 µl. After coincubation for 8 h, 50 µl of the supernatant were transferred to a Luma-Plate 96 (Packard), dried overnight, and measured. In experiments using purified NK cells, incubation was done for 4 h. To correct for spontaneous 51Cr release, a control of labeled target cells in medium only was included (0% specific lysis). The maximum 51Cr release possible was determined by addition of Nonidet P-40 (100% lysis).
sHLA-G ELISA
Secreted HLA-G molecules were measured as total soluble HLA
class I molecules in the culture supernatants. MHC class I Ab (W6/32,
concentration 5 µg/ml, volume 100 µl) was coated onto a 96-well
microtiter plate (Nunc, Roskilde, Denmark) and incubated overnight at
4°C. After three washes with PBS containing 0.05% Tween 20
(PBS-Tween), the plate was saturated with 250 µl of PBS containing
2% BSA. After incubation for 2 h at 37°C, the plate was washed
three times with PBS-Tween. Cell culture supernatants (100 µl) were
added to each well at various dilutions and incubated for 2 h at
37°C. Plates were washed another three times with PBS-Tween, and
peroxidase-labeled rabbit anti-mouse 2m Ab
(dilution, 1/500; volume, 100 µl) was added. After incubation for
1 h at 37°C and washing three times with PBS-Tween, 100 µl of
ortho-phenyl-diamine peroxidase substrate was added (Abbott
Diagnostics, Wiesbaden-Delkenheim, Germany) for 30 min at room
temperature avoiding exposure to light. The reaction was stopped by
addition of 50 µl of 2 M
H2SO4. The concentration
was estimated from the absorption measured at 490 nm (triplicate wells)
on an ELISA microplate reader (Wallac).
Reverse transcription and amplification
First-strand DNA synthesis of 1 µg of total RNA primed with oligo(dT)12–18 (Amersham Pharmacia Biotech) and Expand Reverse Transcriptase (Boehringer Mannheim, Mannheim, Germany) was done according to the manufacturer’s recommendations (22). One microliter of cDNA was amplified using the following primer pairs, which coamplify the HLA-G isoforms HLA-G1, -G2/4, and -G3: Ex1 (5'-CAAGGATGGTGGTCATGGCG-3') and Ex6/3'-untranslated (UT) (5'-CAGCTGTTTCACATTGCAGCCTG-3'), 60°C annealing temperature, 40 cycles. PCR products were analyzed on 1.5% agarose gels containing ethidium bromide or on 8% native polyacrylamide gels followed by silver staining (23).
DNA probes and Northern blot analysis
RNA was isolated with the TriPure Isolation reagent (Boehringer Mannheim). A total of 20 µg of total RNA was separated on 1.2% formaldehyde agarose gels, transferred to Hybond-N+ membrane (Amersham Pharmacia Biotech) in 20x SSC overnight and cross-linked by baking at 80°C for 2 h. The filters were hybridized with 10 ng/ml specific digoxigenin-labeled probe and were detected as described (21). Hybridization probes specific for HLA-G transcripts were derived from the 3'-UT regions and were generated by PCR and incorporation of digoxigenin-labeled dideoxyuridine triphosphate (Boehringer Mannheim). The 486-bp HLA-G probe was labeled from a subcloned 287-bp amplificate with the primers M13 (-20) and M13 reverse (25). The 440-bp HLA-B probe was generated from the subcloned 3'-UT PvuII/PstI fragment using SP6 and T7 primers. To detect HLA-G transcripts in HLA-G transfectants, a 787-bp HLA-G4 probe was generated by PCR using the primer pair Ex1 and Ex6/3'-UT.
Immunoblot analysis
Cells were lysed for 30 min at 4°C in lysis buffer (20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 2% trasylol; Bayer, Leverkusen, Germany) and 1 mM PMSF (Sigma-Aldrich). After removal of cellular debris by centrifugation, protein content of the lysates was quantified using a MicroBradford assay (24). Lysates equivalent to 100 µg of protein were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes (Schleicher and Schüell, Dassel, Germany) using standard blotting techniques. HLA-G H chains were detected with the mAb 4H84 and the ECL Western blotting analysis system (Amersham Pharmacia Biotech).
Statistical analysis
All data are representative of experiments performed at least three times with similar results. Significance was assessed by two-sided t test (*, p < 0.05; **, p < 0.01).
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Results |
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Twelve human glioma cell lines were tested for HLA-G mRNA
expression in the absence or presence of IFN- by Northern blotting
using a probe specific for all HLA-G isoforms (Fig. 1
). Six glioma cell lines transcribed
HLA-G constitutively (U251MG, U373MG, D247MG, T98G, LN-428, and A172).
The presence of different alternative splicing patterns were confirmed
by RT-PCR specific for transmembraneous and soluble isoforms of HLA-G
(data not shown). After stimulation with IFN-, HLA-G transcripts
became detectable in 10 of the 12 glioma lines (Fig. 1). Only U87MG and
LN-308 cells were refractory to IFN--mediated induction of HLA-G
expression. PCR analysis paralleled these observations, confirming
strong up-regulation of different HLA-G isoforms after IFN-
induction (data not shown).
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). After stimulation with IFN-, HLA-G
membrane expression increased and was detectable in eight of the 12
tested glioma cell lines, using a cutoff SFI value of 1.3 (Fig. 2). The
expression of class I (HLA-A, -B, -C mAb W6/32; HLA-A2 mAb BB7.2) and
class II (HLA-DR; mAb L243) molecules was assessed in parallel, showing
that all glioma cells expressed HLA class I molecules constitutively.
IFN- enhanced the surface expression of MHC class I in all cell
lines except A172. The MHC class II DR-molecule was expressed
constitutively in four of 12 glioma cell lines. IFN- induced HLA-DR
in all except two cell lines (LN-308 and U138MG). The human
SV40-transformed astrocytic cell line SV40-fetal human astrocytes did
not show HLA-G RNA or protein expression in the absence or presence of
IFN- (data not shown).
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Immunohistochemistry detected HLA-G expression in four of five
brain tumors. Up to 50% of tumor cells expressed HLA-G in three of the
four tested glioblastomas and in one anaplastic oligo-astrocytoma (Fig. 3; staining shown here with mAb BFL1.1.2
and mAb 87G). Positive cells were evenly scattered throughout the
specimens. One glioblastoma was negative for HLA-G with all tested
Abs.
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To elucidate the functional relevance of constitutive and
inducible HLA-G expression by glioma cells, different alloreactivity
assays were performed. Freshly isolated PBMCs were used as effector
cells and incubated with HLA-A2-mismatched glioma targets that had
previously been cultured in the absence or presence of IFN-. The
relevance of HLA-G molecules for the protection of glioma cells from
alloreactive lysis was shown by preincubating glioma cells with
HLA-G-specific Ab 87G (Fig. 4). HLA-G
expressed on U373MG and T98G participated in the protection of glioma
cells against direct cytolytic activity of PBMCs as recorded in an 8-h
51Cr release assay. The MHC class I-specific Ab
W6/32 was used as a control. Although W6/32 blocks alloreactive lysis
by MHC class I-restricted CD8 T cells, the observed net effect on PBMC
lysis in the presence of W6/32 was an increase in specific lysis. MHC
class I expression regulates NK cell lysis via interaction with killer
inhibitory receptors, therefore blocking MHC class I might have
facilitated lysis by NK cells present within the PBMC population.
Because W6/32 cross-reacts with HLA-G and the negative immune
regulatory molecule HLA-E, part of the observed effects of this Ab on
lysis may also have been mediated by an interaction with HLA-E. In
U87MG, a glioma cell line lacking HLA-G expression, neutralization with
87G had no significant influence on lysis by alloreactive PBMCs (data
not shown).
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To elucidate the effects of the two major isoforms, HLA-G1 and
HLA-G5, on antitumoral immune responses, HLA-G transfectants of an
HLA-G-negative glioma line (U87MG) were generated. Transfectants showed
high levels of HLA-G expression as documented by immunoblot, flow
cytometry, and ELISA for soluble HLA-G (sHLA-G) (Fig. 5). The forced expression of HLA-G
isoforms did not alter the expression levels or inducibility by IFN-
of classical MHC class I or II molecules as determined by flow
cytometry (data not shown).
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Gene transfer of HLA-G1 and HLA-G5 rendered U87MG cells less
susceptible to direct alloreactive lytic killing by HLA-A2-mismatched
PBMCs as assessed by 51Cr release of the target
population (Fig. 6A). Glioma
cell death was also monitored by fluorescence microscopy. Modulation of
glioma cell lysis after incubation of tumor targets with PBMCs is
demonstrated by the significantly higher survival rates of the HLA-G
transfectants compared with the vector control transfectants at 48
h (Fig. 6B). To further characterize the effector
populations inhibited by HLA-G expressed on U87MG cells, we performed
primary alloreactivity assays with purified lymphocyte subpopulations
(CD56, CD4, CD8). U87MG parental cells showed resistance to NK cell
lysis (Fig. 6C). Therefore, effects of ectopic HLA-G
expression could not be attributed to NK cell inhibition. In contrast,
MHC class I-negative K-562 cells were clearly protected from NK lysis
by the expression of HLA-G1. Unexpectedly, lytic assays with CD8 and
CD4 effector cell subsets demonstrated that HLA-G1 directly inhibited
lysis mediated by alloreactive CD4 and CD8 effector cell populations
(Fig. 6D).
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To study the effects of HLA-G on the generation of primary and
secondary immune responses, we first quantified the primary
proliferative response of HLA-A2-mismatched responder PBMCs against
growth-arrested glioma cells (U87MG-pEGFP, U87MG-HLA-G1, or
U87MG-HLA-G5) by measuring
[methyl-3H]thymidine incorporation. The
proliferative response toward glioma cells was significantly suppressed
by U87MG-HLA-G1 and U87MG-HLA-G5 cells (Fig. 7A). To assess whether
induction of apoptosis in the effector cells (19)
contributes to these inhibitory effects, the cellular viability within
the effector cell population was assessed during coculture of freshly
isolated lymphocyte subpopulations (CD8, CD4) with U87MG-pEGFP,
U87MG-HLA-G1, or U87MG-HLA-G5 transfectants. HLA-G1 did not induce
apoptosis in the effector cell populations as detected by Annexin V
staining up to 72 h after coincubation of freshly isolated and not
previously stimulated PBMCs or lymphocyte subsets with target cells
(Fig. 7B). As a positive control for the induction of
apoptosis, PHA-stimulated PBMCs were treated with CD95L.
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C). The protective effect of
HLA-G against specific killing by cytotoxic effector cells depended on
the presence of HLA-G in the priming phase. When HLA-G was present in
the target population after Ag-specific cells had been primed against
U87MG-pEGFP control cells, subsequent lysis of U87MG-pEGFP cells was
not significantly different from that of U87MG-HLA-G1 (Fig. 7D). Similar results were obtained for U87MG transfectants
expressing soluble HLA-G (U87MG-HLA-G5; data not
shown). Few HLA-G-positive cells are sufficient to inhibit alloreactive lysis of HLA-G-negative glioma cells
To assess the possible immune modulatory effects of a minor
population of glioma cells expressing HLA-G within a given tumor cell
population, 51Cr-labeled U87MG-HLA-G1 cells were
mixed with 51Cr-labeled vector control
U87MG-pEGFP cells, and lysis of targets was assessed after incubation
with alloreactive, HLA-A2-mismatched PBMCs. Significant inhibition of
lysis was observed when 10% or more of the target cells expressed
HLA-G (Fig. 8A). To delineate
whether the release of soluble HLA-G as a result of shedding into the
supernatant might account for this effect, the levels of soluble HLA
molecules in the supernatants were quantified by ELISA. However, the
levels of sHLA molecules (classical and nonclassical MHC molecules
absorbed by W6/32 mAb) in the supernatant of HLA-G1 cells were
unaltered upon coincubation of PBMCs with U87MG-HLA-G1 (Fig. 8B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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stimulation
(Fig. 1). A weak constitutive surface expression of HLA-G protein was
observed in four cell lines (U373MG, LN-319, T98G, and A172); cell
surface expression of HLA-G protein was detected in eight of 12 cell
lines after IFN- stimulation (Fig. 2). These data confirm and extend
a previous report (25) that provided the first evidence of
HLA-G expression in two glioma cell lines (T98G and N59).
The expression of HLA-G rendered glioma cells less susceptible to
alloreactive cytolytic killing as demonstrated by blocking experiments
using an HLA-G-specific neutralizing mAb (87G) in U373MG and T98G, two
cell lines which constitutively express HLA-G at the cell surface (Fig. 4). Exposure to neutralizing HLA-G Abs in U87MG, a glioma cell
line which does not express HLA-G mRNA or protein (Figs. 1 and 2), had
no such effect.
To further corroborate these results and to delineate the contribution
of the two major HLA-G isoforms, HLA-G1 and HLA-G5, to immunoregulatory
properties of glioma cells, gene transfer studies were performed in the
MHC class I-positive, HLA-A2-positive glioma cell line, U87MG. This
cell line lacks baseline or inducible expression of HLA-G (Figs. 1 and 2). Gene transfer of HLA-G1 and -G5 rendered these glioma cells
resistant to primary and secondary antitumoral immune responses under
three different assay conditions: 1) HLA-G directly inhibited
alloreactive lysis (Fig. 6), 2) HLA-G inhibited the primary
proliferative response to U87MG stimulator cells (Fig. 7A),
and 3) HLA-G prevented efficient priming of cytotoxic effector cells
and protected glioma cells from lytic killing by Ag-specific cytotoxic
lymphocytes (Fig. 7C).
The first descriptions of HLA-G tissue distribution were suggestive of
a limited expression on extravillous cytotrophoblast cells
(5), suggesting an important role in fetal semiallograft
reaction (26). Therefore, HLA-G functional studies were
mainly conducted using HLA class I-negative cell lines such as LCL
721.221 or K-562, that were protected from NK cell-mediated cytolysis
when transfected with HLA-G (15). HLA-G was shown to
interact with several receptors on NK cells, namely KIR2DL4, ILT-2,
ILT-4, and putatively others (15, 16, 17). The hypothesis of
HLA-G expression as a possible tumor immune escape mechanism has
recently been demonstrated in melanoma cells, in which HLA-G surface
expression was shown to inhibit antitumoral NK cell lysis
(9). Our data demonstrate that the immune-paralytic
effects of HLA-G expressed on glioma cells are NK cell independent
(Fig. 6C). We find that HLA-G1 conveys immunoprotection by
direct interaction with CD8 and CD4 alloreactive T cells (Fig. 6D). Furthermore, HLA-G expressed on tumor cells inhibits
primary proliferation in the effector population and prevents efficient
priming of Ag-specific cytotoxic T cells (Fig. 7, C and
D). Because human glioma cell lines constitutively express
MHC class I and some cell lines also express MHC class II on the cell
surface (Fig. 2), our study provides further evidence for an important
immunoregulatory role of HLA-G coexpressed in the presence of MHC class
I and class II (27). The effects can be attributed to
HLA-G rather than being indirectly mediated by up-regulation of other
inhibitory molecules like HLA-E because lysis inhibition conveyed by
the HLA-G transfectants was reversed by adding a neutralizing HLA-G Ab
(87G) (data not shown). Therefore, our observations extend the
hypothesis that HLA-G is capable of modulating Ag-specific (18, 28, 29) as well as non-Ag-specific cytotoxic T cell responses
(19). HLA-G1 and HLA-G5 display similar immune-inhibitory
effects in our experiments. One possible explanation would be binding
to a common receptor(s) expressed on different lymphocyte subsets.
HLA-G1 as well as HLA-G5 inhibited the primary alloproliferative
response in the responder population, but they did not render effector
cells apoptotic (Fig. 7B). Previous studies have suggested
that soluble HLA-G molecules induce apoptosis in CD8 T cells
(19), extending observations of such an effect of soluble
HLA molecules in general (30). However, PBMCs in those
studies had already been prestimulated before encountering the soluble
HLA-G molecules. By coincubating unstimulated responder cells with
HLA-G, our results favor the mechanism of an HLA-G-induced cell cycle
arrest as possible explanation for the effect on effector cells. Of
note, proliferative allogeneic responses in a mixed lymphocyte reaction
induce soluble HLA-G production by CD4 T cells (31), an
observation that underscores that the immune regulatory capabilities of
HLA-G are broader than previously assumed.
To address the question of what frequency of HLA-G-expressing tumor
cells might be relevant for a down-regulation of antitumoral immune
responses, we performed mixing experiments in vitro. Our data indicate
that a low number of HLA-G-positive glioma cells in a population of
glioblastoma cells may be sufficient for an effective suppression of an
antitumoral immune response in vivo (Fig. 8A). A direct
interaction of HLA-G with certain receptors on immune effector cells is
assumed to be the predominant pathway of HLA-G action. Shedding of
HLA-G into the cell suspension would be a possible explanation for some
of our observations. However, we did not detect any release of soluble
HLA-G by the HLA-G1 transfectants, thus making that possibility rather
unlikely (Fig. 8B). Our surprising observation could be
interpreted as a "negative bystander" effect: few HLA-G-positive
cells render neighboring HLA-G-negative cells "resistant" to
immune-mediated killing or induce nonreactivity in cytotoxic cells.
However, the molecular explanation for this phenomenon remains to
be clarified. Whereas high levels of HLA-G in the transfected glioma
cells are possibly required to directly suppress T cell responses, low
levels or HLA-G as expressed constitutively by some tumor cells might
modulate immune responses through another mechanism(s).
Expression of HLA-G in vivo was demonstrated in four of five brain
tumor specimens (Fig. 3), thus strongly corroborating our functional in
vitro observations in glioma. Although to date ectopic expression of
HLA-G has been demonstrated in some tumors (32, 33, 34, 35, 36), the
overall relevance of this molecule as an alternative principle of tumor
immune escape remains controversial (37, 38, 39, 40), an issue
that is further complicated by the limitations of available Abs to
detect HLA-G expression in vivo (41, 42).
Our results provide the first functional analysis of HLA-G in human gliomas in vitro and in vivo. In summary, our data support an important role of this molecule in the immune escape mechanisms of human gliomas. HLA-G displays its negative immunmodulatory effects severalfold on primary and secondary immune reactions: 1) it directly interacts with alloreactive CD4 and CD8 cytotoxic T cells, 2) it prevents efficient antigenic priming of antitumoral cytotoxic effector cells, and 3) it efficiently inhibits alloreactive proliferation without inducing apoptosis. Because minor portions of HLA-G-expressing cells within a population of HLA-G-negative cells are sufficient to convey relevant immune regulatory effects, expression of this molecule may have major implications also in other sites of desirable and undesirable immune reactions.
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Acknowledgments |
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Footnotes |
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2 H.W. and M.M. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Heinz Wiendl, Department of Neurology, University of Tübingen, Hoppe-Seyler-Strasse 3, D-72076 Tübingen, Germany. E-mail address: heinz.wiendl{at}uni-tuebingen.de
4 Abbreviations used in this paper: CD95L, CD95 ligand; 2m, 2-microglobulin; PI, propidium iodide; SFI, specific fluorescence index; sHLA-G, soluble HLA-G; UT, untranslated.
Received for publication December 21, 2001. Accepted for publication March 1, 2002.
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