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Distribution and Phenotype of Epstein-Barr Virus-Harboring Cells
in Hodgkin’s Disease
By Hermann Herbst, Erik Steinbrecher, Gerald Niedobitek, Lawrence S. Young, Louise Brooks,
Nikolaus Muller-Lantzsch, and Harald Stein
Distribution and phenotype of Epstein-Barr virus (EBV)harboring cells were determined in Hodgkin‘s disease (HD)
biopsies by in situ hybridization with [35S]-labeled RNA
probes specific for the small EBV-encoded nuclear RNAs,
EBERl and EBER2, in some instances preceded by immunohistology for CD20, CD30, CD45RO. and CD68 antigens, the
T-cell receptor pchain, and latent membrane antigen (LMP)
of EBV. Twenty-three of 46 HD cases displayed EBER transcripts in all Hodgkinand Reed-Sternberg (H-RS) cells, and 18
of these cases showed LMP expression exclusively in neoplastic cells. EBER+ small reactive cells were present in 39 cases
in low numbers, and in three cases in abundance. Thus,
presence of H-RS cells with or without LMP expression was
not accompanied by an unrestricted proliferation of reactive
EBER+/LMP- lymphoid cells in the majority of HD patients.
Simultaneousin situ hybridization with [35S]-labeledimmunoglobulin light chain (IgLC) gene probes and nonisotopically
labeled EBER probe showed a phenotype of mature B lymphocytes and a polyclonal composition for a large proportion of
the EBER+ small cells. However, in contrast to noninfected
cells, CD20 expression was not detectable in many of these
cells, which may indicate downregulationof certain differentiation antigens in latently EBV-infected small lymphoid cells
in vivo.
o 1992 by The American Society of Hematology.
HE EPSTEIN-BARR VIRUS (EBV) had been associ-
cases, r e ~ p e c t i v e l y .Others
~ ~ , ~ ~ pointed out that most of the
EBV genomes detected in tissue extracts could have been
derived from EBV-carrying cells of the reactive admixt ~ r e . l More
~-~~
than 90% of adults worldwide are latently
infected with this herpesvirus, so that at least some latently
infected lymphocytes are likely to be present in almost any
lymphoid tissue.22 It was thus argued that the sensitivity
limits of in situ hybridization did not permit detection of
EBV DNA in the cellular admixture,14raising the possibility that, although responsible for the frequent detection of
EBV in HD by PCR, an abundance of EBV-infected cells
remained undetected by in situ hybridization for EBV
DNA.
Detection of the small EBV-encoded nonpolyadenylated
RNA transcripts, EBERl and EBER2, by in situ hybridization now provides a way to clarify this issue. This approach
has previously been applied to various EBV-associated
lesions, such as undifferentiated nasopharyngeal carcioral hairy l e ~ k o p l a k i a CD30+
,~~
anaplastic largecell lymphoma,25and to a limited number of H D case^.^^.^'
The EBERs appear to be transcribed in most human
lesions associated with latent EBV infection.28By virtue of
their extreme abundance, estimated to be up to lo6 copies
per viral genome?9 EBERs lend themselves to the application of radioactive and nonradioactive in situ hybridization,
even in routinely formalin-fixed and paraffin-embedded
t i s ~ u e s . ~For
~ - ~the
7 present study, we used in sitti hybridization with EBER-specific single-stranded [35S]-labeledantisense and sense (control) RNA probes to address the
questions as to the presence, number, and distribution of
EBER+ neoplastic and reactive cells in HD tissue biopsies.
In bome cases, we additionally used sequential labeling for
differentiation antigens by immunohistology and EBER by
in situ hybridization, as well as simultaneous in situ hybridization for immunoglobulin light chain (IgLC) RNA transcripts with [35S]-labeled RNA probes and EBER with
nonisotopically labeled RNA probes to characterize the
phenotype of malignant and reactive EBER+ cells.
T
ated with Hodgkin’s disease (HD) by various lines of
serological and epidemiological e ~ i d e n c e l -prior
~
to the
demonstration of EBV antigens and DNA in isolated cases
of HD in 1985 and 1987, re~pectively.~?~
Since then, many
investigators found EBV genomes in up to 25% of DNA
extracts from H D tissue biopsies analyzed by direct filter
hybridization methods.6-10Because of the limited sensitivity
of this approach, these studies were extended by the
application of the polymerase chain reaction (PCR) technique, which showed DNA sequences in considerably
larger proportions of cases, ranging between 40% and 80%
depending on the sensitivity limits applied.1i-18Although
most investigators found approximately 55% to 60% of the
H D cases to contain substantial amounts of EBV DNA, the
significance of these findings remained controversial. Several investigators favored tumor cells, rather than cells of
the reactive admixture, as the most probable origin of the
EBV DNA, and this view was substantiated by the visualization of EBV DNA8-11,i9,20
and of the EBV-encoded latent
membrane protein (LMP) exclusively in Hodgkin and
Reed-Sternberg (H-RS) cells in up to 40% and 48% of H D
From the Institute of Patholoo, Klinikum Steglitz, Free University of
Berlin, Berlin, Germany; the Department of Cancer Studies, University
of Birmingham, Birmingham, UK; and the Division of Kroloo,
Institute of Medical Microbiology and Hygiene, University Hospital,
University of Saarland, HomburglSaar, Germany.
Submitted December 2,1991;accepted March 23,1992.
Supported by grants from the Bundesministerium fur Forschung und
Technologie (Grant No. 01GA8501), Deutsche Krebshilfe (Grunt No.
W91891St2),Deutsche Forschungsgemeinschafr (Grant No. Mu452l2I ) , and the CancerResearch Campaign (to L.S.Y).
This work ispartial fulfilment of M.D. thesis requirements (E.S.).
Address reprint requests to Hermann Herbst, MD, Institute of
Patholoo, Klinikum Steglitz, Free University Berkh, 1 OOO Berlin 45,
Germany.
The publication costs of this article were defruyed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 I992 by The American Sociery of Hematology.
aoo6-4971/92/8OO2-OO19$3.OO/0
484
MATERIALS AND METHODS
Tissues. Lymph node biopsy specimens from 46 HD patients
submitted for histopathological diagnosis were routinely processed
Blood, Vol80, No 2 (July 15). 1992: pp 484-491
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EBV IN HODGKIN'S DISEASE
by formalin fixation and paraffin embedding. In six cases, snapfrozen aliquots of the tissues, stored at -8O"C, were available.
Development of the disease was unrelated to human immunodeficiency virus (HIV) infection, and none of these cases was incorporated into previous studies. Histological typing followed the Rye
classification. Ten lymph nodes, removed during mastectomy or
prostatectomy and showing a regular histology with only moderate
sinus histiocytosis,were used as controls.
Immunohistology. The production and specificity profiles of the
monoclonal antibodies specific for LMP (clones CS1, -2, -3, and
-4)% and of a rabbit antiserum directed against LMP?I are
described elsewhere. Monoclonal antibodies against CD20 (L26),
CD30 (Ber-Hr2), and CD68 (KP-l), as well as CD3-specificrabbit
antibodies, were obtained from Dakopatts (Glostrup, Denmark).
The anti-T-cell receptor P-chain antibody, PF1, was from T-cell
Sciences (Cambridge, MA), and the CD45RO-specific antibody
A633was a kind gift of Dr G.G. Aversa (Stanford, CA). Sections (4
km) of either formol-fixed/paraffin-embedded or snap-frozen
tissue blocks were stained by the immunoalkaline phosphatase
(APAAP)method." Affinity-purifiedmouse anti-rabbit immunoglobulin, rabbit anti-mouse immunoglobulin antibodies, and
APAAP complex (diluted 1:20) were obtained from Dakopatts.
Incubation with the CD3-specific antibodies was performed overnight. Formol-fixed sectionsrequired a proteolytic treatment with 1
mg/mL Streptomyces giseus protease (Sigma, Deisenhofen, Germany) for 10 minutes before incubation with the monoclonal
antibodies PF1 and Ber-H2.
Probes. EBER1- and EBERZspecific fragments were derived
from the plasmids pJJJl and P J J J ~kindly
, ~ ~ provided by Dr J.
Arrand (Manchester, UK), and subcloned into the BamHIIEcoRI
and EcoRIIHindIII sites, respectively, of the transcription vector
pBluescriptKS (Promega-Biotec, Madison, WI). For the preparation of IgLC RNA probes, the 550-bp SstI fragment containing the
human IgLCK gene constant segment?6 and the 600-bp BglIII
BumHI fragment containing the IgLCX gene C2 constant segm e ~ ~ respectively,
t?~
were subcloned into pGEMl (Promega Biotec). Phages with the IgLCK and IgLCX genomic fragments were
kindly provided by Dr P. Leder, (Cambridge, MA). After linearization with the appropriate restriction enzymes (GIBCO-BRL,
Karlsruhe, Germany), [35S]-labeledantisense or sense (control)
run-off transcripts were generated using either SP6, T3, or T7
RNA polymerasesand [35S]-uridine-5'-(u-thio)-triphosphate(1,250
Ci/mmol; New England Nuclear, Dreieich, Germany) or, altematively, digoxigenin-ll-uridine-5'-triphosphate(Boehringer Mannheim, Mannheim, Germany).38A mixture of EBER1- and EBER2specific RNA probes was applied to increase the sensitivity.
In situ hybridization. The hybridization procedure with either
[35S]-labeled,digoxigenin-labeled, or combinations of [35S]-and
digoxigenin-labeledprobes on frozen and paran-sections, washing steps including RNase digestion of nonspecifically bound
probe, and autoradiographywere performed as described.25Immobilized digoxigenin was detected using a monoclonal digoxigeninspecific antibody (Boehringer Mannheim; dilution 1:20) and the
APAAP procedure. All sections were processed in parallel using
the same batches of reagents and probes. Abrogation of specific
signals in hybridizations preceded by micrococcal nuclease digest i ~ verified
n ~ ~ that the targets of the hybridizations were RNA
molecules. The specificity of the EBER probes was further tested
on six HD-derived cell lines: of which only the line L591, known to
harbor EBV,6expressed EBERl and EBER2.
Sequential immunohistochemistryand in situ hybridization. Cryostat sections fixed in 4% paraformaldehyde were incubated in 2
mg/mL predigested pronase/lx phosphate-buffered saline (PBS)
at 37°C for 5 minutes. Slides were rinsed twice with 0.1 mol/L
glycine/lx Tris-buffered saline (TBS). Antibodies were used in
485
freshly prepared RPMI 1640 medium (GIBCO-BRL), pH 7.5,
containing 10 mg/mL bovine serum albumin, 1.0 mg/mL yeast
tRNA, and 5,000U/mL heparin ammonium salt (Sigma) to inhibit
RNase activity.'IQ4*Incubation was 30 minutes at room temperature each with the monoclonal antibody, rabbit anti-mouse immunoglobulin antibody, and APAAP complex (Dakopatts), the latter
two in 1:20 dilution, using RPMI dilution buffer as described
above. The last two incubation steps were repeated once for 10
minutes each. Washing after each incubation step was done in lx
TBS. Immobilized antibodies were visualized as described above
and slides were immediately subjected to the in situ hybridization
procedure. Paraffin sections were similarly treated following dewaxing in xylene and predigestion in 0.5 mg/mL protease VI11
(Sigma) in PBS for 8 minutes at room temperature; these further
procedures did not differ from those for cryostat sections. Autoradiographic exposure was 4 to 6 days. To estimate the RNA loss
during immunostaining procedures, adjacent tissue sections were
subjected to in situ hybridization not preceded by immunostaining.
The autoradiographic exposure times for slides subjected to the
double-labelingprocedure were adjusted accordingly.
RESULTS
Histological diagnoses and phenotype characteristics of
46 HD cases are summarized in Table 1. Twenty-three
cases showed EBER expression in neoplastic cells, while
the other 23 cases did not display an EBER-specific signal
in any of the H-RS cells even after prolonged autoradiographic exposure. The intensity of the EBER-specificin situ
hybridization signal over H-RS cells varied considerably
from cell to cell; short exposure times (6 to 12 hours of
autoradiography) were particularly useful to assess the
atypical nuclear morphologyof EBER+ H-RS cells, whereas
extended exposure times ( > 4 days) resulted in exhaustion
of the capacity of the autoradiographic emulsion over most
EBER+ cells and were useful to establish the total number
of such cells. The number of EBER+ cells was matched by
the number of CD30+cells in adjacent tissue sections. Thus
in these HD cases, virtually all H-RS cells, and not only a
proportion, expressed EBER transcripts. Hybridization
with sense (control) probes resulted in background signals
over all cells. Eighteen of 23 cases with EBER+ H-RS cells
displayed LMP expression in 10% to 80% of the neoplastic
cells, as detected in paraffin sections. In comparison with
the monoclonal antibodies, CS1-4, the LMP-specific rabbit
antiserum stained smaller percentages of neoplastic cells.
Unlike LMP expression, EBER transcripts were not
restricted to H-RS cells. In most cases, small lymphoid cells
with unsuspicious nuclear morphology were found in small
Table 1. Phenotype of Hodgkin and Reed-SternbergCells
in 46 HD Cases
Diagnosis*
EBER
LMP
CD30
CD20
CD3
HDlpn
HDns
HDmc
HDld
Total
2/2t
10124
212
8/24
212
24/24
212
2/24
Ioiia
7\18
miia
1/18
112
23/46
I I2
212
46/46
012
5/46
012
3/24
2/18
012
5/46
18/46
'Histotypes of HD: Ipn, lymphocyte predominance, nodular subtype;
ns, nodular sclerosing: mc, mixed cellularity: Id, lymphocyte depletion.
tNumber of cases with labeled H-RS cells per number of cases
studied.
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HERBST ET AL
486
Table 2 Numben of EBER-Expmsslng Cells In 46 HD Cases With
EBER* or EBER- H-RS Cells
No. of EBER' c e l l s l 0 . k d Saction &ea
0
HD cases with
EBER' H-RS
cells
HD cases with
EBER- H-RS
2
cells
AllHDcases
2
1-20 21-50 51-100 101-150 151-200 201.250 >250
- - - 2 1 1
2
11
5
5
1
1
-
2
-
-
2
2
23
-
21
Elevated numbers of EBER' cells ( > 100) cells were found in 26 of 46
cases: in all those HD cases carrying EBER' H-RS cells (23 cases), as
well as in three cases with EBER- tumor cells.
numbers (Table 2). regardless of whether the neoplastic
cells were EBV-infected (Fig 1). In two cases with EBER+
H-RS cells, no small EBER+ cells could be identified. In
two additional biopsies, no EBER' cells were present at all,
even in serial sections; three cases, two of which did not
show EBV infection of the tumor cells, displayed high
numbers of EBER-expressing small lymphoid cells, often
accumulated in incoherent clusters. Two of the 10 control
tissues with intact nodal architecture displayed only few
interfollicular EBER+ cells (up to 10 per 0.5-cmZ section
area); the other tissues did not show EBER-specific signals.
Hybridizations with IgLC gene probes were performed with
all tissues that did not contain EBER+ cells. These cases
displayed intense labeling of plasma cells within 48 hours of
exposure, thus excluding the possibility of a failure to detect
EBER+ cells because of RNA degradation.
Simultaneous isotopic/nonisotopic in situ hybridization
double-labeling was used to characterize EBV-infected
cells in each six of HDmc and HDns cases with LMP+
tumor cells and larger numbers of EBER+ small cells. With
respect to EBER-specific labeling, the results were identical to those previously obtained with [35S]-labeledEBERspecific probes. In all cases, the tumor cells did not display
any IgLC transcripts, whereas approximately each 30% of
the small EBER+ cells showed IgLCK or IgLCA gene RNA,
respectively (Fig 2).
To further characterize EBER-expressing cells, nine HD
cases, particularly cases with either an EBER- or EBER+/
CD20-/CD45RO-/TcRp- H-RS cell phenotype, were subjected to the sequential immunohistology/isotopic in situ
hybridization double-labeling procedure on frozen and
paraffin sections using antibodies against CD20, CD30,
CD45R0, CD68, LMP, and the TcRp-chain. In cases with
EBER+ H-RS cells, CD30-specific staining virtually matched
with EBER expression (Fig 3, Table 3). LMP staining was
always associated with EBER-specific autoradiographic
signals, and CD68 reactivity was not coexpressed with
EBER in any of the cases. Approximately 6 of 10 EBER+
reactive cells were detected by the CD20 antibody L26 in
Flg 1. Dotodon of EBER hnraipts In th.cl.from HD p.tkntr. (A)
EBER transcripts In H-RS cells In the absence of reactive EBER* cells; (B)
multiple EBER+ small reactahre cells (dot-like labeling, small arrows) in
the absence of EBER transcripts from H-AS (bold arrows); (C) absence
of specific labeling using EBER-specific sense strand (control) probe In
the same case as (A). Paraffin sections, in situ hybridization, PSIlabeled RNA antisense probes, 24 (A) and 48 (B,C) hours of autoradiographic exposure. (Original magnifications x410 AS; x 120 E.)
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Fig 2. Detection of EBER (red substrate) and lgLCK gene transcripts (silver grains) by simultaneous in situ hybridization showing
EBER+/lgLCK- H-RS cells, an EBER+/lgLCK+small B cell (arrow) with
colocalization of both signals, and several EBER-/lgLCK+ small cells.
Paraffin section, in situ hybridization with PsSI-labeled RNA antisense
IgLoC probe and digoxigenin-labeled antisense EBER probes, APAAP
procedure, autoradiographic exposure5 days, original magnification x540.
Fig 3. Detection of EBERl and EBERZ transcripts in tissue from
mixed cellularity type HD. in situ hybridization preceded by i"unostaining with the antibody Ber-HZ (CD30). EBER transcripts are almost
exclusively associated with CD30+ atypical cells. The autoradiographic signal is heterogeneous, ranging from densely labeled cells t o
cells with no or only few grains due t o the short exposure time of 24
hours. (Cryostat section; original magnification x410.)
Fig 4. Detection of EBERl and EBERP transcripts in tissue from
mixed cellularity type HD, in situ hybridization preceded by immunostaining with the antibody L26 (CDZO). EBER transcripts are associated with CD20- atypical cells and few CD20+ small cells (arrows).
Cryostatsections, in situ hybridization, P?+labeled RNAantisenseprobes,
autoradiographic exposure 24 hours. (Originalmagnification x290.1
Fig 5. In situ hybridization with EBER-specific antisense probes
preceded by immunostaining with the antibody L26 (CD20). EBERspecific autoradiographic signal and immunostaining colocalized in a
small lymphoid cell. H-RS cells are EBER- (arrow). HD, nodular
sclerosing type, paraffin section, autoradiographic exposure 48 hours.
(Original magnification x 250.)
Fig 6. In situ hybridization
with EBER-specific antisense probe
preceded by immunostaining with the antibody L26 (CD20) showinga
cluster of EBER+ reactive cells with weak or absent immunohistological signal. HD, mixed cellularity type, paraffin section, autoradiographic exposure 48 hours. (Original magnification
x 120,)
Fig 7. Detection of EBERl and EBERP transcripts in a mixed
cellularity type HD case, in situ hybridization preceded by immunostaining with the antibody A6 (CD45RO). EBER transcripts in H-RS and
reactive cells are absent from CD45RO+ cells. Paraffin section, in situ
hybridization with [35S]-labeled RNA antisense probes, autoradiographic exposure 48 hours. (Original magnification x280.)
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HERBST ET AL
Table 3. Summary of Double-Labeling Results in a Case of Nodular
SclerosingType HD With EBER+ H-RS Cells
cells harbored EBV in H D cases with EBER+ H-RS cells.
In frozen sections, CD30 staining identifies practically all
H-RS cells in nearly all H D
This observation is of
Autoradiographic Exposure Time
importance in the context of previous demonstrations of
24 h
96h
monoclonality of EBV genomes in H D tissue extracts,5Y8-l0
EEER+/CD30+cells
90
96
and further supports the notion that infection of the
EBER+/CD30- cells
2
2
neoplastic cells by EBV must have occurred before their
EBER-/CD30+ cells
8
2
clonal expansion. The EBV infection is thus placed in an
EEER+and/or CD30+ cells
100
100
appropriate time frame to have a role in the etiology of HD.
Per 100 EEER+ and/or CD30+ cells; H-RS cells were identified by
LMP
expression in H-RS cells additionally supports this
morphology and immunophenotype at the first time point, and at the
concept
because of the transforming potential of this gene
second time point by CD3O-specific staining only, because the nuclear
product in several experimental ~ystems.4~
LMP, which is
morphologywas concealed by the autoradiographic signal.
variably expressed during the course of the cell cycle,44
upregulates bcl-2 protein expression and, by this mechacryostat sections (Fig 4). In paraffin sections, the propornism, prevents programmed cell death in B cells.45 The
tion of EBER+/CD20+ small cells was considerably lower
presence of monoclonal EBV genomes and LMP expresand the staining was reduced to weak signal intensity,
sion in H-RS cells is the most frequent abnormality with
whereas neighboring EBER-/CD20+ cells were still strongly
established
oncogenic potential among H D cases and
labeled (Figs 5 and 6). Furthermore, an HD case with
supports the concept of an etiologic role of EBV in the
CD20+/EBER+H-RS cells showed an intense CD20 stainpathogenesis of a significant proportion of H D cases.
ing of the EBER-labeled tumor cells, further excluding a
However, the presence of EBV in H-RS cells is restricted to
mere technical problem resulting in reduced immunoreaca subset of HD, and certainly more than one immortalizing
tivity of EBER+ small cells. Unambiguous reactivity of
or transforming mechanisms are required for the generaEBER+ small cells with the TcRp-specific antibody PF1 or
tion of malignant CD30+ cells. Therefore, more informathe antibody A6 (CD45RO) was not found in any of the
tion, particularly on the interaction of EBV gene products
tissues (Fig 7). EBER+ reactive cells were thus either of
with cellular genes and proteins, is needed to conclusively
B-cell phenotype (CD20+/IgLC+)or could not be assigned
assess the significanceof the presently available data for the
to a particular lineage.
pathogenesis of HD and other EBV-associated malignanDISCUSSION
cies.
EBV was not restricted to neoplastic cells in biopsies
Detection of the highly abundant EBER transcripts
from HD patients. Variable, usually small numbers of
represents a considerable technical advancement in the
EBER+ reactive lymphoid cells were found in HD cases
study of EBV infection, because it permits visualization of
with EBER+ or EBER- H-RS cells. Absolute numbers
practically all latently EBV infected cells in tissue sections.
could be determined precisely in cases without EBV infecIn contrast to DNA-DNA in situ hybridization, use of
tion of H-RS cells. In addition to the cytomorphologic
single-stranded antisense EBER-specific RNA probes and
aspect, sequential double-labeling using markers not exremoval of nonspecifically bound probe by ribonuclease
pressed by the neoplastic cell population proved helpful to
digestion result in low background signal and facilitate
estimate the frequency of EBER+ reactive cells in tissues
discrimination between EBV+ and EBV- ~ e l l s . 2It~ is
- ~thus
~
with EBER+ H-RS cells. Some cases did not display any
possible to address some of the questions that remained
EBER+ cells even in serial sections. This absence of EBER
open in previous studies, eg, as to the presence of EBV in
expression did not represent a problem of tissue preservamainly neoplastic or reactive cells in HD tissue biopsies,
and their number, distribution, and phenotype.
tion, because IgLC gene transcripts could be detected in
plasma cells and B cells. The presence of EBER+ reactive
In situ hybridization for the detection of EBER transcripts showed the presence of EBV in H-RS cells of 23 of
cells in only very small numbers, or even their absence, in
most cases is likely to explain why EBER+ reactive cells
46 H D cases that were not included in any of our previous
were not described in a previous report on EBER expresreports. Except for the two cases of HDlp, which may be
not representative, HDmc was the histological type of the
sion in HD.26 With respect to the normal lymph node
tissues, similar results were obtained in a study focusing on
disease most frequently associated with the virus, followed
by HDns. These results are well in agreement with a
nonmalignant lymphoid lesions.& The observation of
recently published study describing EBER+ H-RS cells in
EBER+ cells in only a low proportion of normal lymph
nodes is in apparent contradiction to the fact that world11 of 23 cases of typical HD.” LMP was detectable by
immunohistologyin variable proportions of EBER+ neoplaswide more than 90% of the population is seropositive for
tic cells of 18 cases. This figure supports the view that
EBV-specific antibodies. Quantitative evaluation of EBVprevious studies using EBV DNA in situ h y b r i d i z a t i ~ n ~ - ~ l Jinfected
~
peripheral blood cells, on the other hand, showed a
or LMP i m m u n o h i ~ t o l o g yslightly
~ ~ ~ ~ underscored
~~~~
the
very low frequency of EBV+ cells (1 to 5 infected cells per
exact proportion of HD cases with EBV-harboring tumor
lo7 mononuclear leukocytes) in healthy donors,” which is
cells, whereas PCR studieW3 may have overestimated this
in good agreement with the histomorphological findings.
figure. Extension of the autoradiographic exposure time in
Furthermore, antibody titers are unlikely to be determined
combination with CD30-specific immunostaining verified
only by EBV infection of lymphoid cells, because epithelial
that virtually all, and not only a subset, of the neoplastic
cells also represent targets for this herpes
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EBV IN HODGKIN’S DISEASE
489
capsid a~~tigen,l~?~O
and gp250/350 membrane antigen13)
Counting of EBER+ cells per tissue section area showed
from any cells in HD tissues.
elevated numbers ( > 100) of such cells in all cases with
EBER+ H-RS cells and, additionally, in three cases with
In general, the majority of EBER+ cells were of (CD20+)/
EBER- neoplastic cells, ie, in 26 of 46 HD cases (56%;
IgLC+/LMP- phenotype, whereas EBER+/TcRP+ or
EBER+/CD45RO+ cells, ie, mature T cells, were not
Table 2). This proportion is in good agreement with the
This raises the possibility that immature lymresults of several semiquantitative PCR s t u d i e ~ ~ ~ J ~ detectable.
J~J~
phoid cells, ie, cells with incomplete or missing antigen
showing increased numbers (104 internal repetitive fragreceptor gene rearrangements cells, were among the
ment target sequence^^^,^^) of EBV genomes in 55% to 60%
EBER+/IgLC- subp~pulation.~~
However, our estimate of
of the cases. The results presented here support the
the proportions of EBER+ B and T cells is largely based on
previous interpretation of PCR data that an increased EBV
cases with elevated numbers of EBER+ reactive cells,
genome content of HD tissues is most likely related to the
presence of EBV in tumor cells, and in only rare instances
which may not be representative for HD cases with small
due to proliferations of EBV-infected reactive cells.1*
numbers of EBER+ reactive cells. The presence of CD43+
Simultaneousdouble-labelingexperiments helped to charEBV-infected cells in addition to occasional B cells was
acterize the majority of EBER+ reactive cells as IgLC+
recently observed in some cases of HD, and interpreted as
mature B lymphocytes. The presence of both types of IgLC,
evidence for a T-cell nature of these CD43+ cells.27However, CD43 antigen is not lineage-specific; moreover, it is
K and A, indicates an oligoclonal or polyclonal composition
found on many EBV+ lymphoblastoid B-cell lines.52Thus,
of these cells; however, it does not provide information
about the size of individual clones. H-RS cells did not
whereas EBER+/TcRP+/CD45RO+small cells arising in
certain pediatric immunodeficiencysyndromes can be idendisplay IgLC transcripts, which supports previous observatified with our methodology (H. Herbst, unpublished obsertion~:~suggesting their origin from immature B cells, T
vation), it must be concluded that such EBER+ mature T
cells, or other cells6 Immunophenotyping of reactive
EBER+ cells was problematic and, particularly in paraffin
cells, if present at all, appear to be extremely rare in lymph
nodes from normal adults and HD patients.
sections, virtually impossible for the vast majority of these
The frequency of EBER+ cells in the reactive admixture
cells. In frozen sections, most of the small EBER+ cells
was slightly higher when compared with nonhyperplastic
could be stained with the antibody L26 (CD20). However,
control lymph nodes, but their phenotypic properties were
in paraffin sections, the number of these cells was reduced.
similar. The two groups of HD, with or without EBVCD20+/EBER- reactive cells and CD20+/EBER+ tumor
harboring H-RS cells, respectively, did not differ from each
cells, on the other hand, could be detected easily with our
other with regard to the occurrence, frequency, and phenoimmunohistological procedure. These observations, in contype of reactive EBER+ cells. Thus, in the majority of HD
junction with the IgLC+ phenotype of most EBER+ small
patients, the postulated specific isolated immune defect
cells and the established immunophenotype of resting and
that permits proliferation of tumor cells expressing a viral
activated, noninfected B lymphocytes as constantly CD20+
neo-antigen, LMP, is obviously not accompanied by an
cells,@ led us to conclude that expression of the CD20
unrestricted proliferation of reactive EBER+ lymphoid
antigen may be downregulated in latently EBV-infected
cells. This is in sharp contrast to the pattern seen during
small lymphoid cells in vivo. The CD20 antigen directly
infectious mononucleosis,53 as well as in lymph nodes of
regulates transmembrane ion flux in B lymphocytes and is a
key structure for antigen-independent B-cell activati0n.4~ immunosuppressed transplant recipients or HIV-infected
individuals,who generally display larger numbers of EBER+
Downregulation of this activation-associated antigen may
cells.&It is thus likely that there are distinct immunological
thus have specific advantages for EBV-harboring cells. To
differences between patients with global immunodeficientest this hypothesis, it will be necessary to quantitate the
cies, which are at risk to develop EBV-induced oligoclonal
density of CD20 and other antigens on suspended cells, and
or monoclonal non-Hodgkin’s lymph0ma,5~9~~
nonisotopic EBER-specific labeling may facilitate the idenon the one
tification of EBV-infected cells in such experiments. LMP
side, and HD patients on the other.
expression was not observed in any of the reactive cells,
ACKNOWLEDGMENT
which is in keeping with published data,13.z1and may
facilitate evasion of these cells from immune recognition.
We thank H. Protz, T. Finn, and B. Nolte for technical
This interpretation is also supported by the previously
assistance, and L. Oehring for phototechnical assistance. Dr J.
established absence of the EBNA2 latent gene prodArrand kindly supplied us with EBER plasmids, and Dr P. Leder
generously provided phages with IgLC gene constant segments.
U C ~ ~and
~ EBV
, ~ ~lytic
, ~cycle
~ antigens (early antigen,5O viral
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1992 80: 484-491
Distribution and phenotype of Epstein-Barr virus-harboring cells in
Hodgkin's disease
H Herbst, E Steinbrecher, G Niedobitek, LS Young, L Brooks, N Muller-Lantzsch and H Stein
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