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Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 1020-1030
Antigen Presenting Phenotype of Hodgkin Reed-Sternberg Cells:
Analysis of the HLA Class I Processing Pathway and the Effects of
Interleukin-10 on Epstein-Barr Virus-Specific Cytotoxic T-Cell
Recognition
By
Steven P. Lee,
Christothea M. Constandinou,
Wendy A. Thomas,
Debbie Croom-Carter,
Neil W. Blake,
Paul G. Murray,
John Crocker, and
Alan B. Rickinson
From the CRC Institute for Cancer Studies, University of Birmingham,
Birmingham, UK; the Department of Histopathology, Birmingham Heartlands
Hospital, Birmingham, UK; and the School of Health Sciences, University
of Wolverhampton, Wolverhampton, UK.
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ABSTRACT |
Approximately 40% of Hodgkin's disease (HD) cases in Western
countries carry Epstein-Barr virus (EBV) in the malignant
Hodgkin-Reed-Sternberg (H-RS) cells. HLA class I-restricted cytotoxic
T lymphocytes (CTLs) with specificity for viral antigens expressed in
H-RS cells therefore have therapeutic potential. However, a
prerequisite for CTL therapy is that the tumor target be capable of
processing and presenting endogenously expressed antigens via the
transporter associated with antigen processing
(TAP)-dependent HLA class I pathway. We have assessed the
antigen-presenting phenotype of H-RS cells in two ways. First,
immunohistochemical analysis of 38 HD biopsies showed that H-RS cells
were uniformly TAP1/TAP2-positive and expressed HLA class I in the
majority (18 of 24, 75%) of EBV-positive cases compared with only 4 of
14 (29%) of EBV-negative cases. Second, using a panel of 5 H-RS cell
lines, we showed that 4 of 5 could process and present EBV proteins to
HLA class I-restricted EBV-specific CTL clones. Others have reported
that human interleukin-10 (IL-10), which is expressed by H-RS cells in
the majority of EBV-positive HD cases, can abrogate CTL recognition in
some circumstances. However, IL-10 pretreatment of the H-RS lines or of
the EBV-specific CTLs had no such effect in this system. These results
support the possibility that EBV-specific CTLs may be used to treat
virus-positive HD.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE ASSOCIATION BETWEEN Epstein-Barr
virus (EBV) and Hodgkin's disease (HD) is now well established, with
approximately 40% of HD cases in Western countries carrying EBV in the
malignant Hodgkin-Reed Sternberg (H-RS) cell population.1
In such cases, every malignant cell contains the viral genome, and
where patients possess multiple lesions, the same unique viral episome
is present at every site.2 The precise role of EBV in the
origin of HD is unknown but the oncogenic potential of this virus is
clear from its ability to transform resting B cells in vitro into
permanently growing lymphoblastoid cell lines (LCLs) and from its
association with several human tumors.3
Despite the oncogenic potential of EBV, this virus is widespread in the
human population where it usually persists as an asymptomatic infection
of B cells under the control of cytotoxic T lymphocytes (CTLs).3 EBV is a potent target for HLA class I-restricted CTLs, and such effectors are readily reactivated in vitro by
stimulating peripheral blood mononuclear cells with the autologous
EBV-transformed LCL. Within an LCL, EBV establishes a latent infection
with the expression of six nuclear antigens (Epstein-Barr
nuclear antigens [EBNAs] 1, 2, 3A, 3B, 3C, and -LP) and two latent
membrane proteins (LMPs 1 and 2).3 Of these, the EBNA 3 family (EBNAs 3A, 3B, and 3C) represent the immunodominant target
antigens for CTLs, although subdominant responses to many of the other
EBV latent proteins have been detected.4-7 In clinical
situations where such CTL control is ablated (for instance, in heavily
immunosuppressed transplant patients), EBV-positive immunoblastic
lymphomas arise in which the full spectrum of virus latent proteins are
expressed. Importantly, these tumors remain highly susceptible to
adoptive transfer of EBV-specific CTLs prepared by in vitro
reactivation.8
Within H-RS cells, EBV also establishes a latent infection, but
expression of viral proteins is restricted to just EBNA 1, LMP 1, and
LMP 2.9-12 This alternative latent state may allow the
tumor to evade virus-specific CTL surveillance because the immunodominant EBNA 3 proteins are not expressed. Nevertheless, LMP 1 and LMP 2 are known to be subdominant targets for CTL responses on at
least some HLA backgrounds.4-6 Indeed, a number of CTL target epitope sequences restricted through common HLA alleles have
been identified in LMP 2 and these sequences are conserved in viruses
present within HD tumors.6 Accordingly, there is now
considerable interest in the possibility of treating EBV-positive HD
cases by boosting LMP-specific CTL responses through immunization or by
adoptive transfer of appropriate CTL preparations.
In this context, LMP-specific memory CTLs have been detected in the
blood of at least some HD patients,13,14 yet such
reactivities have clearly failed to control the disease. The
explanation may simply be quantitative in that LMP-specific CTLs almost
always comprise a minor component of the EBV-induced CTL
response5,7; in such circumstances, boosting these
responses should prove an effective therapy for HD. However, there may
be other factors which underlie the emergence of EBV-positive HD in a
virus-immune individual. First, H-RS cells may be defective in the
pathway whereby viral antigens are processed and presented to HLA class I-restricted T cells. In the normal HLA class I processing pathway, endogenously synthesized proteins are first cleaved by the proteasome complex into peptide fragments. These peptides are then delivered into
the endoplasmic reticulum via the heterodimeric transporter associated
with antigen processing (TAP1/TAP2) and compete for binding to nascent
HLA class I molecules; the most stable peptide: HLA class I complexes
are then transported to the cell surface for recognition by specific
T-cell receptors.15 Defects in this pathway have been
identified in several human malignancies including small cell lung
carcinoma16 and the EBV-associated tumor, Burkitt's lymphoma (BL).17 In both cases, the defect involves
downregulated expression of the TAP transporters with reduced
expression of HLA class I on the cell surface. Immunohistochemical
studies of HLA class I expression in HD have so far yielded conflicting
results. Poppema and Visser18 reported that the majority of
H-RS cells lack surface HLA class I expression, whereas Oudejans et
al19 found that most EBV-positive H-RS cells are HLA class
I-positive.
In addition to antigen-processing defects, it has been postulated that
H-RS cells may evade T-cell recognition through the expression of
interleukin-10 (IL-10). This cytokine is known to suppress cellular
immune responses20-22 and a recent study has shown its
expression by H-RS cells in the majority of EBV-positive HD
cases.23 Furthermore, there is independent evidence to
suggest that local suppression of the CTL response does occur in
EBV-positive HD. Thus, by screening both the circulating and
tumor-infiltrating T cells from HD patients, Frisan et al13
found that EBV-specific CTLs could be isolated from the blood of a
virus genome-positive HD patient but not from the biopsy itself. Not
only might IL-10 suppress the CTL response, but it has also been
reported in model systems to protect target cells from CTL recognition
by downregulating surface HLA class I expression.24
In the present study we have sought to address these issues (1) by
analyzing TAP1, TAP2, and surface HLA class I molecule expression in HD
tumor biopsies, (2) by assaying a panel of H-RS-derived cell lines in
vitro for their capacity to present antigen to EBV-specific CTLs, and
(3) by determining whether exogenously added IL-10 can act either on
the H-RS lines or on the CTL effectors to prevent CTL-mediated lysis of
H-RS targets.
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MATERIALS AND METHODS |
HD biopsy samples and H-RS cell lines.
Lymph node biopsy samples from 38 cases of HD were obtained from
Birmingham Heartlands Hospital and Good Hope NHS Trust Hospital (Birmingham, UK). Hematoxylin- and eosin-stained sections from each
biopsy were examined and patients classified histologically according
to the Rye convention.25 The H-RS cell lines used in this
study were kindly provided by Dr J. Wolf and Dr V. Diehl (Cologne,
Germany), and Dr H. Kamesaki (Kyoto, Japan) and have all been described
previously.26-29 HDLM2, L540, and L428 were derived from
advanced cases of nodular sclerosis subtype, whereas KMH2 and L1236
were derived from advanced cases of mixed cellularity subtype; all of
the lines are EBV genome negative. H-RS lines were HLA typed by M. Bunce (The Oxford Transplant Centre, Churchill Hospital, Oxford, UK)
using polymerase chain reaction-based DNA typing. Cells were cultured
in RPMI 1640 (Life Technologies, Paisley, Scotland) containing 10%
fetal calf serum, 2 mmol/L glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin (growth medium).
In situ hybridization.
The EBV status of HD biopsies was determined by in situ hybridization
using a fluorescein-conjugated oligonucleotide probe specific for the
abundantly expressed EBV-encoded EBER RNAs (Vector Laboratories, Inc,
Burlingame, CA). Formalin-fixed paraffin-embedded sections were mounted
on slides coated with 3-amino-propyl-triethoxy-saline (APES;
Sigma-Aldrich Co Ltd, Poole, Dorset, UK), deparaffinized with xylene,
hydrated in absolute alcohol, and immersed in phosphate-buffered saline
(PBS). Sections were digested with pronase E (6.25 ng/µL) for 5 minutes, washed in PBS, dehydrated in absolute alcohol, and left to air
dry. The probe hybridization solution was added to each section and
incubated at 55°C overnight in a humidity chamber. Slides were then
washed for 5 minutes each in 2 × standard saline citrate
(SSC) buffer (twice), 0.1 × SSC, and then PBS. Sections were blocked
using 10% normal rabbit serum and bound probe was detected using
rabbit anti-fluorescein isothiocyanate (FITC) conjugated to alkaline
phosphatase (diluted 1:100). Slides were incubated for 30 minutes at
room temperature, then washed in PBS followed by alkaline phosphatase
substrate buffer (pH 9.0) for 5 minutes. Alkaline phosphatase activity
was demonstrated with chromogen overnight. Nuclei were counterstained
with hematoxylin. The EBV-positive cell line B95.8 was used as a
positive control.
Immunohistochemistry.
Tissues were cut to 5-µm thick sections and mounted on vectorbonded
slides (Vectorbond reagent, Vector Laboratories Inc). The expression of
LMP 1 was detected using the monoclonal antibody (MoAb) CS1-4 (kindly
provided by M. Rowe, University of Cardiff, Cardiff, UK) as described
previously.30 HLA class I levels were assessed using the
antibody HC10 at a dilution of 1:300. This antibody, kindly provided by
H. Ploegh (M.I.T., Cambridge, MA) preferentially recognizes HLA B/C
locus products.31 TAP 1 and TAP 2 expression were measured
using anti-C-terminal peptide immune rabbit sera32 (kindly
supplied by J. Trowsdale, Cambridge University, UK) diluted 1:500 and
1:1,000, respectively. Bound antibody was detected using a standard ABC
immunoperoxidase method (Universal VECTASTAIN Elite ABC kit, Vector
Laboratories Inc) and visualized with the diaminobenzidine-based
detection reaction (Vector Laboratories Inc). Nuclei were
counterstained with hematoxylin.
Western blotting.
Cells for Western blot analysis were lysed by sonication in 8 mol/L
urea; 10 mmol/L Tris buffer, pH 7.0; and the protein concentration determined using a Bio-Rad DC protein assay kit (Bio-Rad
Laboratories, Richmond, CA). The lysate was diluted in electrophoresis
sample buffer and 50 µg protein was loaded per track of a
discontinuous sodium dodecyl sulfate (SDS)-polyacrylamide gel with a
7.5% acrylamide resolving gel. The electrophoresed proteins were
transferred to a nitrocellulose membrane and TAP 1 and TAP 2 proteins
were detected using anti-C-terminal peptide immune rabbit
sera32 (anti-TAP 1 diluted 1:400; anti-TAP 2 diluted 1:500)
and a chemiluminescence detection protocol.
Immunofluorescence.
Surface expression of HLA class I molecules on H-RS cell lines was
measured by incubating viable cells with MoAb W6/3233 or
BB7.234 for 30 minutes on ice. After washing with PBS
containing 10% (vol/vol) normal goat serum (NGS), bound antibody
was detected by incubation with FITC-labeled goat anti-mouse IgG
(Sigma-Aldrich Co Ltd) for 20 minutes on ice. After four washes in
PBS + 10% NGS, cells were fixed with 1% (wt/vol)
paraformaldehyde and analyzed using a FACScan (Becton-Dickinson,
Mountain View, CA). W6/32 (ascites) and BB7.2 (culture supernatant)
were diluted 1:200 and 1:5, respectively, using PBS + 10% NGS, and
FITC-labeled goat anti-mouse IgG was diluted 1:50 using PBS + 10% NGS + 10% EBV-seronegative human serum.
CTL clones and cytotoxicity assays.
LCLs were generated in vitro by EBV transformation of B cells from
healthy laboratory donors of known HLA type and cultured in growth
medium. EBV-specific CTLs were reactivated from the peripheral blood of
healthy laboratory donors by in vitro stimulation with the autologous
LCL (irradiated at 4,000 rads; responder to stimulator ratio = 40:1)
in growth medium containing 1% human AB serum (Sigma-Aldrich Co Ltd).
After 7 days, fresh medium and autologous LCL (irradiated) were added,
and on day 14 cells were cloned by limiting dilution at 0.3 cells/well
and maintained in IL-2-conditioned medium as described
previously.6
In cytotoxicity assays, targets were incubated with
[51Cr]O4 for 1 to 2 hours, washed, and then
incubated in 96-well V-bottom plates (Life Technologies) at 2.5 × 103 cells/well in 100 µL of growth medium. CTLs in growth
medium (100 µL/well) were then added at a known effector:target (E:T) ratio. Spontaneous and maximum levels of cell lysis were measured by
culturing targets with growth medium alone or 1% (wt/vol) SDS solution, respectively (100 µL/well). After 5 hours incubation, 100 µL of culture supernatant was obtained from each well and levels of
[51Cr]O4 measured using a gamma counter
(Packard, Pangbourne, Berks, UK). All tests were conducted in
triplicate and the percentage specific lysis was calculated as (release
by CTL  spontaneous release) × 100/(total release in 1%
SDS spontaneous release). In some assays, BL cell lines were used
as targets. The isolation and culture of the BL cell line DH has been
described previously35; OKU is a group I BL cell line
established in the same manner from an African BL patient
(A.B.R., unpublished data). Where assays involved the use
of recombinant vaccinia viruses, target cells were preinfected for 1 hour with virus at a multiplicity of infection of 10, followed by 4 to
15 hours incubation in growth medium. Targets were then labeled with
[51Cr]O4 and used in the assay as described
above. The vaccinia recombinant expressing HLA B*3501 was constructed
using the protocol described by Blasco and Moss.36 The cDNA
for B*3501 was cloned into the transfer plasmid pRB21 and then
transfected into CV-1 cells. After infection of these cells with vRB12,
a recombinant vaccinia expressing HLA B*3501 was rescued, and this was
then plaque purified and titred on BSC-1 cells. Other vaccinia
recombinants used in this study have all been described
previously.7,37 Where assays involved the use of synthetic
peptides, [51Cr]O4-labeled targets were
incubated with peptide at 0.01 µg/mL for 1 hour at 37°C, washed,
counted, and added to the assay plate as described above. None of the
peptides used in this study mediated target cell lysis in the absence
of CTLs. Peptides were synthesized using fluorenylmethoxycarbonyl
chemistry by J. Fox (Alta Bioscience, University of Birmingham,
Birmingham, UK). They were dissolved in dimethylsulphoxide (DMSO) and
protein concentrations measured using a modified biuret assay.
IL-10.
Recombinant human IL-10 (hIL-10) was kindly provided by Schering-Plough
Research Institute (Kenilworth, NJ). Biological activity was assessed
using a method based on that described by Hsu et al.38
Briefly, peripheral blood mononuclear cells (PBMCs) (106
cells/mL) were cultured in AIM-V serum free medium (Life Technologies) containing phytohemagglutinin (PHA, 10 µg/mL) with or without hIL-10
(200 U/mL) for 3 days at 37°C in 5% CO2. Cultures were set up in triplicate in 96-well flat-bottomed plates (Life
Technologies) at 200 µL/well. After 3 days, 50 µL of culture
supernatant was obtained from each well and levels of interferon-
(IFN-) measured using the Quantikine human IFN- assay (R & D
Systems Europe Ltd, Abingdon, Oxford, UK) according to the
manufacturer's instructions.
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RESULTS |
TAP 1, TAP 2, and HLA class I expression in H-RS cells within HD
biopsies.
Biopsy material from 38 HD patients was studied for expression of HLA
class I molecules and the peptide transporter proteins TAP 1 and TAP 2 by H-RS cells as markers of their antigen-processing phenotype. The
results are shown in Table 1, in each case
alongside the histological subtype of the tumor and its EBV status
(based on in situ hybridization for EBERs and staining for the EBV
protein LMP 1). Of the 24 EBV-positive tumors, 18 (75%) expressed
detectable surface HLA class I on the H-RS cells, usually at medium to
high levels. In contrast, of the 14 EBV-negative cases, only 4 (29%) showed clear HLA class I staining of H-RS cells and the majority of
tumors appeared to be HLA class I negative. In three cases, one of
which was an EBV-positive tumor, the biopsy contained a mixture of
class I-positive and class I-negative H-RS cells. By comparison,
immunohistochemical staining for the TAP proteins gave a much more
uniform pattern of results. Thus TAP 1 and TAP 2 were detected in H-RS
cells in all of the tumors, irrespective of their EBV genome status,
although in three cases expression of one or other of these proteins
was weak.
Figure 1
shows staining for HLA class I and TAP 1 and 2 in two representative HD
cases (Table 1, cases 4 and 35). Figure 1A shows H-RS cells from
HD case 4 that are HLA class I positive (graded as "+++" in Table
1); note that the infiltrating lymphocytes that surround the H-RS cells
are also HLA class I positive, but that the H-RS cells stand out due to
the increased level of expression of class I on these cells. The
expression of TAP 1 and TAP 2 in H-RS cells from this same biopsy is
shown in Fig 1B and C, respectively. Figure 1D shows the results of HLA
class I staining of HD case 35 where the surrounding lymphocytes
clearly express class I molecules but the H-RS cells do not. This same
H-RS cell population did nevertheless express TAP 1 and TAP 2, as shown
in Fig 1E and F, respectively. As controls for the TAP-specific
reagents, we stained the T2 mutant LCL which lacks the genes coding for
TAPs 1 and 2 and its TAP-positive parent LCL T1.39,40 T2
cells did not stain for TAPs (Fig 1G and H), whereas the T1 line shows
positive staining with both the anti-TAP 1 and the anti-TAP 2 sera (Fig 1I and J).
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| Fig 1.
Two representative HD cases stained for HLA
class I, TAP 1, and TAP 2. Biopsy material from HD case No. 4 expresses
high levels of HLA class I (A), as well as TAP 1 (B) and TAP 2 (C). HD
case No. 35 shows no detectable HLA class I (D), whereas TAP 1 (E) and
TAP 2 (F) are expressed. Sections were counterstained with hematoxylin.
T2 (TAP-negative cell line) and T1 (TAP-positive cell line) were used
as controls for TAP 1 (G, I) and TAP 2 (H, J) staining. Original
magnification × 672 (A, D) and × 420 (B, C, E-J).
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TAP 1, TAP 2, and HLA class I expression in H-RS cell lines.
We then extended our analysis to study the class I processing pathway
of five H-RS cell lines. Using Western blotting, we measured the levels
of expression of TAP 1 and TAP 2 and compared them with a Burkitt tumor
cell line DH and with a matched LCL established by EBV transformation
of normal B cells from the same individual. As reported
previously,17,41 and as shown here, BL cell lines
express little or no TAP 1 and TAP 2, whereas both of these
proteins are expressed at high level in LCLs (Fig
2A). Levels of TAP expression in the H-RS
lines showed some variation, but in general they were equivalent to or
greater than that seen in the LCL. Using
fluorescence-activated cell sorter (FACS) analysis we also compared
expression of HLA class I molecules on the surface of the
H-RS cell lines and an LCL. The results shown in Fig 2B show that cell
lines KMH2, HDLM2, L1236, and L540 express high levels of HLA class I
(comparable with that seen on an LCL), whereas L428 expresses very low
levels.
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| Fig 2.
Expression of TAP 1, TAP 2, and HLA class I in HD-derived
cell lines. Five H-RS lines plus an LCL and BL cell line from donor DH
were analyzed by Western blotting for expression of TAP 1 and TAP 2 (A). Viable cells from the H-RS cell lines plus an LCL were also
analyzed by FACScan for surface expression of HLA class I using the
MoAb W6/32 (B). Clear profile, staining with second step antibody
alone; shaded profile, W6/32 staining. Results shown are representative
of those observed in several repeated assays.
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CTL recognition of H-RS cell lines.
Studying the H-RS cell lines, we tested their ability to process and
present EBV proteins to HLA class I-restricted CTL clones. All of the
H-RS lines are EBV genome negative and therefore EBV proteins were
expressed in the cells using recombinant vaccinia vectors and their
processing monitored in standard cytotoxicity assays using as effectors
HLA class I-restricted CTL clones appropriate to the class I type of
the H-RS target line. As a positive control, H-RS cell lines were
preloaded with synthetic peptides representing the relevant CTL target
epitope. Once again, results were compared with those obtained using BL
and LCL targets in the same assays. For example, HDLM2 (HLA A1,2 B8,44)
was tested for its ability to present a B8-restricted epitope in EBNA
3A (amino acids 158-166). As shown in Fig
3A, a B8-restricted CTL clone specific for
this epitope mediated clear lysis of HDLM2 expressing EBNA 3A from a
vaccinia vector, whereas HDLM2 infected with a control vaccinia vector
was not lysed. Note that the level of lysis was comparable with that
achieved by preloading HDLM2 directly with the target peptide epitope.
The same pattern of results was observed when a B8-positive LCL was
used as the target cell.* In contrast to
both HDLM2 and the LCL target, a B8-positive BL line (OKU) gave
negative results in this antigen processing assay. Thus, the line was
recognized by the B8-restricted CTL clone when preloaded with the
peptide epitope but was unable to process and present EBNA 3A protein
endogenously expressed from a vaccinia vector (Fig 3A). In the same
way, we showed that HDLM2 could also process and present a defined
B44-restricted epitope in EBNA 3C (residues 161-171; data not shown).
Similar results were obtained with the three other H-RS cells in the
panel which showed easily detectable levels of surface HLA class I
expression. Thus, KMH2 (HLA A11,24; B51,62) and L540 (HLA A3,11; B51)
were able to present a defined A11-restricted epitope in EBNA 3B
(residues 416-424) (Fig 3B), and L1236 (HLA A2; B51) could present a
defined A2-restricted epitope in LMP2 (residues 426-434) (Fig 3C).
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| Fig 3.
CTL recognition of BL, HD, and LCL lines expressing EBV
protein antigens from recombinant vaccinia vectors. (A) A B8-restricted EBNA 3A-specific CTL clone tested against OKU BL, HDLM2, and a B8-matched LCL target. (B) An A11-restricted EBNA 3B-specific CTL clone
tested against KMH2 and L540. (C) An A2-restricted LMP 2-specific CTL
clone tested against L1236. All targets had either been preincubated
with the cognate peptide epitope or preinfected with a recombinant
vaccinia vector expressing the target EBV protein. As controls, targets
were preincubated with an equivalent dilution of DMSO solvent
(peptide) or with the vaccinia vector alone (control vacc). E:T
ratios ranged from 5:1 to 10:1. All results are expressed as % specific lysis + 1 SD and are representative of several repeated assays.
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The analysis was then extended to L428, a HD line which in its trace
levels of surface HLA class I expression may serve as an in vitro
counterpart of those HD cases where the tumor cells are classified as
HLA class I-negative by immunohistochemical staining. This line (HLA
class I genotype A3; B35) was assayed using CTL clones recognizing a
defined B35-restricted epitope in EBNA 3A (amino acids 458-466). As
shown in Fig 4A, L428 was able to present
the exogenously loaded epitope peptide but not the EBNA 3A protein
endogenously expressed from a vaccinia vector. In this respect L428
resembled the processing defective B35-positive BL line (DH) and was
clearly distinct from a processing competent LCL. To investigate the
L428 phenotype further, we attempted to increase surface HLA class I
levels. This could not be achieved by IFN- treatment (data not
shown) and therefore we infected the cells with recombinant vaccinia
vectors expressing either HLA B35 or HLA A2. However, FACS analysis
revealed that vaccinia-mediated expression of these HLA alleles
resulted in only a small increase in the levels of surface class I
molecules in L428 cells compared with that produced by the same vectors
in LCL cells (Fig 4B; data shown for HLA A2 expression only).
Furthermore, when L428 was coinfected with vaccinias expressing both
HLA B35 and EBNA 3A, there was still no detectable CTL recognition (Fig
4C) even though these coinfected targets were clearly sensitized to
lysis if pre-exposed to the epitope peptide before testing in the
cytotoxicity assay. Therefore, we wondered whether there might be an
additional lesion in the HLA class I processing pathway in L428 cells
at the level of the TAP transporter function, even though TAP protein
expression was detectable. This possibility was tested using CTL
recognition of an HLA A2-restricted epitope in the LMP 2 protein (amino
acids 426-434) which is very unusual in being processed by a
TAP-independent route.42 In this case, L428 cells (and a
control LCL from an A2-negative donor) were coinfected with vaccinia
vectors expressing HLA A2 and LMP 2 and tested for lysis by
A2-restricted CTLs specific for the above epitope. Despite clear lysis
of the coinfected LCL target there was no recognition of coinfected
L428 cells (Fig 4D) unless these cells were additionally exposed to the
synthetic epitope peptide before testing in the assay.
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| Fig 4.
L428 is unable to present endogenously synthesized
antigens to HLA class I-restricted CTLs. (A) A B35-restricted EBNA
3A-specific CTL clone tested against DH BL, L428, and a B35-matched LCL
target. All targets had either been preincubated with the cognate
peptide epitope (EBNA 3A residues 458-466) or preinfected with a
recombinant vaccinia vector expressing EBNA 3A. As controls, targets
were preincubated with an equivalent dilution of DMSO solvent
(peptide) or with the vaccinia vector alone (control vacc). (B)
Expression of HLA A2 from a recombinant vaccinia vector was tested in
L428 and an HLA A2-negative LCL. Viable cells were analyzed by FACScan for surface expression of HLA A2 using the MoAb BB7.2. Solid line, BB7.2 staining; dotted line, staining with second step antibody alone.
(C) A B35-restricted EBNA 3A-specific CTL clone tested against a
B35-negative LCL and L428. Targets were infected with vaccinia
recombinants expressing HLA B35 and/or EBNA 3A and in one case
targets were also preincubated with the cognate peptide epitope (EBNA
3A residues 458-466). (D) An A2-restricted LMP 2-specific CTL clone
tested against an A2-negative LCL and L428. Targets were infected with
vaccinia recombinants expressing HLA A2 and/or LMP 2 and in one
case targets were also preincubated with the cognate peptide epitope
(LMP 2 residues 426-434). E:T ratios = 5:1 ( ) and 1:1 ( ).
Results of cytotoxicity assays are expressed as % specific lysis + 1 SD and are representative of several repeated assays.
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The effects of IL-10 on CTL recognition of HD cell lines.
A previous report by Matsuda et al24 described the ability
of IL-10 to block antigen-specific CTL-mediated lysis of human melanoma
cells and allo-specific CTL-mediated lysis of LCLs by downregulating
the surface expression of HLA class I molecules. Because IL-10 is
expressed by tumor cells in some cases of HD, we sought to determine
whether this cytokine has a similar effect on the ability of H-RS cells
to be recognized and lysed by CTL. Using recombinant human IL-10 we
first confirmed that this preparation was biologically active by
demonstrating its ability to inhibit IFN- production by
phytohemagglutinin-treated PBMCs (Fig 5A). We then studied the effect of pretreating the H-RS cell lines with
IL-10, following the procedure described by Matsuda et
al.24 Thus, H-RS cell lines were washed and cultured in
AIM-V serum-free medium (Life Technologies) containing 200 U/mL IL-10
for 3 days. During the last five hours of IL-10 pretreatment, H-RS
lines were infected with recombinant vaccinia vectors expressing EBV
proteins and were then tested for recognition by EBV-specific CTL
clones in a chromium release assay. As shown in Fig 5B, HDLM2
expressing EBNA 3A was clearly recognized by a B8-restricted EBNA
3A-specific CTL clone, whereas HDLM2 infected with a control vaccinia
vector was not. However, IL-10 pretreatment had no obvious effect on the levels of lysis observed. In the same way, L1236 expressing LMP 2 from a vaccinia vector was efficiently lysed by an A2-restricted LMP
2-specific CTL clone even after IL-10 pretreatment (Fig 5C). Furthermore, the same result was obtained with L540, KMH2, and an
A11-positive LCL when tested with an A11-restricted CTL clone specific
for EBNA 3B (Fig 5D).
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| Fig 5.
CTL recognition of H-RS lines pretreated with recombinant
human IL-10. (A) The biological activity of the human IL-10 was shown
by its ability to inhibit IFN- production by PHA-treated PBMCs.
However, IL-10 pretreatment had no inhibitory effect on CTL-mediated
lysis of H-RS lines or an LCL (B-D). (B) A B8-restricted EBNA
3A-specific CTL clone tested against HDLM2 infected with a vaccinia
vector expressing EBNA 3A (vE3A) or infected with the vector alone
(vTK). (C) An A2-restricted LMP 2-specific CTL clone tested against
L1236 expressing LMP 2 from a vaccinia vector (vLMP2) or the vector
alone (vTK). (D) An A11-restricted EBNA 3B-specific CTL clone tested
against L540, KMH2, and an A11-matched LCL expressing EBNA 3B from a
vaccinia vector (vE3B) or the vector alone (vTK). E:T = 5:1 ()
and 1:1 (). All results are expressed as % specific lysis + 1 SD
and are representative of several repeated assays.
|
|
In parallel experiments, we assessed whether pretreatment of the CTL
effectors with IL-10 could inhibit their ability to lyse the H-RS cell
lines. CTL clones were pretreated with IL-10 at 20 U/mL and 200 U/mL in
AIM-V serum-free medium for 3 days and then tested for their ability to
lyse H-RS lines expressing the relevant EBV protein from a recombinant
vaccinia virus. Testing a B8-restricted EBNA 3A-specific CTL clone
against HDLM2 and a B8-positive LCL, we observed no significant effect
of IL-10 pretreatment on the levels of CTL-mediated lysis (Fig
6A). In addition, we obtained the same
result testing an A2-restricted LMP 2-specific CTL clone against both
L1236 and an A2-positive LCL (data not shown). IL-10 pretreatment of an
A11-restricted CTL clone specific for a defined epitope in EBNA 3B
(residues 416-424) again failed to inhibit lysis of KMH2, L540, and an
A11-positive LCL expressing EBNA 3B from a vaccinia vector (Fig 6B).
Indeed, in this case IL-10 pretreatment appeared to increase the levels
of CTL-mediated target cell lysis in a dose-dependent manner. This
result was obtained with two different CTL clones specific for this
particular epitope and was observed in four of five replicate
experiments.
View larger version (45K):
[in this window]
[in a new window]
| Fig 6.
Recognition of H-RS lines and LCLs by CTL clones
pretreated with recombinant human IL-10. (A) HDLM2 and a B8-positive
LCL target were infected with a vaccinia vector expressing EBNA 3A (vE3A) or with the vector alone (vTK) and then tested for
recognition by a B8-restricted EBNA 3A-specific CTL clone AR64.64
pretreated with IL-10 at the concentrations shown. E:T = 5:1 ()
and 2:1 (). (B) KMH2, L540, and an A11-positive LCL target were
infected with a vaccinia vector expressing EBNA 3B (vE3B) or with the
vector alone (vTK) and then tested for recognition by an
A11-restricted EBNA 3B-specific CTL clone CM14 pretreated with IL-10 at
the concentrations shown. E:T = 4:1 () and 1:1 (). All results
are expressed as % specific lysis + 1 SD and are representative of
several repeated assays.
|
|
| |
DISCUSSION |
A prerequisite for any attempt to target EBV genome-positive HD with
virus-specific CTL preparations is that the H-RS cells must be capable
of processing and presenting antigens to HLA class I-restricted T
cells. In this context, two previous immunohistochemical studies of HLA
class I expression by H-RS cells18,19 have produced conflicting results. In the present work we found that the H-RS cell
population was uniformly HLA class I positive in at least 75% of cases
of EBV genome-positive HD, whereas the figure was much lower in EBV
genome-negative tumors; more than half of the EBV genome-negative
tumors had no detectable HLA class I expression in tumor cells. Because
LMP 1 is known to activate HLA class I (and TAP) gene expression in B
cells,41 it is tempting to speculate that a similar
influence is occurring in the context of HD. However, there clearly are
a minority of EBV/LMP1-positive HD cases that lack HLA class I
expression. The present findings are in agreement with Oudejans et
al19 who reported that 23 of 25 EBV-positive HD cases
express medium to high levels of HLA class I (based on staining with
the HC10 MoAb) compared with only 5 of 38 EBV-negative cases. The
contrast between these findings and the results of Poppema and
Visser18 may be more apparent than real because the latter
authors did not assess the EBV status of HD biopsies. Thus it is
conceivable that the large number of HLA class I-negative cases that
they identified (22 of 28) may have reflected an unusually large
proportion of EBV-negative biopsies studied. Our analysis of TAP
expression in H-RS cells found that almost all HD cases, regardless of
EBV status, were positive for TAP 1 (again as reported by Oudejans et
al19) and for TAP 2 (Table 1
and Fig 1). Thus, unlike some carcinomas, the lack of HLA class I
molecules on the surface of some H-RS cells is not due to the
downregulation of either TAP 1 or TAP 2, but rather, must involve an
alternative mechanism.
Although our results indicated that the majority of EBV-positive H-RS
cells express HLA class I molecules, this did not prove that the class
I processing pathway is intact because some processing-defective BL
cells also express relatively high levels of HLA class I molecules on
the cell surface.41,43 To begin to analyze this question experimentally, we used a panel of five cell lines derived from HD
biopsies. These lines have been extensively analyzed and carry many
phenotypic markers characteristic of H-RS cells including CD15, CD30,
and CD71.29,44 L1236 is a HD line of particular interest
because it has also been shown to carry the same immunoglobulin gene
rearrangement sequences as H-RS cells in the bone marrow of the
original HD patient.29 Analyzing H-RS cell lines for HLA
class I and TAP expression showed that all expressed high levels of TAP
1 and TAP 2 and that, with the exception of L428, all expressed high
levels of surface HLA class I molecules (Fig 2). Using EBV-specific HLA
class I-restricted CTL clones, we demonstrated that all 4 HD-derived
lines with high HLA class I expression were able to process and present
endogenously synthesized EBV proteins (Fig 3). Moreover, in the case of
L1236 we were able to show that one of the EBV proteins known to be
expressed in some H-RS cells, namely LMP 2, can be processed and
presented through the HLA class I pathway. Note that, in this
particular case, although TAP expression in L1236 is not downregulated,
processing of the LMP 2-derived target epitope might also have occurred
via a TAP-independent route.42
The only HD line which failed to present endogenously synthesized
antigen to a class I-restricted CTL was L428 (Fig 4A). This cell line
expresses only trace levels of surface HLA class I molecules but high
levels of TAP 1 and 2 (Fig 2B) and may therefore be representative of
HLA class I-negative H-RS cells present in some HD cases (Table 1). In
this case there seemed to be a fundamental lesion in the antigen
processing pathway because it was not possible to achieve CTL
recognition even when both the necessary HLA class I heavy chain and
the necessary EBV target antigen were expressed within the cells from
vaccinia vectors. This was the case whether the target in question was
a conventional TAP-dependent epitope in EBNA 3A (Fig 4C) or an
unconventional TAP-independent epitope in LMP 2 (Fig 4D). These
negative results were not due to an inability to infect L428 cells with
vaccinia viruses because immunofluoresence screening on fixed cell
populations showed that two independent indicator proteins
( -galactosidase and LMP 2) were efficiently expressed in L428 cells
when delivered using vaccinia vectors (S.P.L., data not
shown). A particularly interesting result was the very low level of
surface HLA class I molecules that appeared at the L428 cell surface
despite efficient infection with vectors expressing HLA A2 and B35
heavy chains. This suggests a profound block to mature HLA class I
assembly in L428 cells and is consistent with the functional data from
antigen presenting assays. More work is required to determine the
nature of this defect and its relevance to the situation in HLA class
I-negative H-RS cells in vivo.
The balance of findings from immunohistochemical staining of H-RS cells
in EBV genome-positive HD and from the functional assays with HLA class
I-positive H-RS lines would suggest that in most cases there is no
fundamental block to recognition and lysis of such cells by
EBV-specific effectors. However, it remains possible that IL-10 (which
is expressed by H-RS cells in vivo in the majority of EBV
genome-positive cases) might offer the tumor a means of local immune
evasion. Thus it has been reported that IL-10 pretreatment of target
cells protects them from lysis by tumor- and allo-specific CTLs in a
dose-dependent manner.24 However, we followed this protocol
precisely and were unable to show any change in the sensitivity of the
H-RS cell lines or of a reference LCL target to CTL lysis. Furthermore,
and again in contrast to this earlier report, IL-10 pretreatment had no
effect on the surface expression of HLA class I molecules by either
type of line (SPL, data not shown). Indeed, at least one of the H-RS cell lines (L1236) constitutively expresses IL-1045 and yet is clearly susceptible to CTL-mediated lysis (Fig 3C). IL-10 is also
known to suppress the induction of cellular immune responses by
inhibiting IFN- and IL-2 production by the Th1 subset of
T-helper cells20; this could be explained by an effect on
antigen presentation because IL-10 is known to downregulate major
histocompatibility complex class II expression and cytokine synthesis
by macrophages21 and may also inhibit the recruitment and
maturation of dendritic cells.46 Whether this cytokine
might also impair CTL recognition at the effector phase is perhaps a
more relevant question in the present context, because CTL therapy for
HD would involve the adoptive transfer of effector cells that had
already been activated in vitro. We examined this question in our in
vitro system and found that IL-10 did not inhibit H-RS target cell
lysis by preactivated CTL clones; in fact, in some cases IL-10
pretreatment of effector cells increased the levels of killing
observed. Such results are consistent with an earlier finding that
EBV-specific CTLs reactivated in the presence of viral IL-10, a
homologue of the human cytokine and encoded by an EBV lytic cycle gene,
mediated increased levels of LCL lysis when tested 14 days
later.47
In summary, several of the requirements for successful T-cell-based
tumor therapy seem to be satisfied in at least the majority of cases of
EBV genome-positive HD. First, at least one of the three viral antigens
expressed in tumor cells, namely LMP 2, is a known target for CTL
responses on several common HLA class I alleles, including HLA A*0201,
A*2402, and B*4001, that together cover greater than 50% of the
white population.48 Transcripts for LMP 2 are
detectable in most cases of EBV-positive HD and, even using MoAbs of
limited sensitivity, it is often possible to detect the protein in H-RS
cell by immunohistochemical staining. Second, circumstantial evidence
from HLA class I staining of biopsy material and from functional assays
on cell lines suggests that the class I processing pathway is usually
intact. Third, there is no indication from in vitro work that IL-10,
endogenously produced by H-RS cells, could protect the tumor from
virus-specific CTL recognition. Therefore, the challenge is to optimize
ways of generating and expanding LMP-specific CTL preparations in vitro
and of targeting them to the tumor site in vivo.
| |
FOOTNOTES |
Submitted February 2, 1998;
accepted March 27, 1998.
*
Note that, as described previously, many EBV-specific CTL clones
reactivated in vitro with the autologous LCL mediate little or no lysis
of the LCL in a 5-hour chromium release assay.49 Using such
clones throughout the present study ensured that background levels of
lysis of the LCL alone were low even though this target constitutively
expresses EBV proteins.
Supported by the Medical Research Council and by the Cancer Research
Campaign, UK. S.P.L. is supported by a Medical Research Council Career
Development Award.
Address reprint requests to Steven P. Lee, PhD, Institute for Cancer
Studies, University of Birmingham, Vincent Dr, Edgbaston, Birmingham
B15 2TA, UK; e-mail: s.p.lee{at}bham.ac.uk.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr M. Rowe, Dr J. Trowsdale, and Dr H. Ploegh for
generously providing reagents for immunohistochemistry; and Dr J. Wolf,
Dr V. Diehl, and Dr H. Kamesaki for supplying us with the H-RS cell
lines. We also thank Dr M. Kurilla and Dr J. Yewdell for providing the
vaccinia recombinants, Dr B. Moss for vRB21 and pRB21, and Dr E. Scotet
for HLA B*3501 cDNA.
| |
REFERENCES |
1.
Glaser SL,
Lin RJ,
Stewart SL,
Ambinder RF,
Jarrett RF,
Brousset P,
Pallesen G,
Gulley ML,
Khan G,
O'Grady J,
Hummel M,
Preciado MV,
Knecht H,
Chan JC,
Claviez A:
Epstein-Barr virus-associated Hodgkin's disease: Epidemiologic characteristics in international data.
Int J Cancer
70:375,
1997[Medline]
[Order article via Infotrieve]
2.
Boiocchi M,
Dolcetti R,
Dere V,
Gloghini A,
Carbone A:
Demonstration of a unique Epstein-Barr virus-positive cellular clone in metachronous multiple localizations of Hodgkin's disease.
Am J Pathol
142:33,
1993[Abstract]
3.
Kieff E:
Epstein-Barr virus
, in Fields BN,
Knipe DM,
Howley PM
(eds):
Fields Virology.
Philadelphia, PA, Lippincott
, 1996
, p 2397
4.
Khanna R,
Burrows SR,
Kurilla MG,
Jacob CA,
Misko IS,
Sculley TB,
Kieff E,
Moss DJ:
Localization of Epstein-Barr virus cytotoxic T cell epitopes using recombinant vaccinia Implications for vaccine development.
J Exp Med
176:169,
1992[Abstract/Free Full Text]
5.
Lee SP,
Thomas WA,
Murray RJ,
Khanim F,
Kaur S,
Young LS,
Rowe M,
Kurilla M,
Rickinson AB:
HLA A2.1-restricted cytotoxic T cells recognizing a range of Epstein-Barr virus isolates through a defined epitope in latent membrane protein LMP2.
J Virol
67:7428,
1993[Abstract/Free Full Text]
6.
Lee SP,
Tierney RJ,
Thomas WA,
Brooks JM,
Rickinson AB:
Conserved CTL epitopes within EBV latent membrane protein 2A potential target for CTL-based tumor therapy.
J Immunol
158:3325,
1997[Abstract]
7.
Murray RJ,
Kurilla MG,
Brooks JM,
Thomas WA,
Rowe M,
Kieff E,
Rickinson AB:
Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV)Implications for the immune control of EBV-positive malignancies.
J Exp Med
176:157,
1992[Abstract/Free Full Text]
8.
Rooney CM,
Smith CA,
Ng CC,
Loftin S,
Li CF,
Krance RA,
Brenner MK,
Heslop HE:
Use of gene modified virus-specific T lymphocytes to control Epstein-Barr virus-related lymphoproliferation.
Lancet
345:9,
1995[Medline]
[Order article via Infotrieve]
9.
Deacon EM,
Pallesen G,
Niedobitek G,
Crocker J,
Brooks L,
Rickinson AB,
Young LS:
Epstein-Barr virus and Hodgkin's diseaseTranscriptional analysis of virus latency in the malignant cells.
J Exp Med
177:339,
1993[Abstract/Free Full Text]
10.
Pallesen G,
Hamiltondutoit SJ,
Rowe M,
Young LS:
Expression of Epstein-Barr virus latent gene products in tumor cells of Hodgkin's disease.
Lancet
337:320,
1991[Medline]
[Order article via Infotrieve]
11.
Grasser FA,
Murray PG,
Kremmer E,
Klein K,
Remberger K,
Feiden W,
Reynolds G,
Niedobitek G,
Young LS,
Muellerlantzsch N:
Monoclonal antibodies directed against the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1)Immunohistologic detection of EBNA1 in the malignant cells of Hodgkin's disease.
Blood
84:3792,
1994[Abstract/Free Full Text]
12.
Niedobitek G,
Kremmer E,
Herbst H,
Whitehead L,
Dawson CW,
Niedobitek E,
vonOstau C,
Rooney N,
Grasser FA,
Young LS:
Immunohistochemical detection of the Epstein-Barr virus-encoded latent membrane protein 2A in Hodgkin's disease and infectious mononucleosis.
Blood
90:1664,
1997[Abstract/Free Full Text]
13.
Frisan T,
Sjoberg J,
Dolcetti R,
Boiocchi M,
Dere V,
Carbone A,
Brautbar C,
Battat S,
Biberfeld P,
Eckman M,
Ost O,
Christensson B,
Sundstrom C,
Bjorkholm M,
Pisa P,
Masucci MG:
Local suppression of Epstein-Barr virus (EBV)-specific cytotoxicity in biopsies of EBV-positive Hodgkin's disease.
Blood
86:1493,
1995[Abstract/Free Full Text]
14.
Sing AP,
Ambinder RF,
Hong DJ,
Jensen M,
Batten W,
Petersdorf E,
Greenberg PD:
Isolation of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes that lyse Reed-Sternberg cells: Implications for immune-mediated therapy of EBV(+) Hodgkin's disease.
Blood
89:1978,
1997[Abstract/Free Full Text]
15.
York IA,
Rock KL:
Antigen processing and presentation by the class I Major Histocompatibility Complex.
Annu Rev Immunol
14:369,
1996[Medline]
[Order article via Infotrieve]
16.
Restifo NP,
Esquivel F,
Kawakami Y,
Yewdell JW,
Mule JJ,
Rosenberg SA,
Bennink JR:
Identification of human cancers deficient in antigen processing.
J Exp Med
177:265,
1993[Abstract/Free Full Text]
17.
Khanna R,
Burrows SR,
Argaet V,
Moss DJ:
Endoplasmic reticulum signal sequence facilitated transport of peptide epitopes restores immunogenicity of an antigen processing defective tumor cell line.
Int Immunol
6:639,
1994[Abstract/Free Full Text]
18.
Poppema S,
Visser L:
Absence of HLA class I expression by Reed-Sternberg cells.
Am J Pathol
145:37,
1994[Abstract]
19.
Oudejans JJ,
Jiwa NM,
Kummer JA,
Horstman A,
Vos W,
Baak JA,
Kluin PM,
Vandervalk P,
Walboomers JM,
Meijer CM:
Analysis of major histocompatibility complex class I expression on Reed-Sternberg cells in relation to the cytotoxic T cell response in Epstein-Barr virus-positive and Epstein-Barr virus-negative Hodgkin's disease.
Blood
87:3844,
1996[Abstract/Free Full Text]
20.
Fiorentino DF,
Zlotnik A,
Vieira P,
Mosmann TR,
Howard M,
Moore KW,
Ogarra A:
IL-10 acts on the antigen presenting cell to inhibit cytokine production by Th1 cells.
J Immunol
146:3444,
1991[Abstract]
21.
Malefyt RD,
Haanen J,
Spits H,
Roncarolo MG,
Tevelde A,
Figdor C,
Johnson K,
Kastelein R,
Yssel H,
Devries JE:
Interleukin-10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen presenting capacity of monocytes via down-regulation of class II major histocompatibility complex expression.
J Exp Med
174:915,
1991[Abstract/Free Full Text]
22.
Taga K,
Mostowski H,
Tosato G:
Human interleukin 10 can directly inhibit T cell growth.
Blood
81:2964,
1993[Abstract/Free Full Text]
23.
Herbst H,
Foss HD,
Samol J,
Araujo I,
Klotzbach H,
Krause H,
Agathanggelou A,
Niedobitek G,
Stein H:
Frequent expression of interleukin 10 by Epstein-Barr virus-harboring tumor cells of Hodgkin's disease.
Blood
87:2918,
1996[Abstract/Free Full Text]
24.
Matsuda M,
Salazar F,
Petersson M,
Masucci G,
Hansson J,
Pisa P,
Zhang QJ,
Masucci MG,
Kiessling R:
Interleukin 10 pretreatment protects target cells from tumor-specific and allo-specific cytotoxic T cells and down-regulates HLA class I expression.
J Exp Med
180:2371,
1994[Abstract/Free Full Text]
25.
Lukes RJ,
Carver LF,
Hall TC,
Rappaport H,
Ruben P:
Report of the nomenclature committee.
Cancer Res
26:1311,
1966
26.
Diehl V,
Kirchner HH,
Schaadt M,
Fonatsch C,
Stein H,
Gerdes J,
Boie C:
Hodgkin's diseaseEstablishment and characterization of 4 in vitro cell lines.
J Cancer Res Clin Oncol
101:111,
1981[Medline]
[Order article via Infotrieve]
27.
Kamesaki H,
Fukuhara S,
Tatsumi E,
Uchino H,
Yamabe H,
Miwa H,
Shirakawa S,
Hatanaka M,
Honjo T:
Cytochemical, immunological, chromosomal, and molecular genetic analysis of a novel cell line derived from Hodgkin's disease.
Blood
68:285,
1986[Abstract/Free Full Text]
28.
Drexler HG,
Gaedicke G,
Lok MS,
Diehl V,
Minowada J:
Hodgkin's disease derived cell lines HDLM-2 and L-428Comparison of morphology, immunological and isoenzyme profiles.
Leuk Res
10:487,
1986[Medline]
[Order article via Infotrieve]
29.
Kanzler H,
Hansmann ML,
Kapp U,
Wolf J,
Diehl V,
Rajewsky K,
Kuppers R:
Molecular single cell analysis demonstrates the derivation of a peripheral blood-derived cell line (L1236) from the Hodgkin Reed-Sternberg cells of a Hodgkin's lymphoma patient.
Blood
87:3429,
1996[Abstract/Free Full Text]
30.
Murray PG,
Young LS,
Rowe M,
Crocker J:
Immunohistochemical demonstration of the Epstein-Barr virus-encoded latent membrane protein in paraffin sections of Hodgkin's disease.
J Pathol
166:1,
1992[Medline]
[Order article via Infotrieve]
31.
Stam NJ,
Spits H,
Ploegh HL:
Monoclonal antibodies raised against denatured HLA-B locus heavy chains permit biochemical characterization of certain HLA-C locus products.
J Immunol
137:2299,
1986[Abstract]
32.
Kelly A,
Powis SH,
Kerr LA,
Mockridge I,
Elliott T,
Bastin J,
Uchanskaziegler B,
Ziegler A,
Trowsdale J,
Townsend A:
Assembly and function of the 2 ABC transporter proteins encoded in the human major histocompatibility complex.
Nature
355:641,
1992[Medline]
[Order article via Infotrieve]
33.
Barnstable CJ,
Bodmer WF,
Brown G,
Galfre G,
Milstein C,
Williams AF,
Ziegler A:
Production of monoclonal antibodies to Group A erythrocytes, HLA and other human cell surface antigensNew tools for genetic analysis.
Cell
14:9,
1978[Medline]
[Order article via Infotrieve]
34.
Parham P,
Brodsky FM:
Partial purification and some properties of BB7.2A cytotoxic monoclonal antibody with specificity for HLA-A2 and a variant of HLA-A28.
Hum Immunol
3:277,
1981[Medline]
[Order article via Infotrieve]
35.
Finerty S,
Rowe M,
Berry PJ,
Ranson DL,
Mott MG,
Gregory CD,
Rickinson AB:
Burkitt-like lymphoma in an English childCharacterization of tumor biopsy cells and of the derived tumor cell line.
Br J Cancer
54:385,
1986[Medline]
[Order article via Infotrieve]
36.
Blasco R,
Moss B:
Selection of recombinant vaccinia viruses on the basis of plaque formation.
Gene
158:157,
1995[Medline]
[Order article via Infotrieve]
37.
O'Neil BH,
Kawakami Y,
Restifo NP,
Bennink JR,
Yewdell JW,
Rosenberg SA:
Detection of shared MHC-restricted human melanoma antigens after vaccinia virus-mediated transduction of genes coding for HLA.
J Immunol
151:1410,
1993[Abstract]
38.
Hsu DH,
Malefyt RD,
Fiorentino DF,
Dang MN,
Vieira P,
Devries J,
Spits H,
Mosmann TR,
Moore KW:
Expression of interleukin 10 activity by Epstein-Barr virus protein BCRF1.
Science
250:830,
1990[Abstract/Free Full Text]
39.
Salter RD,
Howell DN,
Cresswell P:
Genes regulating HLA class I antigen expression in T-B lymphoblast hybrids.
Immunogenetics
21:235,
1985[Medline]
[Order article via Infotrieve]
40.
Cerundolo V,
Alexander J,
Anderson K,
Lamb C,
Cresswell P,
McMichael A,
Gotch F,
Townsend A:
Presentation of viral antigen controlled by a gene in the major histocompatibility complex.
Nature
345:449,
1990[Medline]
[Order article via Infotrieve]
41.
Rowe M,
Khanna R,
Jacob CA,
Argaet V,
Kelly A,
Powis S,
Belich M,
Croom-Carter D,
Lee S,
Burrows SR,
Trowsdale J,
Moss DJ,
Rickinson AB:
Restoration of endogenous antigen processing in Burkitt's lymphoma cells by Epstein-Barr virus latent membrane protein-1: Coordinate up-regulation of peptide transporters and HLA-class I antigen expression.
Eur J Immunol
25:1374,
1995[Medline]
[Order article via Infotrieve]
42.
Lee SP,
Thomas WA,
Blake NW,
Rickinson AB:
Transporter (TAP)-independent processing of a multiple membrane-spanning protein, the Epstein-Barr virus latent membrane protein 2.
Eur J Immunol
26:1875,
1996[Medline]
[Order article via Infotrieve]
43.
Rooney CM,
Rowe M,
Wallace LE,
Rickinson AB:
Epstein-Barr virus-positive Burkitt's lymphoma cells not recognized by virus-specific T cell surveillance.
Nature
317:629,
1985[Medline]
[Order article via Infotrieve]
44.
Drexler HG:
Recent results on the biology of Hodgkin and Reed-Sternberg cells.2. Continuous cell lines.
Leuk Lymphoma
9:1,
1993[Medline]
[Order article via Infotrieve]
45.
Wolf J,
Kapp U,
Bohlen H,
Kornacker M,
Schoch C,
Stahl B,
Mucke S,
Vonkalle C,
Fonatsch C,
Schaefer HE,
Hansmann ML,
Diehl V:
Peripheral blood mononuclear cells of a patient with advanced Hodgkin's lymphoma give rise to permanently growing Hodgkin Reed-Sternberg cells.
Blood
87:3418,
1996[Abstract/Free Full Text]
46.
Qin ZH,
Noffz G,
Mohaupt M,
Blankenstein T:
Interleukin 10 prevents dendritic cell accumulation and vaccination with granulocyte-macrophage colony-stimulating factor gene-modified tumor cells.
J Immunol
159:770,
1997[Abstract]
47.
Stewart JP,
Rooney CM:
The interleukin 10 homolog encoded by Epstein-Barr virus enhances the reactivation of virus-specific cytotoxic T cell and HLA-unrestricted killer cell responses.
Virology
191:773,
1992[Medline]
[Order article via Infotrieve]
48.
Gojobori T:
W15.1 reference tables
, in Tsuji K,
Aizawa M,
Sasazuki T
(eds):
HLA-1991.
Oxford, UK, Oxford University Press
, 1992
, p 1066
49.
Hill AB,
Lee SP,
Haurum JS,
Murray N,
Yao QY,
Rowe M,
Signoret N,
Rickinson AB,
McMichael AJ:
Class I major histocompatibility complex-restricted cytotoxic T lymphocytes specific for Epstein-Barr virus (EBV) nuclear antigens fail to lyse the EBV-transformed B lymphoblastoid cell lines against which they were raised.
J Exp Med
181:2221,
1995[Abstract/Free Full Text]
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83(12):
6192 - 6198.
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[Full Text]
[PDF]
|
|
|
|
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60(12):
1342 - 1349.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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J. Immunol.,
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179(11):
7506 - 7513.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Niens, R. F. Jarrett, B. Hepkema, I. M. Nolte, A. Diepstra, M. Platteel, N. Kouprie, C. P. Delury, A. Gallagher, L. Visser, et al.
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November 1, 2007;
110(9):
3310 - 3315.
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|
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August 15, 2007;
110(4):
1326 - 1329.
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|
|
|
|
|
|
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25(21):
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[Full Text]
[PDF]
|
|
|
|
|
|
|
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15(11):
2280 - 2284.
[Abstract]
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|
|
|
|
|
|
|
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October 1, 2006;
177(7):
4897 - 4906.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. P. Lee, J. M. Brooks, H. Al-Jarrah, W. A. Thomas, T. A. Haigh, G. S. Taylor, S. Humme, A. Schepers, W. Hammerschmidt, J. L. Yates, et al.
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J. Exp. Med.,
May 17, 2004;
199(10):
1409 - 1420.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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February 1, 2004;
10(3):
803 - 821.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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78(2):
868 - 881.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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April 15, 2003;
101(8):
3150 - 3156.
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|
|
|
|
|
|
|
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August 1, 2001;
61(16):
6219 - 6226.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
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Mol. Pathol.,
October 1, 2000;
53(5):
248 - 254.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
K J Flavell and P G Murray
Hodgkin's disease and the Epstein-Barr virus
Mol. Pathol.,
October 1, 2000;
53(5):
262 - 269.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
P. Steigerwald-Mullen, M. G. Kurilla, and T. J. Braciale
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74(15):
6748 - 6759.
[Abstract]
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|
|
|
|
|
|
|
P. G. Stevenson, S. Efstathiou, P. C. Doherty, and P. J. Lehner
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PNAS,
July 5, 2000;
(2000)
150240097.
[Abstract]
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|
|
|
|
|
|
|
K. Willenbrock, A. Roers, B. Blohbaum, K. Rajewsky, and M.-L. Hansmann
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July 1, 2000;
157(1):
171 - 175.
[Abstract]
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|
|
|
|
|
|
|
H. Chambost, N. Van Baren, F. Brasseur, D. Godelaine, L. Xerri, S. J. Landi, I. Theate, J. Plumas, G. C. Spagnoli, G. Michel, et al.
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95(11):
3530 - 3533.
[Abstract]
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|
|
|
|
|
|
|
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J. Virol.,
September 1, 1999;
73(9):
7381 - 7389.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
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April 15, 1999;
93(8):
2688 - 2696.
[Abstract]
[Full Text]
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|
|
|
|
|
|
|
P. G. Murray, C. M. Constandinou, J. Crocker, L. S. Young, and R. F. Ambinder
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Blood,
October 1, 1998;
92(7):
2477 - 2483.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. G. Stevenson, S. Efstathiou, P. C. Doherty, and P. J. Lehner
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PNAS,
July 18, 2000;
97(15):
8455 - 8460.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract