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Department of Pathology, Academic Hospital, Vrije Universiteit, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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release enzyme-linked immunospot assay. LMP1 derived LALLFWL peptides
showed strong inhibition of T cell proliferation and NK cytotoxicity,
while acetylated LALLFWL peptides had an even stronger effect. In
addition, Ag-specific IFN- release was severely inhibited. To exert
immunosuppressive effects in vivo, LMP1 has to be excreted from the
cells. Indeed, LMP1 was detected in supernatant of EBV-positive B cell
lines (LCL), and differential centrifugation in combination with
Western blot analysis of the pellets indicated that LMP1 is probably
secreted by LCL in the form of exosomes. The amount of secreted LMP1 in
B cell cultures is well below the immunosuppressive level observed with
rLMP1. Our results demonstrate direct immunosuppressive properties of
LMP1 (fragments) and suggest that EBV-positive tumor cells may actively
secrete LMP1 and thus mediate immunosuppressive effects on
tumor-infiltrating lymphocytes. Moreover, we demonstrate, for the first
time, that transmembrane protein-mediated immunosuppression is not
solely restricted to RNA tumor viruses, but can also be found in DNA
tumor viruses. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-herpes virus family, is associated with
various lymphoproliferative and epithelial malignancies, such as
Hodgkin’s disease
(HD),2 Burkitt’s
lymphoma, B and T cell non-Hodgkin lymphoma, and nasopharyngeal
carcinoma (1, 2, 3, 4, 5). In EBV-positive lymphomas in
immune-competent patients, a restricted set of latent viral genes is
expressed, i.e., latent membrane proteins 1, 2A, and 2B (LMP1, -2A, and
-2B), Epstein-Barr nuclear Ag (EBNA1) and the nontranslated RNAs EBER1
and EBER2 (6, 7). A key protein in EBV-mediated
oncogenesis of lymphoproliferative disorders is LMP1, a protein with
transforming capacities (8). The neoplastic cells in
EBV-positive HD, called Hodgkin Reed-Sternberg (H-RS) cells, express
exceptionally high levels of LMP1. Although LMP1 is a target for CTL
recognition in the context of different MHC class I restriction
elements in vitro, it seems to be subdominant (9, 10, 11).
Surprisingly, only few EBV-positive HD patients have a weak, but
detectable, Ab response against LMP1 despite high Ab levels against
other EBV-encoded proteins (12). This underlines the
inability of the host to mount appropriate immune responses against
LMP1, which is suggestive of immune escape. Because eradication of tumor cells is largely dependent on NK cell- and activated CTL-mediated killing, their presence in the reactive infiltrate is considered to be of major importance. Indeed, we previously found that the tumor-infiltrating lymphocytes in EBV-positive cases of HD contained significantly higher numbers of activated CTLs and NK cells compared with EBV-negative cases (13, 14, 15). Although in the neoplastic cells of EBV-positive HD cases potentially immunogenic viral proteins are expressed (1, 3), MHC class I molecules and TAP are present on the neoplastic cells (13), which have been shown susceptible to T cell-mediated lysis in vitro, and they are inadequately eliminated in vivo, possibly due to local immune suppression.
In a recent study we demonstrated the presence of human IL-10 expression in H-RS cells in EBV-positive cases of HD (15). This, however, did not influence T cell phenotypical markers in infiltrating lymphocytes compared with EBV-negative cases. Furthermore, van den Berg et al. showed that TARC, a T lymphocyte-attracting chemokine, is preferentially expressed in neoplastic cells of HD, but despite the influx of activated cells (granzyme B positive), the EBV-positive H-RS cells are inadequately eliminated (16). In support of this, Frisan et al. described a lack of functional CTL activity in HD-derived tumor-infiltrating lymphocytes (17). In line with this finding we described that high numbers of phenotypically activated CTLs in HD were associated with an adverse clinical outcome (14). Therefore, current data suggest that in HD mechanisms are operational to circumvent the immune system.
The presence of high levels of LMP1 in neoplastic cells of EBV-positive
cases of HD suggest a direct role for LMP1 in local immune suppression,
similar to that of retrovirally transformed cells. In the early 1960s
it was shown that murine retroviruses (Gross, Friend, Rauscher, and
Moloney) exerted various immunosuppressive influences during infections
in vivo (18, 19, 20, 21). These studies showed, among others, a
decrease in cell-mediated immunity, reduced levels of circulating
cytotoxic Abs, and the inability of cells to produce IFN-
(15, 16, 17). Snyderman et al. showed that the transmembrane
protein p15E was responsible for the immunosuppressive properties of
these retroviruses (22). Subsequently, a 17-aa region
named CKS 17 within the p15E protein was identified that was highly
conserved among murine, feline, and human retroviruses
(23). In vitro, CKS 17 was able to inhibit both the
proliferation of T cells and NK cell-mediated cytotoxicity (20, 24, 25). Furthermore, Oostendorp et al. showed that the
immunosuppressive effects of the 17-aa-long CKS17 could be narrowed to
a conserved hexapeptide with an amino acid sequence LDLLFL (24, 25). The mechanism by which these retrovirally encoded peptides
employ their effects is not completely clarified. It is suggested that
these peptides directly inhibit intracellular signaling pathways in T
cells by interfering with protein kinase C (PKC) and adenylate cyclase
(26). Thus, this effect is both MHC class I and MHC class
II independent.
In this paper we demonstrate that low concentrations of
immunoaffinity-purified recombinant LMP1 (rLMP1) have strong immune
suppressive properties on T cells activated by mitogen, Ag, or CD3/CD28
stimulation; NK cytotoxicity; and Ag-induced IFN- release. By
sequence alignment we found that the first transmembrane domain of LMP1
contained two sequences, LALLFWL and LLLLAL, highly homologous to the
retrovirally encoded peptide LDLLFL, part of the retroviral
transmembrane protein p15E. Only LALLFWL and several derivatives
containing this sequence have strong inhibitory effects on T cell
proliferation and NK cytotoxicity in vitro. To exert the
immunosuppressive effect in vivo, LMP1 or LMP1-derived peptides have to
be excreted from the cells. Using a quantitative LMP1
ELISA,3 LMP1 can be
detected at low concentrations in the supernatants of EBV-positive
lymphoblastoid cell lines, suggesting that LMP1 is actually secreted by
these cells. Therefore, we next investigated the mechanism by which
LMP1 is excreted, either passively by means of cell death and
subsequent cell lysis or actively by secretion of LMP1 alone or in the
form of LMP1-containing membrane vesicles.
Our results suggest that LMP1 may be actively secreted from EBV-positive tumor cells to mediate immunosuppressive effects on tumor-infiltrating lymphocytes surrounding the neoplastic cells in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EBV-positive (JY, RAJI) and -negative (BJAB, RAMOS) cell lines were cultured in RPMI 1640 (BioWhittaker, Verviers, Belgium) medium supplemented with 25 mM HEPES, 2 mM L-glutamine, 10% FCS (Integro, Zaandam, The Netherlands), 100 IU of streptomycin, and 100 µg of penicillin (referred to as complete medium) at 37°C in 5% CO2. Sf9 insect cells, derived from the fall army worm Spodoptera frugiperda, infected with either wild-type or rLMP1 baculovirus (provided by Dr. F. Grässer, Universitäts Kliniken des Saarlandes, Hamburg, Germany), were cultured in Sf900-II synthetic serum-free medium (Life Technologies, Grand Island, NY) containing 100 IU of streptomycin and 100 µg of penicillin at 27.5°C at normal air atmosphere.
Preparation of B cell lymphoblastoid cell lines was performed as described previously (27). PBMCs were isolated from heparinized blood of healthy volunteers by density centrifugation using Ficoll-Hypaque (Pharmacia, Uppsala, Sweden).
Recombinant LMP1
Sf9 cells expressing the full-length LMP1 in baculovirus under the control of a polyhedrin promotor and baculo wild type were cultured to the log phase (1 x 106 cells/ml) and infected with one of the baculovirus constructs at a multiplicity of infection of 0.1. After 72 h cells were harvested and washed with PBS.
Recombinant LMP1 was extracted from the cells by a 1-h incubation in sodium acetate (pH 6.0) containing 0.22 M octyl glucoside and 3 M ureum at 4°C. Recombinant LMP1 was purified using mAb-based immunoaffinity chromatography as described previously (see Footnote 3), finally reaching a concentration of 0.41 µg/ml purified protein.
Sequence alignment
Amino acid sequence homology search and alignment were performed using FASTA and PALIGN, which were included in the PC/Gene sequence software package (IntelliGenetics, Mountain View, CA).
Peptide synthesis
Peptides representing different domains of the LMP1 protein and
various control peptides were synthesized using a 433 A peptide
synthesizer (Applied Biosystems, Foster City, CA). The F-moc amino
acids used were purchased from Bachem (Bubendorf, Switzerland). For
some peptides the N-terminus was blocked by acetylation to neutralize
the N-terminal charge. The peptides were purified using reverse-phase
HPLC chromatography (System Gold, Beckman, Mijdrecht, The Netherlands).
Peptides were dissolved in DMSO at a stock concentration ranging from
10–40 mM. An overview of the peptides used in this study is given in
Table I
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PBMCs were cultured in a 96-well round-bottom culture plate (Nunc, Copenhagen, Denmark) at 2 x 105 cells/well for Ag-induced proliferation and 5 x 104 cells/well for mitogen-induced proliferation suspended in complete medium containing 10% human pooled serum (CLB, Amsterdam, The Netherlands) instead of FCS. Soluble Ag (tetanus toxoid, RIVM, Bilthoven, The Netherlands) was used in a final concentration ranging from 25–6.25 µg/ml, whereas the mitogen PHA was used at 5 µg/ml, and anti-CD3/anti-CD28 were both used at 2.5 µg/ml. Peptides were added at a final concentration of 10 µM or lower. As a control, the cells were cultured in the presence of 0.1% DMSO. All cultures were performed in triplicate.
Cells were cultured for 3 days at 37°C in a humidified atmosphere of 5% CO2 in air for mitogen-induced proliferation and 6 days for Ag-induced proliferation. During the last 4 h 14.8 x 103 Bq [3H]thymidine was added per well, and the cells were harvested onto fiberglass filters. [3H]Thymidine incorporation was determined by liquid scintillation counting, and subsequently the stimulation index was determined according to the following formula: S.I. = [(cpmmintogen/Ag - cpmbackground)/(cpmuntreated - cpmbackground)]. Peptide-induced inhibition of proliferation was calculated as the percent inhibition in relation to the proliferation in the presence of DMSO without peptide.
NK cytotoxicity assay
The leukemia cell line K562 was used as target at a concentration of 5000 cells/well. PBMCs were washed twice, resuspended in complete medium, and seeded in 96-well round-bottom plates at different concentrations (E:C cell ratios ranging from 80:1 to 2.5:1). K562 cells were labeled with 51Cr (sodium chromate, 0.5 x 106 cells/14.8 x 106 Bq of 51Cr; Malinckrodt, Petten, The Netherlands) for 90 min at 37°C. Thereafter, the target cells were washed three times and resuspended in complete medium. To each well 50 µl of peptide solution (100 µM) or medium containing DMSO was added to correct for possible effects of DMSO in which the peptides were solubilized. Maximum 51Cr release was measured in the presence of 1% Triton X-100. Plates were centrifuged for 2 min at 100 x g and incubated for 4 h at 37°C in 5% CO2. NK cell cytotoxicity was determined by measuring the 51Cr release in the supernatant using a gamma counter (Wallac, Turku, Finland). All determinations were performed in triplicate. For each sample the percentage of specific killing was calculated as follows: % specific killing = [(cpmsample - cpmbackground)/(cpmmax release - cpmbackground)] x 100. Subsequently, the peptide-induced inhibition of NK cytotoxicity was calculated with respect to control wells without peptides.
Enzyme-linked immunospot assay for single cell IFN- release
This assay was performed as previously described to detect
Ag-specific T cells in fresh PBMCs (28). Ninety-six-well,
polyvinylidene difluoride-backed plates (Millipore, Bedford, MA) were
precoated with 15 µg/ml anti-IFN- mAb 1-DIK (MABTECH,
Stockholm, Sweden). PBMCs were added in duplicate wells at 5.0 x
105 cells/well in the presence of 2 µM peptide
(GLCTLVAML (29), A2-resticted epitope of BMLF1, one of the
lytic cycle Ags of EBV, and two HLA-A2 restricted LMP1-derived
epitopes, YLLEMLWRL and YLQQNWWTL (10). PHA (10 µg/ml)
was used as a positive control. The plates were incubated overnight at
37°C in 5% CO2. The following day, cells were
discarded, and the plates were incubated with the second biotinylated
anti-IFN- mAb 7-B6-1 (MABTECH) at a concentration of 1 µg/ml
for 3 h at room temperature. This was followed by incubation with
streptavidin-conjugated alkaline phosphatase (MABTECH) for an
additional 2 h. Individual cytokine-producing cells were detected
as dark spots after a 30-min reaction with 5-bromo-4-chloro-3-indolyl
phosphate and nitro blue tetrazolium using an phosphatase-conjugate
substrate kit (Bio-Rad, Richmond, CA). The spots were counted under a
dissection microscope, and the number of specific T cell responders was
calculated after subtracting negative control values.
Isolation of LMP1 from supernatants of EBV-positive B cell lines
All cell lines were cultured in fresh medium for 2 days at 1 x 106/ml, and subsequently the supernatant was fractionated by differential centrifugation as described recently (30). Briefly, supernatant was centrifuged for 10 min at 300 x g to remove cells. After a second centrifugation at 300 x g for 10 min, the supernatant was centrifuged twice at 1,200 x g (10 min each time), then at 10,000 x g (30 min), 70,000 x g (60 min), and 100,000 x g (60 min), respectively, using a Beckman ultracentrifuge with a Ti40 rotor. Following each centrifugation step, the pellet was collected for further analysis, and the supernatant was used for subsequent centrifugation.
Gel electrophoresis and immunoblotting
The pellets obtained after differential centrifugation were solubilized and sonicated in sample buffer (0.2 M Tris-HCl (pH 6.8), 4% SDS, 18% glycerol, 2% 2-ME, and 0.004% bromophenol blue) and boiled for 5 min. Samples were separated by SDS-PAGE, using a stacking gel of 3% acrylamide and a linear separation gel of 10% acrylamide (MiniProtean II electrophoresis system, Bio-Rad, Hercules, CA). Polypeptides were transferred onto 0.22-µm pore size nitrocellulose filters (Schleicher & Schuell, Hertogenbosch, The Netherlands) by Western blotting (Mini Trans-blot cell, Bio-Rad) using standard techniques. Nonspecific binding sites on the nitrocellulose were saturated with blocking buffer (5% nonfatty milk powder and 5% FCS in PBS). Subsequently, mAbs directed against the C- and N-terminal domains of LMP1 (OT22C and OT 22CN, respectively (Organon Teknika, Boxtel, The Netherlands)) in optimal dilution (0.1–1 µg/ml) were added and incubated for 1 h at room temperature. In addition, mAbs against CD86 (B7.2, Ancell, Bayport, MN) and HLA-DR (Dako, Carpenteria, CA) were applied to confirm the presence of membrane-associated vesicles, and EBNA1 (OT1X, Organon Teknika) was used as a control for the presence of the remaining nuclear fragments. After washing three times with 0.05% Tween in PBS, specifically bound IgG was detected with a HRP-conjugated second Ab, diluted in blocking buffer. After two washes in 0.05% Tween in PBS and two washes in PBS, HRP activity was visualized by electrochemiluminescence (Roche, Mannheim, Germany).
Statistical analysis
For statistical analysis of the data, a two-sided Mann-Whitney U test was conducted. A p value <0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preliminary T cell proliferation experiments suggested that LMP1-containing cell extracts might affect T lymphocyte function directly. Therefore, we were interested in whether highly purified rLMP1 was able to directly influence polyclonal T cell proliferation induced by PHA-mitogen or anti-CD3/anti-CD28 mAb stimulation of PBMC in vitro. For this purpose rLMP1 was purified to near homogeneity (>95%) from Sf9 cells infected with LMP1-expressing baculovirus using an mAb-based immunoaffinity purification.
Recombinant LMP1 was added during activation of PBMC at various
concentrations ranging from 0.2 to 20.5 ng/ml (4.9–490 fmol/ml). As a
control, the buffer in which rLMP1 was dissolved was added. After 3
days of incubation, both PHA and anti-CD3/anti-CD28-induced
proliferation was measured by [3H]thymidine
incorporation. In the presence of the highest concentration of rLMP1
(20.5 ng/ml) the maximal inhibition was >98%, whereas addition of
buffer had no effect. Inhibition of proliferation gradually decreased
when lower amounts of rLMP1 were added (Fig. 1). Even in the presence of the highest
concentration of rLMP1, PBMC remained viable, as evaluated visually by
trypan blue exclusion. Visual evaluation of this phenomenon revealed
that the increase in the number of activated cell clumps in the wells
and proliferation as measured by [3H]thymidine
incorporation inversely correlated with the amount of rLMP1 present in
the wells (Fig. 2). Thus, LMP1 exerts a
direct inhibitory effect on T cell proliferation by interfering with
the cell-cell contact required during the activation stage.
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Tetanus toxoid-induced proliferation was analyzed showing similar
suppression as described above. Subsequently, we analyzed whether rLMP1
was able to inhibit Ag-specific cytokine production of T cells. The
effect of rLMP1 on IFN- production of T cells reactive with an
immunodominant EBV-peptide GLCTLVAML (BMLF1-derived epitope) and two
HLA-A2-restricted LMP1-derived epitopes (YLLEMLWRL and YLQQNWWTL) was
investigated using an enzyme-linked immunospot assay. In all
experiments rLMP1 was added overnight at a final concentration of 20.5
ng/ml.
The frequencies of T cells reactive with the GLCTLVAML epitope derived
from the lytic BMLF1 decreased by 70% (from 121 ± 20
spots/105 PBMC to 31 ± 17
spots/105 PBMC; n = 3).
Furthermore, IFN- production in T cell precursors reactive with
LMP1-derived epitopes YLLEMLWRL and YLQQNWWTL, was inhibited under the
influence of rLMP1 by 59% (68 spots/105 PBMC to
28 spots/105 PBMC) and 62% (52
spots/105 PBMC to 20
spots/105 PBMC), respectively. IFN- production
in PHA-induced PBMC was used as a positive control and was also
suppressed at least 81% (>600 spots/105 PBMC to
114 ± 60 spots/105 PBMC) when rLMP1 was
added (n = 3). These data clearly show that besides
inhibition of the Ag-, mitogen-, and mAb-induced T cell proliferation,
rLMP1 is also able to suppress both Ag- and mitogen-induced IFN-
production in vitro.
Identification of potentially immunosuppressive domain in LMP1
In an attempt to find a rationale for the observed T cell effects of LMP1, we explored possible homologies with other viral proteins possessing immunosuppressive activity, such as retroviral transmembrane glycoprotein p15E. To reveal possible homologies with the p15E-derived immunosuppressive domain we used the CKS-17 sequence for alignment analysis. Surprisingly, within the first transmembrane domain of LMP1 two small domains with strong homology to the CKS-17 minimal functional domain were found (LALLFWL and LLLLAL, respectively). No homology was found with other transmembrane proteins or with LMP2 or other EBV-encoded proteins. To further test whether LMP1-derived peptides exhibited immunosuppressive properties, we investigated their effects on Ag- and mitogen-induced T cell proliferation and NK cytotoxicity.
Effects of LMP1-derived peptides on T cell proliferation in vitro
To explore the effects of LMP1 on T cell function in more detail,
we first investigated whether LMP1-derived peptides were able to
inhibit T cell proliferation. To examine potential immunosuppressive
effects we evaluated the Ag-, mitogen-, and
anti-CD3/anti-CD28-induced proliferation of PBMCs in the
presence and the absence of putative immunosuppressive peptides of LMP1
with homology to retrovirus-derived peptides. Retroviral peptides
(CKS17 and LDLLFL), previously shown to inhibit mitogen- and Ag-induced
T cell proliferation, were used as a positive control (21, 23, 24). Various irrelevant peptides, either LMP1 derived or
obtained from unrelated proteins, were used as a control for T cell
proliferation assays (see Table I
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PHA-induced proliferation could be inhibited up to 80% by addition of
10 µM LMP1-derived peptide. When long and short LMP1-derived peptides
were tested the latter showed a stronger inhibition of T cell
proliferation (47%) compared with the longer peptides (29%; Fig. 3A). These peptides were also
able to significantly inhibit tetanus toxoid-induced proliferation up
to 70% (data not shown). When acetylated peptides were used to
neutralize the N-terminal charge of the peptides to mimic, more
closely, their hydrophobic character in the membrane, inhibition was
even stronger, being >90% for acetylated LALLFWL (Table I and Fig. 3B). Again, the shorter domain of LMP1, LALLFWL, gave the
strongest inhibition of proliferation compared with the longer peptides
(Table I). Although both LALLFWL and LLLLAL showed strong homology with
the immunosuppressive retroviral peptides CKS 17 and LDLLFL, the latter
did not show immunosuppressive properties in proliferation assays
(Table I and Fig. 3A).
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). In all experiments LMP1-derived
peptides showed a considerable stronger inhibition of T cell
activation. Effects of LMP1-derived peptides on NK cytotoxicity
Next we were interested in the effect of LMP1-derived peptides on
NK cytotoxicity (21, 22). In all experiments, peptides
were continuously present during the effector phase of the NK
cytotoxicity assays. No direct effect of any of the peptides on the
viability of the PBMCs or K562 was observed. The results, depicted in
Table I and Fig. 4, show that all
LMP1-derived peptides containing the LALLFWL sequence were able to
inhibit NK cytotoxicity up to 77% at a concentration of 
100 µM.
In contrast, the second domain (LLLLAL), with homology to the
retroviral immunosuppressive domain LDLLFL, showed no inhibition of NK
cytotoxicity.
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) showed inhibition of
cytotoxicity. The retrovirus-derived peptides (LDLLFL and CKS17)
clearly showed inhibition of cytotoxicity up to 57% comparable to the
results obtained by Oostendorp et al. (22) (Table I). Detection of LMP1 in supernatant of EBV-positive and -negative cell lines
An LMP1 capture ELISA recently developed by Meij et al. (see
Footnote 3) was used to analyze whether LMP1 could be detected in the
supernatant of EBV-positive cell lines. Using this assay soluble LMP1
was detected in the supernatant of EBV-positive lymphoblastoid cell
lines (JY, RAJI, B95-8), whereas no LMP1 was detected in EBV-negative
cell lines. Expression of LMP1 increased during culture, and after 4–5
days the highest levels of LMP1, reaching up to 25 pg/ml (0.6 fmol),
were observed, while cell viability remained >95% (see Fig. 5). Although LMP1 is detectable in
supernatant of EBV-positive cell lines, the concentration seems to be
insufficient to induce local T cell suppression in vitro.
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Detection of LMP1 in exosomes
To assess whether LMP1 is secreted as single protein or in a
membrane-associated form, the supernatant was fractionated by
differential centrifugation. LMP1 was mainly detected in the 300
x g (whole cells), 10,000 x g, and
70,000 x g pellet fractions of the EBV-positive cell
lines JY and B95-8 (Fig. 6A).
Moreover, the presence of both MHC class II molecules and CD86,
previously shown to be present in exosomes, was detected in the same
fractions of EBV-positive cell lines, confirming the data reported by
Raposo et al. (Fig. 6B) (26). In addition, the
EBV-encoded nuclear Ag EBNA1 was tested to exclude the possibility of
nuclear fragments during the exosome preparation and was found to be
absent (data not shown). LMP1 was detected in none of the fractions of
the EBV-negative cell line BJAB, whereas in these cells MHC class II
molecules were present. Together these data indicate that LMP1
colocalizes with MHC class II molecules and is possibly secreted from
EBV-positive B cells in the form of small vesicular structures known as
exosomes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a recent study by Meij et al. (12), it was further substantiated that LMP1 is a subdominant component in the humoral immune response. In patients with EBV-associated malignancies, only a small percentage showed LMP1-directed Abs at a very low level (33% HD of cases and 8% of nasopharyngeal carcinoma patients) despite the fact that LMP1 is expressed at high levels in most of these cases. In addition, the cell-mediated immune response against LMP1 is subdominant compared with the readily detectable response against epitopes such as the EBNA3 family, since it has proven to be difficult to obtain CTL clones directed against LMP1 (27).
In this study we demonstrate, for the first time, that intact LMP1 as well as peptides derived from the first transmembrane region directly inhibit T cell activation and NK cytotoxicity in vitro. This provides a novel mechanism, namely direct immunosuppression mediated through (fragments of) LMP1, by which EBV-positive cases of Hodgkin lymphomas may circumvent the immune system. Not only is LMP1 capable of suppressing the strong mitogen and anti-CD3/anti-CD28-induced T cell activation, but it also suppresses functionally relevant Ag-specific memory T cell responses, as indicated in this study. LMP1 contains a domain in its first transmembrane region closely homologous to retrovirally encoded immunosuppressive peptides that are known to inhibit lymphocyte functions (inhibition of T cell proliferation and NK cytotoxicity) and modulation of cytokine expression in favor of immunosuppressive cytokines (induction of IL-10 and inhibition of IL-2 and IL-12) (26). Moreover, the fact that LMP1 can be secreted from EBV-positive cell lines in vitro suggests that this mechanism might be of relevance for immune escape of EBV-positive neoplastic cells in vivo. It may be speculated that H-RS cells, which are considered to be of B cell origin and display high LMP1 expression levels, are able to secrete vesicles (exosomes) containing LMP1 in vivo.
EBV-transformed B cell lymphoblastoid cell lines are extremely potent stimulators of T cell proliferation and powerful inducers of CTL responses in vitro, despite the expression and secretion of LMP1. This phenomenon can be explained by the fact that LMP1 secretion in supernatants is only found at extremely low concentrations (<0.6 fmol), well below the immunosuppressive limit. In vivo, however, LMP1 might still exhibit its local immunosuppressive effect caused by the inability to diffuse and thereby reach much higher concentrations in close vicinity of the neoplastic cells.
Our findings indicated that at the peptide level, the amino acid sequence LALLFWL might be responsible for the observed immunosuppressive effects. The amount of peptide used to induce these effects is much greater compared with the amount of rLMP1 protein used. This phenomenon is possibly due to the hydrophobic nature of these peptides, whereby aggregation of these peptides might induce masking of the effective immunosuppressive domains.
The mechanism by which p15E, LMP1, or their related peptides exhibit
their actions is not fully understood, but Haraguchi et al.
(18) proposed a mechanism by which these immunosuppressive
peptides directly modulate important molecules in the signal
transduction pathway leading to a imbalance of cytokine production. It
has been shown that upon retroviral infection, transcription of IL-12
and IL-2 is strongly decreased, whereas IL-10 transcription is
augmented under the influence of CKS17 (18). The proposed
model of modulation of transcription of cytokines might occur via two
different pathways: 1) induction of high concentrations of cAMP via
activation of adenylate cyclase, and 2) either direct inhibition of PKC
activity or indirect inhibition via suppression of phospholipase C1
(18, 23). Both proposed mechanisms lead to enhanced
expression of Th2-associated cytokines, IL-10 and TGF-ß, with
concomitant down-regulation of the Th1-like cytokines, IL-2, IL-12, and
IFN-.
We and others have previously shown that in EBV-positive cases of HD, high numbers of cells (either neoplastic or reactive cells) express IL-10, whereas low numbers of cells showed IL-2 expression (14, 32, 33). This is supported by in vitro data showing that LMP1 is able to up-regulate the expression of IL-10 upon transfection into LMP1-negative Burkitt’s lymphoma cell lines (34). Furthermore, EBV-positive cases of post-transplant lymphoproliferative disorders, expressing high levels of LMP1, are associated with elevated levels of serum IL-10 which further substantiates the hypothesis that LMP1 or LMP-derived peptides may induce local immunosuppression by induction of certain cytokines. Moreover, preliminary data show that addition of LMP1-derived peptides to activated PBMC and subsequent intracellular detection of cytokines by FACS analysis resulted in down-regulation of IL-2 expression (data not shown).
We hypothesize that LMP1 and hydrophobic transmembrane domain-derived peptides can insert directly into the membrane. This hypothesis is supported by the fact that the effective LMP1-derived peptides are very hydrophobic and, moreover, the inhibitory effects are more pronounced when charged groups on the peptides are neutralized by acetylation, thereby facilitating entry into the membrane. Recently, Busson et al. showed that in lymphoid and epithelial cells LMP1 is associated with glycosphingolipid-rich domains (GSL domains) in the plasma membrane (35, 36). These domains are clustering sites for heterodimeric G proteins, PKC, and adenylate cyclases. It is suggested that these GSL domains are important for trafficking and probably are also required for the inhibitory function of LMP1 (35, 36). LMP1 itself contains six hydrophobic membrane-spanning domains with which it is likely to penetrate the membrane at the GSL domains and subsequently can bind to and induce adenylate cyclase and inhibit PKC.
In conclusion, we have presented evidence that EBV, a human DNA tumor virus, is capable of inducing T cell anergy via a novel direct route involving LMP1, possibly mediated by secretion of EBV-encoded (fragments of) LMP1. These peptide fragments are possibly involved in circumvention of the local immune system in EBV-positive cases of HD by virtue of the induction of immunosuppressive cytokines. This is the first time that direct immunosuppression, previously thought to be restricted to RNA viruses, has been described in a DNA virus.
We recently developed a novel mAb with specificity for the N-terminal region of LMP1 (amino acid sequence 1–13) (12). Using this reagent and cloned subfragments of LMP1, in particular the first transmembrane region, the hypothesized localization and secretion as well as the role of this region in immune suppression may be further delineated.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: HD, Hodgkin’s disease; LMP, latent membrane protein; EBNA, Epstein-Barr nuclear Ag; H-RS cells, Hodgkin Reed-Sternberg cells; PKC, protein kinase C; GSL domain, glycosphingolipid-rich domain; EBER, EBV-encoded small RNA.
3 P. Meij, M. B. H. J. Vervoort, C. J. L. M. Meijer, E. Bloemena, J. M. Middeldorp. Production monitoring and purification of EBV-encoded latent membrane protein I expressed and secreted by recombinant baculovirus-infected cells. Submitted for publication.
Received for publication December 27, 1999. Accepted for publication April 24, 2000.
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A. Clayton, J. P. Mitchell, J. Court, M. D. Mason, and Z. Tabi Human Tumor-Derived Exosomes Selectively Impair Lymphocyte Responses to Interleukin-2 Cancer Res., August 1, 2007; 67(15): 7458 - 7466. [Abstract] [Full Text] [PDF]
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M. K. Gandhi, E. Lambley, J. Duraiswamy, U. Dua, C. Smith, S. Elliott, D. Gill, P. Marlton, J. Seymour, and R. Khanna Expression of LAG-3 by tumor-infiltrating lymphocytes is coincident with the suppression of latent membrane antigen-specific CD8+ T-cell function in Hodgkin lymphoma patients Blood, October 1, 2006; 108(7): 2280 - 2289. [Abstract] [Full Text] [PDF]
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R. H. Edwards, D. Sitki-Green, D. T. Moore, and N. Raab-Traub Potential Selection of LMP1 Variants in Nasopharyngeal Carcinoma J. Virol., January 15, 2004; 78(2): 868 - 881. [Abstract] [Full Text] [PDF]
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J. Flanagan, J. Middeldorp, and T. Sculley Localization of the Epstein-Barr virus protein LMP 1 to exosomes J. Gen. Virol., July 1, 2003; 84(7): 1871 - 1879. [Abstract] [Full Text] [PDF]
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N. A. Marshall, M. A. Vickers, and R. N. Barker Regulatory T Cells Secreting IL-10 Dominate the Immune Response to EBV Latent Membrane Protein 1 J. Immunol., June 15, 2003; 170(12): 6183 - 6189. [Abstract] [Full Text] [PDF]
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S. Gottschalk, O. L. Edwards, U. Sili, M. H. Huls, T. Goltsova, A. R. Davis, H. E. Heslop, and C. M. Rooney Generating CTLs against the subdominant Epstein-Barr virus LMP1 antigen for the adoptive immunotherapy of EBV-associated malignancies Blood, March 1, 2003; 101(5): 1905 - 1912. [Abstract] [Full Text] [PDF]
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A. Leen, P. Meij, I. Redchenko, J. Middeldorp, E. Bloemena, A. Rickinson, and N. Blake Differential Immunogenicity of Epstein-Barr Virus Latent-Cycle Proteins for Human CD4+ T-Helper 1 Responses J. Virol., September 15, 2001; 75(18): 8649 - 8659. [Abstract] [Full Text] [PDF]
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)
proliferation by rLMP1. Proliferation was measured by
[3H]thymidine incorporation after 3-day culture of PBMCs
with 5 µg/ml PHA or 2.5 µg/ml of both anti-CD3/antiCD28 in the
presence or the absence of rLMP1. The data are the median ± SD of
five different donors. The median counts per minute in controls in the
absence of LMP1 were 7900 and 9700 for PHA and
anti-CD3/anti-CD28, respectively.
